摘要:

Analyst, January 1996, Vol. 121 (37-41) 37 Study of the Chemiluminescent Characteristics of Ninhydrin and its Application Guo Nan Chen, Xue Qin Xu and Fan Zhang Department of Chemistry, Fuzhou University, Fuzhou, Fujian, 350002, China Ninhydrin was found to exhibit chemiluminescence (CL) when it was oxidized with hydrogen peroxide in neutral or weakly acidic solution. Copper(n) and Con can catalyse the CL reaction, on the basis of which ppb levels of CU" and Co" can be determined. In the presence of Cu", some amino acids enhanced the CL reaction, whereas others inhibited the CL reaction in the presence of Co". A flow-injection system with CL detection was developed for the determination of some amino acids. The mechanism of the CL reaction is discussed. Keywords: Ninhydrin; chemiluminescence; amino acid; flow injection Introduction Ninhydrin has been widely used as a reagent for the determina- tion of amino acids by spectrophotometry;1,2 however, its chemiluminescent characteristics have not been studied. It was found that when ninhydrin was oxidized with hydrogen peroxide in neutral or weakly acidic solution, chemilumines- cence (CL) was observed with a peak at 436 nm.This CL reaction proceeded very rapidly and its intensity was pH- dependent. An Na2HP04-KH2P04 buffer solution was used in this work, and the CL intensity was found to be constant between pH 6.7 and 7.0. In this pH range, the effects of more than 30 metal ions on the CL reaction were investigated; it was found that only Cu" and Co" could catalyse the CL reaction, and that the catalytic CL intensity was proportional to the concentra- tion of Cu" and Co".A CL method could, therefore, be established for the determination of Cull and Co" at the ppb level. Chemiluminescence is one of the most sensitive methods for the determination of amino acids, and many CL systems have been used for this purpose, the peroxyoxalate and luminol systems being the most common. As ninhydrin reacts with amino acids quantitatively under suitable conditions, proce- dures for the determination of amino acids were developed based on the CL reaction of ninhydrin. In addition, a flow- injection (FI) system with CL detection was developed for the determination of some amino acids. Experimental Apparatus A Lambda 9 spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) and an RF-540 fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) were used. The FI system for the determination of amino acids consisted of an HFC-1 chem- iluminescent detector, an LZ-2000 Flow Injection Processor and a recorder. Details of the HFC- 1 chemiluminescent detector and LZ-2000 Flow Injection Processor can be found in refs.3 and 4, respectively. The FI manifold is shown in Fig. 1. One of the dual peristaltic pumps (A) was used to pump the reagents (only three channels were used), while the sample solution was delivered to the injection valve by pump B. Reagents All reagents were of analytical-reagent grade or better and water doubly distilled in a fused-silica apparatus was used through- out. Ninhydrin solution. A stock solution (1 X 10-2 moll-l) was prepared by dissolving 1.7814 g of ninhydrin (AR, Shanghai Chemical) in 100 ml of water and diluting to 1000 ml with water.This solution was diluted further as required. The amino acids used were Asp, Asn, Thr, Glu, Gly, Val, Met, Ile, Leu, Tyr, Phe, His, Lys, Trp, Pro, Gln, Hyp and Arg (Shanghai Chemical). These amino acids were dissolved in water and diluted to 1 X 10-2 mol 1-1 to give a stock solution. Procedure Procedure for investigating the CL characteristics of ninhydrin Ninhydrin, buffer (or NaOH), Cu" (or Co") solutions and water were added to the reaction cell in turn by pipette to make the total volume up to 2.05 ml and mixed. The cell was then placed in the detector chamber. The shutter was opened and the zero point of the recorder was adjusted.Then, 0.3 ml of H202 solution was injected into the reaction cell and the CL signal was recorded. Procedure for determination of amino acids The procedure consisted of three steps; step 1: buffer + Cut' (or Co") (Rl), H202 (I) (R2) and H202 (11) (R3) were continuously pumped into the manifold by pump A at flow rates of 4.2, 5.1 and 3.75 ml min-1, respectively, for 20 s (during this step pump B was stopped); step 2: pump A was stopped, and pump B was used to load ninhydrin + amino acid (R4) into the injection valve at a flow rate of 3.75 ml min-1 for 15 s; step 3: pump B was stopped, and R1, R2 were pumped into the CL detector by pump A for 20 s, at the same time R3 was pumped into the injection A Fig. 1 injection valve; R, recorder; W, waste The FI manifold.A, Pump A; B, pump B; CD, CL detector; V,38 Analyst, January 1996, Vol. 121 valve as the carrier to take & in the injection valve loop to the CL detector, and the CL signal was then recorded. Results and Discussion Initial Investigation of the Ninhydrin-HzOz CL Reaction The initial investigation showed that when ninhydrin was oxidized by H202 in a suitable medium, CL was observed. The reaction was instantaneous; the maximum CL intensity was reached in 4 s. The effect of pH on the CL reaction of ninhydrin was examined, and it was found that the reaction was pH- dependent. H3B03-KC1-Na2CO3 buffer solution (pH 7.4-1 1 .O), Na2HP04-KH2P04 buffer solution (pH 5.2-8.3) and 0.5 moll-1 NaOH solution were used to examine the variation in the CL of the reaction.It was found that CL could be observed over a wide pH range; however, the CL intensity was strongest under neutral or weakly acidic conditions. The Na2HP04- KH2P04 buffer solution was therefore used as the reaction medium and the pH was fixed at 6.1 as this gave the strongest CL intensity. The experiment also showed that the optimum concentration of H202 was 0.223 mol 1-l (concentration in cell). The effects of more than 30 metal ions on the CL reaction were examined under the above-mentioned conditions. The results are shown in Table 1 from which it can be seen that only Cu" and Coil can catalyse the CL reaction effectively; therefore, a detailed investigation of the effects of Curl and CO" on the CL reaction was carried out and the results are presented below.Catalytic Effect of Cu" and Co" on the Ninhydrin-H202 CL Reaction As mentioned above, the reaction between ninhydrin and H202 gave CL over a wide pH range, which was called the Table 1 Effect of metal ions on the ninhydrin-H202 CL reaction* Ion c/mg 1-1 h/mm h/ho Ion c/mg 1-I hlmm h/ho Bim Tall Au"I Pd" Al"' Rh" u022+ VV Fellr SnIv Pt" Hg" AsV TeIV Smrrr YbUi CU" Pb" ym 0.37 1.72 0.138 0.357 3.75 0.25 0.357 0.357 0.212 0.106 0.212 0.106 0.212 0.212 1.06 0.021 0.212 35.7 31.7 37.5 0.75 54.5 1.09 37.5 0.75 39.5 0.79 37.0 0.74 39.0 0.78 46.5 0.93 4.10 0.82 4.10 0.82 45.9 0.92 28.0 0.56 48.5 0.97 43.0 0.86 46.5 0.93 51.0 1.02 59.0 1.18 167.8 3.35 56.0 1.12 30.5 0.61 562 0.69 3.73 1.78 0.357 0.212 0.357 0.357 0.2 12 1.06 0.106 0.212 0.2 12 0.2 12 0.02 1 0.212 0.25 16.3 33.5 0.67 34.5 0.69 31.0 0.62 37.5 0.75 36.5 0.73 36.5 0.73 51.0 1.02 34.0 0.68 36.0 0.72 38.5 0.77 20.5 0.41 44.0 0.88 31.0 0.62 52.5 1.05 52.0 1.04 73.0 1.46 39.0 0.78 616.0 12.3 * c: concentration of metal ions; h: CL peak height; ho: background CL peak height, ho = 50.0 mm in this experiment.background CL. When the catalytic effect of metal ions on the CL reaction is to be examined, it is advantageous to decrease the background CL in order to emphasize the catalytic effect of the metal ions. Therefore, the conditions for catalytic CL will be different from the optimum conditions mentioned above for the initial investigation. Three main factors, viz., pH, concentration of H202 and ninhydrin, were examined at this stage. In consideration of the interdependence of these factors, they could not easily be optimized by traditional methods.The modified simplex method, however, can readily be adapted to solve such problems, and was therefore used for this purpose. The optimum conditions found for the Cu" and Corl systems using the modified simplex method are listed in Table 2. Mechanism of the Chemiluminescent Reaction Treatment of ninhydrin by HMO method In order to probe the mechanism of CL, ninhydrin was treated by the Huckel Molecular Orbital (HMO) method. For conven- ience in the discussion, the carbon and oxygen atoms of ninhydrin are numbered as follows: lo The n-electron density distribution and the bond strength are shown in Table 3, from which it can be seen that the x-electron density at C-2 is the lowest; therefore, the radical anion 0 2 - * would attack the C-2 position first.Moreover, the calculation showed that the bond strength between C-1 and C-2 was the smallest; thus after 02-• attacks C-2, it is easier to form a peroxide ring between C-1 and C-2. In general, the occurrence of CL from organic compounds is due to decomposition of a peroxide ring. CL, fluorescence and UV spectra Fig. 2 shows the CL spectra of ninhydrin with the maximum at 436 nm [see Fig. 2, A]; after the addition of Cu" or Co", the CL Table 2 Optimum conditions for Cu" and Co", linear response range and detection limits* 410-3 CJ Ion pH moll-' moll-' LRR/gml-I DL/g ml-l Co" 6.9 3.45 0.388 1.0 X 10-9-1.0 X 10-8 5.0 X 10-10 Cull 6.7 3.0 0.378 5.0 x 10-9-1.0 x 10-6 1.0 x 10-9 * cl: concentration of ninhydrin; c2: concentration of H205 LRR: linear response range; and DL: detection limit.Table 3 n-electron density distribution and bond strength of ninhydrin* Atom No. 1 2 3 4 5 6 7 8 9 10 11 12 x-edd 0.6290 0.5626 0.6290 0.9248 0.9099 0.9100 0.9100 0.9099 0.9247 1.5936 1.5025 1.5936 Atom No. 1-2 1-9 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9 4 10-1 11-2 12-3 Bond strength 0.2432 0.4314 0.2432 0.4314 0.6148 0.6612 0.6362 0.6612 0.6148 0.4854 0.7087 0.7931 0.7087 * x-edd: n-electron density distribution. Parameters: ho = 2; h, = 0.2; K, = 1.4.Analyst, January 1996, Vol. 121 39 intensity increased, but there was no change in the shape and peak position of the spectra [see Fig. 2, B and C]. The fluorescence emission spectra of the product are shown in Fig.3. Fig. 3, A, shows the fluorescence spectrum of ninhydrin, its peak maximum is at 438 nm; Fig. 3, B, is the fluorescence spectrum of the product after adding Hz02; the fluorescence intensity is greatly increased in the presence of Co'I or Cu" [see Fig. 3, C]. From a comparison of Figs. 2 and 3, it was found that the CL spectrum of ninhydrin was very similar to that of the product. 14 10 c .- C c 2 p tu % a 6 a, C v c .- c - 2 C \ 360 400 440 480 520 560 Wavelength/nm Fig. 2 Chemiluminescent spectra of ninhydrin. A, Ninhydrin (3.45 X 10-3 mol 1-')-buffer solution (pH 6.90); and B, A + H202 (0.382 moll-'); and C, B + Col* (20 mg 1-I). C 80 h c .- 5 60 P s c 2 v .- 40 c C - 20 0 400 450 500 550 600 Wavelength/nm Fig. 3 Fig. 2. Fluorescent emission spectra of the product he, = 350 nm, A-C as The UV spectra of ninhydrin and its oxidation product were measured and are shown in Fig.4. There are two characteristic absorption bands at 232 and 255 nm before the addition of H202 [see Fig. 4, A], which are mainly due to the presence of a five- membered ring with ketone groups. However, the characteristic absorption bands at 232 and 255 nm gradually disappear after the addition of H202, and only the absorption band of the benzene ring can be observed [see Fig. 4, B], which indicates that the five-membered ring is oxidized by H202. Mechanism Based on the results of the treatment by the HMO method and the investigation of the CL and UV spectra, a possible mechanism for the CL reaction of ninhydrin can be proposed. The radical anion 0 2 - * attacks C-2 first, a peroxide ring between C-1 and C-2 is then formed.The peroxide is further decomposed by H202 to produce an excited intermediate, which will emit light when it returns to the ground state. The mechanism can be shown as follows: a t 8-H i- c@ ---+ + hv b Application to the Determination of Amino Acids Ninhydrin can react with amino acids under heating to give a blue-violet product, Ruhemann Violet, which has a maximum absorption at 570 nm; therefore, ninhydrin has been widely used as a reagent for the determination of amino acids. We found that the product, Ruhemann Violet, did not give CL when it was oxidized by H202; hence, in the presence of excess of ninhydrin, amino acids can be determined indirectly by examining the A 0.48 h c v) .- 5 0.32 c $ g) 5 a 2 m v f! 0.16 200 240 280 320 Fig.4 The UV spectra of ninhydrin and its oxidation product. A, Ninhydrin (5.0 X 10-3 mol l-')-buffer solution (pH 6.90); and B, A + H202 (0.382 mol 1-I).40 Analyst, January 1996, Vol. 121 variation in the CL intensity of the reaction between ninhydrin and the amino acids. Moreover, we also found some interesting phenomena, which are described below. 1. In the presence of Cu", when some amino acids were added to and heated with ninhydrin, and the resulting solution was reacted with H202, it was found that the CL intensity of the system was greatly enhanced, and that the enhancement was proportional to the concentration of amino acids. 2. In the presence of Co", when some amino acids were added to and heated with ninhydrin, and the resulting solution was reacted with H202, it was found that the CL intensity of the system was greatly inhibited, and that the inhibition was proportional to the concentration of amino acids.Based on these properties of ninhydrin, the possibility of the determination of amino acids by using these CL reactions was investigated, and procedures for the determination of amino acids by FI with CL detection were developed. Ninhydrin-H202-Cu11 system for determination of some amino acids Treatment of sample. A 1 ml volume of amino acid solution was placed in a 15 ml test-tube, and 1 ml of 1 X moll-' ninhydrin solution was then added and mixed. The test-tube was plugged with cotton, and placed in a boiling-water bath for 15 min; it was then cooled with water and set aside for 5-10 min, after which the contents were diluted to 5 ml with water.The solution obtained was used as the sample solution for FI with CL detection. Selection of conditions. It was found that Trp, Gly, Glu, Phe, Gln, Pro, Asp, Leu, Thr, Ile, Val and Lys exhibited an enhancement for this CL system, and that the enhancement was dependent on the pH of the buffer solution, and on the concentrations of H202, Cu" and ninhydrin. Trp is taken as an example for selection of the optimum conditions. The effect of pH on the determination of Trp is shown in Fig. 5, from which it can be seen that the enhancement of the amino acid CL is pH-dependent, and that the optimum enhancement is obtained at pH 6.70. The optimum concentration of Cu" is 20 mg 1-1 (see Fig.6). As regards the concentration of H202, good results were obtained between 0.29 and 0.59 mol 1-l; 0.44 mol 1-1 H202 was chosen subsequently (see Fig. 7). Fig. 8 shows that the enhancement of amino acid CL is proportional to the concentration of ninhydrin; therefore, the concentration of ninhydrin can be selected based on the concentration of amino acid in the sample. In general, ninhydrin should be in excess; a 10-fold excess would be suitable. Relative standard deviation, linear response range and detection limit. Under the selected conditions, the s, (n = 10) for 4.0 X 10-6 moll-1 of Trp was 1.7%. The linear response range and the detection limit of 12 amino acids were examined and the results are shown in Table 4. 6 6:3 6.5 6.7 6.9 7.2 7.5 PH Fig.5 Effect of pH on determination of Trp. 10, background CL of ninhydrin; I, enhanced CL, A I = I - I. [Cu"] = 20 mg 1-1; [ninhydrin] = 3.33 X 10-3 moll-1; [Trp] = 2.0 X 10-4 moll-'; and [H202] = 0.294 mol 1-1. Ninhydrin-H202-Co1[ system for determination of some amino acids For the ninhydrin-H2O2-CoI1 system, His, Arg, Tyr, Asn, Met and Hyp were found to inhibit the CL reaction; the inhibition was dependent on the pH of the buffer solution, and on the concentrations of H202, Co" and ninhydrin. The treatment of the sample solution was the same as that for the ninhydrin-H202-Cu11 system. Taking His as an example for - 0 0 2i 5 14 20 25 30 35 2 [Cu"]/mg I-' Fig. 6 Effect of Cu" on determination of Trp. Io, background CL of ninhydrin; I, enhanced CL; AI = I - I,; pH 6.70; [ninhydrin] = 3.33 X 10-3 moll-1; [Trp] = 2.0 x 10-4 moll-l; [H202] = 0.294 moll-'.l o r - o 0.144 0.3 0.384 0.408 0.6 7 [H,O,]/mol I-' Fig. 7 Effect of H202 on determination of Trp. 10, background CL of ninhydrin; I, enhanced CL; A I = I - IO [Cu"] = 20 mg 1-l; [ninhydrin] = 3.33 x moll-l; [Trp] = 2.0 x moll-I; pH 6.70. 12 r & In 10 .- ? * c S 6 = 4 2 0 I A1 0 1 2 3 4 5 6 7 [Ninhydrin]/mmol 1-l Fig. 8 Effect of ninhydrin on determination of Trp. lo, background CL of ninhydrin; I, enhanced CL; A I = I - IO [Cu'l] = 20 mg 1-I; pH 6.70; [Trp] = 2.0 x 10-4 mol 1-1; [H202] = 0.441 mol 1-1. Table 4 Linear response range and detection limit of 12 amino acids Amino acid Trp GlY ASP Phe Gln Leu Glu Pro Ile Thr Val LYS Linear range/mol 1- 1.0 x 10-6-1.0 x 10-4 1.0 x 10-5-1.0 x 10-4 1.0 x 10-6-1.0 x 10-4 1.0 x 10-5-1.0 x 10-4 1.0 x 10-6-1.0 x 10-4 2.0 x 10-5-2.0 x 10-4 1.0 x 10-5-1.0 x 10-4 2.0 x 10-6-1.0 x 10-3 1.0 x 10-6-1.0 x 10-3 1.0 x 10-5-1.0 x 10-4 1.0 x 10-6-1.0 x 10-4 1.0 x 10-6-1.0 x 10-4 Detection limit/mol I-' 2.7 X 6.8 x 10-6 2.1 x 10-6 5.4 x 10-6 4.8 x 10-7 5.9 x 10-7 - 4.5 x 10-7 4.2 x 10-7 4.2 x 10-7 5.1 x 10-7 2.9 xAnalyst, January 1996, Vol.121 41 Table 5 Linear response range and detection limit of six amino acids Amino acid Linear range/mol1-1 Detection limit/moll-l His 2.0 x 10-6-1.0 x 10-3 6.1 x 10-7 A% 1.0 x 10-54.0 x 10-4 4.0 x 10-6 Asn 1.0 x 10-6-4.0 x 10-4 - Met 2.0 x 10-5-1.0 x 10-4 9.2 x 10-6 TYr 1.0 x 10-5-1.0 x 10-4 4.2 x 10-6 2.0 x 10-6-8.0 x 10-4 4.5 x 10-7 HYP selecting the optimum conditions, the results showed that the optimum conditions for the inhibition of the CL reaction were: pH 6.9; [HZ021 = 0.29 mol 1-1; [ninhydrin] = 3 X 10-3 moll-1; and [Cotl] = 14 mg 1-1. Under the optimum conditions, the s, found (ten replicates) for 6 X 10-6 mol 1-1 of His was 1.7%. The linear response range and the detection limit of six amino acids were examined and the results are shown in Table 5. This work was financially supported by the State Education Commission, China, and the Natural Sciences Foundation of Fujian Province, China. References 1 Zarkadas, C. G., Can. J . Biochem., 1975,53, 96. 2 Zhong, H. S., and Guang, J. S., Biochemistry, Gaodeng Jiaoyu Press, Beijing, 1990, p. 64. 3 Chen, G. N., Duan, J. P., and Hu, Q. F., Anal. Chim. Acta, 1994,292, 159. 4 Chen, G. N., Duan, J. P., and Hu, Q. F., Mikrochim. Acta, 1994, 116, 227. Paper 5l047.586 Received July 19, 1995 Accepted September 7, I995

ISSN:0003-2654    

DOI:10.1039/AN9962100037

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, January 1996, Vol. 121 (43-48) 43 Existence of Two Basic Sites in Triazolo-I ,4=diazepines: Determination of Two p& Values for a Model Compound in Water Beatrice Legouin and Jean-Louis Burgot UFR des Sciences Pharmaceutiques et Biologiques, Dkpartement d' Etudes Physicochimiques et BiocinCtiques des Pharmacosyst2mes, Laboratoire de Chimie Analytique, 2 av. du Pr Lkon-Bernard, 35043 Rennes-Cedex, France By a UV/VIS spectrophotometric study in the pH range -1.6 to 10.1 and by a polarographic study of a water soluble model compound, the occurrence of two basic sites in water has been ascertained for triazolo-1,4-diazepines. The pK, values found for this model were -0.24 and +1.81. Owing to the overlapping of the two pK, values, microforms exist simultaneously. Corresponding ionization microconstant values have been tentatively assigned. Keywords: Triazolo-I ,4-thienodiazepine; ultraviolet-visible spectrophotometry ; polarography; pK,; ionization microconstant Introduction 1,4-Benzodiazepines are compounds of important phar- maceutical interest.' It is now well known that their 4,Sazome- thine bond suffers hydrolysis in acidic media to give the corresponding aminobenzophenones, which reversibly cyclize into the original closed forms by elevating the pH of the solution.As a result, 1,4-benzodiazepines exist in such media as pH-dependent, equilibrated mixtures of ring opened and closed forms.2-12 Given the fact that these compounds generally possess multibasic sites, this hydrolytic process is accompanied by acid-base reactions. Hence, the knowledge of the different ionization constants is of utmost importance both for carrying out mechanistic studies concerning their hydrolytic process and for firmly grounding their pharmacological properties.Some of these diazepine derivatives have their 1,2-bond fused to a 1,2,4-triazolo ring. This is the case with alprazolam, estazolam and triazolam, which are triazolobenzodiazepines, and also with brotizolam, etizolam and of the We'973 compound of Gallo et al.,l which are triazolothienodiaze- pines. R I = H R,=H ESTAZOLAM R, = CH3 R2 = Br BROTIZOLAM RIeCHj Rt=Cl TRIAZOLAM R, = C6H1, R2 = Bt We'973 R, = CH3 R2= H ALPRAZOLAM R, = CH, Rt = CzHs ETEOLAM The pK, values in water of these triazolodiazepines (2.76, 2.76,2.84 and 5.9,2.26 and 1.52 for brotizolam,8,11 etizolam,*O estazolam10.12 and triazolam,6.9 respectively) are systematically attributed to the conjugated acid form of the imine group.It is important to recall that these determinations were performed with low percentages of methanol in water. Surprisingly, no PKa data concerning the basic character of the triazolo ring have been given in the aforementioned works, although the conju- gated acids of simple derivatives of this heterocycle exhibit pK, values falling in the 2.2-3.4 range.13 As a result, owing to the overlapping of these two ranges of pK,, the attribution of the above pKa values to the azomethine group of triazolodiazepines that has been carried out so far is questionable. In this paper we demonstrate that triazolodiazepines actually possess two pK, values.We used for our demonstration a UV/ VIS spectrophotometric study of the following compound: 4,7,8,10-tetrahydro- 1 -methyl-6-(2-chlorophenyl)-[4',3'- 4,5]pyrid0-[3,2--thieno- 1,2,4[4,3-a]-triazolo- 1,4-diazepine (NHPTT)14: NHm 3.5-DLM~HYL4-PHENYL-l.2.4-TRIAZOLE The pK, value of the protonated imine group of this model compound was tentatively assigned by a polarographic study and that of the triazolo ring by an independent determination of the pKa value of 3,5-dimethyl-4-phenyl- 1,2,4-triazole. The aqueous solubility of NHPTT, conferred by the protonation of the piperidino moiety in the pH range of our study, allowed us to work without using any additional organic solvent. Experimental Apparatus Measurements of pH were performed by use of a Tacussel LPH430T pH meter that was calibrated daily with six NBS buffers (commercial buffers manufactured according to the National Institute for Standards and Technology recom- mendations) and using an Ingold 9811 (pH 0-14) glass electrode.All UV/VIS spectra were recorded by using a Uvikon 930 spectrophotometer with 1 cm silica cells. Dc and differential-pulse polarograms were recorded on a Tacussel EPL3. Potentials were measured versus a saturated44 Analyst, January 1996, Vol. 121 calomel electrode (Tacussel XR 100). The following conditions were used: scan rate, 5 mV s-l; drop time, 2 s; pulse duration, 60 ms; pulse amplitude, 60 mV. Reagents The water used throughout this work was de-ionized by a set of ion exchanging columns (Bioblock Scientific, Illkirch, France) to p > 2 MQ cm-1.NHPTT was kindly purchased for us by Beaufour-Ipsen Industry.14 3,5-Dimethyl-4-phenyl- 1,2,4-triazole was synthe- sised according to Reiter's method.15 Physical properties were in full agreement with those given in the literature. The buffers for solutions of pH >2 were the Britton- Robinson buffers.16J7 Table 1 Determination of the pKa of the piperidino moiety : results obtained by treating spectrophotometric data for pH values from 6.51 to 10.1 (spectra recorded immediately after the preparation of solutions) C/moll-' A = 2 4 0 m A = 275 nm 10-4 ~Ka3 EB" EBH+ 1.5 x 10-4 p~~~ EB EB (experimental) EBH+ (experimental) * E in 1 mol-1 cm-1. 7.96 r7.86. 8.111 14 000 [13 900; 14 1001 16 200 [16 000; 16 4001 8.14 [8.10; 8.171 14 550 [13 500 13 6001 15 650 [ 15 600; 15 7001 13 850 16 500 8.25 [8.18; 8.331 6750 [6700; 68001 5400 [5350; 55001 8.09 [8.07; 8.121 6550 [6500; 66001 5380 [5360; 54001 6200 5100 BH 3'.For solutions of pH < 2 Bascombe and Bell's acidity functions18J9 (aqueous sulfuric acid solutions) were used. Methods All measurements were performed at 25 "C. Care was taken to avoid the opening of the benzodiazepine ring (by hydrolysis) in acidic media. Preliminary polarographic studies had indicated that, in acidic media (--1 < pH < 3), no 'opened' product could be detected for time durations below 1 min from the beginning of the preparation of solutions ('closed' and 'opened' products exhibited two very well differentiated waves). For UV/VIS studies we selected two working wavelengths, which differed from the pH ranges of the solutions studied: for -1.6 < pH < 3.1, h = 250 nm and h = 300 nm, and for 6.5 < pH < 10.1, h = 240 nm and h = 275 nm.Two wavelengths were selected for the sake of comparison of the K, values which, of course, had to be the same. For the same reason, we selected two analytical concentrations: 1 X mol I-' and 1.5 X 10-4 moll-'. In the two ranges -1.6 < pH < 3.1 and 6.5 < pH < 10.1, 19 and 16 working pH were used, respectively. Moreover, for each pHj value, 6 measurements on theoretically identical (but prepared in a totally independent way) solutions were replicated. Solutions were prepared by mixing aliquots of 2 ml of a stock solution of NHPTT (1 X 10-3 or 1.5 X 10-3 moll-1) with 18 ml of the appropriate buffer.Spectra were recorded in the 190-300 nm range at fast scan (scan speed, 2000 mm min-1) against the corresponding blank. The whole process (mixing of solution and recording) did not exceed 1 min. The fact that with our data treatment we obtained by calculation (see below) the same molar absorptivities of species BH+ for the most acidic media as those found experimentally at higher pH values was a strong argument in favour of the existence of only the closed form in the experimental conditions. In our calculations, unknowns were K, values and, when the species could not exist alone in solution, E values. Moreover, in some cases, we deliberately considered E values as supplementary unknowns. This provided a way of checking the accuracy of our data and calculations by comparison of the experimental and H' "p Ka, &" BH' OC' B &"' B*H,*+ Fig.1 Investigated ionization pathways of NHPTT. * The site of protonation on this nitrogen atom of the triazole was arbitrarily chosen.Analyst, January 1996, Vol. 121 45 I I -0.4 calculated E values. Treatment of experimental data was performed by two non-linear squares procedures. In the first one, data which were simultaneously treated by the regression procedure were chosen in such a way that for each pHi, one absorbance AFp was kept at random among the six replicates. All the selected data allowed the determination of the search for parameters by minimization of the cost function: I I 3.00 I 2.00 1 .oo 0.00 200 250 300 350 Wavelengthhm Fig.2 Absorption spectra of NHPTT in solutions of different pH: - 1.6; -1; -0.5; 0; +0.5; +l; +1.5; +2; +2.5; +3. (Scan speed 2000 nm min-I; path length 1 cm; C,,, = mol I-'). Table 2 Determination of the pKa of the imino and triazolo groups: results obtained for the first ionization scheme at different concentrations and wavelengths for pH between - 1.6 and +3.1 (same experimental conditions as in Table 1) EBH+* 1.5 x 10-4 PK,, EBH+ EBH+ (experimental) (experimental) EBH33+ * E in 1 mol-1 cm-1. h = 250nm -0.36 [-0.45; -0.261 1.70 [1.63; 1.791 16 050 [15 900 16 2001 12 400 [ 12 250; 12 5001 9 600 [9450; 97001 -0.28 [-0.39; -0.151 1.69 [1.59; 1.801 15 550 [14 480; 15 6501 12 450 [12 250; 12 6001 9 750 [9650; 99001 15 850 9 550 A = 300nm -0.10 [-0.18; 01 1.92 [1.89; 1.941 2 650 [2600; 27001 9 350 [9200; 95001 13 OOO [12 850; 13 2001 -0.24 [-0.32; -0.131 1.82 [1.79; 1.851 2 700 [2650; 27501 9 600 [9400; 98001 13 250 [13 100; 13 4001 2 500 13 300 where A?'" stood for the absorbance calculated according to the model and wi was a weighting factor defined classically by wi = 1/02, a? being the variance of the whole of the replicates of absorbance measurements for a given pHi value.Strictly, the same process was repeated seven times. Variances for the parameters were calculated from the preceeding results in the usual manner. In the second procedure, pooling of the 19 X 6 and 16 X 6 data was performed. The objective function was calculated according to Weighting factors were the same as above. The variances of the search for parameters were calculated through the variance- covariance matrix which takes place naturally during the process of minimization of the U function.It is relevant to note that the variances found with the two procedures (see results) were the same. The algorithm of minimization of the U function that we used was a direct one, according to the Hooke and Jeeves20 principle. For polarographic studies, systematic deoxygenation of solutions was accomplished by the bubbling of purified nitrogen through the buffer and through the stock solutions separately and by passing nitrogen over the solution in the cell throughout the experiments. The concentration of solutions in the cell was maintained at 1 X 10-3 moll-'. The lack of electroactivity of 3,5-dimethyl-4-phenyl- 1,2,4-triazole in our experimental con- ditions had been previously ascertained by an independent polarographic study.The triazolo ring of estazolam has also been found to be inactive on the mercury drop12 by other authors. Determination of the pK, of 3,5-dimethyl-4-phenyl- 1,2,4-triazole was performed potentiometrically by titration of 5 X mol 1-1 solutions with 2.5 X 10-2 moll-' hydrochloric acid. Results and Discussion Since the goal of this work was either to confirm or to invalidate the existence of a basic site on the triazolo ring, two ionization v) z? E I -0.6 1 PH Fig. 3 EIl2 versus pH. Experimental conditions: EO = -0.3 V; 'c - 2 s; t = 25 "C; scan rate, 5 mV s-1; pulse duration, 60 ms; pulse amplitude, 60 mV. Table 3 Half-wave potential values at pH between - 1 and +1 PH -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 E l f l versus SCE -0.46 -0.46 -0.47 -0.48 -0.49 -0.50 -0.52 -0.53 -0.55 -0.56 -0.5746 Analyst, January 1996, Vol.121 sequences were investigated: on the one hand, the three Acid-Base Pairs BHz2+IBH+ and BH$+lBH22+ (or equilibria sequences B‘Hz2+IBH+) K” 1 K”2 K“3 BH33+ BH22+ + H+ BH22+ BH++H+ BH+ B + H+ which took into account the protonation of the triazolo ring; on the other, the two equilibria sequences K’a , B’H22+ BH+ + H+ Ultraviolet-visible spectra (of NHPTT) recorded immediately after preparation of solutions (see Experimental) exhibited a continuous evolution with the pH in the range -2-4 (Fig. 2). The absence of an isosbestic point in the pH range was, at first sight, already an argument in favour of the occurrence for more than one equilibrium.Nevertheless, we performed a non-linear squares treatment of the experimental UV-visible data accord- ing to the two ionization schemes. Hence, the two mathematical models allowing the calculation of absorbances A:a1c were: Acalc - C&a 1 Ka2 - &BH+ Ka1 Ka2 + Ka, (H+) + (H+I2 K“3 COKa, (H+) BH+ B + H+ + &BH22+ Kal Ka2 + Ka1 (H+) + which did not (Fig. 1). In this work, no activity correction was performed because of the necessary use of acidity functions. c o (H+>2 K.1 Ka2 + Ka1 (H+) + (H+I2 + &BH33+ CO K’ai co (H+) Acid-Base Pair BH+IB In order to be sure that overlapping between the acidities of BH+ and of BH33+, BH22+ (or of B’H22+) does not exist, we (2) pale - - &B’H22+ + EBH+ K’ai + (H+> K’ai + (H+) determined the pKa of the acid-base pair BH+/B which intervenes in the two ionization schemes.The values found for the two concentrations at 240 and 275 nm, together with those of molar absorptivities of BH+ and B obtained simultaneously (see experimental part), are given in Table 1. The values are self-consistent. We note that calculated EBH+ and EB are in good agreement with the experimentally obtained values. The pKa found is somewhat lower than the pKa values of benzylamines.2l A pKa value (BH+/B) = 8.10 allowed us to be sure that in the pH range of study of the pairs BH22+/BH+ and BH33+/BH22+ (or B’H22+/BH+) (see below) all the piperidino moiety was protonated and, hence, no overlapping conferred by this acidity and no concentration problems could exist.which result from the laws of matter conservation and of equilibria in water.22 In expression (l), the only unknowns were Ka1, Ka2 and &BH22+, this last one being experimentally unattainable because of the low ratio of the two Ka values. The consequence is that the species BH22+ did not exist alone whatever the pH of the solution was. As above, &BH+ and &BH33+ were also considered as supplementary unknowns for the sake of comparison with their experimental values. Alternatively, in expression (2), the only unknown was Pa!. For the same reason as previously discussed, we also considered &B’H22+ and &BH+ as supplemen- tary unknowns. The results for the first ionization scheme are given in Table 2. B 1 ~ , 2 + N’ H *. BH+ B~H:+ Fig.4 Ionization microconstants scheme.Analyst, January 1996, Vol. 121 47 It appears that pKal and pKa2.values did not vary significantly with the analytical concentrations and with the wavelengths. The same was true for the &BH33+, &BH22+ and EBH+ values for each wavelength. Moreover, the molar absorptivities obtained were in agreement with the experimental values. Therefore, this ionization process was satisfactory. A supplementary study of these results was also carried out. The experimental absorbances were treated by pooling all their values (again according to the same ionization scheme). This was possible because, for each pH value, six replicates were available. Generally, whatever the nature of the experimental data studied, such a treatment allows the calculation of the particular cost function Uo, which takes into account only random errors.Then, comparison of Uo and U provides an estimate of the lack of fit of the mode1.23 For the investigated ionization pathway, the values were Uo = 95 and U(minimum minimorum)3oonm - 166 and U(minimum minimor~m)~5~ nm = 1 10. These values indicated the quality of the model and confirmed the preceeding results. The likelihood of the pKal value being -0.24 and the pKa2 value 1.81 is reinforced by the pKa value of 3.7 for 3,5-dimethyl-4-phenyl- 1,2,4-triazole that we determined by pH measurement and by polarographic study of NHPTT. Its polarograms exhibited only one wave, the half-wave potentials of which shifted in the range -0.46 to -0.57 V versus SCE with pH (Fig.3 and Table 3). A plot of EIR versus pH exhibited a break at pH -0.5. Therefore, the pKa value of NHPTT was close to this pH. The results obtained with the second ionization process (B’H22+/E3H+) are given in Table 4. Discrepancies found for pK’,,, EB’H22-b and EBH+ values, according to the analytical concentrations and the wavelengths used, as well as the very Table 4 Determination of the pKa of the imino and triazolo groups: results obtained according to the second ionization scheme at different concentra- tions and wavelengths Clmol l-1 h = 250nm h = 300nm 10-4 +0.66 EBH+* 15 000 &B‘HZ2+ 10 000 lJ 965 1.5 x 10-4 PK’,, +0.84 EBH+ 15 200 U 1018 &B’H22+ 10 600 * E in 1 mol-1 cm-1. +1.59 3 000 11 000 4 000 3 000 11 500 4 224 +1.55 1.8 T 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 0.5 1 1.5 2 4 1 k,, - k,, -0- 4 2 - k22 -I- Fig.S Ionization microconstants kij versus kl I for the range kl < K,,. high objective function, are obvious, which strongly indicates a lack of fit of the model. These results confirm, a contrario, the preceeding ones. As a result, it can be concluded that the basic character of the triazolo ring does exist. Assignment of K,, and K,, Values to the Ionization Sites By comparison with the polarographic behaviour of a reference compound (chl~ramphenicol~~) it was found that the observed polarographic wave corresponded to a two-electron transfer. Owing to the well known fact that the protonated imines are reduced by a two-electron transfer,2325 we can deduce that the reduced function was the imine.Since a pKa = -0.5 value was found by the study of the displacement of the half-wave potential ,5112 versus pH, it could be deduced that pKal = -0.24 was the ionization constant of the protonated imine and hence a pKa2 value of 1.8 1 was that of the protonated triazole. Actually, this IS not quite exact because the ratio Ka!/Ka2 = 112 indicates that ionization of the two conjugated acids occurs somewhat simultaneously. The two species B1H22+ and BZH22+ coexist and the four ionization microconstants, kl 1, kzl, kI2 and k22 are effective (Fig. 4). (By accident, the microform B2Hz2+ was identical to the conjugated acid B’H22+ considered above in the second model.) As a result the K,, and Ka2 values found previously were only the macroscopic ones. They correspond to the ionization steps Ka , Ka2 BH33+ B1H22+ + B2H22+ + H+ BH+ + H+ I BH2*+ and the species we symbolized above by BH22+ were actually a mixture of the microforms B1H22+ and B2H22+ in a constant ratio.26 Between macroscopic and microscopic constants only three independent relations can be drawn: (4) k l l k12 = k21 k22 (5 1 Owing to this fact, it was not possible to obtain microscopic kj,j values without supplementary assumptions, nor was it possible to assign the found Kal and Ka2 values to a definite ionization scheme, i.e., to assimilate K,, to kll (or k21) and K.2 to k12 (or k22).We tentatively assigned the ranges of kj, values in the following manner. In a first step we plotted different values of kll, k21, k12 and k22 versus kl1 for the range of values kll < K.1 (Fig.5). In a second step we accepted the assumption that kl1 > kz1 owing to the preceding discussion. This allowed us to exclude from the range of interest values k l l < 0.8689, k21 > 0.8689, k12 > 0.031 1 and k22 < 0.031 1. Finally, considering that k22 > k21 and that k l l > k12 (for the same reason as in the second step) limits the range of interest to values 1.5678 < k l l < 1.7000 3.78 X 10-2 < k2l < 0.1692 1.58 X < k12 < 1.75 X 10-2 0.1692 < k22 < 0.7120 These values are satisfactory from a chemical standpoint. The triprotonated imine, BH33+, is a stronger acid than the diprotonated one B2H22+ and likewise for the triprotonated triazole BH33+ and the diprotonated one B1HZ2+. This can, indeed, be well explained by inductive effects induced by supplementary positive charges.The range -0.23 < pKa1, < -0.20 confirms the polarographic results. It is worth noting that48 Analyst, January 1996, Vol. 121 the pKI 1 and pK22 values are considerably lower than the known pK, values of N-alkylamines.27 To conclude, triazolodiazepines must be absolutely con- sidered as dibases, the protonation occurring on the azomethine group and on one of the nitrogen atoms of the triazolo ring. For our model compound, we found values of -0.24 and + 1.8 1 for the macroscopic ionization constants which corre- spond mainly to the ionization of the protonated imine group and of the protonated triazolo ring. Plausible ranges of microscopic constants values have been defined.The authors are grateful to Guy Bouer for his technical assistance. References Yang, S. K., J. Pharm. Sci., 1994, 83, 898. Triballet, C., Boucly, P., and Guemet, M., Bull. Chem. Soc. Fr., 1981, 2-3, 113. Pfendt, L. B., and Popovic, G. V., J. Chem. SOC., Perkin Trans. 2, 1994, 1845. Gallo, B., Alonso, R. M., Vicente, F., Ortiz, I., Irabien, A., Patriarche, G. J., and VirC, J.-C., Pharmazie, 1988, 43, 212. VirC, J.-C., Gallo Hermosa, B., and Patriarche, G. J., Analusis, 1987, 15, 499. JimCnez, R. M., Dorninguez, E., Badia, D., Alonso, R. M., Vicente, F., and Hernandez, L., J. Heterocyclic Chem., 1987, 24, 421. VirC, J.-C., and Patriarche, G. J., J. Electroanal. Chem., 1986, 214, 275. Gallo, B., Alonso, R. M., Madariaga, J. M., Patriarche, G. J., and Vir6, J.-C., Anal. Lett., 1986, 19, 1853. Konishi, M., Hirai, K., and Mori, Y., J. Pharm. Sci., 1982, 71, 1328. 10 I1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Inotsume, N., and Nakano, M., Chem. Pharm. Bull., 1980, 28, 2536. Gallo, B., Alonso, R. M., Lete, E., Badia, M. D., Patriarche, G. J., and GebeIke, M., J. Heterocyclic Chem., 1988, 25, 867. Jimenez, R. M., Alonso, R. M., Oleaga, E., Vicente, F., and Hernandez, L., Fresenius Z. Anal. Chem., 1987,329,468. Barton, D., Ollis, W. D., Comprehensive Organic Chemistry, Pergamon, Oxford, 1979, vol. 4, p. 365, Ipsen-Beaufour Industry, 35, rue Spontini, 75 116 Paris, France. Reiter, A. L., and Berg, E. G., Heterocycles, 1992, 34, 771. Perrin, D. D., and Dempsey, B., in Buffers for pH and Metal Zon Control, Chapman and Hall, London, 1974, P. 155. Britton and Robinson, J. Chem. SOC., 1931, 458. Rochester, C. H., Acidity Functions, Academic Press, London, 1970, vol. 17, p. 155. Bascombe, K. N., and Bell, R. P., J. Chem. Soc., 1959, 1096. Hooke, R., and Jeeves, T. A., J. Assoc. Comput. Mach., 1961, 8, 212. Blakwell, L. F., Fischer, A., Miller, I. J., Topsom, R. D., and Vaughan, J., J. Chem. SOC., 1964, 3588. Butler, J. N., Ionic Equilibrium. A Mathematical Approach, Addison- Wesley, Reading, MA, USA, 1964, p. 61. Draper, N., and Smith, H., Applied Regression Analysis, John Wiley, New York, USA, 2nd edn., 1981, pp. 33-40. Fossdal, K., and Jacobsen, E., Anal. Chim. Acta, 1971, 56, 105. Lund, H., Acta Chem. Scand., 1959, 13, 249. Edsall, J. T., Martin, R. B., and Hollingworth, B. R., Proc. Natl. Acad. Sci., 1958, 44, 505. Bouzard, D., Weber, A., and Le Henaff, P., Bull. SOC. Chim. Fr., 1972, 9, 3385. Paper 5104881 H Received July 24, 1995 Accepted September 20, I995

ISSN:0003-2654    

DOI:10.1039/AN9962100043

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, January 1996, Vol. 121 (49-54) 49 Sensitive Peroxyoxalate Chem il uminescence Determination of Psychotropic lndole Derivatives Juana Cepas, Manuel Silva and Dolores Pkrez-Bendito Department of Analytical Chemistry, Faculty of Sciences, University of Chrdoba, E-14004 Cbrdoba, Spain The continuous-addition-of-reagent (CAR) technique was used for the determination of hallucinogenic alkaloids such as N-methyltryptamine, 5-methyltryptamine, a-methyltryptamine, bufotenine and psilocin by measuring the peroxyoxalate chemiluminescence produced on reaction with the bis(2,4,6-trichlorophenyl)oxalate- hydrogen peroxide system following derivatization with dansyl chloride. This is the first reported use of chemiluminescence for the determination of these compounds. The special ability of the CAR technique to suppress background emission in peroxyoxalate chemiluminescence reactions provides a highly sensitive means of determination (the response to indole alkaloids is linear over two orders of magnitude and detection limits are in the picomole range).The proposed method compares favourably with existing alternatives in terms of sensitivity and was validated in the determination of psilocin in mushroom samples. Key words: Chemiluminescence; peroxyoxalate, continuous addition; indole derivatives Introduction Hallucinogens make up a heterogeneous group of compounds primarily resulting in altered cognitive and perceptual states that cannot otherwise be experienced. 192 Psychotropic indole deriva- tives with a chemical structure derived from tryptamine are a major group of hallucinogenic alkaloids of considerable biological interest as they block the neural uptake of noradre- naline and have complex effects on reflex activity in the spinal cord.3 The psychogenic NJV-dimethylated tryptamines are of special interest as they have been shown to occur in urine from schizophrenic patients and autistic ~hildren.~.~ Several thin- layer and gas chromatographic methods for the separation and identification of these tryptamines have been rep0rted.~.6>7 Also, psilocin (5-hydroxy-NJV-dimethyltryptamine) and its ana- logues have been widely determined by high-performance liquid chromatography (HPLC), especially in mushroom sam- ples of the genus Psilocybe using UV (254 nm),8 variable- wavelength UV,9JO photodiode-array,ll fluorimetric8JO and voltammetric8.12 detection.These methods are not very sensi- tive; their limits of detection are typically several micrograms per millilitre, except for electroanalytical detection, which affords sub-microgram per millilitre concentrations. More sensitive methods are therefore required for the determination of these psychotropic compounds. This paper reports a simple, sensitive method for the determination of psychotropic indole derivatives following derivatization with dansyl chloride; the method uses the continuous-addition-of-reagent (CAR) techniquel3,14 and per- oxyoxalate chemiluminescence (PO-CL) detection. The dansy- lated derivatives exhibit a high CL intensity on reaction with the bis(2,4,6-trichlorophenyl)oxalate (TCP0)-hydrogen peroxide system.The CAR technique was used owing to its ability to suppress background emission15 on account of the special way in which the sample and reagents are mixed (the TCPO solution is added to the reaction vessel containing hydrogen peroxide and the dansylated derivative). Under these conditions, high hydrogen peroxide to TCPO concentration ratios can be used and background emission can thus be suppressed. The proposed method is the first attempt at the determination of these psychotropic indole derivatives using a PO-CL reaction. The high sensitivity achieved makes it a useful alternative to other HPLC detection systems. The method was validated in the determination of psilocin in mushroom samples. Experimental Reagents All chemicals used were of analytical-reagent grade.Psycho- tropic indole derivatives were obtained from Sigma (St. Louis, MO, USA) with the exception of a-methyltryptamine, which was purchased from Aldrich (Milwaukee, WI, USA). All were used as received. Standard solutions containing 5 X 10-3 moll-' of each drug were prepared by dissolving the required amount in acetone (chromatographic grade, Romil Chemicals), and stored at 4 "C in a refrigerator. A 10-2 mol 1-1 bis(2,4,6-trichloro- pheny1)oxalate solution was made by dissolving 449 mg of the chemical (Aldrich) in 100 ml of ethyl acetate. A 0.7 mg ml-1 5-dimethy laminonaphthalene- 1 -sulfonyl chloride (dansyl chlo- ride, Sigma) solution was prepared in acetone and stored refrigerated. A 4 X 10-2 mol 1-l tris(hydroxymethy1)methyl- amine (Merck, Elmsford, NY, USA) buffer solution was prepared by dissolving 121.1 mg of reagent in water and adding enough hydrochloric acid to adjust the pH to 9.0 in a final volume of 25 ml.A 9 X 10-2 moll-' sodium carbonate buffer solution was made by dissolving 954 mg of salt (Merck) in water and adjusting the pH to 10.5 with HC1 in a final volume of 100 ml. Apparatus Chemiluminescence was measured on a Perkin-Elmer 650- 10s spectrofluorimeter with its light source off, the sample holder of which was replaced with a small magnetic stirrer holding a cylindrical glass reaction vessel; in order to acquire as much emitted CL as possible, an Oriel 44132 (1 in diameter) mirror was placed in front of the photomultiplier tube. The TCPO solution was added from a Metrohm Dossimat 665 autoburette.Kinetic data were acquired and processed by a NEC/Multisync 2A 33 MHz compatible computer equipped with a PC-Multilab 8 12 PG analogue-to-digital converter and running a program written by the authors in QBasic V.4.0.50 Analyst, January 1996, Vol. 121 Derivatization of Psychotropic Indole derivatives A volume of 200 pl of standard psychotropic solution containing between about 0.6 and 140 nmol of the hallucinogen, depending on its dynamic range (see Table I), was derivatized by adding 200 pl of 9 X mol 1-l carbonate buffer (pH 10.5) and 65 pl of 0.7 mg ml-1 dansyl chloride over 15 min at room temperature. Then, 45 pl of 0.13 mol 1-1 proline solution were added and the mixture was heated at 55 "C for 20 min. Next, 450 pl of doubly distilled water were added and the mixture was allowed to cool to room temperature.The derivative was extracted into 250 pl of chloroform and an aliquot of the organic phase was used for the subsequent CL determination. PO-CL Determination of Psychotropic Indole Derivatives To the reaction vessel of the CAR system were added 6.0 pl of derivatized psychotrope solution in chloroform, 65 pl of concentrated hydrogen peroxide (33% v/v, aqueous solution), and 20 1-11 of 4 X 10-2 mol I-' TRIS buffer (pH 9.0). The mixture was accurately diluted to 1 .O ml with acetone and 1 .O X moll-' TCPO was added at 8 ml min-1 with continuous stirring. The CL signal was monitored throughout the reaction and kinetic data (relative CL intensity versus time) were acquired at a rate of 10 ms per point, the maximum reaction rate being measured within about 1.5 s.The observed maximum reaction rate was corrected by subtracting the maximum reaction rate measured using the same procedure for a reagent blank (containing no psychotrope). Preparation of Mushroom Samples An amount of between 10 and 50 mg (accurately weighed) of ground, dried mushrooms was allowed to macerate in 10 ml of dilute acetic acid (pH 4.0) for 1 h, after which the mixture was heated at 70 "C for 30 min. Once cool, the mixture was filtered through glass-wool and the filtrate was adjusted to pH 8 with concentrated ammonia solution. The solution was extracted with 15 ml of chloroform for 10 min and the organic phase was then evaporated to dryness. The residue was dissolved in 200-300 pl of acetone and a 50 pl aliquot was subjected to the derivatization and PO-CL determination of psilocin in the mushroom sample. Results and Discussion The CAR technique is an effective means for increasing the signal-to-noise ratio in peroxyoxalate chemiluminescence reac- t i o n ~ ~ ~ and hence to ensure the sensitive determination of analytes.In this work, it was used to develop a PO-CL method for the determination of psychotropic compounds following the formation of dansylated derivatives using the TCPO-hydrogen peroxide system. Five hallucinogens were used, namely N- methyl tryp tamine, 5 -me thyltryp tamine, a-me thy ltryp tamine, bufotenine (4-hydroxy-NJV-dimethyltryptamine) and psilocin (5-hydroxy-NJV dimethyltryptamine); the structural formulae of their dansylated derivatives and the CL reaction scheme are shown in Fig.1. Their CL versus times curves, recorded under suitable experimental conditions, are shown in Fig. 2. As can be seen, the maximum response was exhibited by N-methyl- tryptamine. Optimization of the Dansylation Reaction Primary and secondary amines and phenols are known to react quantitatively with dansyl chloride (DNS-CI) to yield sulfo- namides or phenolic esters that exhibit intense yellow fluores- cence.I6 The dansyl derivatives are formed under different experimental conditions depending on the particular starting compound. Thus, the derivatization reaction usually takes place in alkaline medium at room temperature for about 30-40 min and some heating is occasionally required. In this work, we optimized the experimental conditions for the derivatization reaction since the psychotropic indole compounds studied had never been dansylated previously.We chose N-methyltrypta- mine (NMT) as the psychotropic compound to be used to investigate the effect of experimental variables on the deriva- tization reaction and the ratio between the maximum reaction rates (MRR) measured in the presence and absence of hallucinogen (MRRsam,1JMRRbl~) as the measured analytical parameter. All concentrations quoted are referred to the final volume in the reaction medium (1 .O ml). The effect of DNS-C1 on the formation of dansylated hallucinogens is shown in Fig. 3A; as can be seen, about 170 nmol of DNS-C1 ([DNS-Cl]/[NMT] = 25) ensured the maximum difference between the observed maximum reaction rate for the sample and blank reactions.We chose this measured analytical parameter since the MRRSmple/MRRblank was in- creased by low DNS-C1 concentrations owing to the low MRRblank levels, and under these conditions the dansylation reaction was not quantitative. The decrease in the observed analytical signal at high concentrations of the labelling reagent can be ascribed to an increase in MRRblank that resulted in a virtually negligible difference between the sample and blank signals. From these results, a DNS-C1 amount of 168.8 nmol was chosen as optimum, which was obtained by placing 65 pl of 0.7 mg ml-1 dansyl chloride in the reaction solution. As in most reported procedures, the alkaline medium for the derivatization reaction was provided by carbonate buffer; the optimum buffer concentration and pH were 9 X 10-2 moll-' and 10.5, respectively (Fig.3B). These values resulted in a maximum MRRsamplJMRRblank ratio of approximately 1.2. The effects of temperature and time on the dansylation reaction were also tested in the light and in the dark. The temperature was varied between 20 and 60 "C and the time over the range 5-150 min, both in the light and in the dark. Based on the results, the dansylation reaction was quantitative when the psychotropic solution was treated with alkaline DNS-C1 for 15 min at room temperature in the light. At that point, the derivatization reaction was optimized; however, the ratio between the measured maximum reaction rates of sample and blank was not very high (approximately 1.2) Table 1 Figures of merit of the calibration plots for the PO-CL determination of psychotropic indole derivatives Correlation coefficient LOD/ s,+ Alkaloid Linear range/nmol Linear regression equation* ( n = 15) pmol (%) N-Methyltryptamine 0.6-40 MRR = (4.4 X + 3.1 X 10-3C 0.9994 138 1.53 5-Methyltryptamine 0.6-40 MRR = -(2.0 x 10-3) + 2.0 x 10-3c 0.9986 184 1.90 a-Methyltryptamine 3.0-70 MRR = -(2.3 X lop3) + 7.1 X 10-4C 0.9950 830 2.20 Bufotenine 0.640 MRR = -(4.2 X lop4) + 1.1 X 10-3C 0.9981 230 2.00 Psilocin 10-140 MRR = -(1.1 X + 9.0 X 1O-T 0.9935 2920 1.66 * MRR = maximum reaction rate; C in nmol.Analyst, January 1996, Vol.121 51 owing to the high contribution of excess of DNS-CI to the analytical signal. Two alternative procedures have been re- ported for removing unused DNS-Cl, namely the selective extraction of dansylated derivatives in an organic solvent such as toluene17J8 or benzene,19920 and the addition of proline before the extraction.21-22 On the basis of the experimental results, we used proline to remove excess of DNS-CI and chloroform for the selective extraction of dansylated halluci- nogens.Under these conditions, the MRRsmplJMRR~l~ ratio was considerably increased (to approximately 3 .O). Optimization of the PO-CL Reaction Although the PO-CL reaction between TCPO and hydrogen peroxide has previously been used by the authors for the determination of phenothiazine derivatives using the CAR technique,15 re-optimization was required on account of the different spectrofluorimetric features of the analytes in this work.All experiments were carried out in an acetone-water- chloroform (91.0 + 8.5 + 0.5) medium with a minimum water content (water strongly decreases the CL signal) that would be exclusively supplied by the hydrogen peroxide and TRIS solutions added to the reaction vessel; the chloroform solvent contained the dansylated psychotropic indole derivatives and the acetone acted as co-solvent to ensure miscibility in the reaction vessel (between water, chloroform and ethyl acetate, the last of which was provided by the TCPO solution added from the autoburette). The effect of the concentration and pH of the TRIS buffer is shown in Fig. 4A; as can be seen, the maximum analytical signal SO, I 0 H Dansylated psilocin CI CI Dansylated methyltryptarnines R, R2 R3 NMethyltryptamine CH, H H 5-hhthyltryptrmine H H CH, CI H Dan sy I ated bufotenin e 0 0 c - c II II I I 0 - 0 C1 CI L J TCPO l.2-Dioxeth.~-3.4-dio~ r o o l * * Dansylated + hv hallucinogen + 2 co, --+ Da nsy la t ed Da nsy lated + ha1 lucinogen - hallucinogen Lo -O_I Fig.1 Structures of the psychotropic dansylated derivatives assayed and CL reaction scheme.52 Analyst, January 1996, Vol. 121 was obtained with 4 X 10-2 mol 1-1 TRIS buffer (pH 9.0), a volume of 20 pl of which was used in the reaction vessel. The effect of the hydrogen peroxide concentration was studied over the range 0.13-0.7 mol 1-1; the maximum response was obtained with a 0.7 moll-' concentration (65 pl of concentrated 1 .o 0.0 0.5 1 .o 1.5 2.0 2.5 3.0 Time/s Fig.2 Relative PO-CL intensity versus time plots for the psychotropic indole derivatives studied: 1 N-methyltryptamine; 2,5-methyltryptamine; 3 psilocin; 4, bufotenine; and 5, or-methyltryptamine. All compounds at 3.0 nmol except psilocin (30 nmol). For conditions, see under Experimental. [DNS-Cl]/[NMT] 0 1 0 2 0 3 0 4 0 5 0 6 0 I 1 I I 1 1 1 A 0 1 0 0 2 0 0 3 0 0 4 0 0 DNS-Cllnmol hydrogen peroxide added to the reaction vessel). This concen- tration was a compromise between the increased PO-CL signal obtained by raising the hydrogen peroxide concentration and the decreased signals resulting from the increased amounts of water contained in the aqueous H202 solution. The effects of the TCPO concentration and its addition rate are illustrated in Fig. 4B. A 1 X 10-2 moll-' TCPO solution in ethyl acetate added at 8 ml min-1 from the autoburette was selected for subsequent experiments.Higher TCPO concentrations could not be used owing to the low solubility of this reagent in ethyl acetate. Determination of Psychotropic Indole Derivatives Under the selected working conditions, psychotropic indole derivatives were determined at the nanomole level by using the proposed PO-CL method. Data relevant to the calibration graphs for these hallucinogens are summarized in Table 1. The limits of detection (LODs) were calculated following IUPAC recommendations;23 the precision, as the relative standard deviation, was checked on 11 samples containing between 3.0 and 10 nmol of psychotropic indole derivative (psilocin was tested at 40 nmol owing to its lower sensitivity).The over-all time required to perform three replicate analyses (excluding that required for the derivatization reaction) and sample changeover in the CAR system was 45 s, so the throughput was about 80 samples h-1. The different sensitivities achieved depend on the structure of each hallucinogen, which can influence the CL signal of the dansylated derivative. Buffer pH 7 8 9 10 11 12 1.3 8 0.0 0.1 0.2 0.3 0.4 [ Carbonate buffer ]/mot I-' Fig. 3 hallucinogens. [NMT] = 7.0 nmol. Conditions for PO-CL reaction as described under Experimental. MRR, maximum reaction rate. Effect of, A, the DNS-Cl concentrations; and B the carbonate buffer concentration (filled circle) and pH (open circle) on the dansylation of the TRIS pH 7 8 9 10 11 12 Addition Rate/ml min-' 0 2 4 6 8 1 0 1 2 1 4 3.5 3.0 2.5 2.0 0.00 0.05 0.10 0.15 [TRIS]/mol I-' 3.5 3.0 25 2.0 Fig.4 circle) on the PO-CL determination of 7.0 nmol of dansylated NMT. Other conditions as described under Experimental. MRR, maximum reaction rate. A, Influence of the TRIS concentration (filled circle) and pH (open circle); and B of the TCPO concentration (filled circle) and addition rate (openAnalyst, January 1996, Vol. 121 53 At this point, it is of interest to compare the analytical features of the proposed PO-CL method for the determination of psilocin with existing alternatives. Because such alternatives are chromatographic (HPLC) and use various detection system, we chose LODs for comparison and assimilated the amount of psilocin injected into the chomatograph with the LOD of the proposed method, in addition to the amount of derivatized psilocin used for the PO-CL determination in the CAR system.Based on these considerations, the proposed method has an LOD of approximately 15 ng for psilocin, which is similar to the LODs for the cromatographic methods using voltametric d e t e c t i o n , g ~ ~ ~ , ~ ~ but one to two orders of magnitude lower than those using optical (spectrophotometric or spectrofluorimetric) detection.*,l0,25 In view of these results, the proposed method is an excellent choice for the sensitive determination of psilocin. Its use as a CL detection system in HPLC is bound to introduce significant improvements in the determination of these hallucin- ogens and related compounds. Determination of Psilocin in Fungi of the Genus Psilocibe The above results suggested that the proposed method could be applied to the determination of hallucinogenic alkaloids in real samples.Because the recreational use of hallucinogenic mush- rooms is a matter of growing concern in some countries, we applied the proposed method to the determination of psilocin in this type of sample; among hallucinogenic mushrooms, several Psilosyhe species contain indole alkaloids (mostly psilocybin and smaller amounts of psilocin).I0 The proposed method is useful for the determination of psilocin in the presence of psilocybin since the hydroxyl group at which dansylation takes place is phosphorylated in psilocybin, which therefore produces no CL signal. However, psilocybin can readily be converted into psilocin by a rapid dephosphorylation reaction with alkaline phosphatase,* so the proposed method can be used to determine both indole alkaloids in two sample aliquots (the Table 2 Recovery of psilocin added to mushrooms samples Sample Cap 1 Cap 2 Cap 3 Stem Psi 1 oc in/pg Recovery Found Added I-18 % 3.75 - 12.25 20.40 12.25 20.40 12.25 20.40 12.25 20.40 5.78 - 6.53 - 5.72 - - 16.10 24.15 17.72 26.83 19.03 26.88 18.12 25.99 - - - - 100.6 100.0 98.2 102.5 101.3 99.8 100.8 99.5 Mean: 100.3 - - - SD: 1.28 Table 3 Determination of psilocin in Psilocyhe Semilanceata (Fr.) Kumm.by use of the proposed PO-CL method Sample Amount/mg Psilocin (%)* Average recovery (%) Cap 1 49.0 0.046 f 0.001 100.3 Cap 2 12.1 0.195 f 0.015 100.3 Cap 3 35.4 0.11 1 k 0.003 100.6 Stem 48.9 0.070 f 0.002 100.1 * Average of three determinations k standard deviation.psilocybin concentration would obviously be obtained by difference). A fungal species of the genus Psilocybe, viz., Psilocybe semilanceata (Fr.) Kumm., was collected at Puerto de 10s Anclares, Lug0 (Spain), in 1994. Several samples of caps and stems were subjected to the procedure described under Experimental, which is based on the aqueous-organic extrac- tion method for the isolation of psilocin from hallucinogenic mushrooms proposed by Casale26 (chloroform was used instead of diethyl ether, however). In order to evaluate potential adverse effects from other components, the recovery of psilocin added to these samples was also determined by comparing the results obtained before and after adding the psilocin standard solution.The recoveries obtained are given in Table 2. Table 3 shows the results provided by the proposed method, which were consistent with reported contents of psilocin in hallucinogenic mushroom samples. lo The authors gratefully acknowledge financial support from the Direcci6n General Interministerial de Ciencia y Tecnologia (DIGICyT) for the realization of this work as part of Project PB9 1-0840, and Dr. F. Infante of the Departamento de Biologia Vegetal y Ecologia of the University of C6rdoba (Spain) for kindly supplying the mushroom specimens. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Haddad, L. D., and Winchester, J. F., Clinical Management of Poisoning and Drug Overdose, Saunders, Philadelphia, 2nd edn., 1990.Wingard, L. B., Jr., Brody, T. M., Lamer, J., and Schwartz, A., Human Pharmacology: Molecular-to-Clinical, Mosby Year Book, St. Louis, MO, 1991. Bowman, W. C., and Rand, M. J., Textbook of Pharmacology, Blackwell, Oxford, 2nd edn., 1980. Faurbye, A., and Pind, K., Nature (London), 1968, 220,489. Himwich, H. E., Jenkins, R. L., Fujirnori, M., Narasimhachari, N., and Ebersole, M., J. Autism Child. Schizophr., 1972, 2, 114. Baumann, P., and Narasirnhachari, N., J. Chromatogr., 1973, 86, 269. Narasimhachari, N., and Hirnwich, H. E., Life Sci., 1973, 12, 475. Christiansen, A. L., and Rasmussen, K. E., J. Chromatogr., 1983, 270, 293. Vanhaelen-FastrC, R., and Vanhaelen, M., J. Chromatogr., 1984,312, 467. Wurst, M., Semerdzieva, M., and Vokoun, J., J. Chromatogr., 1984, 286, 229. Wurst, M., Kysilka, R., and Koza, T., J. Chromatogr., 1992, 593, 201. Kysilka, R., and Wurst, M., J. Chromatogr., 1989, 464,434. Mhrquez, M., Silva, M., and PCrez-Bendito, D., Analyst, 1988, 113, 1733. Velasco, M., Silva, M., and Perez-Bendito, D., Anal. Chem., 1992,64, 2359. Cepas, J., Silva, M., and Ptrez-Bendito, D., Anal. Chem., 1994, 66, 4079. Del Castillo, B., Alvhrez-Builla, J., and Lerner, D. A., in Luminis- cence Techniques in Chemical and Biochemical Analysis, ed. Baeyens, W. R. G., De Keukeleire, D., and Korkidis, K., Marcel Dekker, New York, 1991, ch. 5 , pp. 99-100. Ibe, A., Saito, K., Nakazato, M., Kikuchi, Y., Fujinura, K., and Nishima, T., J. Assoc. Off. Anal. Chem., 1991, 74, 695. Barrett, D. A., Shaw, P. N., and Davis, S. S., J. Chromatogr., Biomed. Appl., 1991, 104, 135. Cann-Moisan, C., Caroff, J., and Girin, E., J. Chromatogr., Biomed. Appl., 1992, 112, 134. Desiderio, M. A., Davalli, P., and Perin, A., J. Chromatogr., Biomed. Appl., 1987, 63, 285. Lindsay-Smith, J. R., Smart, A. U., Hancock, F. E., and Twigg, M. V., J. Chromatogr., 1991, 547, 447.54 Analyst, January 1996, Vol. 121 22 Lindsay-Smith, J. R., Smart, A. U., Hancock, F. E., andTwigg, M. V., Chem. Ind. (London), 1989,11, 353. 23 Long, G. L., and Winefordner, J. D., Anal. Chem., 1983, 55, 712A. 24 Kysilka, R., J. Chromatogr., 1990, 534, 287. 25 Bomer, S., and Brenneisen, R., J. Chromatogr., 1987, 408, 402. 26 Casale, J. F., J. Forensic. Sci., 1985, 30, 247. Paper 5104356E Received July 5 , I995 Accepted September 12,1995

ISSN:0003-2654    

DOI:10.1039/AN9962100049

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, January 1996, Vol. 121 (55-59) 55 Microbore Liquid Chromatography-Electrospray Mass Spectrometry of Selected Synthetic Pyrethroid Insecticides Ian A. Fleeta, John J. Monaghanajb, Derek B. GordonQ'c and Gwyn A. Lorda,= a Michael Barber Centre for Mass Spectrometry, Department of Chemistry, UMIST, Sackville Street, Manchester, UK M60 I QD b Department of Chemistry, The University of Edinburgh, West Mains Road, Edinburgh, UK EH9 3JJ c Department of Biological Sciences, Manchester Metropolitan University, Chester Street, Manchester, UK MI 5GD The pyrethroid insecticides kadethrin, a-cypermethrin, flucythrinate and SSI-116 have been studied by positive-ion electrospray mass spectrometry (+ESMS) in the presence of ammonium acetate and formic acid. Ammoniated molecule base peak ions [M + NH4]+ were observed for all the insecticides studied at low electrospray source sampling cone voltages.The effect of increasing the cone voltage (40-120 V) and its influence on the extent of fragmentation experienced by each insecticide were studied. A number of these key fragment ions found in +ESMS spectra of a-cypermethrin have been examined by MS-MS under low-energy collisional activation (CA) conditions. On-line microbore reversed-phase liquid chromatographic separations were performed on mixed pyrethroid standards. The eluates were analysed by +ESMS to establish the lower limits of detection using full-scan and selective-ion recording (SIR) modes. Limits of detection (signal-to-noise ratio better than 3 : 1) for each component of the pyrethroid mixture, injected on column, were in the range 120-300 pg (0.30-0.77 pmol) using full-scan mode and 12-60 pg (0.03-0.15 pmol) by SIR.Keywords: Microbore liquid chromatography; electrospray ionization mass spectrometry; electrospray source sampling cone voltage; tandem mass spectrometry; collisional activation; selective-ion recording; ester and non-ester pyrethroid insecticide Introduction Pesticide research over the last thirty years has yielded many structurally diverse insecticides which are classed as synthetic pyrethroids. This research effort was instigated by the in- secticidal properties and low mammalian toxicities of the natural pyrethrins' and a desire to produce synthetic analogues having improved photo-stability and potency, while undergoing faster biodegradation and photodegradation, than the more persistent chlorinated pesticides such as DDT.Research by Elliott2 and co-workers, involving the correlation of stereo- chemical structure with insecticidal activity, led to the discov- ery of permethrin, cypermethrin and many other important pyrethroids which are insecticidal analogues of cyclopropane- carboxylic acid esters. Further efforts by various research groups worldwide, involving a sequence of isosteric replacements of groups originally present in pyrethrin I, have yielded a number of new pyrethroids of commercial interest.3-7 These new pyrethroids lack the cyclopropanecarboxylic acid ester bond and are mainly achiral. They are broadly insecticidal, have low mammalian toxicities and show markedly lower toxicities towards fish than pyrethroids containing the cyclopropanecarboxylic acid ester bond.These structurally diverse pyrethroids are applied to crops, forests, soils, animal feeds and in household use. The resulting loss of these compounds as the intact molecules themselves, their degradation products and/or metabolites to the environ- ment requires the detection of these compounds at the microgram and sub-microgram levels. While the combination of positive-ion electron ionization (EI) and positive/negative-ion chemical ionization (+/-CI) mass spectrometry offers the analytical chemist specificity in the detection of certain synthetic pyrethroids,*99 no single ionization method provides the desired sensitivity for the detection of all members of this structurally diverse insecticide class at the trace level.The fissile nature of the bond between the cyclopropanecarboxylic acid ester oxygen and the alpha- carbon of the benzylphenoxy-containing pyrethroids lo leads to low abundance or non-existent molecular ions in EIMS. Because the central linkages of the non-ester-type pyreth- roides' 1312 show similar facile cleavage, these compounds also yield molecular ions of low abundance. Cyclopropanecarboxylic acid ester pyrethroids'3 and non- ester types yield predominently protonated and/or ammoniated molecule ions when studied by +CI (ammonia). Using -CI (methane) the mass spectra of ester-type pyrethr~ids'~ show almost exclusive formation of the corresponding carboxylate anion. Under similar conditions, the non-ester pyrethroid types cannot form stable carboxylate anions, and yield poor spectra.The development of electrospray ionization14 has revolu- tionized the mass spectrometric analysis of high relative molecular mass compounds. More recently electrospray ioniza- tion has been successfully ultilized to analyse many compounds of lower molecular mass including pharmaceuticals, drugs and sulfonylurea herbicides.15 In this work we have studied a number of structurally diverse pyrethroids using positive-ion electrospray mass spectrometry (+ESMS) in the presence of ammonium acetate and formic acid at low electrospray source sampling cone voltages. The structures of each insecticide are shown in Scheme 1. Kadethnn and a-cypermethrin are representative of the cyclopropanecarboxylic acid ester-type pyrethroids, flucythrinate is an ester type, lacking a cyclopro- pane ring, and SSI- 1 16 is a silicon-containing non-ester-type pyrethroid.The effect of increasing the cone voltage and its influence on the extent of fragmentation experienced by each56 Analyst, January 1996, Vol. 121 insecticide were investigated. A number of key fragment ions found in +ESMS spectra of a-cypermethrin, at higher cone voltages, have also been examined by electrospray tandem mass spectrometry, (+ESMS-MS) under low-energy collisional acti- vation (CA) conditions. Additionally we have investigated the use of on-line microbore reversed-phase HPLC coupled with +ESMS at low cone voltages to separate and detect these structurally diverse pyrethroid insecticides at the trace level.Microbore reversed- phase HPLC offers higher column efficiencies than conven- tional packed columns, drastically reduced flow rates (compati- bility with mass spectrometric techniques) and the ability to work with a smaller sample volume, thus allowing scope for sample enrichment by concentration.16 Experimental A VG Quattro tandem quadrupole mass spectrometer (VG Organic, Altrincham, Cheshire, UK) fitted with an electrospray ionization source, interfaced to a tri-axial probe held at 4.4 kV, was used to carry out various +ESMS and +ESMS-MS experiments. The electrospray source high voltage lens was held at 0.55 kV, for all experiments, and the sampling cone voltage was varied between 40 and 120 V. Mass spectrometry and MS-MS experiments were carried out with the resolution set such that the peak width at half height of the ammoniated molecule ion (a-cypermethrin) was <0.45 u.The mass spectrometer was calibrated over the desired mass range, using poly(ethy1ene glycol). The first quadrupole analyser (MS) was used to study +ES sampling cone voltage induced fragmentations while tandem Kadeth ri n a-Cypermethrin v Flucythyrinate Y FN Scheme 1 Pyrethroid structures. experiments (MS-MS) were performed on selected key ions observed following cone voltage induced fragmentation studies. Individual 25 pg ml-l pyrethroid standards were infused through the tri-axial probe, using a 100 pl syringe and syringe infusion pump at a rate of 5 pl min-1 (Model 22, Harvard Apparatus South Natick, MA, USA). A 'make-up' flow consisting of 70 + 30 propan-2-ol-H20 containing 10 mmol l-1 CH3COONH4 and 22 mmol 1-l HCOOH at a flow rate of 5 pl min-1 was pumped via a micro-gradient syringe pump (Brownlee Labs., Santa Clara, CA, USA) to the tri-axial probe as shown in Fig.1. The tri-axial probe comprises of two concentric stainless-steel capillaries and an inner 700 mm X 75 pm id (375 pm od) uncoated (inner wall) fused-silica capillary (Polymicro Technologies, AZ, USA) which terminates approx- imately 0.5 mm beyond the inner steel capillary. On-line isocratic reversed-phase HPLC experiments were performed by injecting, via a Valco C14W injector with a 0.06 pl rotor volume (Valco, Houston, TX, USA), mixed pyrethroid standards (1-250 pg ml-l) using a 25 cm X 0.5 mm id Spherisorb ODs2 microbore poly(etherether ketone) (PEEK) column.A micro-gradient syringe pump, Fig. 1, was used to pump a mobile phase of 70 + 30 propan-2-ol-H20 containing 10 mmol l-1 CH3COONH4 and 22 mmol l-1 HCOOH at a flow rate of 10 p1 min-1. All MS-MS experiments were obtained using argon gas in the radiofrequency only, hexapole collision cell. The gas pressure was adjusted within the cell so that 50% suppression of the selected ion was obtained. The collision energy was 25 eV in the laboratory frame of reference for each experiment. The mobile phase solvents, propan-2-01 and water, were HPLC grade (Rathburn Chemicals, Walkerburn, Scotland, UK). Ammonium acetate and formic acid were of ACS quality (Sigma-Aldrich, Poole, Dorset, UK). Individual and mixed pyrethroid standards were freshly prepared in the range 1-250 pg ml-1, by serial dilution of their respective stock standards, using 70 + 30 propan-2-ol-H20 containing 10 mmol 1-1 CH3COONH4 mobile phase.All standards were stored in amber 1 cm3 glass vials at 5 "C. All pyrethroid samples used in this study were of research grade. Common names or research codes of each pyrethroid have been used throughout this paper: kadethrin, CAS Registry voltage \Analyst, January 1996, Vol. 121 57 No. 58769-20-3, 5-benzyl-3-furylmethyl(E)-( lR)-cis-2,2-di- methyl-3 -(2-oxothiolan-3-ylidenemethyl)cyclopropanecar- boxylate; a-cypermethrin, CAS Registry No. 67375-30-8, a racemate comprising (R ,S)-a-cyano-3 -phenoxybenzyl-( 1 S&)- cis-3-( 2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxy- late; SSI- 1 16, CAS Registry No.99503- 10-3, (4-ethoxyphe- ny1)-dimethyl- { [ (3-phenoxyphenyl)methoxy]methyl} -silane; flucy-thrinate, CAS Registry No. 70 124-77-5, (+)-a-cyano(3- phenoxyphenyl)methyl(+)-4-(difluoromethoxy)-a-( 1 -methyl- ethy1)benzeneacetate. Flucythrinate (Cybolt) and SSI- 1 16 were donated by Cyanamid, Gosport, Hampshire, UK and Shionogi & Co. Shiga, Japan, respectively. Samples of kadethrin and a- cypermethn were purchased from Promochem (Welwyn Garden City, Hertfordshire, UK). Results and Discussion We have studied a number of structurally diverse pyrethroids using +ESMS in the presence of ammonium acetate and formic acid at low-electrospray-source sampling cone voltages. The structures of kadethrin, a-cypermethrin, flucythrinate and SSI- 116 are shown in Scheme 1.The effect of increasing the cone voltage and the influence on the extent of fragmentation experienced by each insecticide were investigated. A number of key fragment ions found in +ESMS spectra of a-cypermethrin have been examined by +ESMS-MS under low-energy CA. We have also investigated the use of on-line microbore reversed- phase HPLC coupled with +ESMS at low cone voltages to separate and detect these structurally diverse pyrethroid in- secticides at the trace level. Sampling Cone Voltage-induced Fragmentation Experiments The positive-ion electrospray spectra of cypermethrin, at sampling cone voltages between 40 and 120 V (in increments of 20 V), were acquired by scanning the mass range, m/z 50-500, at a rate of 8 s per scan.The resulting spectral data for each scan were stored as an averaged spectrum. At lower cone voltages the positive-ion electrospray spec- trum of a-cypermethrin consists of predominantly ammoniated molecule ions [M + N&]+, in their predicted isotopic abun- dances of 9 : 6 : 1 for a species containing two chlorine atoms, Fig. 2. Progressively higher cone voltages resulted in a simultaneous diminution of the [M + NH4]+ population, with a concomitant increase in the abundance of the protonated molecule ion [M + HI+ and of diagnostically useful fragment ions, Fig. 3. Additionally, the electrospray spectra of a- cypermethrin show that the [M + NH4]+ and [M + HI+ ions (for cone voltages up to 100 V) both yield the correct isotopic abundances for a species containing two chlorine atoms. Many fragment ions observed in the +ES spectra of a-cypermethrin are commonly observed in the corresponding +EIMSl0 and/or +CI (ammonia) MS spectra.l3 Kadethrin shows a similar behaviour to a-cypermethrin, yielding predominantly [M + NH4]+ ions (m/z 414), at lower cone voltages (cone voltage 40 V), in its +ES spectra. Progressively higher cone voltages produce a simultaneous diminution of the [M + NH4]+ population, with a concomitant increase in the abundance of [M + HI+ (m/z 397) and the fragment ions. The +ES spectra of flucythrinate and SSI-116 (cone voltage 40 V), also show the same trends in fragment ion production at progressively higher cone voltages. However, these two pyrethroids do not yield protonated molecule ions [M + HI+ when undergoing transition from low to higher cone voltages.A positive-ion electrospray spectrum of the mobile phase in the absence of analyte, at a cone voltage of 40 V, is dominated by an intense protonated propan-2-01 solvent dimer ion at m/z 12 1 (base peak). At higher cone voltages the protonated propan- 2-01 solvent dimer ion diminishes in abundance. Low cone voltage +ES spectra of the mobile phase also show a minor artifact ion at m/z 391 which has been attributed to the protonated molecule ion of bis(2-ethylhexyl) phthalate. At higher cone voltages, the bis(2-ethylhexyl) phthalate ion undergoes fragmentation with subsequent loss of the side chains to form the ions at m/z 279 and m/z 149 (protonated phthalic anhydride). MS-MS of Some Key Ions Observed Following Sampling Cone Voltage-induced Fragmentation of a 25 pg ml-1 a-Cypermethrin Standard Many fragment ions observed in the +ES spectra of each pyrethroid, at higher sampling cone voltages, are identical to fragment ions observed in either their +EIMS and/or +CI (ammonia) MS spectra.We have investigated at a source sampling cone voltage of 70 V a number of key fragment ions observed in the +ES of a-cypermethrin. Individual +ES product-ion spectra of the ammoniated molecule ions of a-cypermethrin, m/z 433 (2 X 373) and m/z 435 (1 X W I , 1 X 37Cl), show direct,connectivity between these ions and the product ions m/z 4 16 [M + HI+ (2 X 3Tl); m/z 418 [M + HI+ (1 X 3T1, 1 x 37C1), and m/z 191 (2 X T 1 ) ; m/z 193 (1 X 3Tl, 1 X 37Cl), respectively. Product-ion spectra of the ions at m/z 416 and 418 also show connectivity between these ions and the product ions m/z 191 and 193, respectively. The fragment ions m/z 191 and 193 are observed in the +CI (ammonia) spectra of a-cypermethrin but not in their +EI spectra.The formation of protonated molecule ions at higher sampling cone voltages from their ammoniated molecule ions reflects the dissociation of [M + NH4]+ to [M + HI+ + NH3, Scheme 2. Confirmation that NH3 has been lost directly as a neutral species from the [M + NH4]+ ion is provided by a constant neutral loss scan of 17 Da. A product-ion scan of the 359 m/z Fig. 2 Positive-ion electrospray spectrum of Cypermethrin (cone voltage 40 V). 193 I m/z Fig. 3 Positive-ion electrospray spectrum of Cypermethrin (cone voltage 100 V).58 Analyst, January 1996, Vol.121 m/z lBylBWl95 Scheme 2 Formation of a dichlorovinyldimethylcyclopropane acylium ion, [C8HgCl20]+, following homolytic cleavage of the carbon-oxygen bond alpha to the ester carbonyl group. m/z 191 ion (2 X 35C1), obtained at the same cone voltage, shows connectivity between this ion and the fragment ions at m/z 163,127 and 91. The ions, m/z 191,193 and 195, have been attributed to the formation of a dichlorovinyldimethylcyclo- propane ac ylium ion, following homolytic cleavage of the carbon-oxygen bond alpha to the C=O group, Scheme 2. Subsequent loss of CO from the m/z 191 ion yields a dichlorovinyldimethylcyclopropane carbenium ion [C7H935C12]+ at m/z 163, which undergoes further successive loss of molecules of HC1 to form m/z 127 and 91 ions.1° The ions, m/z 163,127 and 91, are also observed in the +EI spectrum of a-cypermethrin.Tandem electrospray mass spectrometry experiments at higher sampling cone voltages on key ions indicate that a- cypermethrin undergoes similar fragmentations to those ob- served under EIMS (70 eV) and/or +CI (ammonia) ionization conditions. This suggests that the fragment ions produced in each ionization process have similar internal energies. Low Sampling Cone Voltage, Microbore Isocratic Reversed-phase HPLC Separation of Mixed Pyrethroid Standards On-line reversed-phase liquid chromatography positive elec- trospray ionization experiments were performed on mixed pyrethroid standards to establish the lower limits of detection. Individual mixed standards were chromatographed through a 25 cm X 0.5 mm id Spherisorb ODs2 microbore PEEK column, Fig.1, and the resulting eluates were subjected to +ES ionization. Mass spectral data were acquired by scanning either v la*] Kndethrin .- 5 Tl<MS,Ps+).4 s 0-1 ’ I . ’ - ’ ‘ ’ ’ ” I . ’ . I ’ ” ‘ ‘ Time/min Fig. 4 standard, and narrow scan range (m/z 380-480). LC-(+ESMS) reconstructed mass chromatograms of individual pyrethroid ammoniated molecule ions, using a 10 pg ml-l mixed pyrethroid 18Q 541M ES+), 418 S414C ES+, - 4 4 h lea! Kadethrin 43 Time/min LC-(+ESMS) single-ion chromatograms of individual pyrethroid ammoniated molecule ions, using a 1 pg ml-l mixed pyrethroid standard. Fig. 5Analyst, January 1996, Vol. 121 59 ~~ Table 1 Limits of detection* for individual components of a pyrethroid mixture using full scant and SIR$ modes Pyrethroid M, Full scan/pg SIR/pg SSI-116 392 300 30 (4 10)s Kadethrin 396 120 12 (414)s a-Cypermethrin 415 300 60 (433)s) Flucythrinate 451 240 18 (469)s * Signal-to-noise ratio better than 3 : 1 for individual components of a pyrethroid mixture injected on column.t Using 10 pg ml-1 mixed standard of individual pyrethroids. * Using 1 pg ml-l mixed standard of individual pyrethroids. Monoisotopic mass of ammoniated molecule ion, [M + N&]+. a narrow mass range between m/z 380 and 480 (full-scan mode) at a rate of 3 s per scan or by monitoring single ions (selective- ion recording, SIR) characteristic of the ammoniated molecule ions for each pyrethroid (dwell time 0.2 s, inter-scan delay 0.02 s and 1 Da span).Full-scan experiments were performed using mixed pyrethroid standards in the range 10-250 yg ml-l and mixed pyrethroid standards in the range 1-10 yg ml-l were used for SIR experiments. Loss of chromatographic resolution (band broadening) is observed, Figs. 4 and 5, compared with off-line experiments using isocratic reversed-phase HPLC with a standard size analytical column, 25 cm X 4.6 mm id Shandon ODS column, and UV detection (not shown). The band broadening is attributed largely to post-column dispersion in the length of unpacked capillary necessary for interfacing to the mass spectrometer. ‘Memory’ effects in the electrospray source region may also contribute. Although band broadening is observed, the specificity of mass spectrometry obviates the need to chromatographically resolve every single component by detection of unique ammoniated molecule ions and/or fragment ions (higher cone voltages). However, this does not apply in the case of isobaric compounds, including diastereoisomers, where chromatographic separation is necessary.Using full-scan mode, limits of detection at a signal-to-noise ratio better than 3 : 1 were calculated from the LC-(+ESMS) reconstructed mass chromatograms for each ammoniated mole- cule ion, Fig. 4, and found to be in the range 120-300 pg (0.30-0.77 pmol) injected on column, Table 1. Limits of detection at a signal-to-noise ratio better than 3 : 1 were calculated from the LC-(+ESMS) single-ion chromatograms of each ammoniated molecule ion, Fig. 5, and found to be in the range 12-60 pg (0.03-0.15 pmol) injected on-column, Table 1.Conclusion This study provides evidence that microbore reversed-phase LC-(+ESMS) at lower sampling cone voltages, in the presence of ammonium acetate and formic acid, is a sensitive analytical technique for the separation and unambiguous detection of structurally diverse pyrethroid insecticides at the trace level. Ammoniated molecule base peak ions were observed for all the insecticides studied at low cone voltage. Progressively higher cone voltages resulted in a diminution of the [M + NH4]+ population, with a concomitant increase in the abundance of diagnostically useful fragment ions. At higher sampling cone voltages the +ES spectrum of a- cypermethrin was found to yield fragment ions in common with the compound’s +EIMS and/or +CIMS spectra.Tandem electrospray mass spectrometry experiments, performed on key ions observed in the spectra of a-cypermethrin, have provided evidence that the fragment ions produced in each ionization process have similar internal energies. Limits of detection at a signal-to-noise ratio better than 3 : 1 for each individual component of a pyrethroid mixture injected on column were in the range 120-300 pg using full-scan mode and 12-60 pg by SIR. Where tandem mass spectrometry is available further improvements may be made to achieve ultimate sensitivity, especially in the presence of high chemical noise, by monitoring in selective-reaction monitoring mode (SRM) key ions which have a product/precursor ion relationship. The authors thank Dr.T. Takahashi of Shionogi & Co. and R. E. Hyson of Cyanamid for the donation of research-grade samples and provision of technical help. We also thank Dr. P. Myers of Phase Separations, for the kind donation of the microbore HPLC column and Professor S. Gaskell and colleagues at the Michael Barber Centre for Mass Spectrometry, UMIST, for their help and support. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Pyrethrum-The Natural Insecticide, ed. Casida, J. E., Academic Press, New York, 1973. Elliott, M., in Recent Advances in the Chemistry of Insect Control, Special Publication No. 53, ed. Janes, N. F., The Royal Society of Chemistry, London, 1985, pp. 73-102. Udagawa, T., Numata, S., Oda, K., Shiraishi, S., Kodaka, K., and Nakatani, K., in Recent Advances in the Chemistry of insect Control, Special Publication No.53, ed. Janes, N. F., The Royal Society of Chemistry, London, 1985, pp. 192-204. Bushell, M. J., in Recent Advances in the Chemistry ofinsect Control, Special Publication No. 79, ed. Crombie, L., The Royal Society of Chemistry, London, 1989, pp. 125-141. Sieburth, S. McN., Lin, S. Y., Engel, J. F., Greenblatt, J. A., Burkart, S. E., and Gammon, D. W., in Recent Advances in the Chemistry of insect Control, Special Publication No. 79, The Royal Society of Chemistry, London, 1989, pp. 142-149. Gordon, R. F. S., Bushell, M. J., Pascoe, R., and Enoyoshi, T., Pests and Diseases: Flufenprox-A New insecticide for Rice, Proceedings of an International Conference sponsored by the British Crop Protection Council, Brighton, November 23-26, vol. 1, 1992, pp. 81- 88. Bushell, M. J., and Salmon, R., in Advances in the Chemistry of insect Control 111, Special Publication No. 147, ed. Briggs, G. G., The Royal Society of Chemistry, Cambridge, 1994, pp. 103-1 16. Class, T. J., Znt. J . Environ. Anal. Chem., 1992, 49, 189. Class, T. J., J . High Resolut. Chromatogr., 1991, 14, 446. Fleet, I. A., Tetler, L. W., and Monaghan, J. J., Org. Mass Spectrom., 1993, 28, 626. Fleet, I. A., Tetler, L. W., and Monaghan, J. J., J . Mass Spectrom., 1995,30, 617. Fleet, I. A., and Monaghan, J. J., unpublished work. Lidgard, R. O., Duffield, A. M., and Wells, R. J., Biomed. Environ. Mass Spectrom., 1986, 13, 677. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M., Mass Spectrom. Rev., 1990, 9, 37. Volmer, D., Wilkes, J. G., and Levsen, K., Rapid Commun. Mass Spectrom., 1995, 9, 767. Ahuja, S., Trace and Ultratrace Analysis by HPLC, Wiley, New York, 1992. Paper 51051 77K Received August 3, 1995 Accepted September 20, I995

ISSN:0003-2654    

DOI:10.1039/AN9962100055

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, January 1996, Vol. 121 (61-66) 61 Investigation of On4 i ne Reversed-phase Liq u id C h r o mat og rap h y-Gas Chromatography-Mass Spectrometry as a Tool for the Identification of Impurities in Drug Substances Elise C. Goosensa, Karel H. Stegmana, Dirk de Jonga, Gerhardus J. de Jong" and Udo A.Th Brinkmanb a Solvay Duphar, Analytical Development Department, P.O. Box 900, 1380 DA, Weesp, The Netherlands Boelelaan 1083, I081 HV, Amsterdam, The Netherlands Free University, Department of Analytical Chemistry, De The potential of on-line reversed-phase (RP) LC-GC-MS for the identification of impurities in pharmaceutical products has been investigated. The technical aspects of the system were studied using the potential drug eltoprazine as test compound. After LC separation on a 2 mm id RPLC column, using 5 mmol 1-1 methanesulfonic acid in acetonitrile-water (84 + 16 v/v) as eluent, the eltoprazine-containing fraction of 200 p1 was introduced on-line into the GC-MS, the mass spectrometer being a magnetic sector instrument.Before this introduction, the methanesulfonic acid was removed on-line from the LC eluent via an anion-exchange membrane. Most of the solvent introduced into the GC-MS was evaporated via a solvent vapour exit installed in front of the capillary GC column. As an application, impurity profiling was performed on the drug substance mebeverine which contained mebeverine amine as test impurity. The addition of acetonitrile was necessary before introduction into the GC; therefore only about 10% v/v of the mebeverine amine LC peak was transferred to the GC-MS.Nevertheless, electron impact and chemical ionization spectra of the impurity could be obtained at a level of 0.1% with respect to the drug. Keywords: Impurity profiling; reversed-phase liquid chromatography; gas chromatography-mass spectrometry, drug substance Introduction During the various stages of developing new pharmaceutical products, such as impurity profiling of the drug substance, metabolic studies and stability studies of the drug substance and the formulated product, unknown compounds are often ob- served in liquid chromatography (LC). These impurities have to be characterized and identified if they are present at a level exceeding 0.1 %area with respect to the drug substance. In general, these unknowns are isolated by preparative LC and subsequently characterized by NMR, IR and MS.This is often a time-consuming process, mainly because of the required isolation procedure. Moreover, artefacts can be formed during isolation, which often includes evaporation and storage. It would therefore be highly desirable to couple the LC system directly to these spectroscopic devices. The practicality of LC- MS for drug identification, impurity and metabolite profiling and identification has been demonstrated'-3 and its potential has increased now that several interfaces have become available. LC-MS still has its limitations when the LC eluent contains non-volatile buffer salts or other additives. In addition, sufficient structural information of the compound of interest is not always provided, unless LC-MS-MS is performed.Another approach is to couple LC on-line with capillary gas chromatog- raphy (GC)-MS. The advantage of this approach is that GC-MS is a routine procedure which is highly sensitive and provides adequate structural information when using standard ionization techniques such as electron impact (EI) and chemical ionization (CI). Moreover, the extra separation step introduced in LC-GC- MS increases the over-all efficiency. On the other hand, application of the technique is limited to compounds amenable to direct GC analysis. In 1987 Raglione et al.4 described the use of LC-GC-MS for the analysis of solvent-refined coal. Liquid chromatography was used for ring-size separation, with subsequent GC-MS for isomer separation and identification.On-line LC-GC-MS was succesfully applied by Ostman and Nilssons and Vreuls et a1.6 for the determination of polycyclic hydrocarbons in urban air and vegetable oils, respectively. They all used normal-phase LC systems, i.e., apolar mobile phases. Ogorka and co-workers,7.8 on the other hand, used reversed-phase (RP) LC-GC-MS for the identification of unknown compounds observed in RPLC chromatograms during the development of new drug sub- stances. The authors circumvented the problems arising from the high water content of the LC eluent and the presence of buffer salts by inserting a liquid-liquid extraction step between the RPLC and GC parts of the system. In this way, the impurity of interest was extracted from the aqueous LC fraction into an organic solvent.Subsequently, 500 p1 of the extract were transferred on-line to the GC-MS part of the system. Depending on the analyte and composition of the LC eluent, dichloro- methane, n-pentane or n-hexane was used as the extraction solvent. In earlier papers we reported on the feasibility of the direct coupling of RPLC with GC.9-11 Acetonitrile-water mixtures containing methanesulfonic acid (MSA) as an ion-pair reagent were used as the eluent in a RPLC-GC set-up in which MSA was removed by an anion-exchange membrane prior to introduction into the GC.' The potential drug eltoprazine was used as the test compound, and LC columns with an id of 2 mm and eluent flow rates up to 200 p1 min-1 were used. Insertion of a solvent vapour exit in front of the capillary GC column allowed an introduction volume into the GC of 200 pl at an introduction rate of 200 pl min-1.The technique was found to be limited to acetonitrile-water mixtures in which the percent-62 Analyst, January 1996, Vol. 121 age of water does not exceed that of the azeotrope (84 + 16 v/v) . 9 3 10 In this paper the use of RPLC-GC-MS for the identification of impurities and degradation products of pharmaceutical products was studied using a magnetic sector mass spec- trometer; eltoprazine was selected as the test compound. The drug substance mebeverine and some minor impurities were selected as model compounds for the impurity profiling study. Mebeverine amine was selected as the 'unknown impurity' which had to be identified at a level of 0.1% of mebeverine.Experimental Chemicals Eltoprazine, mebeverine, mebeverine amine, butoverine, verat- ric acid and 3-desmethyl-mebeverine were from Solvay Duphar (Weesp, The Netherlands). Tetrabutylammonium hydroxide 30-hydrate was purchased from Fluka (Buchs, Switzerland). Potassium hydroxide, potassium dihydrogenphosphate and sulfuric acid were from J.T. Baker (Deventer, The Netherlands) and methanesulfonic acid from Merck (Darmstadt, Germany). All other reagents were of analytical-reagent grade. Before use, the eluents and regenerant solutions were filtered through a Millipore (USA) HA membrane (0.45 mm) to remove small particles. Apparatus and Procedures The LC system consisted of a Model 260D syringe pump (ISCO, Lincoln, NE, USA), a 25 cm X 2 mm id Chromspher poly-cls LC column from Chrompack (Bergen op Zoom, The Netherlands), a Rheodyne (Cotati, CA, USA) injection valve with an internal loop volume of 1 pl (Type 7413) and a UV detector from Jasco (Tokyo, Japan).The GC part of the system consisted of two GCs, a Mega GC (Carlo Erba Strumentazione, Milan, Italy) with on-column injector and autosampler (ASSSO), in which the retention gap (10 m X 0.53 mm id CPWax52CB, d f = 0.02 pm, Chrompack) was installed, and a second Mega GC for the capillary analytical column (30 m X 0.32 mm id DB-1, d f = 0.25 pm; J&W, Folsom, CA, USA). The two GCs were connected via a heated interface with a length of 70 cm (Horst, Lindenfels, Germany) to control the temperature of the final part of the retention gap. A solvent vapour exit (SVE) was installed in the second GC between the retention gap and the analytical column via a Y- shaped glass press-fit.A 10 cm X 0.32 mm id fused silica capillary connected this Y-piece with another glass Y-piece which was connected with an on-off pinch solenoid valve (Sirai, Pioltello, Italy) and a restrictor (1 m X 0.05 mm id X 0.36 mm od deactivated fused silica, LC-Service, Emmen, The Netherlands). Apart from the valve, the whole SVE assembly was installed in the GC oven. A cation micromembrane suppressor (CMMS-2 mm, Dio- nex, Sunnyvale, CA, USA) was inserted in the eluent stream between the LC column and the UV detector. The regenerant was delivered by an SSI (State College, PA, USA) pump. If necessary, acetonitrile was added to the LC eluent via a Spectroflow 400 pump (Kratos Analytical, Ramsey, NJ, USA).Electron impact and CI spectra were recorded on a Kratos (Manchester, UK) Concept 1 S double focusing mass spec- trometer, which was coupled to a Sun computer with Mach3 software for computing data. Perfluorokerosine was used as the reference compound for calibration. A schematic presentation of the total RPLC-GC-MS system is given in Fig. 1. After sample introduction into the LC system and separation on the LC column, the eluent flows through the CMMS device which contains the anion-exchange membrane. The regenerant solution is continuously pumped along the other side of the membrane in a direction opposite to that of the LC eluent. MSA in the eluent is exchanged with the hydroxide ions from the regenerant. After the passage through the CMMS, acetonitrile can be added to the eluent in order to achieve the azeotropic acetonitrile-water ratio (84 + 16 v/v).The eluent is then flushed through the syringe needle of the on-column autosampler and subsequently led to waste. Transfer of an LC fraction is accomplished by introducing the needle for a few seconds, the transfer time, into the retention gap. The transfer volume, i.e., the volume introduced into the GC, can be calculated from the introduction rate and transfer time. As soon as the transfer is started, the temperature programmes of both GCs are started, the valve of the SVE is opened and most of the solvent is discharged to the atmosphere. Following evaporation of the solvent, the SVE valve is closed and gas flows through the retention gap and the analytical GC column to the MS, purging the remaining solvent in the SVE line via the restrictor.10 By starting the temperature ramp of the retention gap (after 3 min) prior to that of the GC column (after 8 min), a cold trapping effect can be created at the top of the analytical column; as a result, narrow reconcentrated peaks are obtained.gJ0 The mass spectrometer started to acquire data after a typical solvent delay time (4 min for eltoprazine and 2.5 min for mebeverine amine).The mass range mlz 50-500 was scanned at 0.8 s decade-]. The transfer line temperature was 280 OC, the source temperature 100 "C, the electron current 500 pA and the electron energy 70 eV. In the CI mode, ammonia-methane (90 + 10 v/v) was used as the reagent gas. All further RPLC-GC-MS conditions for the determination of eltoprazine and mebeverine amine are given in Table 1.Results and Discussion Development of the RPLC-GC-MS system In a previous paper we described the on-line coupling of RPLC and GC for the determination of eltoprazine." A 2 mm id LC eluent pump UV detector ionisation valve MS Fig. 1 Schematic diagram of the RPLC-GC-MS instrumentation.Analyst, January 1996, Vol. 121 63 column was used and the eluent was acetonitrile-water (84 + 16 v/v) which contained 5 mmoll-1 methanesulfonic acid (MSA), the flow rate being 200 p1 min-1. By using a thin film coated Carbowax retention gap, acetonitrile-water mixtures can be introduced into the GC at introduction rates of up to 200 p1 min-l and introduction volumes up to 200 p,l with a restriction to the water content of the RPLC eluent which should not be higher than about 16% v/v.9JO However, because any addition of buffer components or additives, even volatiles, ruined the retention gap or distorted the peak shape of the analyte," MSA had to be removed from the eluent before introduction of the eltoprazine-containing fraction into the GC.About 99.9% of MSA was removed from the LC eluent via an in-line coupled anion-exchange micromembrane inserted be- tween the LC and GC parts of the system. MSA ions were exchanged with hydroxide ions present in large excess in the regenerant. The regenerant also contained 84% v/v acetonitrile, in order to prevent diffusion of acetonitrile from the eluent to the regenerant side of the membrane, which would disturb the percentage of water in the eluent.A 200 p1 volume of LC effluent, free of MSA, was introduced into the Carbowax- coated retention gap using an SVE in front of the capillary GC column to eliminate most of the solvent. No losses of eltoprazine were observed (recovery, 99%) and repeatability was satisfactory (relative standard deviation at the 150 pg ml- level, 3%). In the present study, this RPLC-GC system was coupled with a magnetic sector mass spectrometer instead of a flame ionization detector. Fig. 2 shows a total ion current (TIC) chromatogram as well as an EI spectrum of eltoprazine obtained after a 1 p1 injection of a 150 pg ml-1 eltoprazine standard solution. Of the other peaks shown in the chromatogram, the last two are siloxane-containing compounds due to stripping of the stationary phase of the GC column.The first peak is tributylamine; probably this is an impurity from the regenerant. Obviously, the EI-spectrum of eltoprazine obtained by means of LC-GC-MS, with the molecular ion at m/z 220 and its relevant fragments at m/z 178 and m/z 163, is fully comparable with the library spectrum. Although this is a satisfactory result, there is one main aspect that has to be considered when coupling the MS to the RPLC-GC system: the MS has to tolerate the introduction of large volumes of acetonitrile-water vapour. In our RPLC- GC system, about 95% of the LC eluent is eliminated via the solvent vapour exit. This means that, out of the introduction volume of 200 pl of acetonitrile-water (84 + 16 v/v), 10 pl will be transferred to the MS.Preliminary experiments, however, showed that the maximum volume of solvent that can be introduced into the ion source is about 3 p1. The introduction of more solvent disturbed the electronics and vacuum of the system. The problem was solved by temporarily closing the isolation valve between the ion source and the mass analyser of the MS (see Fig. 1) during solvent introduction, which was indicated by a pressure increase in the ion source. The valve was re-opened when the vacuum had been restored and data acquisition was started after the solvent delay time. In other words, the introduction of 200 yl of acetonitrile-water into the GC-MS apparently does not create problems if an SVE is used and the spectrometer is protected during solvent introduction into the MS. For comparison, Ogorka and c o - w ~ r k e r s ~ ~ ~ transferred LC fractions of 500 p1 apolar solvents to a GC-MS, with the difference that they used a quadrupole-type MS instead of a magnetic sector instrument.In order to avoid malfunctioning of the MS, next to an SVE they also used a GC-MS open-split interface (ratio about 1 : 11) and the filament was switched off for about 10 min. Coupling LC-GC to a magnetic sector MS has some advantages over a quadrupole MS; for example, high resolution MS reveals the exact mass of the mass peaks and can therefore facilitate the elucidation of the structure or identifica- tion of unknown compounds. On the other hand, the scanning rate that can be used with a quadrupole MS is higher compared with a magnetic sector instrument.In our system we have tried to find an optimum between the number of scans per peak and the ion intensity by varying the scanning rate from 0.3 s per decade (decrease in ion intensity) to 2 s per decade (fewer scans per peak, leading to the risk of missing the peak). As a compromise, 0.8 s per decade was selected for our experiments. Application: Impurity Profiling A mixture of mebeverine and some of its possible impurities (butoverine, veratric acid, mebeverine amine and 3-des- methylmebeverine) was selected as the test mixture to study the potential of RPLC-GC-MS as an identification technique for impurity profiling. Mebeverine amine was selected as the test impurity. Because of its low thennostability, mebeverine itself ~~ - ~~ ~~ ~ Table 1 RPLC-GC-MS conditions for the determination of eltoprazine and mebeverine amine Conditions Eltoprazine Mebeverine amine LC System Eluent Injection volume Flow rate UV detection Acetonitrile-water (84 + 16 v/v) + 5 mmol 1-1 Acetonitrile-water (50 + 50 v/v) + 5 mmol 1-1 MSA MSA or 1 mmol 1-1 MSA 1 Pl 1 czl 200 yl min-1 254 nm 200 p1 min-1 220 nm LC-GC Interface Membrane CMMS-2 mm CMMS-2 mm Regenerant Flow rate 2 ml min-1 2 ml min-1 Acetonitrile addition - 500 pl rnin-1 60 mmol 1-l TBAOH in acetonitrile-water 100 mmol 1-1 KOH in acetonitrile-water (84 + 16 v/v) (50 + 50 v/v) GC System Introduction rate 200 pl min-1 Temperature GC 1 Temperature GC2 SVE vent-time 90 s Inlet pressure 150 kPa He Introduction volume 200 p1 Temperature interface 200 "C 95 "C (3 min), 30 "C min-1,200 "C 80 "C (8 min), 30 "C min-1,280 "C 700 pl min-1 60 pl 95 "C (3 rnin), 30 "C min-I, 280 "C 80 "C (8 rnin), 30 "C min-',280 "C 150 "C 15 s 150 kPa He64 Analyst, January 1996, Vol.121 cannot be analysed by GC. Therefore, LC acts as a pre- separation step, separating the main product and the impurities from each other. The first step was to develop an LC procedure involving the use of an eluent that is compatible with the GC system. In the existing LC procedure, acetonitrile-50 mmol 1-1 potassium phosphate buffer (pH 6) (40 + 60 v/v) was used as the eluent. An attempt was made to remove this non-volatile additive from the eluent by coupling an anion- and cation-exchange membrane in series between the LC and GC parts of the system.However, the attempts failed because the phosphate ions were not completely removed by the CMMS device (a removal of 75% of the phosphate ion, determined by ion chromatography). Therefore, we preferred to add MSA to the eluent and to use the CMMS for anion removal only. Fig. 3 shows the LC separation of the mixture before and after passage through the CMMS, using acetonitrile-water (50 + 50 v/v) containing 5 mmol 1-1 MSA as eluent. The peak areas of both veratric acid (a) and 3-desmethylmebeverine (c) are seen to be distinctly smaller after passage through the membrane. For the former analyte, with its acidic nature, this is according to expectation. However, the loss observed for the tertiary amine (c) can not easily be explained. The peak area of the test impurity, mebeverine amine (b), remained the same.Passing the membrane device did cause some peak broadening, as was already observed in a previous study." t 6.44 9.29 12.13 14.58 17.43 Time - 178 i 9 m/z 80 io 80 80 loo lb lu, Is0 leo 2.00 820 m/z Fig. 2 (a) TIC chromatogram and (b) EI spectrum of eltoprazine obtained by LC-GC-MS of a standard solution containing 150 pg ml-1 of eltoprazine; (c) library spectrum of eltoprazine. *, Siloxane-containing compounds; t tributylamine. LC injection volume, 1 p1; transfer volume, 200 p1. For further conditions, see Table 1. Since the maximum percentage of water in an acetonitrile- water mixture that can be introduced into the GC system is 16% v/v, our aim was to minimize the percentage of water in the eluent, while still maintaining sufficient separation between mebeverine and each of the impurities. By using a Chromspher Poly C18 LC column, the minimum percentage of water was found to be 50% v/v.Because acetonitrile can diffuse through the membranes,ll 90% v/v of acetonitrile was added to the regenerant solution to provide acetonitrile diffusion to the eluent side. Unfortunately, the increase of the acetonitrile content of the mobile phase was only minor (2% v/v). Consequently, after passage through the CMMS, acetonitrile still had to be added to the LC eluent in order to achieve the required 84 + 16 (v/v) acetonitrile-water ratio. By adding 500 pl min-1 of acetonitrile to the LC eluent [acetonitrile-water (50 + 50 v/v); flow rate, 200 pl min- 13, the required 84 : 16 ratio was obtained. A main consequence of this addition was that the introduction rate into the retention gap increased from 200 to 700 yl min-1 , while the evaporation rate during introduction into the GC (175 pl min-I), of course, did not change. The rather large difference between the introduction and the evaporation rate caused a serious reduction of the maximum introduction volume, as is shown in Table 2.From the maximum introduction volume of 58 pl, 2/7 (16 pl) originates from the LC eluent. As the mebeverine amine- containing LC fraction is about 160 yl, only 10% v/v of the LC peak can be introduced into the GC-MS. If the 16 p1 heart-cut is taken at the peak maximum, the mass percentage of impurity that is transferred will be about 20%. Fig. 4 shows a TIC of the LC-GC-MS transfer of mebeverine amine obtained with a 0.2 mg ml-1 mebeverine amine standard solution (1 pl injected on the LC) and the pertinent EI spectrum.Because of extensive d - e 1 9 b a I. Time/min 10 Fig. 3 of mebeverine (d) and 25 pg ml-l of mebeverine amine (b) and some other impurities [veratric acid (a) 3-desmethylmebeverine (c) butoverine (e)]; (a) before CMMS passage, (h) after CMMS passage. LC injection volume, 1 pl; eluent, acetonitrile-water (50 + 50 v/v) containing 5 mmol 1-1 of MSA; flow rate eluent, 200 pl min-I; regenerant, 100 mmol 1-1 KOH in acetonitrile-water (50 + 50 v/v); flow rate regenerant, 2 ml min- l . LC-UV chromatogram of a standard mixture containing 1 mg ml- Table 2 Effect of raising the introduction rate into the GC Introduction rate 700 p1 min-' Evaporation rate 175 pl min-' Maximum volume of liquid in 10 m retention gap (9) 45 p1 Maximum transfer time 5 s Maximum introduction volume 58 plAnalyst, January 1996, VoE.121 65 50 I fragmentation into fragments with m/z 149, 121 and 72, the molecular ion of the compound (m/z 193) could not be detected. In a second run, a CI spectrum was therefore also recorded. This distinctly shows the protonated molecular ion m/z 194 (Fig 4). Obviously, using both ionization modes, EI and CI, in tandem, is an interesting tool for obtaining structural information. The mixture shown in Fig. 3 contained 2.5% m/m of mebeverine amine (25 pg ml-1) compared with mebeverine. After transfer of the mebeverine amine fraction to the GC-MS, about 5 ng of this compound is finally detected, which amount was considered to be close to the identification limit (see below). Since it was our aim to identify impurities as low as 0.1 % m/m of the main compound, the mebeverine concentration in the mixture had to be increased from 1 mg ml-1 to 25 mg ml-1.Maintaining the same mebeverine amine concentra- tion (25 pg ml-I), a 0.1% m/m level was then obtained. Introduction of this mixture into the LC system gave a mebeverine peak that was strongly overloaded; consequently, mebeverine amine co-eluted with the main peak. Decreasing the 100 50 0 11 ' I CH, Mebeverine amine I_ 501 I 121 ,., , . , , / ' , , , ;, . 60 80 100 120 140 160 180 200 220 240 0 1 J , I , , , , , , , , , , , I , m/z 194 100 3 I 1 72 I MSA concentration from 5 to 1 mmol 1-1 improved the separation of mebeverine amine from mebeverine sufficiently [(Fig.5(a)]. On-line transfer of the mebeverine amine fraction (about 5 ng) to, and analyses by, GC-MS yielded the EI and CI spectra shown in Fig. 5(b), which shows the two most important fragments (EI: m/z 72 and 121) and the protonated molecular ion (CI: m/z 194), respectively. The total and the extracted ion chromatograms in Fig. 5(a) show that this amount is very close to the identification limit of the system. If necessary, single ion monitoring at m/z 121 and 72 will increase sensitivity and therefore will contribute to confirm the presence of these ions in the peak. Obviously, an identification level of 0.1 % with respect to the drug substance can be achieved, despite the relative inefficiency of the LC separation, the unfavourable degree of fragmentation of the test impurity and the relatively high water percentage of the LC eluent.b I 0 28 Ti me/m in 5.1 3 7.57 10.42 13.26 16.11 18.55 Time + '"1 50 4 72 121 60 80 100 120 140 160 180 200 0 m/z 194 50 60 80 100 120 140 160 180 200 220 01 m/z Fig. 5 (a) LC-UV chromatogram of a mixture containing 25 mg ml-1 of mebeverine (d) and 25 pg ml-I (0.1% m/m) of mebeverine mine (b) and veratric acid (a) after CMMS passage with a TIC and extracted ion chromatograms (mlz 72 and mlz 121) of the transferred mebeverine amine fraction (about 5 ng) obtained by LC-GC-MS. (b) The pertinent EI and CI spectra of the transferred mebeverine amine fraction. LC injection volume, 1 y1; eluent, acetonitrile-water (50 + 50 v/v) containing 1 mmol I-* MSA; flow rate eluent, 200 yl min-1; regenerant, 100 mmol 1-1 KOH in acetonitrile-water (50 + 50, v/v); flow rate regenerant, 2 ml min-l; acetonitrile addition, 500 p.1 min-'; transfer volume, 58 pl.For further conditions, see Table 1.66 Analyst, January 1996, Vol. 121 Conclusions The present work gives a fair idea of the potential and limitations of on-line RPLC-GC-MS. No technical problems are encountered when 200 pl LC eluent fractions are used for analysis, and additives such as MSA can be removed by means of a membrane-based anion exchange. If precautions are taken, such as solvent venting in the GC part of the system and protection of the MS analyser during solvent introduction, MS detection does not meet with any serious problems, even when using a magnetic sector instrument.Finally, the sequential recording of EI and CI spectra adds to the identification power of the total LC-GC-MS set-up. The main limitation of the present system is the maximum percentage of water in the LC eluent of 16% v/v. As most RPLC separations require the use of distinctly higher water contents, post-LC addition of acetonitrile is necessary which adversely affects the maximum allowable introduction volume into the retention gap and, thus, the sensitivity of the total procedure. The use of LC columns with smaller internal diameter (0.32-1 mm) will not really be beneficial because of the negative influence on the loading capacity. One recommendation should be to select an LC stationary phase which enables separations of the analytes of interest with an LC eluent which is rich in organic modifier.Another aspect of some concern is the ion- exchange membrane device, which gets clogged rather easily. It is therefore important to filter all solutions prior to use. Besides, one should always be aware that analyte losses may occur as a result of (largely unknown) analyte-membrane interactions. As regards a comparison with LC-MS, both techniques can obviously play a complementary role in impurity profiling. LC- MS is a powerful technique for the determination of molecular ions of relatively polar and high relative molecular mass compounds that are not amenable to direct analysis by GC. However, for all compounds that can be subjected to direct GC analysis, LC-GC-MS is the method of choice because of the improved separation efficiency, the higher sensitivity as well as the much more versatile and powerful identification.It should be noted that the GC behaviour is unknown prior to the identification. Finally it should be admitted that, because of the in-between GC step, two typical problems of LC-GC-MS, viz., the presence of buffer salts and the percentage of water in the eluent, are less stringent in LC-MS. Here, volatile buffer constituents like ammonium acetate and fonnate can often be tolerated or are even necessary, and water does not create problems at all. In conclusion, the present study illustrates that RPLC-GC- MS can serve highly useful purposes in areas of applications such as impurity profiling, where qualitative (structural in- formation) rather than quantitative analysis is of primary importance. Further optimization should mainly be directed at improving RPLC-GC interfacing for aqueous mobile phases. The authors like to thank the students W. Salburg and L. Maslam for their experimental work and P. Scherpenisse from the AM1 group at Solvay Duphar for stimulating discussions. References 1 2 3 4 5 6 7 8 9 10 11 Emi, F., J . Chromatogr., 1982, 251, 141. Tomer, K. B., and Parker, C. E., J . Chromatogr., 1989, 492, 189. Qin, X., Ip, D. P., Chang, K. H.-C., Dradransky, P. M., Brooks, M. A., and Sakuma, T., J. Pharm. Biomed. Anal., 1994,12,221. Raglione, J. T. V., Troskosky, A., and Hartwick, R. A., J . Chromatogr., 1987, 409, 213. Ostmann, C., and Nilsson, U., J . High Resolut. Chromatogr., 1992, 15, 745. Vreuls, J. J., de Jong, G. J., and Brinkman, U. A. Th., Chromato- graphia, 1991, 31, 113. Ogorka, J., Schwinger, G., Bruat, G., and Seidel, V., J . Chromatogr., 1992, 626, 87. Wessels, P., Ogorka, J., Schwinger, G., and Ulmer, M., J. High Resolut. Chromatogr., 1993, 16, 708. Goosens, E. C., de Jong, D., van den Berg, J. H. M., de Jong, G. J., and Brinkman, U. A. Th., J . Chromatogr., 1991, 552,489. Goosens, E. C., de Jong, D., de Jong, G. J., and Brinkman, U. A. Th., J . Microcol. Sep., 1994, 6, 207. Goosens, E. C., Beerthuizen, I. M., de Jong, D., de Jong, G. J., and Brinkman, U. A. Th., Chromatographia, 1995,40, 267. Paper Sl05009J Received July 28, 1995 Accepted September 15, I995

ISSN:0003-2654    

DOI:10.1039/AN9962100061

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, January 1996, Vol. 121 (67-69) 67 Establishing the Cut-off Concentration for the Detection of Etorphine in Horse Urine Robert F. Smitha, Laurence S. Jackson4 and Andrew Moorec a Division of Biomedical Science and Health Research Institute, ShefSield Hallam University, Shefield UK S I 1 WB. E-mail: R.F.Smith@shu.ac.uk c Pain Relief Unit, The Churchill, Oxford UK OX3 7L.J Horse Racing Forensic Laboratory, Newmarket UK CB8 7DT An 1251 radioimmunoassay to determine the pattern of urinary excretion of etorphine (a semisynthetic opiate agonist) after its administration to horses is described. Three thoroughbred horses were each given 5,15,30 and 100 pg of etorphine intramuscularly. Urine was collected for up to 72 after administration. The maximum etorphine concentration after administration of a dose of 5 pg was 711 pg ml-1 (concentrations were greater than 100 pg ml-1 after 23 h in all three horses); a 15 yg gave 2661 pg ml-1 (levels remained above 100 pg ml-l for more than 44 h in each horse); a 30 yg dose gave a maximum of 3344 pg ml-l (levels were above 100 pg ml-l for 24,72 and 72 h); and 100 pg gave in excess of 10 000 pg ml-1 (levels were greater than 300 pg ml-1 for up to 70 h).Forty-eight urine samples from horses not given etorphine all had levels of etorphine less than 100 pg ml-1. There was no increase in apparent etorphine concentrations after hydrolysis of samples with p-glucuronidase and aryl sulfatase. The half-lives of etorphine equivalents (calculated with a mono-exponential equation after the 100 pg dose) in the urine of the three horses were 569,803 and 821 min, respectively.We conclude that radioimmunoassay can provide a useful first line screening procedure for the assessment of etorphine use in racing horses. Keywords: Etorphine; kinetics; equine; radioimmunoassay Introduction Etorphine (4,5-epoxy-3-hydroxy-6-methoxy-a,l7-dimethyl-a- propyl-6,14-ethenomorphinan-7-methanol), is a potent semi- synthetic opiate agonist, derived from thebaine, a phar- macologically inactive opium alkaloid. A characteristic of etorphine is its high potency, its rapid onset and short duration of action. In human beings the effects of etorphine are similar to those of morphine.' Etorphine is used in veterinary practice combined with a phenothiazine (Large Animal Immobilin) for use in restraining large animals, or with methotrimeprazine (Small Animal Immobilin) where it is used for sedation and analgesia in small-animal surgery.In horses, low doses of etorphine induce a locomotor stimulant response similar to that noted for other opiates such as morphine and fentanyl.2 This combination of locomotor effect and low dose has made etorphine eminently suitable for illegal and inappropriate use to influence the outcome of horse races. Etorphine has been successfully measured using a variety of techniques: GC-MS;3 HPLC;4 ELISA;5 and radioimmunoassay (RIA).6 The high sensitivity and ability to analyse large numbers of samples in a relatively short time has made immunoassay a useful tool for the screening of large numbers of samples for drugs before confirmation by other techniques.This paper describes the use of commercially available reagents to screen horse urine for the presence of etorphine and considers the setting of an appropriate cut-off point to determine positives from negatives. Experimental Etorphine Administration and Sample Collection Etorphine were administered to each of three thoroughbred horses on four separate occasions. Doses of 5,15,30 and 100 pg of etorphine were administered intramuscularly (IM) to each horse and urine was collected, whenever voided, for up to 72 h after the dose. In addition, 48 urine samples were collected, without preservative, from horses not given etorphine and stored frozen at -20 "C until analysis. Radioimmunoassay The RIA reagents were used as supplied by the manufacturer (EuroDPC, Glyn Rhonwy, Llanberis, UK).A volume of 25 yl of a standard solution of etorphine in urine (0, 100, 500, 1000, 2500,5000 pg ml-I), control urine (300 and 3000 pg ml-l) or sample urine were incubated, in duplicate, for 1 h at room temperature with 100 p1 of 1251 of etorphine solution and 100 yl of rabbit anti-etorphine solution. Following this primary incubation, 1 ml of cold precipitating solution (donkey anti- rabbit antiserum in 8% m/v polyethylene glycol-saline) was added, and the tubes incubated for a further 10 min before centrifugation at 3000 rpm for 20 min at 4 "C. After centrifuga- tion the supernatant fluid was decanted and the bound radioactivity in each tube was counted for 60 s in a gamma counter. The data were plotted using a 4-parameter logistic- curve fit of the percentage : bound by bound zero ratio of the response variable against the logarithm of the etorphine concentration.The etorphine concentration of the unknown samples was determined as etorphine equivalents by inter- polation from the standard curve. Urine Hydrolysis In order to assess whether the RIA cross-reacted with conjugated metabolites of etorphine, 45 samples found positive for etorphine were analysed before and after incubation with 8- glucuronidase and aryl sulfatase. Urine samples (100 pl) were diluted (1 + 1) with citrate-phosphate buffer (pH 5) containing 200 U ml-1 (1 U = 16.67 nkat) of P-glucuronidase (EC 3.2.1.3 1, Type HI, from Helix pomatia; Sigma, Poole, Dorset, UK) and 80 U ml-1 of aryl sulfatase (EC 3.1.6.1, type V, from Patella vulgata; Sigma).Samples were incubated for 1 h at 37°C in a water-bath before analysis for etorphine by using RIA. The results were corrected for dilution.68 Analyst, January 1996, Vol. 121 Results The etorphine RIA gave a standard curve between 100 and 5000 pg ml-*. Assessment of the assay precision was made by selecting three samples to give a range of values throughout the standard curve (220, 1008, and 2006 pg ml-1) and measuring the mean, s and relative standard deviation for 20 pairs of tubes in a single assay (intra-assay) and as a duplicate pair in 20 separate assays (inter-assay). The precision was found to be < 10% for both intra- and inter-assay. Sensitivity was assessed as the apparent etorphine concentration equivalent to the mean minus 2 s of the response (given by measurement of the zero calibrator 40 times) and was determined as 15.3 pg ml-l.The assay was shown to cross-react with N-dealkyl etorphine (77%) and diprenorphine (7 1 %). There was no significant increase in the apparent etorphine concentration after hydrolysis of samples with P-glucuronidase and aryl sulfatase. Assessment of Optimum Cut-off The forty-eight samples from horses not given etorphine all had levels c 100 pg ml-1. Etorphine was detectable in the urine of each animal given etorphine after each dose of drug (Figs. 1-4), although the duration of all positive tests varied with dose and with the choice of cut-off point. These findings are summarized in Table 1. 1000 Etorphine dose 5 pg 0 0 on v 100 Tirne/h Urinary etorphine concentrations against time for each of the three Fig.1 horses for a 5 yg dose. Etorphine dose 15 pg k‘ 0 0 0 A” 0 a 100 I I I I I I I 0 10 20 30 40 50 60 Time/h Urinary etorphine concentrations against time for each of the three Fig. 2 horses for a 15 yg dose. After administration of 5 pg of etorphine the maximum urinary etorphine concentration was 7 1 1 pg ml- and remained above 100 pg ml-l for more than 23 h in all three horses; after 15 pg of etorphine the maximum level was 2661 pg ml-1 and the etorphine remained above 100 pg ml-1 for more than 44 h in each horse; and after 30 pg of etorphine the maximum level was of 3344 pg ml-l and levels were above 100 pg ml-1 for 24, 72 and 72 h for each of the three horses, respectively. Early samples collected after the 100 pg dose contained etorphine concentrations greater than 5000 pg ml-1 and were still in excess of 300 pg ml-1 for up to 70 h.The half-lives of etorphine equivalents (calculated with a mono-exponential equation from the 100 pg dose) for the three horses were 569, 803 and 821 min. 10000 I - E a g 1000 e 5 cr, 1 .- c 1 I ) , I 100 I 0 10 20 30 40 50 60 70 80 Tirne/h Urinary etorphine concentrations against time for each of the three Fig. 3 horses for a 30 yg dose. 10000 - I E 0, Q -. .- r“ 1000 c P 5 100 Etorphine dose 100 I I I I I I I 0 10 20 30 40 50 60 70 80 Time/h Urinary etorphine concentrations against time for each of the three Fig. 4 horses for a 100 yg dose. Table 1 Range of times in the three horses given etorphine at each dose where urinary etorphine concentrations remained above the putative cut-off value Duration of positive test/h Dose/pg 500/pg ml-’ 250/pg ml-l lOO/pg ml-l 5 3 4 18-46+ 2446+ 15 10-18 2 w + 44-46+ 30 18-25 22-26 24-72+ 100 52-65 68+ 68+Analyst, January 1996, Vol.121 69 Discussion Etorphine has three important effects in the horse. It is a locomotor stimulant, an analgesic, and may extend the time to exha~stion,~ making it suitable for abuse in horse-racing. The low levels of drug found in the urine of horses administered etorphine and the large number of samples to be tested presents a considerable analytical challenge. Immunoassay combines the advantages of excellent sensitivity and high sample throughput; features which make the technique ideal for screening applica- tions before confirmation with other analytical techniques. The problem for any screening procedure lies in the establishment of an appropriate cut-off point which can accurately determine true positives from false positives.In the case of immunoassay this is not just a question of sensitivity but of the background noise or ‘matrix effect’ caused by use of undiluted, unextracted biological samples; a problem which is well known as a cause of false positives particularly affecting equine samples. The choice of cut-off point will always be a trade off between maximizing true positives and minimizing false positives, although in the case of drug abuse some false negatives may be tolerated in order to avoid the consequences which can ensue from false positives, even given that all positive samples will be further investigated. For etorphine to be effective a horse would need to receive a dose of drug very shortly before a race and sampling would be expected to take place as soon as possible after the race and in any case within 24 h.For these reasons a cut-off point of 100 pg ml-1 appears to be a level suitable for the screening of samples for the illegal use of etorphine. All 48 samples from horses not given etorphine had apparent etorphine concentrations below this level and, even at the lowest dose of etorphine, levels remained above 100 pg ml-1 for between 24 and 46 h. The incubation of etorphine positive samples with hydrolysing enzymes did not significantly increase the concen- tration of etorphine equivalents found in the urine.This is likely to be because the antiserum was equally cross-reactive to both conjugated and free etorphine. The long (> 10 h) half-life of etorphine and etorphine glucuronides in equine urine found in this study accords well with the long half-life of etorphine reported in human plasma,8 and with other potent opiates (such as buprenorphine) which are also known to be excreted into urine over many hours and days post-administration. We conclude that a simple radioimmunoassay using com- mercially available reagents is of value in the screening of urine samples for etorphine abuse in racing horses before confirma- tion with reference analytical techniques. References Jasinski, D., Griffith, D. R., and Cam, D. B., Clin. Pharmacol. Exp. Ther., 1979, 17, 267. Tobin, T., in Drugs and the Performance Horse, Charles C . Thomas, Springfield, IL, USA, 198 1. Bonnaire, Y., Plou, P., Pages, N., Boudene, C., and Jouany, J. M., J . Anal. Toxicol., 1989, 13(4), 193. Glasel, J. A., and Venn, R. F., J . Chromatogr., 1981, 213(2), 337. Stanley, S., Jeganathan, A., Wood, T., Henry, P., Turner, S., Woods, W. E., Green, M., Tai, H-H., Watt, D., Blake, J., and Tobin, T., J . Anal. Toxicol., 1991, 15, 305. Tai, C. L., Wang, C. J., Weckman, T. J., Popot, M. A., Woods, W. E., Yang, J. M., Blake, J., Tai, H. H., and Tobin, T., Am. J. Vet. Res., 1988,49(5), 622. Suann, C., Rose, R., Plummer, C., Knight, P., Proc. 8th Zntl. Con$ Racing Analyst Vet 1990. Friedrich, G., Braunstein, P., Friedrich, M., Vach, W., Beitr. Gerichtl. Med., 1991, 49, 111. Paper 5105353F Received August 10,1995 Accepted September 20, I995

ISSN:0003-2654    

DOI:10.1039/AN9962100067

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, January 1996, Vol. 121 (71-73) 71 Amperometric Biosensor for the Determination of the Artificial Sweetener Aspartame With an Immobilized Bienzyme System Shu-Fen Chou Department of Food Health, Chia-Nan College of Pharmacy, Tainan Hsien, Taiwan A biosensor using an immobilized bienzyme system was developed to determine the artificial sweetener aspartame in foodstuffs. The bienzyme system, consisting of a-chymotrypsin and alcohol oxidase, was co-immobilized by a covalent cross-linking method with glutaraldehyde on a dissolved oxygen electrode. The optimum operating conditions for the biosensor were pH 6.5-8.5 and 30 "C. A linear relationship was observed between the decrease in dissolved oxygen and the aspartame concentration in the range 0.33-2 mmol l-1. When the enzyme electrode was stored in dithiothreitol solution, the biosensor was stable for more than 50 d and 450 assays.An amperometric biosensor method was established for the assay of aspartame in commercial products without the need for sample pre-treatment or special reagents. Such a development should facilitate rapid quality control testing in the food industry. Keywords: Aspartame; amperometric biosensor; covalent cross-linking method Introduction Aspartame (L-aspartyl-L-phenylalanine methyl ester; Nutra- sweet) is a low calorie artificial sweetener with a pronounced sucrose-like taste and which, organoleptically, is about 180 times sweeter than sucrose. Recently, it has been approved for use in various foods such as breakfast cereals, table-top sweeteners and soft drinks as well as other low calorie and therapeutic diets.It is also rapidly replacing saccharin which has a bitter aftertaste and has been implicated in causing cancer in laboratory animals. l ~ 2 Various chromatographic methods for the determination of aspartame in foodstuffs such as HPLC, GC, gel-permeation chromatography and TLG6 have been devel- oped. These methods, although having been properly optimized for routine applications, suffer from lengthy procedures. In 1985, a simpler method, viz., a microbial amperometric sensor for measuring the change in respiration rate (oxygen change) caused by uptake of substrate, was described.7~8 The device achieved reasonable sensitivity and stability; however, glucose and the amino acid constitutents of aspartame interfered. An attempt has been made to establish an enzyme sensor which exploits immobilized L-aspartase to convert the aspartame molecule into an electrode-detectable species (ammonium).9 Despite requiring no sample pre-treatment or special reagents, the sensor system was adversely affected by L-aspartate (usually present in beverages).In order to overcome this drawback, an amperometric enzyme sensor for aspartame, composed of a- chymotrypsin and alcohol oxidase, was developed. lo The bienzyme system was immobilized by an entrapping method with a dialysis membrane, and was combined with dissolved oxygen electrode equipment. The bienzyme system was able to convert the aspartame molecule into an oxygen electrode- detectable species, but is was only stable for about 7 d.In this work, the aforementioned bienzyme system was co- immobilized by a covalent cross-linking method with glutar- aldehyde and combined with electrochemical equipment in order to improve the operational stability of the biosensor. The results obtained indicate that the biosensor approach should be an attractive alternative for aspartame testing in the food industry. Experimental Reagents and Standards a-Chyrnotrypsin (EC 3.4.2 1.1 ., from bovine pancreas), alcohol oxidase (EC 1.1.3.13., from Hansenula sp.) and dithiothreitol were obtained from Sigma (St. Louis, MO, USA). Aspartame was obtained from Tokyo Kasei Chemical (Tokyo, Japan). Standard solutions of aspartame were prepared weekly and stored at 5 "C. All chemicals used were of analytical-reagent grade.Apparatus The sensor system (Fig. 1) consisted of a standard bath assembly [Yellow Springs Instruments (YSI) 5301, Yellow Springs, OH, USA], a standard oxygen probe (YSI 5357,5331), a biological oxygen monitor (YSI 5300), a circulator (Type BL20, Yihder Instruments, Taipei, Taiwan) and a recorder (Type BD40, Gilson, The Netherlands). Preparation of Immobilized Enzyme Electrode Enzymes were immobilized by a covalent cross-linking method with glutaraldehyde.9 No bovine serum albumin was used, because of the high protein content of a-chymotrypsin com- bined with alcohol oxidase. The lyophilized enzymes {a- chymotrypsin [250 mU (1 VU = 16.67 nkat)] and alcohol oxidase (250 mu)} were deposited on a dialysis membrane, and 20 yl of phosphate buffer were added in order to solubilize the two enzymes. Next, 2 pl of 6.25% m/v glutaraldehyde solution were gently added and mixed.The resulting membrane was allowed to dry at room temperature for 2 h, and was mounted against a gas-selective membrane, taking care to avoid bubble entrapment. The electrode was equilibrated in distilled water for at least 5 h at 30 "C before use. The electrode was stored in 10 mmol 1- dithiothreitol solution. All measurements were taken at 30 & 1 "C. Samples All samples were purchased from local supermarkets, Measure- ments were made after direct dissolution of the dry powdered72 Analyst, January 1996, Vol. 121 samples or dilution of Diet Coke with de-ionized water or phosphate buffer (pH 6.5-8.5). Results and Discussion Several enzymic systems might be able to convert the aspartame molecule into an electrode-detectable species,g for example: ( 1) with L-aspartase, liberation of ammonium ions from aspartame can be monitored potentiometrically with an ammonium ion- selective electrode.However, aspartate, which is a breakdown product of aspartame in stored soft drinks, can interfere. (2) Use of a direct process involving L-amino acid oxidase which might recognize aspartame. This enzyme might be able to convert the aspartame molecule into an oxygen electrode-detectable spe- cies. The limitations of this approach are low enzymic activity and poor selectivity. (3) Aspartame can be cleaved by a- chymotrypsin to give methanol, which is detectable with a second enzyme, alcohol oxidase. This bienzyme system can convert the aspartame molecule into an oxygen electrode- detectable species.A study was undertaken of the bienzyme system (3) in order to overcome the disadvantages of systems (1) and (2) and establish an aspartame biosensor. It has been demonstrated that aspartame can hydrolyse slowly in the low pH range used in soft drinks; hence the aspartame products become less sweet on prolonged storage.3 Thus, this biosensor might be used to monitor the deterioration in the quality of aspartame in stored soft drinks. However, the bienzyme system, which has reasonable sensitivity and high enzymic activity, could be used to analyse several types of foodstuffs. C Fig. 1 Schematic diagram of enzyme sensor system. A, Oxygen electron, B, standard bath assembly (1, exit port; 2, inlet port; 3, magnetic stirrer; 4, sample chamber; 5 , brake); C , biological oxygen monitor; D, recorder; and E, circulator. c z T l , , l , , l 3 4 5 6 7 8 9 PH Fig.2 Effect of pH on the aspartame biosensor. Circles, 0.05 mol 1-l acetate buffer; and triangles, 0.05 mol 1-1 phosphate buffer. Reaction temperature 30 "C. Optimization of Operating Conditions for the Biosensor Analytical parameters such as pH, temperature and linear range of substrate concentration were studied. Fig. 2 displays the optimum pH values of the biosensor with the immobilized bienzyme system [containing a-chymotrypsin (250 mu) and alcohol oxidase (250 mu)], and clearly indicates that the optimum pH values are in the range 6.5-8.5. Moreover, because the optimum pH values of this system covered a wide range (pH 6.5-8.5), de-ionized water could be substituted for buffer solution in the reaction (this was demonstrated experimentally).Thus, experimental procedures could be simplified. In order to study the effect of temperature, the decrease in dissolved oxygen (DO) was measured at 25,30,40,50 and 60 "C 9. The results demonstrated that the higher the temperature the larger the decrease in DO; however, the decrease in DO appeared to be slower above 50 "C. Hence, 30 "C was selected as the optimum temperature for reasons of economy and to improve the stability of the biosensor. Fig. 3 shows that under the optimum conditions, a linear relationship is observed between the decrease in DO and the aspartame concentration in the range 0.33-2 mmol 1-1.Interference Study The effects of some interferents which might be present in commercial low calorie foods on the aspartame biosensor were tested. The results demonstrated that, at a concentration of 0.66 mmol 1- l , most of the ingredients in foodstuffs such as sucrose, lactose, citric acid, succinic acid, silicon dioxide, sodium benzoate, dextrin and dextrose, and the amino acid breakdown products of aspartame, did not interfere; however, TRIS buffer did interfere because the propane-1,3-diol part of the molecule was also oxidized by alcohol oxidase." Hence, TRIS buffer could not be used as the buffer system. Biosensor Stability A prerequisite consideration for the possible application of the biosensor in industry is its operational stability. The operational 0 ' ' ' I r I 0.5 1.0 1.5 2.0 Concentration/rnrnol I-' Fig.3 Calibration curve of aspartame concentration for the biosensor. a a 150 .- - G o 1 ; ; ; : ; ;j ; ' 2 Ti rn e/d ays Fig. 4 Operational stabilities of the aspartame biosensor. Stored in (a) dithiothreitol solution; and (b) buffer solution. Reaction temperature 30 "C.Analyst, January 1996, Vol. 121 73 Table 1 Determination of aspartame in commercial products Aspartame content (%) Sample* Nominali Previous method* Proposed methods A 3.8 3.70 B 3.5 3.56 C 2.7 2.58 3.82 3.60 2.65 compared with a calibration graph for aspartame. The results obtained with the proposed biosensor closely corresponded to the labelled values of aspartame for the commercially available products (storage time of the commercial products was less than 1 month).Relative errors were within 3%. Because primary alcohols interfere,12 the determination of aspartame in alcoholic foodstuffs cannot be carried out with the proposed biosensor. Conclusions A biosensor composed of an immobilized bienzyme system was developed for the efficient assay of aspartame in several low calorie foodstuffs (excluding alcoholic products). The biosensor might also be used to monitor the deterioration in the quality of aspartame in stored soft drinks. The device exhibited reasonable sensitivity and excellent stability of operations. * A: Aspartame + lactose + silicon dioxide; B: aspartame + dextrin; C: t Labelled values provided by the manufacturer. * The previous enzyme sensor was composed of a bienzyme system (containing cu-chymotrypsin and alcohol oxidase) immobilized by an entrapping method with a dialysis membrane and was combined with a dissolved oxygen electrode. l o Diet Coke.3 n = 3, standard deviation: 1-2%. References stability of the biosensor was tested by storing the enzyme electrode in 10 mmol 1-l dithiothreitol solution, i.e., an antioxidant which can prevent the -SH group of the enzymes from being oxidized. Fig. 4 shows the stability of the biosensor with and without storage in the dithiothreitol solution. The biosensor was stable for more than 50 d when stored in the dithiothreitol solution and could be used for 450 assays. Without storage in the dithiothreitol solution, the biosensor was only stable for up to 2 d. Determination of Aspartame in Various Commercial Products Aspartame was determined in three types of commercial low calorie foods using the proposed biosensor and the previously described aspartame enzyme sensor.10 Table 1 summarizes the data obtained for these samples.Two types of dry powdered mixtures were analysed: the first was a mixture of aspartame and dextrin, while the second was a mixture of lactose, aspartame and silicon dioxide. In addition, Diet Coke, which contains citrate, carbonate, sodium benzoate, caffeine, caramel and aspartame, was also analysed. Measurements were taken after direct dissolution of the samples. No pre-treatment or special reagents were required, and the electrode response was 1 2 3 4 5 6 7 8 9 10 1 1 12 Cloninger, M. R., and Baldwin, R. E., J . Food Sci., 1974,39, 347. Cloninger, M. R., and Baldwin, R. E., Science, (Washington, D.C., Tsang, W.-S., Clarke, M. A., and Parrish, F. W., J . Agric. Food Chern., 1985,33,734. Hussein, M. M., D’Amelia, R. P., Manz, A. L., Jacin, H., and Chen, W.-T.C., J . Food Sci., 1984, 49, 520. Homler, B. E., Food Technol., 1984,38(5-8), 50. Issaq, H. J., Weiss, D., Ridlon, C., Fox, S. D., and Muschik, J. B., J . Liq. Chromatogr., 1986, 9, 1791. Renneberg, R., Riedel, K., and Scheller, F., Appl. Microbiol. Biotechnol., 1985, 21, 180. Rechnitz, G. A., Science, (Washington, D.C., 1883-), 1981, 214, 287. Guilbault, G. G., Lubrane, G. J., Kauffmann, J.-M., and Patriarche, G. J., Anal. Chim. Acta, 1988, 206, 369. Chou, S. F., Chen, J. H., Chou, L. W., Fan, J. J., and Chen, C. Y., J . Food Drug Anal., 1995, 3(2), 121. Janssen, F. W., and Ruelius, H. W., Biochim. Biophys. Acta, 1968, 151, 330. Bergmeyer, H. U., Methods of Enzymatic Analysis, Verlag Chemie, Weinheim, 3rd edn., 1984, vol. 2, p. 143. Paper 5f04889C Received July 24, 1995 Accepted September 11,1995 1883-), 1970, 170, 81.

ISSN:0003-2654    

DOI:10.1039/AN9962100071

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, January 1996, Vol. 121 (75-78) 75 Determination of Low Concentrations of Nickel and Aluminium in Membrane Electrolyser Liquors Michael Cullen and Susan Lancashire ICI Chemical and Polymers Ltd, R & T Department, The Heath, Runcorn, Cheshire, UK WA7 4QD Differential-pulse adsorptive stripping voltammetry (DPASV) has been used to determine nickel and aluminium concentrations in electrolyser cell liquors; these are composed of both NaCl and KCI brines of various concentrations up to saturated (30% m/v) and NaOH and KOH liquors up to 32% m/m. In the analysis procedure, nickel is complexed with dimethylglyoxime at pH 8.8 k 0.1; the complex is then adsorbed on a hanging mercury drop electrode (HMDE) at -0.8 V. In a separate procedure, aluminium is complexed with 1,2-dihydroxyanthraquinone-3-sulfonic acid at pH 7.5 k 0.1 and adsorbed on the HMDE at -0.85 V.The detection limits are 0.1 pg 1-1 nickel (60 s adsorption) and 0.2 pg 1-1 aluminium (30 s adsorption) in saturated brine. The linear working range is up to 20 pg 1-1 for nickel and up to 100 pg 1-1 for aluminium. Concentrations greater than this range have been determined by taking a smaller sample volume. In both procedures, hydrazinium sulfate is used to remove interference from free chlorine prior to analysis. These methods are in routine use for the quality control of ICI's FM21 membrane electrolyser brines with nickel and aluminium concentrations typically below 5 pg 1-1 and 30 pg 1-1, respectively. Keywords: Nickel; aluminium; membrane electrolyser liquors; differential-pulse adsorptive stripping voltammetry Introduction This paper describes the methods used to detect low concen- trations of nickel and aluminium in electrolyser liquors of the FM21 membrane electrolysis cells used for the manufacture of chlorine, and sodium hydroxide or potassium hydroxide.During the normal operation of the FM21 electrolysis cells, it was found that low concentrations of trace metals, such as nickel and aluminium, in the feed brine had an adverse effect on cell performance. In order to monitor these effects, sensitive procedures for the determination of nickel and aluminium in saturated brine were required. Our initial investigations using inductively coupled plasma optical emission spectroscopy (ICP-OES) and ETAAS could not achieve the required detection limits in the presence of very high levels of dissolved solids.ETAAS could only achieve a minimum quantification limit of 50 yg 1-1, with a detection limit of 10 yg 1-1 for nickel. Aluminium in a variety of matrices has been routinely measured in our laboratory by a colorimetric method using Solochrome Cyanine R, but the procedure was not sensitive enough for this application. Work has previously been reported using adsorptive stripping voltammetry to detect nickel using dimethylglyoxime (DMG)'.* and aluminium with 1,2-dihy- droxyanthraquinone-3-sulfonic acid (DASA)1,3.4 in sea-water and freshwater. The detection limits reported for sea-water are 0.1 nmol l-1 of nickel (6 ng 1-1) and 1 nmol l-1 of aluminium (27 ng 1-1) for a 60 s deposition time.However, the procedures described could not be directly applied to membrane electro- lyser liquors as these samples often contain free chlorine, have wide variations in pH, brine strengths up to saturated NaCl (30% m/v) and caustic strengths up to 32% m/m NaOH. The aim of this work was to produce a standard method for the determination of nickel and aluminium concentration in electrolyser liquors, which would be both sensitive and precise enough to correlate with variations in electrolysis cell perform- ance. A further requirement was that a short over-all analysis time was achieved in order to allow routine application of the method to large numbers of samples. Consequently, it was important to establish the optimum conditions for the analysis of nickel and aluminium in electrolyser solutions.This paper describes adsorption stripping voltammetry procedures which can achieve detection limits of 0.1 f 0.025 pg 1- * for nickel and 0.2 f 0.05 pg 1-1 for aluminium in electrolyser feed and exit brines. External standard calibrations are performed in blank brine solutions and the interference caused by free chlorine in the electrolysis cell exit brine is removed by adding hydrazin- ium sulfate prior to addition of the other reagents. Experimental Equipment The following Metrohm (Herisau, Switzerland) systems were used: the VA646 processor, VA675 sample changer, multi- mode electrode (MME), Ag/AgCl reference (3 mol 1-1 KC1) and platinum auxiliary electrodes. The MME was set to a hanging mercury drop mode with a surface area of 0.55 mm2 and a -75 mV pulse amplitude was used unless otherwise stated.A fresh mercury drop was used for each scan. All adsorption steps were carried out with stirring using the medium stirring rate on the VA646 followed by an unstirred 10 s equilibration time. A scan rate of 10 mV s-1 was used with a 6 mV step and a pulse time of 0.6 s. All glassware was stored in approximately 10% v/v hydrochloric acid and rinsed well with de-ionized water prior to use. Reagents De-ionized water obtained via a Milli-Q water system was used in the preparation of all solutions. Working standard nickel and aluminium solutions were prepared daily by serial dilution of the nickel and aluminium 1000 mg 1-1 Spectrosol standard solutions (Merck, Poole, Dorset). The hydrochloric acid used was Aristar grade (d 1.18, Merck) and the ammonia was a PrimaR ammonia solution (d 0.88; Fisons, Loughborough, Leices ters hire).Blank saturated brine (30% m/v NaCl). This solution was prepared by dissolving 30 g of solid NaCl (Aristar grade) in 100 ml of de-ionized water.76 Analyst, January 1996, Vol. 121 Ammonium buffer (0.8 mol l-l)-DMG solution. This was prepared by dissolving 0.1 g of DMG (AnalaR Grade, Merck) in 10 ml of methanol then diluting to 500 ml with 0.8 mol 1-1 ammonia-ammonium chloride buffer (adjusted to pH 8.8). BES buffer (N,N'- bis-(2- hydroxyethy1)-2 -aminoethanesul- fonic acid), 1 mol 1-l. This buffer was prepared by dissolving 21.3 g of BES buffer solid (Aldrich, Gillingham, Dorset) in 100 ml of water, adjusting the pH to 7.5 ( f O .l ) using ammonia solution or hydrochloric acid as required and then adding 0.01 g of manganese(1v) oxide in order to remove trace metals.3 DASA solution, 1 X 10-3 moll-'. This reagent was prepared by dissolving 0.032 g of DASA (Aldrich) in 100 ml of de- ionized water. Hydrazinium sulfate (1% m/v). This was prepared by dissolving 1 g of the AnalaR grade solid in 100 ml of de-ionized water. This solid is extremely toxic and all the necessary safety measures were applied. Starch iodide paper (Merck). This was used for checking chlorine removal. Procedure for the Determination of Nickel in Electrolyser Brine Calibration Hydrazinium sulfate (0.1 ml of a 1% solution), 10.0 ml of de- ionized water and 10.0 ml of ammonia-DMG solution (pH 8.8) were added to a polarographic cell and mixed.The solution was de-oxygenated by a purge with pure nitrogen for 4 min prior to the application of -0.8 V for 60 s stirred adsorption time followed by 10 s equilibration time. A differential-pulse stripping scan was carried out to a final potential of -1.3 V. Standard additions of nickel to the cell produced a linear calibration up to 20 pg 1-1 of nickel. The nickel peak occurred at -0.99 V and a typical cell blank of 0.2 pg 1-I was found. Standard scans are illustrated in Fig. 1. Sample preparation Hydrazinium sulfate (0.1 ml of a 1% solution) and 10.0 ml of brine sample were added to a clean polarographic cell and Z a n -300 W > a 3 0 -250 >E -200 0 v) I I I -150 7 1 > -100 E 7 -50 t- W 2 0 v! = 00 Fig. 1 Typical nickel scans: 0, 1 and 10 pg 1-l spiked Ni cell concentrations.mixed; the pH was then adjusted to 1 or less by adding hydrochloric acid. Ammonia-DMG solution ( 10.0 ml) was then added and the solution pH adjusted to 8.8 f 0.1. For samples with a nickel concentration greater than the linear range, the sample was diluted with water and then 10 ml of the diluted sample were taken into the cell. The prepared sample solution was next scanned under the same instrumental conditions as used for the nickel calibration standards, allowing the nickel concentration to be determined from the calibration graph after correcting for any dilution used. Procedure for the Determination of Aluminium in Electrolyser Brine Calibration Hydrazinium sulfate (0.1 ml of a 1 % solution), 1 .O ml of blank 30% m/v NaCl solution, 18.6 ml of water and 0.2 ml of 1 moll-' BES buffer (pH 7.5) were added to a polarographic cell.DASA (0.1 ml of a 1 X 10-3 moll-' solution) was added and the cell solution de-oxygenated for 4 min using a pure nitrogen purge. An initial potential of -0.85 V with a stirred adsorption time of 30 s was then applied, followed by a 10 s equilibration time before starting the scan. A differential-pulse stripping scan was then carried out to a final potential of -1.25 V. The aluminium peak occurred at - 1.08 V with a typical cell blank of less than 1 pg 1-1 of aluminium. The calibration was linear up to 100 pg 1-1 of aluminium (for 2 ml of a 30% m/v NaCl sample) using the above conditions. Typical standard scans are illustrated in Fig. 2. Sample preparation Two millilitres of sample plus 0.1 ml of hydrazinium sulfate were added to a clean polarographic cell and then mixed to destroy any chlorine; starch iodide paper was used to check for completion of the reaction.Water (17.6 ml) and 0.2 ml of BES buffer were then added and the pH adjusted to pH 7.5 f 0.1 using ammonia solution or hydrochloric acid as required. The prepared solution was examined under the same instrumental conditions as for the calibration standards and the aluminium concentration determined using the calibration graph. For samples with concentrations greater than the linear range (100 pg 1-1) the sample was diluted with water and then 2 ml of the diluted sample were taken and analysed. The sample concen- tration was then corrected for dilution as appropriate.-70 7 Fig. 2 concentrations. Typical aluminium scans: 0, 5 and 50 pg 1-1 spiked A1 cellAnalyst, January 1996, Vol. 121 77 Procedure for the Determination of Nickel and Aluminium in Caustic Liquors Calibration The calibration procedures for nickel and aluminium in caustic liquors (KOH, NaOH) are the same as those detailed for electrolyser brine solutions. Sample preparation For 32% m/m caustic liquors, 25.0 ml of sample were pipetted into a clean 150 ml beaker and then water added to give a total volume of approximately 50 ml. Next, the pH of the solution was adjusted to pH 1 by using hydrochloric acid and the solution transferred into a 100 ml calibrated flask and diluted to the mark with water. The prepared solution was then analysed following the procedure described for electrolyser brine samples.A correction for the dilution used (X4) was applied to obtain the concentration of nickel or aluminium in the sample before acid treatment and dilution. Results Optimized Conditions The optimum conditions for the analysis of electrolyser liquors described in the procedures were chosen after investigation of the effects of pH, adsorption potential, drop size and pulse amplitude on both the Ni and A1 responses. Effect of Sodium Chloride Concentration on the Nickel Response The different types of electrolyser brine contain different concentrations of NaCl ( e . g . , cell feed brine is 30% m/v NaC1; cell exit brine is about 20% m/v NaCl). The effect of changes in sodium chloride concentration on the nickel response was investigated by carrying out nickel calibrations in firstly 10 ml ammonia-DMG solution plus 10 ml of water, and secondly 10 ml of ammonia-DMG solution plus 10 ml 30% m/v NaCl solution.In both instances the experimental conditions were -0.8 V stirred adsorption potential for 30 s, -75 mV pulse amplitude and a final pH of 8.8. Known additions of 2 mg 1-l nickel standard were made and the nickel response recorded after each addition. Little variation in the slope of the nickel calibration was found with changes in NaCl concentration. Hence, for the nickel method, an external calibration could be carried out in a solution of 10.0 ml water and 10.0 ml of ammonia-DMG solution (pH 8.8) and for the sample analysis up to 10.0 ml of 30% m/v NaCl could be used in the cell. Effect of Sodium Chloride Concentration on the Aluminium Response Solutions of sodium chloride with concentrations from 0 to 15% m/v in the cell, 0.1 ml of DASA, 0.2 ml of BES, at a final pH of pH 7.5, were studied using a 30 s adsorption time at -0.85 V.In each experiment a calibration graph was established and the gradients compared. Unlike the small effect that changes in brine strength had on the nickel response, a reduction of approximately 25% was found in the aluminium response when 1% m/v NaCl was present in the cell as compared with the response with no added NaC1. As is shown in Fig. 3, calibration curves obtained in the range 1.54% m/v of NaCl in the cell gave similar slopes and provided sufficient sensitivity for the analysis of electrolyser liquors.Above 6% m/v of NaCl in the cell, it was found that the aluminium response decreased significantly. Hence, based on these results, a brine strength range of 1 . 5 4 % m/v in the cell was chosen for the optimized procedure. Precision and Limit of Detection for Nickel and Aluminium For nickel, following the optimized procedure, the limit of detection (LOD) for saturated brine (30% m/v of NaCl) at 3 times the standard deviation of the blank was 0.1 f 0.025 pg 1-l (for n = 5). Using a nickel cell concentration of 1 pg 1-l the relative standard deviation was 1% (n = 5). For aluminium, following the optimized procedure, the LOD for saturated brine (30% m/v NaC1) at 3 times the standard deviation of the blank (n = 5) was 0.2 A- 0.05 pg 1-1. For a 5 pg 1-1 aluminium cell concentration, the relative standard deviation was 0.6% (n = 5).In both the nickel and aluminium procedures, the LOD could have easily been improved by using an increased adsorption time, but at the cost of a reduction in the linear range. Thus, the choice of optimum conditions is the best compromise between sensitivity, linear range and speed of routine analysis for nickel and aluminium in electrolyser samples. Samples of brine were spiked with 5 pg 1-1 additions of both nickel and aluminium and analysed by the optimized proce- dures. From this, the over-all recovery of both methods was found to be > 95%. Typical Results A set of typical membrane electrolyser sample results for nickel and aluminium using the optimized procedures is given in Table 1.Interferences For electrolyser brine analysis, the most significant interference in both the nickel and aluminium methods is from dissolved chlorine. This was easily removed by the addition of hydrazin- ium sulfate which, at the levels described in the procedures, did not have any noticeable effect on the response of either method and could be omitted if the samples were known not to contain free chlorine, bromine or iodine. 100 80 a 5 60 c m al c Y a .- 40 20 0 a / 0 2 4 6 8 10 12 14 16 Al concentration/pg I-’ Fig. 3 NaC1. Calibration curves for A1 in (a) 0, (b) 1.5, (c) 3, (d) 6, (e) 15% m/v Table 1 Typical membrane electrolyser results Sample No. Sample matrix Al/pg 1-1 Ni/pg 1 - 1 1 30% m/v NaCl 60 12 2 18% m/v NaCl 30 3 3 30% m/v NaCl 10 3 4 32% m/m NaOH 50 100 5 32% m/m NaOH 80 1000 6 32% m/m NaOH 80 8078 Analyst, January 1996, Vol.121 A high level of Zn is a potential interferent in the aluminium method but it can be masked by the addition of EDTA.3 However, no interference was seen at the levels of Zn (< 0.05 mg 1-1) normally found in membrane cell electrolyser brine. Components commonly found in electrolyser brine and which do not interfere are Ca at 0.05 mg l-l, Mg at 0.05 mg l-l, 0.5 mg 1-1 of Sr, 1 mg 1-1 of Ba, 10 mg 1-1 of soluble silica, 10 mg 1-1 of total I, 10 mg 1-1 of sulfate, 0.5 mg 1-1 of Hg and 0.05 mg 1-1 of Mn, Sn, Ti or Pb. Electrolyser cell exit brine can contain up to 20 g 1-1 of sodium chlorate, 1 mg 1-1 of fluoride and 1 g 1-1 of active chlorine, but following the procedures described in this paper no interference was observed. Conclusions The methods described above have been found to be suitable for the determination of nickel and aluminium in membrane electrolyser liquors and are routinely applied in our laboratory for monitoring their effect on FM2 1 electrolyser performance. In addition to NaCl brines and NaOH liquors, these methods have also been successfully applied to various KCl brines up to 30% m/v of KC1, and KOH electrolyser process liquors. The authors thank the members of the ICI Electrochemical Technology Business in their support of this work. References 1 van den Berg, C. M. G., Chem. Oceanogr., 1988,9, 197. 2 Pihlar, B., Valenta, P., and Nurnberg, H., J . Electroanal. Chem., 1986,214, 157. 3 van den Berg, C. M. G., Anal. Chim. Actu, 1986, 188, 177. 4 Hemhndez-Brito, J. J., Gelado-Caballero, M. D., Ptrez-Pefia, J., and Herrera-Melirin, J. A., Analyst, 119, 1994, 1593. Paper 51033563 Received May 25, 1995 Accepted September 19, I995

ISSN:0003-2654    

DOI:10.1039/AN9962100075

出版商:RSC    

年代:1996

数据来源: RSC

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Analyst, January 1996, Vol. 121 (79-81) 79 Electrochemical Trace Analysis of Gold in Ore Jyotsna Shukla and K. S. Pitre Department of Chemistry, Dr. Hari Singh Gour University, Sagat (M.P.) 470 003, India Differential-pulse polarography (DPP) has been successfully used for the simultaneous determination of some trace metal ions in ore. Ammonium tartrate (0.1 mol 1-1) was used as supporting electrolyte and 0.01% gelatin as maximum suppressor. The sample produced eight well defined polarographic peaks. The first signal with E , = -0.08 V versus SCE in the polarogram may be due to Agl or Ad1'. To confirm the presence of Agl and/or Ad1' in the sample, EDTA was used as a complexing agent, which resulted in this single peak splitting into two, with E , equal to -0.04 V and -0.2 V versus SCE, confirming the presence of both Agl and AulI1 in the sample. The observed voltammetric results were compared with those obtained by using AAS.Keywords: DifSerential pulse polarography; trace metal; chalcopyrite; gold Introduction Chalcopyrites are a known source of gold.'-2 Often, these ores are also associated with galena and sphalerite.34 Galena is a known source of silver.5-6 Thus, the ore may contain silver and gold and also other trace metals. In order to determine Au and Ag at trace levels, various electrochemical methods have been employed.7-11 The accuracy and reliability of trace analysis in chalcopyrite by using voltammetric methods has been investi- gated. Experimental Apparatus Polarographic measurements were made on an Elico Model CL- 90 (Hyderabad, India) pulse polarograph.The electrode system consisted of a dropping-mercury electrode (DME) as working electrode, a coiled platinum wire as auxiliary electrode and a saturated calomel electrode (SCE) as reference electrode. All pH measurements were made on an Elico Model LI- 120 digital pH-meter. Chemicals and Reagents Merck (Darmstadt, Germany) analytical-reagent grade chem- icals were used. Stock solutions of ammonium tartrate (1 mol 1-I), Ag, Au, Cu, Cd, Pb, Zn, Fe and Cr (0.01 mol 1-l) and EDTA Na2 (0.01 mol 1-l) were prepared by dissolving their requisite amounts in doubly distilled water. Gelatin solution (0.01%) was prepared in hot (5040°C) distilled water. The solutions were standardized and diluted as required. Preparation of the Sample Solution Chalcopyrite ore (containing sphalerite and galena) was obtained from Rajpur Dariba, Udayapur, Rajasthan, India.Finely pulverized ore sample (1 g) was dissolved in 10 ml of hydrobromic acid and evaporated to dryness. The dry residue was dissolved in 10 ml of 16 mol 1-1 nitric acid and again evaporated to dryness. This dry residue was dissolved in 10 ml of ammonium hydroxide containing 3 g of tartaric acid. The final volume was made up to 100 ml with distilled water.12 Preparation of the Analyte and Recording of the Voltammogram The sample solution (10 ml) was mixed with 10 ml of 1.0 moll-' ammonium tartrate as supporting electrolyte and 10 ml of 0.01% gelatin as maximum suppressor in a polarographic cell. The final volume was made up to 100 ml with distilled water.The pH of the test solution was adjusted to 9 k- 0.02, with ammonium hydroxide. The analyte was placed in a polarographic cell. Pure hydrogen gas was bubbled through the test solution for 15 min and the pH of the test solution was checked before recording the polarograms. Results and Discussion The differential pulse polarograms of the sample solution (Fig. 1) showed eight well defined peaks with Ep = -0.08; -0.28; -0.4; -0.63; -0.92; - 1.14; -1.34; and -1.44 V versus SCE indicating the presence of Ag1/Au1I1; Cu"; Pb"; Cd"; Nil'; Zn"; Fell1; and Cr"', respectively. Table 1 Instrumental parameters Parameter Value Parameter Value Initial applied voltage Sensitivity Charging current compensation IR compensation Height of Hg column Time constant Pulse amplitude Temperature 0.0 V versus SCE 10 PA 3.5 5.0 140.0 cm 5.0 ms 50 mV 25 f 2 "C Drop time 1 s Scan rate 12 mV s-1 Aquisition Fast (6 mV s-I) Output Zero* 5.0 PH 9.00 * 0.02 On Polarocard x-Axis 200 mV cm-1 y-Axis 200 mV cm-' * The value was fixed according to the instruction manual.200 mV H Fc c C 3 p! 0 Voltage Fig. 1 Differential-pulse polarogram of 100 mg per 100 ml of ore sample (including chalcopyrite, galena and sphalerite) in 0.1 mol 1-l ammonium tartrate + 0.01% gelatin at pH 9 k 0.02.80 Analyst, January 1996, Vol. 121 To confirm the presence of the above-mentioned metal ions in the sample, a known quantity of standard solution of each metal ion was added to the analyte and the resulting polarogram was recorded (which increased the observed wave height of each metal ion signal without any change in E , values).After confirming the presence of these metal ions in the sample, some synthetic samples of various ion concentrations were prepared (Table 2) and their polarograms were recorded under identical conditions. The results indicated no change in Table 2 Analysis of synthetic samples* Composition of synthetic sample- Cu 0.47 0.63 0.95 1.90 Pb 2.33 3.10 4.66 9.32 Cd 0.01 0.02 0.03 0.07 Ni 0.07 0.09 0.14 0.29 Zn 0.10 0.14 0.21 0.43 Fe 0.43 0.48 0.72 1.45 Cr 0.06 0.07 0.10 0.21 Amount found by using DPP- Cu 0.46 0.62 0.96 1.89 Pb 2.32 3.11 4.65 9.31 Cd 0.01 0.02 0.03 0.07 Ni 0.07 0.09 0.15 0.29 Zn 0.10 0.14 0.23 0.43 Fe 0.42 0.48 0.71 1.45 Cr 0.05 0.07 0.11 0.20 * Average of four determinations ( 10W2 pg I-').Voltage Fig. 2 Ag in 0.1 mol 1-1 ammonium tartrate +0.01% gelatin at pH 9 f 0.02. Differential-pulse polarogram of Na2EDTA-Au and Na2EDTA- Table 3 Minimum investigated detection limits Metal ion DPP/pg ml-1 Individual Combined Individual Combined Individual Combined Individual Combined Individual Combined Individual Combined Individual Combined Individual Combined Individual Combined 0.10 0.10 0.19 0.19 0.063 0.063 0.26 0.26 0.06 0.06 0.06 0.06 0.64 0.64 0.56 0.56 0.52 0.52 half wave potential (Fig. 3) of the above-mentioned metal ions. The linear relationship between the concentration of each metal ion and the corresponding wave height/peak height was also unchanged confirming the possibility of accurate simultaneous, multi-element qualitative and quantitative determination of the metal ions in the sample.The concentrations of each metal ion (taken/found) in synthetic samples by using DPP is given in Table 2. Minimum Investigated Detection Limit The minimum investigated detection limits (smallest quantities that have been used) for measurement of the individual and combined metal ions are given in Table 3. Except for Ag' and Au"', all metal ions in the sample could be determined in one run. For Ag' and Au"' differential complexation of the metals with Na2EDTA was used. The detection limits were examined by preparing synthetic samples. Analysis of Gold and Silver The peak potential at -0.08 V may have been due to either Agl or Au"'. According to the literature13-14 this value is neither that of Ag and Au. To confirm the presence of Ag and Au, differential complexation of the metals with 10 ml of 0.01 moll-' Na2 EDTA, was used.As a result, the single peak split into two, with E, equal to -0.04 V and -0.2 V versus SCE, indicating the presence of both ions, i.e., both Ag and Au were present in the sample (Fig. 2). Ag-Na2EDTA (1 + 1) and Au- Voltage Fig. 3 Differentialpulse polarogram of a synthetic sample containing Cu" (1.26 mg); Pb" (6.15 mg); Cd" (0.05 mg); Nil' (0.67 mg); Zn" (0.22 mg); Fern (0.95 mg); and Cr"' (0.19 mg) per 100 ml, in 0.1 mol I-' ammonium tartrate +0.01% gelatin at pH 9 f 0.02. Table 4 Results of ore sample (mixture of chalcopyrite, galena and sphalerite) analysis for metal ions (pg 1-1). Metal Standard ion Added Found Recovery (%) deviation Ag Au c u Pb Cd Ni Zn Fe Cr - 2.16 5 .OO 190.50 828.80 6.70 29.30 45.00 140.00 20.70 - - - - - - - - 2.40 4.54 4.00 8.95 190.50 380.00 932.24 1760.00 7.00 13.65 29.70 59.00 43.20 88.10 145.00 284.00 21.30 42.00 99.6 99.4 99.7 99.9 99.6 100 99.8 99.6 100 0.02 0.01 0.01 0.02 0.04 0.08 0.03 0.02 0.02Analyst, January 1996, Vol.121 81 NaZEDTA (1 + 1) complexes were separately prepared and their polarograms were recorded. They produced well defined peaks with E , equal to -0.04 V and -0.2 V versus SCE, respectively. Similar signals were also observed using a mixture of the two complexes. After confirming the presence of Ag and Au in the sample, quantitative analysis was carried out on these two ions. Quantitative Analysis of the Sample by Using DPP Quantitative analysis of Ag, Au, Cu, Pb, Cd, Ni, Zn, Fe and Cr in the sample at pH 9 A- 0.02 was carried out by using DPP.Spiked samples were prepared in order to evaluate the concentration of each metal ion. The results are given in Table 4. The results indicate that the percentage recovery is over 99% for all metal ions, with high accuracy and precision of determination. Table 5 shows the final analysis results for the sample. These results were compared with those obtained by using AAS. The comparative data are also shown in the table. Although the Table 5 Comparison of AAS and voltammetric trace analysis data on the ore sample (mixture of chalcopyrite, galena and sphalerite) Amount found/lO-2 pg g-l Metal ion Au"' CU" Pb" Cd" Nilf Zn" Fell1 Cr"' Ag' AAS Not reported Not reported 186.00 922.00 5.00 28.00 39.00 139.00 21.00 Voltammetry 2.40 4.00 190.50 932.24 7.00 29.70 43.20 145.00 21.30 polarographic and AAS data are in good agreement for the Cu, Pb, Cd, Ni, Zn, Fe and Cr content of the sample, AAS failed to determine Ag and Au.Statistical data supports the superiority of the polarographic method for such an analysis. The authors thank Professor S. P. Banerjee, Chemistry Department, Dr. Hari Singh Gour University for providing laboratory facilities. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Rao, S. K. L., Ind. Miner., 1974, 15, 28. Khetegurov, G. V., Geokhimiya, 1969, 2, 1362. Poddar, B. S., Econ. Geol., 1965, 60,636. Rao, R., Poddar, B. C., Mathur, R. K., and Dhara, M. K., GSI Miscellaneous Publication, 1977, 27, 347. Ganzha, T. I., Tarasenko, Pilat, V. Z., Panchenko, B. V., and Sondar, V. M., Tsvetn. Met. (Moscow), 1972, 45(6), 67. Khetreyarv, K. K., and Tallinsk, T. V., Politekhn. Inst. Ser., 1965, 228, 141. Verma, N., and Pitre, K. S., Indian J. Chem., Sect. A: Inorg. Bio- inorg., Phys., Theor. Anal. Chem., 1992, 31, 210. Verma, N., and Pitre, K. S., Analyst, 1993, 118, 65. Bharathibai, J., Rajagopalan, S. R., and Padma, D. K., Indian J . Chem., Sect. A: Inorg. Bio-inorg., Phys., Theor. Anal. Chem., 1995, 34, 320. Verma, N., and Pitre, K. S., J. Indian Chem. Soc., 1994, 71, 129. Shukla, J., and Pitre, K. S., Indian J. Chem., Sect. A: Inorg. Bio- inorg., Phys., Theor. Anal. Chem., 1996, in the press. Babaeva, Z. E., Issled. 061. Neorg. Fiz. Khim., Chem. Abstr., 91348W. Vogel, A. I., Text Book of Quantitative Inorganic Analysis, ELBS Longman, London, 1978,4th edn., p. 324. Meites, L., Polarographic Techniques, International Scientific, N.Y., 1965, 2nd edn., p. 661. Paper 51029.581 Received May 10, 1995 Accepted August 17, I995

ISSN:0003-2654    

DOI:10.1039/AN9962100079

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, January 1996, Vol. 121 (83-88) 83 Certified Reference Materials (CRMs 479 and 480) for the Quality Control of Nitrate Determination in Freshwater Ph. Quevauvillera, M. Valcarcelb, M. D. Luque de Castrob, J. Cosanob and R. Moselloc a European Commission, Standards, Measurements and Testing Programme (BCR), Rue de la Loi, 200, B-1049 Brussels, Belgium b Universidad de Cdrdoba, Depto. de Quimica Analitica, E-14004 Cdrdoba, Spain c Istituto Italian0 di Idrobiologia, Viale Vittorio Tonolli 50-52, I-28048 Pallanza, Italy Monitoring of freshwater and groundwater is routinely performed to control the level of contamination by a wide variety of trace and major constituents, including nitrates. In order to verify the quality of such determinations, the Standards, Measurements and Testing Programme (formerly BCR) has started a project, the first step of which was to evaluate the performance of European laboratories in an interlaboratory study and the second phase was a certification campaign of nitrate in two artificial freshwater samples.This paper presents an overview of the results of the interlaboratory evaluation and gives details of the preparation of the candidate certified reference materials (CRMs), the verification of their homogeneity and stability, the techniques used in the certification and the certified values. Two CRMs were produced: CRM 479 containing a low nitrate content (214 * 4 pmol kg-1 or 13.3 fr 0.3 mg kg-1) and CRM 480 containing a high nitrate content (885 & 13 pmol kg-1 or 54.9 fr 0.8 mg kg-1). Keywords: Interlaboratory study; certified reference material; quality control; nitrate; artificial freshwater Introduction A number of EC Directives related to water have been issued over the years, dealing with the quality of drinking water (75/440/CEE, 79/869/CEE, 80/778/CEE) or underground water (80/68/CEE) or with the protection of fish life (78/659/CEE).They prescribe the determination of a wide variety of elements, ions and compounds, including nitrate. The need to improve and to control the quality of water analysis has justified the organization of several interlaboratory studies within the Community Bureau of Reference (BCR) programme (now renamed Standards, Measurements and Testing Programme), which enabled the production of certified reference materials (CRMs) of, for example, freshwater (CRMs 398 and 399)' and artificial rainwater (CRMs 408 and 409).* Whereas the amounts of nitrate could be certified in the CRMs of rainwater, their certification in the freshwater CRMs was not possible owing to the stabilization procedure used (involving the addition of nitric acid).Consequently, it was decided to organize a separate interlaboratory study, the final aim of which was to produce freshwater CRMs certified for their contents of nitrate, one with a low nitrate content (CRM 479) and one with a high nitrate content (CRM 480). These CRMs correspond to values below and slightly above the maximum permissible nitrate content (approximately 50 mg 1-1 or 806 mol 1-1) mentioned in the drinking water Directive. The present certification follows a successful interlaboratory study, the aim of which was to improve the quality control of nitrate determination in freshwater.3 It was possible to use the group of laboratories participating in this study to certify CRMs 479 and 480 on a collaborative basis.Certification Procedure After a preparatory meeting at which all the requirements for certifying reference materials were discussed, the candidate CRMs were shipped to the participating laboratories (see acknowledgements). Each laboratory that took part in the certification exercise was requested to make a minimum of five independent replicate determinations on at least two different ampoules of each CRM on different days. The results were statistically evaluated, presented in the form of bar-graphs and discussed at a technical meeting with all the participants.In order to obtain the accuracy required for certification it is necessary to ensure that no substantial systematic error is left undetected. i. In a meeting of the laboratories participating in the certification, the sources of error and the measures taken to eliminate them were discussed. Errors related to a particular technique or analytical step could not be detected. The evidence given by the bar-graphs led to the conclusion that there was no substantial difference between the methods and that, therefore, the probability of an over-all systematic error would be low. ii. The laboratories participating in the certification exercise applied their methods correctly, i.e., the determinations were performed only when the method was under statistical con- trol.The simplest way of producing synthetic freshwater CRMs would have been to certify the nitrate contents on a gravimetric basis. It was, however, decided to proceed with a certification on a collaborative basis in order to obtain detailed information on the techniques routinely used for nitrate determinations, in particular on the within- and between-laboratory relative standard deviations [coefficients of variation (CV)]. This procedure logically followed the improvement scheme initiated by the first interlaboratory study (see below). Preliminary Investigations A feasibility study was carried out to investigate the optimum conditions for the preparation of the candidate CRMs of artificial freshwater to be certified for their nitrate contents.3 Solutions were prepared at three levels of concentration, viz., 0.5, 8 and 53 mg kg-1, and their stability was verified at 20 and 40 "C over a period of 6 months.84 Analyst, January 1996, Vol.121 ~~ Possible procedures for achieving a good stability of natural freshwater samples were discussed in a preparatory meeting with the participants in the project. Freezing was not considered to be a suitable procedure for the long-term storage of CRMs of natural water as it may lead to irreversible physico-chemical changes (e.g., formation of insoluble Ca salts) and would necessitate the use of special containers and ways of dispatch- ing. Freeze-drying would also be susceptible to physico- chemical difficulties.Consequently, in view of the risks of instability of natural waters and of the high variability in composition, it was decided to prepare artificial solutions containing known concentrations of nitrate and other major constituents, if necessary with addition of stabilizing agents. The pH of the solution was considered to be critical for the stability and subject to changes when the sample is in contact with CO2 (e.g., during bottling). The evaporation of C02 as well as the precipitation of CaC03 were considered to be possible sources of inhomogeneity and instability. Problems were considered to be less if samples could be equilibrated with air and if a carbonate buffer would be added in order to avoid major changes. On the basis of the chosen procedure, Na2C03 was added up to a concentration of 530 mg kg-l to maintain the pH against air; the resulting pH ranged between 9 and 9.5, which is higher than the pH observed in natural water.The pH was therefore lowered by adding acid (0.5 ml of HCl, 10 mol kg-1 in a litre of solution) in order to achieve a pH value of 6.8. Implicitly, Ca could be present to such an extent only that precipitation would not occur and all other ions could be added with the exception of, for example, phosphate and ammonium that might provoke microbiological growth. The addition of UV absorbing organic matter was also discussed to match the presence of humic acids; as it was found difficult to prepare water samples containing humic acids, the use of lauryl sulfate having a wide absorption range in the UV and known to be stable at concentrations in the range 1-3 mg 1-l was recommended; 2 mg I-' of lauryl sulfate was therefore added to the samples.Finally, phenylmercury acetate (C8H8Hg02) was added to avoid the development of moulds. Different concentrations were studied and an undesirable white precipitate was observed in the samples for concentrations of 100 mg 1-1 of phenylmercury acetate a well as deleterious effects on the redox copper-coated Cd column at the determina- tion step in the photometric method based on reduction to nitrite; this effect was not noticeable at the 10 mg 1-1 level which was therefore used for the sample preparation. The levels of nitrate concentrations were chosen to be in the range of levels found in the environment, considering the maximum value specified in the EC Directive (50 mg 1-1 or 806 moll-I).For the first interlaboratory study, the participants were instructed to prepare samples with concentrations of about 1, 10 and 50 mg kg-1 (respectively solutions A, B and C). The concentra- tions of the other compounds were CaC12.4Hz0 (9 1.7 mg 1-I), MgS04.7H20 (123 mg 1-1) and Na2C03 (530 mg 1-l) to simulate the hardness of a natural water. Nitrates were added in the form of high-purity salts (KNO3) dissolved in water obtained from a Millipore water-purification system (Milli-Q water). The water was boiled, homogenized by mechanical shaking using a magnetic stirrer and filtered with sterile filters to 0.2 pm. The different compounds were added by pumping. In order to verify the long-term stability of the samples on storage, two types of ampoules of 200 ml were selected, in white and brown glass, respectively.The short-term stability (3 months) of the three sets of samples was verified at 20 and 40 OC. The homogeneity was verified prior to the stability experiments by performing five measurements in 15 ampoules (for each of the three concentrations considered) randomly selected during the filling procedure. No instability was demonstrated over a period of 3 months for the three sets of solutions, both in white and brown glass ampoules. Conse- quently, the white glass ampoules were selected for the storage of the solutions and a temperature of 20 "C was adopted for the storage conditions. Interlaboratory Study The three above-described solutions were subsequently shipped to a group of about 30 laboratories for an interlaboratory study which enabled variations in standard deviations for ion chromatography due to, for example, the application of different columns, different eluents, and the use of chemical or electronic suppression (conductivity detection) to be explained.This intercomparison allowed a group of experts to be constituted and prepared them for the certification campaign. In addition, this study enabled the suitability of the procedure used for the preparation of candidate CRMs of verified homogeneity and stability to be confirmed. The results of this exercise are described in detail elsewhere.3 Preparation of the Candidate Reference Materials Two 150 1 PVC containers (one for each solution) to be used for the preparation and homogenization of the candidate reference materials were cleaned with a detergent, rinsed with distilled water and further rinsed with ultrapure water.For each of the reference materials, the ampoules were cleaned in a similar way. The ampoules were (air) dried for 2 d and conditioned for at least 24 h with the solution that they would contain. The preliminary investigations had shown that this procedure was adequate to bring the walls into adsorption equilibrium with the solution. The sample preparation followed the procedure used in the preliminary investigations. The two reference materials were prepared from ultrapure water to which freshly prepared solutions of the different substances described in Table 1 were added (amounts in grams added to approximately 150 1 of ultrapure water).All reagents were of pro analysi quality. The final pH of the solutions was about 6.8. The homogenization of the solutions was achieved by maintaining a constant agitation (with a mechanical shaker) during the addition of the solutions. The (conditioned) ampoules were filled with the CRM solutions and immediately heat-sealed. The ampoules were then stored at ambient temperature in the dark. Precautions were taken to avoid contamination during the ampouling procedure. Homogeneity Study One ampoule was selected out of each 50 ampoules prepared to verify the between-ampoule homogeneity of each CRM. Thus, a total of 25 ampoules was analysed per CRM, each ampoule being analysed in triplicate. The method repeatability was determined by ten replicate analyses of one sample of each candidate CRM.In order to establish the homogeneity and stability of candidate CRMs, a highly repeatable FI method was optimized.' Photometric detection was found to give the most repeatable results. The method was based on the reduction of nitrate to nitrite and the formation of a diazo compound which Table 1 Amounts of substances (g) added to 150 1 of water for the preparation of the two candidate CRMs KN03 3.670 and 12.475 Na2C03 79.500 CaC12.4H20 13.755 MgS04.7H20 18.450 Potassium phthalate 0.750 Lauryl sulfate 0.300 Phenylmercury acetate 1 S O 0Analyst, January 1996, Vol. 121 1.12 ..................................................................................... 85 0 0.08 z 0.80 is coupled to sulfanilamide (SPA) and N-( 1 -naphthyl)ethylene- diamine (NED) to yield an azo derivative with a maximum absorption at 540 nm.After mixing with the carrier (N&C1- Na2B407-EDTA), the sample reaches the redox Cd column (copper-coated with EDTA-CuS04) where nitrate is reduced to nitrite. Then, the reduced analyte solution is sequentially mixed with the SPA and NED solutions to yield the azo compound, which is monitored at 540 nm as it passes through the flow cell of the photometric detector. The measured signal is the height of the FI peak. The CVs obtained for nitrate are listed in Table 2. As shown, the overlap was within the uncertainty U,, of the CV (an estimation of the uncertainty U,, of the CV is calculated as follows: U,, =: CV/v2n, with n = number of replicates).Consequently, it was concluded that both batches of CRMs were homogeneous, within the uncertainty of the method. - I I I Stability Study The stability tests were performed by analysing randomly selected samples after 1, 3, 6 and 12 months of storage at 4, 20 and 40 "C. One replicate analysis of each of three ampoules stored at different temperatures was performed at each occasion of analysis. In this way, the long-term variability included both the analytical uncertainty and the between-bottle variability. The same analytical procedure as for the study of homogeneity was used. Any change in the content of an analyte with time indicates an instability provided that a long-term analytical reproducibility is obtained. Instability would be detected by comparing the contents of nitrate in samples stored at different temperatures with those stored at low temperature at the various occasions of analysis.The solutions stored at 4 "C were used as reference for the samples stored at 20 and at 40 "C, respectively. Fig?. 1 and 2 are plots of the ratios (R,) of the mean value (X,) of three measurements made after a period t at, respectively, 20 and 40 "C, and the mean value (X40c) from three determinations made at each occasion of analysis on samples stored at a temperature of 4 "C: R, = yt/%40c Table 2 Results of the homogeneity tests. CV method * CV between t CRM cv f u,, cv f u,, CRM 479 2.33 k 0.52 3.03 f 0.43 CRM 480 2.54 f 0.57 3.22 k 0.46 *Ten replicate determinations. t One determination in each of 25 different ampoules 120 l- v) a 3 cd 1.04 > m a .- N 0.96 cd rr 1.12 - - g 088 z z .................................................................................. 0.80 I I 1 I 1 I 0.00 2.60 5.20 7.80 10.40 13.00 Time (months) Fig. 1 lines: 1.1, 1.0 and 0.9. Stability study of CRM 479. Circles, 20; squares 40 "C. Dotted the uncertainty U, on the ratio R, was obtained from the CV of three measurements obtained at each temperature: U, = (CV? + CV40~2)''~ . R, In the case of ideal stability or absence of additional changes, R, should be 1. In practice, however, there are some random variations owing to the uncertainty on the measurement. On the basis of these results, it was concluded that no instability could be demonstrated and the materials were considered suitable for certification.The materials will be monitored further at regular intervals. Analytical Methods Used in the Certification Precautions taken by the laboratories participating in the certification are summarized in Table 3 which was used as a check list to avoid sources of error. A summary of the techniques used in the certification is given in Table 4. Technical Scrutiny All the results submitted for certification by the participating laboratories were discussed in a technical meeting to confirm the performance of the methods of analysis and their respective values. All data submitted for certification were obtained from laboratories that had demonstrated good analytical quality control and had fully implemented the analytical precautions outlined in the previous sections.The results accepted after the technical scrutiny were then statistically evaluated. Figs. 3 and 4 show the bar-graphs of the accepted results; each set of results is identified by the code number of the laboratory. Variations of 5% in the efficiency of the Cd column are not uncommon owing to the inhomogeneity of the column; higher variations were, however, found unacceptable for certification. Doubts were expressed on the use of hydrogencarbonate- carbonate buffer in comparison with borate buffer, the latter giving more reliable results. Discussions arose on the apparent discrepancy within the different SPEC (spectrophotometric determination of the diazo compound) results for the CRM 480. No explanation could, however, be found.Since no systematic error was suspected the data were accepted for certification. A further assessment of the sets of data was carried out using a Youden's plot4,5 which enables the results obtained on two solutions with different analyte concentrations to be compared, in order to detect possible systematic bias. Results are plotted in a scatter diagram, in comparison with the expected values or, alternatively, the means of the laboratory means. The diagram is divided into four quadrants in which two straight lines represent the expected values (or the means) for the two samples. If the results are affected by random errors only, they will be spread randomly over the four quadrants. If the results are located in I I86 Analyst, January 1996, Vol. 121 the lower left and upper right quadrants, forming a characteristic elliptical pattern along the 45" line passing through the expected values, one may conclude that systematic errors occurred in the measurements, underestimating or overestimating the concen- trations in both samples.The Youden graph for nitrate in CRMs 479 and 480 (Fig. 5), obtained using the mean values of each laboratory, shows clearly the prevalence of systematic errors since 14 out of 15 data are located in the upper right and lower left quadrants. However, as discussed below, these systematic errors are not statistically significant and no differences could be observed, on statistical grounds, between the different techniques used. With respect to the per cent. CV of the mean of Table 3 Some possible sources of error and measures taken to prevent them Analytical step Systematic error by Preparation Weighing Volumetric manipulation Sample preparation Adsorption/desorption Reagent contamination Contamination by toolslvials Contamination from laboratory air Adsorption/irreversible precipitation Incomplete conversion Calibration Contribution +/- +/- +I- + + + - - +/- Minimized by Calibrated balance Dilutions, etc., carried out with calibrated glassware, Use of distilled water washed non-metallic containers; Reagents of appropriate purity were chosen; verification Distilled water washing; verification with blank Use of clean benches or clean room; care in performing temperature control blank determinations used where appropriate with blank determinations determinations methods under cover or in closed systems; verification with blank determinations pH control Excess of reagents; methods a priori verified Reagents of suitable purity and stoichiometry were chosen Table 4 Summary of techniques and sample masses as applied in the determination of nitrate Sample mass = l o g 100 mg 200 mg 100 mg 50 mg 4 g 10 g 50 Pg 4 g 160 Pg 50 Pg 250 Pi3 Autosampler FI 25 Pg FI Sample pre-treatment and calibration Reduction on Cd column; addition of sulfanilamide and N-( 1- naphthy1)ethylenediamine dihydrochloride.Calibrant: KN03 (purity 399.99%) in water; calibration graph No pre-treatment. Calibrant: KN03 (purity > 99%); calibration graph Reduction on Cd column; addition of sulfanilamide and N-( 1 - naphthy1)ethylenediamine dihydrochloride.Calibrant: KN03 (purity > 99%); calibration graph No pre-treatment. Calibrant: KN03 (purity > 99%); calibration graph No pre-treatment. Calibrant: NaN03 (purity > 99.99%) verified Addition of NH4C1 buffer (pH 8.2); reduction on Cd column; against KN03 (purity 99.99%); calibration graph addition of sulfanilamide and N-( 1 -naphthyl)ethylenediamine. Calibrant: NaN03 (purity > 99.99%) verified against KN03 (purity 99.99%); calibration graph No pre-treatment. Calibrant: NaN03 (purity 299.5%) in water; calibration graph No pre-treatment. Calibrant: KN03 (purity 299.5%) in water; calibration graph Addition of NH4C1 buffer (pH 8.2); reduction on Cd column; addition of sulfanilamide and N-( 1 -naphthyl)ethylenediamine. Calibrant: KN03 (purity > 99%) in water; calibration graph Addition of NH4C1 buffer; reduction on Cd column; addition of sulfanilamide and N-( 1-naphthy1)ethylenediamine.Calibrant: NaN03 (purity 399.99%) in water; calibration graph No pre-treatment for CRM 479; dilution for CRM 480. Calibrant: KN03 (purity > 99%) in water; calibration graph No pre-treatment. Calibrant: NaN03 (purity 299%) in water; calibration graph Reduction by hydrazine addition with a copper catalyst; addition of sulfanilamide and N-( 1-naphthy1)ethylenediamine. Calibrant: KN03 (purity > 99.99%) in water; calibration graph Addition of N h C l buffer: reduction on Cd column; addition of sulfanilamide and N-( 1 -naphthyl)ethy lenediamine dihydrochloride. Calibrant: KN03 (purity 399.5%) in water; calibration graph No pre-treatment.Calibrant: NaN03 (purity > 99.5%) in water; standard additions Addition of NH4Cl buffer; reduction on Cd column: addition of sulfanilamide and N- 1 -( 1 -naphthyl)ethylenediamine dihydrochloride. Calibrant: KN03 (purity 399.5%) in water: calibration graph Final determination* SPEC of diazo compound at 540 nm IC; conductivity SPEC of diazo compound at 540 nm IC; conductivity IC; conductivity SPEC (SFA) of diazo compound at 540 nm IC; conductivity IC; conductivity SPEC (SFA) of diazo compound at 540 nm SPEC (SFA) of diazo compound at 540 nm IC; conductivity IC SPEC of diazo compound at 540 nm SPEC (SFA) of diazo compound at 540 nm IC SPEC (SFA) of diazo compound at 540 nm Series 01 02 03 04 05 05 06 07 08 09 10 11 12 13 14 15 * IC = Ion chromatography; SPEC = visible light or UV spectrometry; SFA = segmented flow analysis.Analyst, January 1996, Vol.121 87 0 N92b n N92a laboratory means, the values obtained for both CRMs were in good agreement with precisions usually obtained in a single laboratory for this range of concentrations. The CVs are plotted in Fig. 6 against the values obtained in the interlaboratory study 845- 01 SPEC 03 SPEC 05 SPEC ' I I 190 200 210 220 230 240 +......+......+......+......+.....+.......~.*....+.*....+......+......+ I +-----.-----> < ---- ---, 841 00 WEC 12 SPEC 13 SPEC 15 SPEC 02 l C 04 1c 05 I C 06 IC 07 I t 10 I C 11 IC 14 1c :MEANS I 1 <-*-, I ! Fig. 3 Laboratory means and 95% CI Nitrate in CRM 479 in pmol kg-1. 01 SPEC 03 SPEC 05 SPEC 09 SPEC 12 SPEC 13 SPEC 15 SPEC 02 IC 00 IC 05 I C 06 IC 07 I C 08 IC 10 I C 11 IC 14 IC ;MEANS: Fig.4 630 850 870 890 910 930 +.....+......+.....+.....+...... +......+......1.....+.....+......+.*...+ i g ....... ........ > I (-*-> I < -_-__-_- t ----.---, I I I <-*I--> ! g-------t------> I 1 <-**-, ! Laboratory means and 95% CI Nitrate in CRM 480 in pmol kg- 1. C R M Nit rate/pmol kg-' g8fi7 9351 Fig. 5 horizontal and vertical lines are the means of the laboratory means. Youden plot. Nitrate in CRM 479 versus nitrate in CRM 480. The (three samples, respectively, BCR-ma, BCR-FWb and BCR- FWc). An improvement in the precision was clearly achieved for concentrations of the order of 200 pmol kg-l (CRM 479 versus BCR-FWb), whereas the difference was lower for samples with concentrations of about 800 pmol kg-1 (CRM 480 and BCR-FWc) since a good precision had already been obtained in the interlaboratory study.Fig. 6 also compares the CVs obtained in other interlaboratory studies: the first group of results (indicated by N followed by the respective year) was obtained in the framework of an international collaborative programme for the assessment and monitoring of the acid- ification of rivers and lakes;6,7 the second group of results (referred to as R1 to R4) corresponds to interlaboratory studies organized by the Italian network for the study of atmospheric deposition chemistry from 1989 to 1992.8.9 The diagram shows a regular decrease in CV values from 2625% for concentra- tions of about 10 pmol kg-1 to 5-10% for concentrations of about 100 pmol kg-l.Statistical Evaluation The sets of accepted results were submitted to the following statistical tests : Kolmogorov-Smirnov-Lilliefors tests to assess the con- formity of the distributions of individual results and of laboratory means to normal distributions; Nalimov test to detect 'outlying' values in the population of individual results and in the population of laboratory means; Bartlett test to assess the over-all consistency of the variance values obtained in the participating laboratories; Cochran test to detect 'outlying' values in the laboratory variances (si*); One-way analysis of variance (F-test) to compare and estimate the between- and within-laboratory components of the over-all variance of all the individual results. The purpose of this statistical examination is essentially to ensure that the population of results accepted for certification has a normal distribution before the 95% confidence interval of the mean of means is calculated.This was true in all the cases (Kolmogorov-Smirnov-Lilliefors tests). In addition, no out- lying mean values were detected (Nalimov test). The set of variances was not homogeneous for both CRMs. As two different methods were used, each having a different repeatabil- ity and reproducibility, this is not surprising and fully acceptable (it is also for the reason that the s, calculated is not really applicable to any particular method; sw is a composite value for the methods used in this particular certification). Comparison of the Methods In the certification exercise laboratories applied two different techniques, namely spectrophotometric determination of the 20 - 8 > 0 v .10 - R3a P R4a R2b BCR-Was 10 100 NO;/pmol kg-' 1000 Fig. 6 frame of various interlaboratory studies on nitrate. Coefficients of variation (%) between laboratories, obtained in the88 Analyst, January 1996, Vol. 121 diazo compound (SPEC) and ion chromatography (IC), which made it possible to compare the results per technique. A grand mean of the means of all the eight laboratories applying either SPEC or IC was calculated. The obtained grand means were then compared in order to investigate whether a particular bias could be attributed to any method. As shown in Table 5, the CVs within SPEC or IC are systematically larger than those between the two techniques.Consequently, it cannot be inferred that the results of SPEC do not agree with those of IC for the certified content of nitrate. It could therefore be concluded that both SPEC and IC gave values that were good approximations of the true value for nitrate and could hence be applied in the certification. Certified Values The certified values as amounts of substance content and as mass fractions of nitrate are presented in Tables 6 and 7. The certified value is the unweighted mean of the accepted sets of Table 5 Results of the evaluation of consistency of the methods used for CRMs 479 and 480 cv (96) between means of Technique laboratories cv (%) of final with the between deter- same No. of sets means of CRM mination* technique of results techniques CRM479 SPEC 3.12 8 1.23 CRM480 SPEC 3.25 8 0.61 IC 3.67 8 IC 1.94 8 * SPEC = Visible light or UV spectrometry; IC = ion chromato- graphy * Table 6 Certified values of nitrate (as amounts of substance content) Certified CRM value Uncertainty Unit P* CRM 479 214 4 pmol kg-1 16 CRM480 885 13 pmol kg-I 16 * Number of data sets.Table 7 Certified values of nitrate (as mass fractions) Certified CRM value Uncertainty Unit P* CRM479 13.3 0.3 mg kg-* 16 CRM480 54.9 0.8 mgkg-* 16 * Number of data sets. results (p). The half-width of the 95% confidence interval of the mean is used as the estimate of the uncertainty. Availability The CRMs are available from the Institute for Reference Materials and Measurements, Retieseweg, B-2440 Geel, Bel- gium. Each bottle is accompanied by a certificate and a report describing the certification campaign.10 The authors thank all the laboratories that participated in the certification campaign: Preparation, homogeneity and stability studies. University of Cbrdoba, Department of Analytical Chemistry (Cbrdoba, Spain). Analyses. Anglian Water (Colchester, UK); Centre d’Estudits AvanCats de Blanes (Girona, Spain); C.N.R., Istituto Italian0 de Idrobiologia (Pallanza, Italy); C.N.R., Istituto di Ricerca sulle Acque (Brugherio, Italy); C.N.R.S., Service Central d’ Analyse (Vernaison, France); Compagnie Gknkrales des Eaux (Maisons- Lafitte, France); Dansk Teknologisk Institut (khus, Denmark); E.C.N., Energieonderzoek Centrum Nederland (Petten, The Netherlands); EPAL, Laboratorios Centrais (Lisboa, Portugal); GSF, Inst. fur Okologische Chemie (Neuherberg, Germany); Instituto Hidrogrhfico (Lisboa, Portugal); K.I.W.A. (Nieuwe- gein, The Netherlands); Ministhe des Affaires Economiques (Brussels, Belgium); Presidio Multizonale di Prevenzione (Venezia, Italy); University of Plymouth (Plymouth, UK). References I 2 3 4 5 9 10 Quevauviller, Ph., Vercoutere, K., and Griepink, B., Mikrochim. Acta, 1992, 108, 195. Reijinders, H. F. R., Quevauviller, Ph., van Renterghem, D., Griepink, B., and van der Jagt, H., Fresenius’ J . Anal. Chem., 1994, 348, 439. Quevauviller, Ph., van Renterghem, D., Valcfircel, M., Luque de Castro, M. D., Cosano, J., and Griepink, B., Anal. Chim. Acta, 1993, 283, 600. Youden, W. J., Ind. Qual. Control, 1959, 15, 24. Youden, W. J., and Steiner, E. H., in Statistical Manual of the Association of Oficial Analytical Chemists, Association of Official Analytical Chemists, Arlington, VA, 1975. Hovind, H., Norwegian Institute for Water Research (“A) Report, Oslo, 1988,40 pp. Hovind, H., Norwegian Institute for Water Research (“A) Report, Oslo, 1993, 50 pp. Mosello, R., Bianchi, M., Geiss, H., Marchetoo, A., Morselli, L., Muntau, H., Serrini, G., Serrini Lanza, G., and Tartari, G. A., Doc. Ist. Ital. Idrobiol, Annual Report, 1992, 35, 49 pp. Mosello, R., Bianchi, M., Geiss, H., Marchetto, A., Morselli, L., Muntau, H., Serrini, G., Semni Lanza, G., and Tartati, G. A., Doc. Ist. Ital. Idrobiol, Annual Report, 1993, 40, 49 pp. Quevauviller, Ph., and Valcfircel, M., EUR Report, European Commission, Brussels, 1995, EUR 16137 EN, p. 27. Paper 5103408F Received May 30,1995 Accepted September 14,1995

ISSN:0003-2654    

DOI:10.1039/AN9962100083

出版商:RSC    

年代:1996

数据来源: RSC