ANALYST, JANUARY 1993, VOL. 118 29 6-Methoxy-2-methylsuIfonylquinoline-4-carbonyl Chloride as a Fluorescence Derivatization Reagent for Amines in Liquid Chromatography Tomohiko Yoshida, Youichi Moriyama, Kayoko Nakamura and Hirokazu Taniguchi Meiji College of Pharmacy, 1-35-23 Nozawa, Setaga ya, Tokyo 154, Japan 6-Methoxy-2-methylsuIfonylquinoline-4-carbonyl chloride was found t o be a sensitive fluorescence derivatization reagent for primary amines. The reagent reacted with the amines in acetonitrile in the presence of potassium carbonate t o give the corresponding fluorescent amides, which could be separated on a TSK,,, ODs-80TM reversed-phase column with aqueous 55% v/v acetonitrile as eluent. Pentylamine, hexylamine, heptylamine and octylamine were used t o investigate the derivatization conditions.The detection limits (signal-to-noise ratio = 3) of these amines were in the range 0.5-1 .O pmol per 20 PI injection volume. Alcohols did not give any fluorescent products under these derivatization conditions. Keywords: 6-Methox y-2-meth ylsulfon ylquinoline-4-carbon yl chloride; primary amine; fluorescence de riva tiza tio n reagent; h ig h -perform an ce liquid ch ro ma tog rap h y Benzoyl chloride, 1 4-nitrobenzoyl chloride2 and 3 ,5-di- nitrobenzoyl chloride,3 having a carbonyl chloride group, are well known derivatization reagents for the determination of amines by high-performance liquid chromatography (HPLC) with spectrophotometric detection. On the other hand, several fluorescent derivatization reagents such as 5-dimethyl- aminonaphthalene-1-sulfonyl chloride,4 phthalimidylbenzoyl chloride derivatives ,5 3,4-di hydro-6,7-dime t hox y-4-me thyl-3- oxoquinoxaline-2-carbonyl chloride6 and 7-dimethylamino- coumarin-3-carbonyl fluoride7 have been developed for the determination of amines.Quinoline derivatives, in addition to naphthalene, quin- oxaline and coumarin derivatives, are also widely known fluorescent compounds, e.g., quinine sulfate. To the best of our knowledge, however, quinoline derivatives (except for 3-benzoylquinoline-2-carboxyaldehyde8) have not been applied as fluorescence derivatizatian reagents to HPLC with fluorescence detection. In a previous paper,Y several 6-methoxyquinoline-4-carb- oxylic acid derivatives containing an S-atom at the 2-position of the quinoline ring were synthesized, and the fluorescence quantum yields of these compounds were measured. Subse- quently, 6-methoxy-2-methylsuIfonylquinoline-4-carboxylic acid (MSQC) was found to have the highest fluorescence quantum yield, and the quantum yields of MSQC-amide derivatives were about the same as those for amide derivatives of 7-methoxycoumarin-4-carboxylic acidl(1 in an ethanol sol- vent.Moreover, the MSQC-amide derivatives showed the highest fluorescence intensity in acetonitrile, which is widely used as a component of the mobile phase in reversed-phase HPLC. This paper describes the synthesis of 6-methoxy-2-methyl- sulfonylquinoline-4-carbonyl chloride (MSQC-CI), according to a previous method," as a fluorescence derivatization reagent for amines in reversed-phase HPLC.In order to investigate the reactivity of amines with MSQC-CI, pentyl- amine, hexylamine, heptylamine and octylamine were used. The MSQC-CI reagent reacted with these amines in aceto- nitrile in the presence of potassium carbonate to produce the corresponding fluorescent amides. The amides were separated on a reversed-phase column with aqueous 55% v/v acetonitrile. Experimental Reagents and Materials All chemicals were of analytical-reagent grade. All amines used were purified by distillation. De-ionized and glass- distilled water was used. Acetonitrile was of HPLC-grade (Kanto Chemicals). All solvents (Luminazol) for measuring the fluorescence quantum yield were purchased from Wako Pure Chemicals. The MSQC-CI solution (1 mmol 1-1) was prepared by dissolving MSQC-CI (3 mg), synthesized from MSQC as described previously,ll in acetonitrile (10 ml).This solution was stable for 10 h. Apparatus All melting-points (uncorrected) were determined with a Yanagimoto micro melting-point apparatus. Infrared (IR) spectra were recorded in KBr discs with a Hitachi 270-30 infrared spectrophotometer. Mass spectra were obtained with a Jeol DX302 spectrometer. Proton nuclear magnetic res- onance (IH NMR) spectra were recorded with a Jeol JNM-GX400 spectrometer using tetramethylsilane as an internal standard. Fluorescence spectra and fluorescence quantum yield (using quinine sulfate solution in 0.05 mol 1-1 H2SOL1 as a standard solution) were measured12 with a Hitachi F-4000 fluorescence spectrophotometer in a 1 cm quartz cell.Fluorescence Quantum Yield12 First, the excitation spectrum (210-606 nm) was calibrated by using Rhodamine B (5 g 1-1, in ethane-1,2-diol) as a quantum counter at the emission wavelength (640 nm) fitted. Next, the emission spectrum was calibrated by using the diffuser and the attenuator by scanning both the excitation and emission wavelengths (210-603 nm). The emission spectra of the sample solution (MSQC-amide solution in various solvents) and the standard solution (quinine sulfate solution in 0.05 moll-' H2S04) were recorded at an excitation wavelength of 366 nm. Each area (F, or F,) at the peak width at half-height on the spectrum (relative light quantum number-wavenum- ber) obtained was calculated. The fluorescence quantum yield (ax) for the unknown compound was then calculated accord- ing to the following equation: ax = X F,IF, X (nx)2/(ns)2 X E,/E, X c,/c, where Q5 is the quantum yield of the standard, quinine sulfate (0.55), n is the refractive index of the solution, E is the molar absorption coefficient at 366 nm and c is the molarity.Subscripts x and s refer to the unknown compound and the standard, respectively. High-performance Liquid Chromatography For HPLC a Model L-6200 high-performance liquid chroma- tograph (Hitachi) equipped with a Rheodyne 7125 injector (2030 ANALYST. JANUARY 1993, VOL. 118 pl loop) (Rheodyne) was used. A Jasco RC-250 recorder, SIC chromatocorder 12 and a Jasco FP-210 spectrofluorimeter fitted with a 12 PI flow cell were used. The spectrometer was set at an excitation wavelength of 342 nm and an emission wavelength of 448 nm.A TSK,,, ODs-80TM column (particle size 5 pm, 150 x 4.6 mm i.d.; Tosoh) was employed at room temperature. Aqueous 55% v/v acetonitrile was used as the mobile phase at a flow rate of 1.0 ml min-1. Preparation of Fluorescent Compounds (1-10) From Amines The acetonitrile solution (15 ml) of MSQC-CI (0.1 g) was added to the acetonitrile solution ( 5 ml) containing each of the amines (1 g). After the mixture had been allowed to stand for 30 min at room temperature, it was evaporated to dryness in vacuo. The residue was dissolved in a small amount of dichloromethane, then subjected to column chromatography on Wakogel C-200 (Wako Pure Chemicals) with hexane-ethyl acetate-ethanol (46 + 3 + 1, v/v). The blue fluorescent eluate was collected and the solvent removed in vacuo. The residue was recrystallized from dichloromethane-hexane to give the corresponding compounds (1-10, Tables 1 and 2).Derivatization Procedure To a 0.2 ml portion of an acetonitrile solution of the amines were added about 10 mg of potassium carbonate and a 0.2 ml portion of 1 mmol 1-1 MSQC-Cl solution in a polypropylene micro-centrifuge test-tube (1 .5 ml volume). The mixture was allowed to stand at room temperature for 5 min. A 0.05 ml portion of the reaction solution was diluted with mobile phase to 1.0 ml and a 20 p1 portion of the resultant solution was injected into the chromatograph. Results and Discussion Structural Assignment and Fluorescence Properties of Compounds 1-10 The elemental analysis data and spectral data are shown in Tables 1 and 2, respectively. Compounds 1-10, except for compound 9, showed an I R absorption band due to the NH of the secondary amide groups at 3292-3308 cm-1.The 1R absorption bands due to the CO of the secondary amide groups or methylsulfonyl groups was observed at 1622-1660 or 1152-1160 and 1304-1316 cm-1, respectively. The 'H NMR signals due to methylsulfonyl and methoxy protons were observed at 3.30-3.34 (3 H, s) and 3.92-3.98 (3 H, s) ppm, respectively. These data, the mass spectra data and elemental analyses were consistent with the assigned structure of the corresponding carboxyamides. The fluorescence quantum yields and corrected fluores- cence emission maxima (excited at 366 nm) of compounds 1-10 in various solvents are shown in Table 3.The fluores- cence quantum yields of compounds 1-6 (aEtOH = 0.25-0.30) were about the same as those of the amide derivatives (QEtOH = 0.25-0.34) of 7-methoxycoumarin-4-carboxylic acid in an ethanol solvent described by Goya et a1.10 Consequently, it was found that MSQC-CI reacted with amines to form the same intense fluorescent amide derivatives as those derived from 7- methoxycoumarin-4-carbonyl chloride. 10 Moreover, the MSQC-amide derivatives showed the highest quantum yields in acetonitrile, which is widely used as a component of the mobile phase in reversed-phase HPLC. The maxima of the fluorescence spectra in acetonitrile were slightly blue-shifted compared with those in methanol for compounds 1-8. The fluorescence intensities of compounds 1-8 in 55% v/v aqueous acetonitrile as mobile phase were slightly lower than those in acetonitrile.However, the fluorescence quantum yields of these compounds in 55% aqueous acetonitrile were higher than those in methanol. The quantum yield of compound 9 derived from diethylamine was below 0.01 and that of compound 10 derived from aniline was not calculated at the concentration which gave an absorbance of 0.02 at the corrected excitation wavelength (366 nm). 12 Separation of MSQC Derivatives The effect of different organic modifiers in the mobile phase was studied by using the four amines listed in Table 4. The log k' value (k' = capacity factor) of all the amines was smaller Table 1 Analytical data for fluorescent compounds 1-10* Analysis (YO) Calc. (Found) Compound 1 2 3 4 5 6 7 8 9 10 * Me1 ting-poi nt/ "C 206-207 I 74- 1 75 222-223 139- 140 138-139 135-136 164-167 234-235 I41 -1 42 245-246 Yield 68 63 64 81 78 76 71 89 83 86 (Yo) C 54.53 55.89 (55.76) 55.89 57.13 (57.08) 58.27 (58.21) 59.32 (59.09) 61.61 (61.62) 59.65 (59.50) 57.13 (57.05) 60.66 (60.68) (54.73) (55.74) H 5.23 5.63 5.63 (5.66) 5.99 (6.00) 6.33 (6.40) 6.64 (6.68) 4.90 (4.80) 6.12 (6.09) 5.99 (6.21) 4.53 (4.40) (5.39) (5.75) N 9.08 (9.02) 8.69 (8.69) 8.69 8.33 7.99 7.69 (7.63) 7.56 (7.53) 7.73 (7.74) 8.33 (8.34) 7.86 (7.86) (8.64) (8.23) (8.00)ANALYST, JANUARY 1993, VOL.118 31 Table 2 Spectral data for fluorescent compounds 1-10 Compound 1 2 3 4 5 6 7 8 9 10 MS. mlz 308 322 322 336 350 364 370 362 336 356 (M+ 1 IR, v,,,/cm-l NH C=O SO2 1H NMR in CDCI, [6 (ppm)] 3308 1642 1158 1.35 (3 H, t, CH2CH3), 3.33 (3 H, s , S02CH3), 3.61 (2 H, m, CH2CH3), 3.98 (3 H, s , OCH,).6.36 1308 (1 H, br, NH). 7.50, 7.71, 8.08 and 8.09 (each 1 H, aromatic H) 3308 1622 1158 1.06 (3 H, t, CH,CH,CH,), 1.71 and 3.54 [each2 H, m, (CH2)2CH3], 3.34 (3 H, s, SO,CH,), 3.97 1316 (3 H, s, OCH3), 6.26 (1 H, br, NH), 7.51, 7.70, 8.09 and 8.10 (each 1 H, aromatic H) 3300 1622 1158 1.36 [6 H, d, CH(CH3)2], 3.34 (3 H, s, S02CH3), 3.97 (3 H, s, OCH3), 4.39 [ 1 H, m, CH(CH3)2], 1310 6.10 ( 3 H, br, NH), 7.51, 7.68, 8.07 and 8.09 (each 1 H, aromatic H) 3304 1640 1158 1.01 [3 H, t, (CH2)3CH3], 1.49, 1.68 and 3.56 [each 2 H, m, (CH2),CH,], 3.33 (3 H, s, S02CH3), 1314 3.97 (3 H, s, OCH,), 6.27 (1 H, br, NH), 7.51, 7.69, 8.08 and 8.09 (each 1 H, aromatic H) 3296 1644 1154 0.95 [3 H, t.(CH&CH3], 1.42, 1.69 and 3.56 [8 H, m, (CHZ)4CH3]. 3.33 (3 H, s, S02CH,), 3.97 1314 (3 H, s, OCH3), 6.28 (1 H, br, NH), 7.51, 7.69, 8.08 and 8.09 (each 1 H, aromatic H) 3292 1644 1156 0.92 [3 H, t, (CH2)5CH3], 1.36.1.43, 1.68and 3.56 [lOH, m, (CH2)sCH3], 3.33 (3 H, s, S02CH,), 1316 3.97 (3 H, s, OCH3), 6.31 (1 H, br, NH), 7.50, 7.69. 8.08 and 8.09 (each 1 H, aromatic H) 3304 1660 1156 3.30 (3 H, s, SOzCH,), 3.92 (3 H, s, OCH3), 4.74 (2 H, d, CH2), 6.65 (1 H, br, CONH). 7.32-7.44 1304 (5 H, m, C6Hs), 7.50, 7.66, 8.07 and 8.10 (each 1 H, aromatic H) 3308 1642 1160 1.21-2.14(10H,m, cyclicH), 3.33 (3H, s,S02CH3),3.96(3H,s,0CH,),4.09(1 H,m,cyclicH), 1312 6.16 (1 H, d, NH), 7.50, 7.66, 8.07 and 8.08 (each 1 H, aromatic H) 1152 1.07 and 1.38 (each 3 H, t , CH2CH3), 3.12 and 3.71 (each 2 H, br, CH2CH3), 3.32 (3 H, s, 1314 S02CH,), 3.93 (3 H, s, OCH?), 7.08, 7.52, 7.99 and 8.14 (each 1 H, aromatic H) 1156 3.34 (3 H, s, S02CH3), 3.96 (3 H, s, OCH,), 7.25-7.74 (5 H, m, ChHs), 7.50,7.73,8.08 and 8.23 1312 (each 1 H, aromatic H), 8.30 (1 H, br, NH) 1638 1652 3296 Table 3 Fluorescence spectral data for compounds 1-10 Compound Quantum yield (A.,","x/nm)* Acetonitrile 55% Acetonitrile 1 0.56 (437) 0.44 (455) 2 0.54 (438) 0.43 (456) 3 0.46 (438) 0.43 (458) 4 0.56 (437) 0.46 (455) 5 0.50 (438) 0.47 (454) 6 0.56 (438) 0.42 (456) 7 0.61 (438) 0.51 (457) 8 0.30 (436) 0.30 (455) 9 -t --t 10 --$ 4 * Excited at 366 nm and corrected by using Rhodamine B. -t Below 0.01.$ Could not be measured. Methanol 0.27 (456) 0.29 (455) 0.24 (453) 0.25 (454) 0.27 (454) 0.28 (454) 0.31 (456) 0.19 (455) --t --$ Chloroform 0.53 (431) 0.44 (431) 0.56 (431) 0.50 (431) 0.53 (431) 0.57 (432) 0.36 (430) -7 -3 0.55 (430) Cyclohexane 0.1 1 (420) 0.10 (421) 0.09 (422) 0.11 (421) 0.10 (420) 0.10 (420) 0.20 (422) 0.06 (419) -7 -3: Table 4 Effect of organic modifier of mobile phase* Acetonitrile Methanol Tetrahydrofuran Amine Log k' h Log k' h Log k' b Pentylamine 2.45 -0.030 3.23 -0.037 2.70 -0.041 Hexylamine 2.88 -0.034 3.93 -0.045 3.22 -0.048 Heptylamine 3.32 -0.039 4.58 -0.051 3.72 -0.055 Oct ylamine 3.74 -0.043 5.30 -0.058 4.20 -0.062 * Log k' = a + h(x), where x is the modifier concentration.with acetonitrile as modifier than with methanol or tetra- hydrofuran as modifier; therefore, acetonitrile was the most suitable organic modifier in the mobile phase.The MSQC derivatives of pentylamine, hexylamine, hepty- lamine and octylamine were subjected to a separation study on a TSK,,, ODS-80TM reversed-phase column using aqueous acetonitrile as the mobile phase. At acetonitrile concentra- tions higher than 60% v/v, the peak for pentylamine was overlapped by the reagent blank peak, whereas at acetonitrile concentrations lower than 45% v/v the pentylamine peak took longer to elute and there was some broadening of the peak. The best separation was obtained with aqueous 55% v/v acetonitrile as the mobile phase. Fig. 1 shows a typical chromatogram of the MSQC derivatives obtained with a mixture of the four amines (concentration, each 0.05 mmol 1-1) under the selected derivatization conditions. Derivatization Conditions and Reaction With Alcohols The use of acetonitrile as a solvent for the derivatization reaction gave the highest detector response. Pyridine, acetone and tetrahydrofuran gave a lower detector response (about 88, 55 and 32%, respectively, of that obtained in acetonitrile).Dimethylformamide and dimethyl sulfoxide did not yield any detector response for the corresponding amines. Hence, acetonitrile was chosen for the recommended procedure. The uncorrected fluorescence-emission maximum of the MSQC derivative of hexylamine was observed at 448 nm with excitation at 342 nm in the mobile phase (55% v/v acetonitrile solution). Potassium carbonate accelerated the derivatization reaction and the most intense peaks for the amines examined were achieved by the addition of 7-15 mg; about 10 mg were added to the reaction mixture. Potassium carbonate was found to be32 ANALYST, JANUARY 1993, VOL.118 0 10 20 Time/mi n A C D Fig. 1 Chromatogram obtained for the reaction of amines with MSQC-CI. A, Pcntylaminc; B, hexylamine; C, hcptylamine; and D, octylaminc. An aliquot (0.2 ml) of a mixturc of thc amincs (each 0.05 mmol 1 - l ) was treated as dcscribcd undcr Dcrivatization Procedure 15 E 2 10 c 0) a, f Y (D .- 2 5 0 1 2 3 MSQC-Cl/mmol I-' Fig. 2 Effect of MSQC-CI concentration on the tluorescence derivatization. A, Pcntylaminc; B , hexylaminc; C, hcptylamine; and D, octylaminc. An aliquot (0.2 ml) of a mixture of the amines (each 0.05 mmol 1-I) was reacted with various concentrations of MSQC-CI as described under Derivatization Procedure more effective than pyridine, quinuclidine and dibenzo-18- crown-6.Even in the absence of potassium carbonate, the reaction was complete within 3 min at room temperature. However, the peak areas of pentylamine, hexylamine, hepty- lamine and octylamine were about 69-70%0 of those obtained in the presence of potassium carbonate. The dcrivatization reaction of the four amines with MSQC- C1 proceeded rapidly, independently of the temperature (0-100°C); maximum and constant peak areas for the corresponding amines were obtained on allowing the reaction mixture to stand at room temperature for about 3 min. Therefore, a reaction time of 5 min was selected. Maximum and constant peak heights for the four amines (concentration, each 0.05 mmol 1 - 1 ) were obtained at Table 5 Retention times and detection limits of the MSQC derivativcs of primary and secondary amines Amine Prop ylaminc Butylamine Pentylamine Hexylamine Hcpt ylaminc Octylamine Benzylamine 4-Methylbenzylamine 2-Phenylethylamine Cyclohexylamine Dipropylamine Di butylamine Detcction limit*/ pmol per 20 pl min injcction volumc Retention time/ 6.8t 10.9-I- 6.1$ 8.73 12.83 19.4$ 13.7t 21.47 18.0-1 17.0-1 17.5-1 11.43 1 .o I .o 0.5 0.5 0.5 1 .o 1 .o 1 .o 1 .0 2.0 100 100 * The amount in the injection volume (20 pl) giving a signal-to- t Mobile phase: acetonitrile-water (40 + 60).$ Mobile phase: acetonitrile-water (55 + 45). noise ratio of 3. MSQC-CI concentrations greater than about 0.6 mmol 1-1; hence a 1 mmol 1 - 1 MSQC-Cl solution was used (Fig.2). The MSQC derivatives in the final solution were stable for at least 10 d in daylight at room temperature. As reported previously,ll MSQC-Cl reacts with primary and secondary alcohols at high temperatures (100 "C) and long reaction times (60 min) to produce the corresponding fluores- cent esters. However, under the derivatization conditions adopted here, the reaction of MSQC-Cl with primary and secondary alcohols gave no fluorescent products. Calibration Graphs, Precision and Detection Limits The relationship between the peak area and the amount of propylamine , butylamine , pentylamine , hexylamine, heptyl- amine, octylamine and benzylamine was linear from 0.5 pmol to 0.15 nmol per 20 pl injection volume (corresponding to 1 ymol 1-1-0.3 mmol 1 - 1 of sample solution).The precision was established by repeated analyses (n = 10) of the above seven amines. The relative standard deviations were 7.0, 4.4 and 3.5% (propylamine), 6.2,4.5 and 3.1% (butylamine), 7.3, 4.2 and 2.9% (pentylamine), 4.3,3.9 and 2.9% (hexylamine), 5.1, 3.8 and 2.7% (heptylamine), 4.5, 3.8 and 2.7% (octyl- amine) and 4.0,3.2 and 3.3% (benzylamine) at concentrations of 0.005, 0.05 and 0.1 mmol 1 - 1 , respectively. The detection limits (signal-to-noise ratio = 3) ( n = 10) for the primary amines were from 0.5 to 1.0 pmol and those for secondary amines were above 100 pmol per 20 yl injection volume (Table 5 ) . Conclusions The reagent MSQC-CI, which is characterized by a methylsul- fonyl group, readily reacted with primary amines at room temperature to produce the corresponding strongly fluores- cent amides, which were detected with a detection limit of 0.5-1 .O pmol per injection volume.The results presented in Table 5 suggest that 2-phenylethylamine in plasma might be determined with MSQC-Cl by reversed-phase HPLC. More- over, the reaction of MSQC-Cl with alcohols gave no fluorescent products under the derivatization conditions employed. The authors are grateful to Professor T. Nakagawa of Kyoto University for his valuable suggestions. Thanks are due to Professor Y. Shibanuma of this College for measurements of IR spectra and for his advice.ANALYST, JANUARY 1993, VOL. 118 33 References 1 2 3 4 5 6 7 Fitzpatrick. F. A., and Siggia, S., Anal. Chem.. 1973,45,2310. Newsome. W. H., and Panopio, L. G., J . Agric. Food Chem., 1978, 26, 638. Suzuki, Y., and Tsuchiya, N., Bunseki Kuguku, 1981, 30, 240. Yamada, K., and Aizawa, Y., J. Pharmacol. Methods, 1983,9, I. Tsuruta, Y., and Kohashi, K., Anal. Chim. Actu. 1987, 192, 309. Ishida, J . , Yamaguchi, M., Iwata, T., and Nakamura, M., Anal. Chim. Actu, 1989, 223, 319. Fujino, H . , and Goya, S . , Yakugaku Zusshi, 1990, 110,693. 8 9 10 11 12 Beale, S. C., Savage, J . C., Wiesler, D., Wietstock, S. M.. and Novotny, M., Anal. Chem., 1988, 60, 1765. Yoshida, T., Moriyama, Y., and Nakano, S . , Chem. Pharm. Bull., 1992,40, 1322. Goya, S . , Takadate, A., Tanaka, T., and Nakashima, F., Yakugaku Zasshi, 1980, 100,289. Yoshida, T., Moriyama, Y.. and Taniguchi, H., Anal. Sci., 1992, 8, 355. Nishikawa, Y., and Hiraki, K., Analytical Methods of Fluores- cence and Phosphorescence, Kyoritu Press, Tokyo, 1984. Paper 2iO4250I Received June 26, 1992 Accepted August 6, 1992