ANALYST, SEPTEMBER 1986, VOL. 111 1039 Spectrophotometric Determination of Some Pharmaceutical Carbonyl Compounds Through Oximation and Subsequent Charge-transfer Complexation Reactions Saied Belal* College of Pharmacy, Department of Pharmaceutical Analytical Chemistry, Alexandria University, Alexandria, Egypt Afaf A. El Kheir, Magda M. Ayad and Sobhi A. Al Ad1 College of Pharmacy, Department of Pharmaceutical Chemistry, Zagazig University, Zagazig, Egypt Two spectrophotometric procedures are presented for the determination of six pharmaceutical carbonyl compounds through charge-transfer complexation of their oximes with either the a-acceptor iodine or the n-acceptor choranil. The optimum assay conditions and their applicability to the determination of the test compounds in pharmaceutical products are described.The results obtained, which compared favourably with those given by a pharmacopoeial method, illustrate the accuracy, sensitivity and simplicity of the developed procedures. Keywords: Pharmaceutical carbonyl compound determination; charge-transfer complexation; oximation; spectrophotometry Progesterone, testosterone propionate and nandrolone phenylpropionate are A4-3-ketosteroid drugs in common use in therapeutics as progestational, androgenic and anabolic agents, respectively. A number of methods have been described for their determination including titrimetric,l col- orimetric,2-7 spectrophotometric,8 polarographic9J0 and chromatographicll-13 procedures. The pharmacopoeial method of analysis for their injectable solutions uses the isoniazide colorimetric14 method.Griseofulvin is a potent antifungal antibiotic; carvone and menthone (constituents of oil of caraway and peppermint, respectively) are used as carminatives and flavouring agents in pharmaceutical preparations. The latter is also used as an antipruritic agent. A review of the assay methods for griseofulvin, carvone and menthone, including titrimetric, spectrophotometric, electrochemical and chromatographic procedures, has been described by the authors,15 and methods of analysis for carbonyl compounds have been surveyed.16 Official compendia describe several different procedures for the determination of carbonyl compounds in volatile oils, including the acid - base titrimetric - hydroxylamine methods of the BPI7 and EP18 and the USPI9 method using the neutral sulphate.Charge-transfer complexation reactions have been exten- sively used for the determination of electron-donating basic nitrogenous compounds using as reagents the a-acceptor iodine or polyhalo or polycyano quinone n-acceptors in organic solvents.2G29 The introduction of a Schiff's base moiety from the reaction of carbonyl compounds with substituted ammonia or hydrazines was first used by the authors30 in the determination of corticosteroid drugs through solvent extrac- tion and charge-transfer complexation of their phenylhydraz- ones. The authors have used the oximation reaction for the indirect titrimetric and/or nitrite - diazo coupling spectropho- tometric determination of carbonyl drugs.7J5 Until now, the analytical use of charge-transfer complexation reactions of oximes or carbonyl compounds amenable to oximation has not been reported.This paper describes the development of a spectrophotometric analysis of ketonic drugs through their oxime formation, followed by solvent extraction and reaction with electron acceptor reagents. * Present address: College of Medicine and Allied Sciences, King Abdulaziz University, Jeddah, Saudi Arabia. Experimental Reagents and Materials All chemicals used were of analytical-reagent grade; solvents were of spectroscopic grade. Stock standard solutions of the drugs were prepared by accurately weighing 100 mg of the cited drugs into 100-ml calibrated flasks, dissolving in absolute ethanol and diluting to volume with the same solvent. Working standard solutions of the drugs were prepared by diluting the stock standard solutions with ethanol to give solutions of 0.1 mg ml-1 concentration.Hy droxylammonium chloride solution, 5% mlV in 60% ethanol. a-Acceptor, 2 x 10-4 M iodine solution in chloroform. n-Acceptor, 2 X 10-4 M chloranil solution in chloroform. Drugs. The drugs determined were progesterone, testo- sterone propionate and nandrolone phenylpropionate powders, griseofulvin powder, carvone and menthone (Aldrich), oil of caraway and oil of peppermint. Instrument A double-beam spectrophotometer (Uvidec-320) with 1-cm quartz or glass cells was used. Procedures Oximation step To an ethanolic solution equivalent to 10 mg of the drug were added 4 ml of hydroxylammonium chloride reagent and a drop of glacial acetic acid, and the mixture was refluxed on a water-bath for 1 h (A4-3-ketosteroids), or 20 min (other ketones).The reaction mixture was then evaporated to dryness at 70 "C and the residue was transferred into a 100-ml separating funnel with the aid of 10 ml of each of chloroform and water. The mixture was then shaken and the chloroform layer separated and transferred into a 100-ml calibrated flask. Extraction was continued with six successive 10-ml portions of chloroform, which were collected in the same calibrated flask. The extract was diluted to volume with chloroform and then subjected to either Procedure A or Procedure B. Colour development Procedure A. A 0.5-1-ml aliquot of the ketoxime extract was transferred into a 10-ml calibrated flask, treated with 2 ml1040 ANALYST, SEPTEMBER 1986, VOL.111 of iodine solution and allowed to stand for 30 min, before diluting to volume with chloroform. The absorbance of the solution was measured at 300 nm (ketosteroids) or 295 nm (griseofulvin, carvone and menthone) in a I-cm cell against a reagent blank. Procedure B. A 1-ml aliquot of the extract was transferred into a 10-ml calibrated flask, treated with 5 ml of chloranil solution and allowed to stand for 45 min at room temperature, or placed in a water-bath at 45 “C for 15 min, cooled and then diluted to volume with chloroform. The absorbance of the solution was measured at 430 nm (ketosteroids) or 440 nm (other ketones) in a 1-cm cell against a reagent blank. The concentration of the drug was calculated from the calibration graph obtained by applying either procedure A or B to standard solutions equivalent to 1-5 mg of drug per 100 ml, or from the corresponding linear equation describing the calibration graph.Application to A4-3-ketosteroid drugs in ampoules An accurately measured volume of the injection equivalent to 10 mg of the drugs was dissolved in 40 ml of light petroleum (40-60 “C, saturated with 90% ethanol) and extracted with four 20-ml portions of 90% ethanol (saturated with light petroleum). The alcoholic extracts were collected in a 100-ml calibrated flask and diluted to volume with 90% ethanol. A portion was subjected to the oximation and colour develop- ment procedures described above. Application to griseofulvin tablets Twenty tablets were finely powdered and an accurately weighed amount equivalent to 40 mg of drug was refluxed with 75 ml of absolute ethanol for 15 min.Sufficient ethanol was added to dilute to 100 ml, the mixture was centrifuged and a 25-ml aliquot of the supernatant liquid was subjected to the oximation and colour development procedures. Application to caraway or peppermint oil A 1-ml volume of the volatile oil was diluted to 100 ml with ethanol and then 1 ml of the resulting solution was subjected to the oximation procedure, followed by procedure A or B. For the standard additions method, a suitable aliquot of the standard solution of carvone or menthone was added to a 1-ml aliquot of the previously analysed diluted oil, then the assay was carried out and the recovery of the added amount was calculated.and griseofulvin contain two keto groups per molecule, they also gave a monoxime, a fact which was noted in the literature15J1 and in this work from molar absorptivity calculations applying Job’s33 method and other experimental observations. Iodine Acceptor Procedure Oximation results in the introduction of a basic centre to the carbonyl drug molecule. Mixing the oxime and iodine in chloroform media resulted in a change of the violet colour of iodine to yellow, owing to a charge-transfer complexation reaction between the ketoxime n-donor and the a-electron acceptor iodine followed by the formation of a triiodide ion pair, as shown by the following suggested routes, which agree with reports on similar reactions in the literature25.26: OH OH OH OH Outer complex Inner complex Triiodide ion pair oLmax.= 295-300 n n The absorption graphs of the reaction products (Figs. 1 and 2) show a high absorption band in the region of 300 nm and a lower band with a maximum at 365 nm, characteristic of the n-donor - iodine charge-transfer complexes. In chloroform, iodine itself has a maximum absorption at about 512 nm and the free oxime has an absorption peak at 34&350 nm. However, the iodine - oxime product has an absorption maximum at 270-300 nm. This variation is due to the modification of the absorption maximum of the triiodide ion formed from the release of iodide ions in the reaction by the accompanying ketoxime cation.25.26.32 o-6 I Results and Discussion Oximation The addition - elimination oximation reaction of the studied compounds is assumed to give an oxime according to the following route: 260 300 340 380 Wavelengthlnm Fig.1. Absorption spectra of oxime - iodine Complexes in chloro- form for: I, griseofulvin (0.019 mg ml-1); 11, carvone (0.016 mg ml-1); and 111, menthone (0.02 mg ml-1) ‘C=O + HzN-OH -‘C=N- OH + H20 / / The authors’ earlier studies7J5 of the optimum conditions for carrying out this reaction have led to the described conditions of oxime preparation and manipulation of the reaction mixture prior to its solvent extraction. Chloroform, the chosen extraction solvent, was also an appropriate medium for the charge-transfer complexation reaction, as it has the desired solubilising and lipotropic characteristics, in addition to a reasonable degree of polarity. Stoicheiometry As carvone, menthone, testosterone propionate and nandrol- one phenylpropionate contain only one carbonyl group, they should give a monoxime on reaction.Although progesterone I I I O ’ 2;o 300 340 380 Wavelengttilnm Fig. 2. Absorption spectra of oxime - iodine complexes in chloro- form for: I, progesterone (0.025 mg ml-I); 11, testosterone propionate (0.028 mg ml-I); and 111, nandrolone phenylpropionate (0.03 mg ml-1)ANALYST, SEPTEMBER 1986, VOL. 111 1041 C hloranil Acceptor Procedure Mixing the chloroform oxime extract (Amax. 340-350 nm) with chloroformic chloranil solution (Amax, 290 nm) resulted in the development of a red chromogen. The colour reached its maximum intensity and stability on standing for 45 min at room temperature or on heating in a water-bath at 45 "C for 15 min.This interaction is due to charge-transfer complexation between the ketoxime, n-donor and the chloranil n-acceptor , which, because of the polarity of the medium, may lead to a radical ion pair28 as shown in Scheme 1. The chloranil radical ion pair had a A,,,, of 430-440 nm (Figs. 3 and 4). The A,,,. of the chloranil radical ion would hence be slightly modified by the accompanying ketoxime cation, otherwise different ketox- ime complexes would have given an identical A,,,, 0 0 Oxime Ch lora n iI n - JC complex / in chloroform / 400 440 480 520 Wavelengthlnm Fig. 3. Absorption spectra of oxime - chloranil complexes in chloroform for: I, progesterone (0.045 mg ml-I); 11, testosterone propionate (0.045 mg ml-1); and 111, nandrolone phenylpropionate (0.044 mg ml-1) I n I I I ' 400 440 480 520 Wavelengthlnm 0- Radical ion pair Scheme 1 Fig.4. Absorption spectra of oxirne - chloranil complexes in chloroform for: I, griseofulvin (0.033 mg ml-l); 11, carvone (0.038 mg ml-l); and 111, menthone (0.035 mg ml-I) Table 1. Beer's plot data for ketoxime charge-transfer complexes in chloroform Iodine acceptor (procedure A) Chloranil acceptor (procedure B) Slope Progesterone . . . . . . . . 0.174 Testosteronepropionate . . . . 0.145 Nandrolone phenylpropionate . . 0.122 Griseofulvin . . . . . . . . 0.242 Carvone . . , . . . . . . . 0.215 Menthone . . . . . . . . 0.198 Drug (b) Intercept 0.04 0 -0.01 0.02 0.02 -0.01 (a) Slope 0.200 0.181 0.154 0.262 0.200 0.162 (b) Intercept -0.02 -0.01 -0.03 -0.03 0 0.02 (a) Table 2.Results of assay of pharmaceutical carbonyl compounds using iodine acceptor charge-transfer complexation method (procedure A) and the BP 1980 method Recovery* k S.D., YO Preparation Proposed method BP method Pure progesterone . . . . . . . . 100.15 f 0.38 100.19 k 0.73 Progesterone (Lutone ampoules) . . 99.99 k 0.41 99.63 k 0.44 Pure testosterone propionate . . . . 100.31 5 0.20 100.47 k 0.51 Testosterone propionate (Testone E. ampoules) . . , . . . 99.97 k 0.39 99.71 2 0.77 Pure nandrolone phenylpropionate . . 100.14 k 0.53 99.96 k 0.49 Nandrolone phenylpropionate (Durabolin ampoules) . . . . . . 99.84 2 0.35 100.02 f 0.63 Pure griseofulvin . . . . . . . . 99.87 f 0.55 100.11 k 0.7 Griseofulvin (Griseofulvin tablets) . . 99.98 f 0.74 100.26 f 0.66 Pure carvone .. . . . . . . . . 100.19 f 0.65 100.52 f 0.88 Carvone (in caraway oil) . . . . . . 99.75 k 0.45 98.98 k 0.55 Pure menthone . . . . . . . . . . 100.21 k 0.62 100.41 k 0.68 Menthone (in peppermint oil) . . . . 99.75 k 0.45 99.55 k 0.52 * Mean of five determinations k standard deviation. t Figures in parentheses are tabulated values of t and F a t the 95% confidence limit. t (2.3)t 0.11 1.38 0.66 0.66 0.54 0.54 0.6 0.62 0.69 2.28 0.49 0.66 F (6.39) 1- 3.65 1.12 6.30 4.0 1.12 3.25 1.63 1.25 1.83 1.5 1.2 1.351042 ANALYST, SEPTEMBER 1986, VOL. 111 Table 3. Results of assay of pharmaceutical carbonyl compounds using chloranil acceptor charge-transfer complexation method (procedure B) and the BP 1980 method Recovery* f S.D., Yo Preparation Proposed method BP method (2.3)t (6.39) t t F Pure progesterone .. . . . . . . 100.20 k 0.66 100.19 k 0.73 0.02 1.20 Progesterone (Lutone ampoules) . . 100.27 f 0.47 99.63 f 0.44 2.28 1.16 Pure testosterone propionate . . . . 100.42 f 0.49 100.47 k 0.51 0.16 1.08 Testosterone propiona te (Testone E. ampoules) . . . . . . 99.77 k 0.50 99.71 k 0.77 0.15 2.40 Pure nandrolone phenylpropionate . . 100.01 f 0.41 99.96 f 0.49 0.18 1.47 Nandrolone phenylpropionate (Durabolin ampoules) . . . . . . 99.76 f 0.44 100.02 f 0.63 0.76 1.95 Pure griseofulvin . . . . . . . . 100.10 k 0.77 100.11 f 0.70 0.02 1.22 Griseofulvin (Griseofulvin tablets) . . 100.34 * 0.60 100.26 f 0.66 0.20 1.22 Pure carvone . . . . , . . . . . 99.95 k 0.52 100.52 k 0.88 1.26 2.85 Carvone (in caraway oil) .. . . . . 99.49 f 0.40 98.98 f 0.55 1.70 1.87 Pure menthone . . . . . . . . . . 100.19 _t 0.49 100.41 k 0.68 0.59 1.96 Menthone (in peppermint oil) . . . . 99.99 k 0.59 99.55 k 0.52 1.29 1.29 * Mean of five determinations ? standard deviation. t Figures in parentheses are the tabulated values o f t and F a t the 95% confidence limit. Assay Parameters Effect of temperature After its full development, the complex may dissociate with a corresponding decrease in intensity on heating above room temperature. Effect of complex formation Leaving the reaction mixture to stand, as described under Procedures, was essential for the complete development of the complexes by transformation of the outer complexes to inner complexes.32 Effect of varying acceptor concentration The concentration of the acceptor should not be excessive in order to avoid the formation of ter-molecules or higher complexes. 32 Linearity of Calibration Graphs A linear relationship was obtained for the absorbance of the ketoxime acceptor reaction products when the concentration of the parent drugs was in the concentration range 0.01-0.05 mg ml-1 in the final measured solutions.The graphs show negligible or zero intercepts and are described by the regression equations A = a + bC (A, absorbance of a 1-cm layer; b, slope; a, intercept; and C, concentration of the measured solution in mg per 100 ml) obtained by the least-squares method.34 The data for the compounds studied (Table 1) that were used for the calibration graphs indicate the sensitivity of the proposed procedures.Quantification, Accuracy and Precision of Procedures A and B The validity of the proposed procedures for the determination of the studied compounds in their pure state and in phar- maceutical forms was tested by analysing these products with the proposed procedures and an official method.17 A standard additions technique was used for the determination of carvone and menthone in volatile oils of caraway and peppermint, respectively, to give the percentage recovery of an added amount. The results obtained (Tables 2 and 3) were compar- able to those obtained by the official method as both the t and Fvalues did not exceed the theoretical values.34 This indicated that the proposed procedures were as accurate and precise as the applied official methods and that no interference from excipients and vehicles was encountered.This was expected because the procedure involves solvent extraction of the oxime formed and a preliminary clean-up step with 90% ethanol was used for the injections. The procedure proved to be directly applicable to volatile oils with no preliminary separation step. The proposed procedures offer the advan- tages of accuracy and simplicity of reagents and apparatus. Among the spectrophotometric methods used to determine ketones in volatile oils, the proposed procedure is one of the most sensitive, simple and accurate and is of potential use as a general method. 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L., “Statistical Manual €or Chemists,” Academic Press, London, 1971, p. 61. Paper A6122 Received January 23rd, 1986 Accepted February 24th, 1986