Chem.Anal. (Warsaw), 41,19 (1996)

Determination ofAntimycotic Substances, Derivatives ofImidazole', by ,Gas Chromatographic Method

by,E. KlJblin andT. Kanjewska DepartmentofChemicalAnalysis, Institute ofDrugResearch and Control, 00-725 Warszawa, Poland

Key words: bifonazole, chlormidazole, econazole nitrate, isoconazole nitrate, metroni­ dazole, miconazole, tioconazole, gas chromatography

Simple method for gas-chromatographic identification of 7, among 9 used, antimycotic compounds, derivatives of imidazole has been elaborated. The conditions have been e~tablished for quantitative determination of selected compounds of this group present insubstlnces and such pharmaceuticals as ointments and creams. The column, packed with UCW-98 on Chromosorb WAW, and flame-ionisation ,detector were used. The, statistical data indicate satisfactory precision of the method, both in the determination ofimidazole derivatives in substances and in preparations.

Opracowano prost<\,m'etod€( identyfikacji za pomoc<\, chromatografii gazowej 7, sposr6d 9 stosowanych, zwi<\,zk6w przeciwgrzybiczych, pochodnych imidazolu. Ustalono wa­ runki oznaczania ilosciowego wybral1ych zwi<\,zk6w z tej grupy w substancjach i pieparatach farmaceutycznych - masciach i kremach. Zastosowano kolumn€( wypetnio­ n<\, UCW-98 Ila Chromosorbie WAWoraz detektor 'ptomieniowo-jonizacyjny.' Dane statystyczne wykazuj<\, dostateczn<\,precyzj€( metody,zar6wno przy oznaczaniu zwi<\,z­ k6w - pochodnych imidazolu, w sUbstancjach, jak i w preparatach.

Imidazole (1,3-diazole)derivatives are used in therapy as lipotropic, thyrostatic, antiulcericand antimycotic remedies, and also as intubation narcotics. The antimy­ cotic derivatives of imidazole are the remedies of wide spectrum ofactivity - they operate against dermatophytes: Trichophyton and Emidermophyton, sacharomycetes: Candida albicans, Malasscnia furfur and Torulopsis and phytomycetes. The most of pharmaceuticals of this group indicate also bactericidal activity, mostly against Gram-positive bacteria ofStreptococcus and Staphylococcus group: and also against Trichomonas vaginalis. These compounds are mostly fbr external use as lotions, gels, creams and ointments, as well as shampoos in cases of skin mycosis. The pharmaceuticals containing imidazole derivatives are also used as vaginal tablets or globules in Iuycotic or mycotic-bacterial infections. The pharmaceuticals'containing ketocona- 20 E. Kublin and T. Kaniewska zole are used internally as tablets and suspensions in mycotic infections after the use of several drugs, as e.g. immunopressive compounds, and in tumor diseases [1-4]. Several papers relative to identification and determination of individual imida­ zole derivatives were found in the literature. In quantitative determination spectro­ photometric [5-19], colorimetric [20-26], high perfonnance liquid chromatographic [27-36], titrimetric [37-39], polarographic [40] and'voltammetric [41,42] methods were used. Moreover the compounds of this group can be detennined densitometri- cally [43,44]. . Not.many reports on the determination of imidazole derivatiyesby $aschroma­ tography are present in the literature. They concern the analysis ofmetronidazole by gas chromatography after its previous convertion in a derivative with trifluorobis (trimethylsilil)acetamide [45]. The preparation of the derivative with very toxic reagent considerably prolong thetitne used for thedetennination. The aim otthis paper was to elaborate a sensitive and simple method for the identification and quantitative determination of imidazole derivative compounds by gas chromatography, without the process of sililation.

EXPERIMENTAL

Reagents solutions and apparatus Standard substances: Metronidazole -Polfa (PoznaI1,Poland); Miconazole - Institute of Pharma­ ceutical Chemistry (Warsaw, Poland); Clotrimazole - Merckle; Ketoconazole - Janssen; Bifonazole ­ Bayer AG; Econazole nitrate - Cil~g AG; Tioconazole -hie. Rockville; Isoconazole nitrate -Schering; Chlormidazole - Polfa (Grodzisk, Poland). Substances and pharmaceuticals investigated: Tioconazole - Pfizer; Econazole nitrate - Cilag AG; Bifonazole - Ricerca; Mitekol Kreme 1%(econazolenitrate) - Lek Ljubljana; Myco~por Creme 1% (bifonazole) - Bayer AG; Gyno-Trosyd - ointment 6.5 % (tioconazole) - Research Center of Biotechnology (Warsaw, Poland), under lice.l1ce of Pfizer. The reagents used were analytically pure. A gas chromatograph Philips Unicam PU 4550 equipped with aflame ionisation detectorand glass column 2 mlong andO.4cminternal diameter, packed with 10 % of UCW-98 on Chromos()rb WAW (80-100 mesh) was used. The temperatures applied were: column - 280°C, injector - 300°C, detector - 320°C. Nitrogen (flow rate 60 ml min-I) was the carrier gas. The standard solutions (1 % methanolic) were prepared, using metronidazole, tioconazole,econa­ zole nitrate, isoconazole nitrate, ketoconazole, bifonazole, c1otrimazole,chlormidazole and miconazole; 3!-d of each sol~tion were introduced to the column. The retention times measured in conditions given above are following (in minutes): metronidazole 0.64; chlormidazole 2.03; tioconazole 5.77; econazole nitrate 6.37; bifonazole 7.03; isoconazole nitrate 7.85, miconazole 8.25. For quantitative determination three compounds were selected: bifonazole, tioconazole, and eco­ nazole nitrate. The method of internal standard was applied; prenylamine lactate in 0.5 mg ml-Isolution was used as the standard; its retention time was 3.58 minute. For each compound investigated 0.2%, 0.15 %, 0.1 % and 0.05 % solutions of the substance in 0.05 % methanolic solution of the internal standard were prepared 3 1-11 of these solutions were injected into the column. For bifonazole, tioconazole and econazole nitrate the calibration graphs of the dependence of peak area of th,c compound determined to the peak area of the internal standard were rectilinear for 0.5 mg ml-I to 2mg ml-I concentration range. The elaborated method was applied for the determination of bifonazole, tioconazole and econazole nitrate content in pharmaceuticals: Mitekol-l % cream; Mycospor -1 % cream; Gyno-Trosyd - 6.5 % ointment. Determination a/derivatives0/ imidazole 21

For statistical evaluation ofthe results the 0.1 % solutions of bifonazole, tioconazole and econazole nitrate (as standards) in q.05 % methanolic solution of internal standard - prenylamine lactate and 0.1 % solutions ofsubstances investigated - bifonazole, tioconazole and econazole nitrate, - in 0.05 % solution of internal standard in methanol, were used.

Proced~

The determinations were performed under elaborated and given above conditions. The samples of creams Mitekol and Mycospor (2 g each) and Gyno-Trosyd ointment (0.3 g) were heated at a water bath (40-50°C) until the base of cream and ointment was dissolved, then the samples were shaken mechan­ ically for 30 min and filtered. Aliquots (3 ml) of the solutions were mixed with 5 ml portions of internal standard (prenylamine lactate solution) and diluted to 10ml with methanol. The samples (20 mg) of standard substances (tioconazole, bifonazole and econazole nitrate) were shaken with 10 011 portions of methanol until dissolved. In volumetric flasks (lO ml capacity) 3 ml portions of the solutions of bifonazole, tioconazole andeconazole nitrate were plac~d, mixed with 5 ml of internal standard solution of prenylamine lactate and diluted to 10 ml with methanol. 3 J.l.1 portions ofinvestigated and standards solutionswere applied on the chromatographic column. For the samples ofcreams· and ointments the c~Hihration graphs for bifonazole, tioconazole and econazole nitrate were plotted; they were rectilinear for 0.5 mg ml-1 to 2 mg ml-1 .concentration range of these compounds. Comparative studies were performed using spectrophotometric method. For that 0.001 % solution of bifonazole, 0.02 % solution oftioconazoleand 0.04 % solution ofeconazole nitrate in methanol were prepared. The samples of Mitecol..cream, Mycospor-cream and Gyno-Trosyd-ointment(O.lg each) were heated at water bath (40°-50°C), extracted with methanol, sha"en mechanically for 1 hand filtered. Solutions were diluted with methanol analogically as reference solutions for chromatographic investigations.

RESULTS AND DISCUSSION

The chromatograms obtained are presented in Figs 1, 2 and 3. Statistiql1 evalu­ ation of the results is given in Tables 1 and 2. Statistical data, presented in these Tables, indicate satisfactory accuracy of themethod,both for the compounds and pharmaceuticals ofointments and creams type. The coefficients ofvariation obtained for 0.6 mg Inl-1to Img ml-1concentrationrangeindicate the precision of the method. The calibration graphs, plotted for standard substances and these isolated from creams and ointments were rectilinear for a.Smg ml-1 to 2 mg ml-1 concentration range. The limit ofdetection was O.3l-lg. Theelaboratedconditio~ were shown ·as a good ones for the quantitative determination of selected compounds of imidazole derivatives by gas-chromato­ graphic method in substances and pharmaceuticals - creams and ointments. The gas-chromatographic method elaborated enables direct and precise determination the content of compounds being investigated in substances and in ointments and creams. The investigations performed using spectrophotometric method for determining active substances in Mitekol- 1 % cream and Mycospor -1 % cream, despite uphill and laborious extraction, gave considerably higher results. It means, that a part of auxilliary substances, which was not eliminated by extraction, is characterised by maxima of similar values. The aim of the further investigations will be the elaboration of gas-chromato­ graphic conditions for the determination of active substances being present beside the products of their decomposition. 22 E. Kublin and T. Kaniewska

A B c

'b b

0 to: b II) a 0 to: a II) a 0 to: at 10 at 0 .~ iii fII) iii '"

Figure 1. Chromat~gram of bifonazole A- standard solution, B- extract obtained from Mycospor­ cream, C - solution of investigated compound; a)prenylamine lactate, b)..... bifonazole

Table 1. Statistical evaluation o{ the results ofthe determination ofthe compounds in substances by gas­ chromatographic, and spectrophotometric method , Method ,Number Average Variance Standard Confidence Variation of content for S2 deviation interval coefficient sam,ples all samples S X±ts % % pu=0.95 % Bifonazole

, Gas 10 99.69 0.263 0.513 99.69 ± 0.367 0.51 chromatography Spectrophotometry 7 100.58 0.314 0.560 loo.58± 0.518 0.56 Tioconazole Gas 10 100.43 0.969 0.984 100.43 ± 0.704 0.98 chromatography Spectrophotometry 7 100.81 0.257 0.507 100.81 ± 0.469 0.50 Econazole nitrate Gas 10 99.88 0.113 0;336 99.88 ± 0.240 0;34 chromatography Spectrophotometry 7 100.47 0.289 0.203 100.47 ± 0.497 0.54 Determination ofderivativesofimidazole 23

A B c

a

a b b a

,..., b .:;;

Figure 2. Chromatogram of tioconazole: A-standard solution, B -extract obtained from Gyno-Trosyd ointment, C- solution of investigated compound, a) prenylamine lactate, b) tidconazole

Table 2. Statistical evaluation of the results of the determination of acive compounds in pharmaceuticals by gas-chromatographic method

Pharmaceutical Number Average VarianceS2 Standard Confidence Variation of result for deviation interval coefficient samples all samples S X± ts % mg pu=0.95 mg

Mitekol-cream (econazole nitrate) 7 10.01 0.001 0.035 10.01 ± 0.032 0.35

Mycospor-cream (bifonazole) 8 10.21 0.010 0.098 10.21 ± 0.082 0.96

Gyno-trosyd-ointment (tioconazole) 8 65.23 0.031 0.177 65.23 ± 0.148 2.71 24 E. Kublin and T. Kaniewska

A B c

a • II GIl II) b cri I b I b 10 10 II) ~ CD CD .... II) CII') CO) cri .0 cri cO cO

Figure 3. Chromatogram of econazole nitrate: A- standard solution, B- extract obtained from Mite­ kol-cream, C -solution ofinvestigated compound, a) prenylamine lactate, b) econazole nitrate

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Received March 1995 AcceptedJuly 1995