Anal. Chem. 1987, 59, 174R-197R (476) Buckleton, J. S.; Axon, 8. W.; Walsh, K. A. J. Forensk Sci. Int. 1886, 32(3), 161. (477) Arora, B. B. Forensic Sci. Inf. 1986. 32(3), 185. (478) Walsh, K. A. J.: Axon, B. W.; Buckleton, J. S. Forensic Sci. Int. 1986, 32(3), 193. (479) Locke, J.; Underhill, N.; Russell, P.: Cox, P.; Perryman, A. C. Forensic Sci. Int. 1886, 32(4), 219. (480) Ulmanskv, M.: Foaelman. M.: Degany, - . I.: Bab, I. Forensic Sci. Int. . 1886, 32(4),.237. (481) Cook, R.: Wilson, C. Forensk Scl. I n f . 1986, 32(4), 267. (482) Jackson, 0.;Cook, R. Forensic Sci. Int. 1086. 32(4), 275. (483) Proceedings of a Forensic Science Symposium on the Analysis of Sexual Assault Evidence; US. Government Printing Office; Washington, DC, 1984. (484) Proceeding of the International Symposium on the Forensic Applications of Electrophoresis; U S . Government Printing Offlce: Washington, Dc, 1985. (485) Proceedings of the International Symposium of the Analysis and Identification of Polymers; US. Government Printing Offlce: washington, DC, 1985. (486) Di Maio, V. J. M. Gunshot Wounds. Practical Aspects of Firearms, Ballistics, and Forensic Techniques; Elsevier Science: New York, 1985.
(487) Fieher, 8. A. J.; Svensm, A.; Wendell, 0. Techniques of Crime Scene Investigation; 4th ed.; Eisevier Science: New York, 1986. (488) Davies, G.: Ed. Forensic Scisnce, 2nd ed : American Chemical Society: Washington, DC, 1986. (489) Barnett, G., Chiang, C. W., Eds. Pharmacokinetics and Pharmacodynamics of psvchoactive Owgs; Biomedlcal Pub.: Foster City, CA, 1985. (490) Moffat, A. C., Jackson, J. V., Moss, M. S., w i m p , B., Eds. “Clarke’s Isolation and Identflicatbn Drugs, 2nd ed.; Pharmaceutical Press: London, 1986. (491) Curry, A. S., Ed. Analytical Methods of Human Toxicology, Vertag Chemie: Weinhelm, Fed. Rep. Ger., 1985. Ed. Methodology of Analyticai Toxicology: CRC Press: (492) Sunshine, I., Boca Raton, FL, 1985. (493) Agureil, S., Dewey, W. L., Eds. CannabinoMs: Chemistry, Pharmacology, and Thereupefic Aspects; Academic: Orlando. FL, 1984. (494) Harvey, D. J., Ed. Marihuana; 84 (Eighty-four). Procedures Oxford Symposum on Cannabis, 7985: IRL: Oxford, U. K., 1985. (495) Munster, B. E.,Henningsen, K., E&. Advances in Forensic Haemogenetics, 7 ; Springer-Verlag: berlin, 1986. (496) Lee, H. C., Gaensslen, R. E., Eds.; Advances in Forensic Science, Volume One-Forensic Seroiogy ; PSG Publishing Co.; Littleton, MA, 1986
Pharmaceuticals and Related Drugs R. K. Gilpin* Department of Chemistry, Kent State University, Kent, Ohio 44242
L. A. Pachla
Warner-LambertlParke-Davis, Pharmaceutical Research, PharmacokineticslDrug Metabolism, Ann Arbor, Michigan 48105
The current review is a survey of pharmaceutical analysis and related methodolo y appearing in Analytical Abstracts or Chemical Abstractsktween July 1984 and October 1986. The article deals exclusively with compounds in unformulated and dosage form and does not cover biochemical and clinical aspects of the subject. Since it is only possible to cite a representative sampling of the total work published, emphasis is placed on references that appear to be significant and are more easily accessible. In an attempt to make the review more inclusive, we have included additional citations in the Nitrogen- and Oxygen-ContainingCompounds section. However, not all of these a p ear in the text. Those not specifically discussed are listelin Table I. The review is divided into 10 major sections: General, Alkaloids, Antibiotics, Inorganics, Nitrogen- and OxygenContaining Compounds, Steroids, Sulfur-Containing Compounds, Vitamins, Techniques, and Miscellaneous. Most of the major sections are divided into subsections. As in the past, because of space limitations and the desire to list as many citations as possible, a reference generally appears only in a single section.
GENERAL Books and reviews of a comprehensive nature published were: “EnantioeelectiveDrug Analysis” (9),“Pharmaceuticals and Related Drugs” (22), “Methods for Analysis of Drugs” (38), “Drugs and the Pharmaceutical Sciences, Vol 11: Pharmaceutical Analysis: Modern Methods, Pt. B” (411, and “Analysis and Control of Protein and Polypeptide Drugs” (55). The book “Drug Analysis by Gas Chromatogra hy” (31) and a review of various chromatogra hic proce&res applied to pharmaceutical formulations ago appeared (14). Specific aspects of performance characteristics related to gas chromatographic methods (26),capillary GC column selection (5), and pharmaceutical application of gas chromatography-mass spectrometry (8) were discussed. Additionally, the use of combined HPLC-MS to determine drugs and hormones was reviewed (56). 174 R
0003-2700/87/0359- 174R106.5010
As in the past, the most widely employed technique in pharmaceutical analysis was liquid chromatography. A major review with over 850 references considered the separation of various pharmacologically active materials (18). General advances in HPLC technology (4,13)as well as specific aspects of high-speed analysis (21) and the precision of various HPLC methods (28) were discussed. The utilization of chiral stationary phases (58) and correlations between reversed-phase retention data and solute hydrophobicity (23) and compound activity (33) also were considered by several investigators. The use of secondary mobile phase modifiers continues to be an important topic. A book (24) and an extensive review (1) devoted to the theory and applications of ion-pair methodology were published. Similarly, ion-pairing techniques were used to enhance detection (11). A more or less generalized scheme for determining 49 drugs from several different therapeutic classes was developed (60). Several halogen-free reversed-phase TLC systems were compared with equivalent liquid chromatographic systems (17). Based on the identity and purity testing of 28 drugs, TLC was reported to have higher separating power. A book on “Plant Drug Analysis: A Thin Layer Chromatography Atlas” was published (57) within the current period. Likewise, a review of the application of thin-layer chromatography for drug identification, stability and purity testing, and the determination of drugs in formulations was written (19). A variety of techniques used to obtain quantitative information from TLC were evaluated (39). Coated glass tubes were used in place of conventional plates to identify vitamins, alkaloids, and other types of compounds (34). Principal component analysis was applied to RF values of nearly 600 basic and neutral pharmaceutically active compounds as an aid in their identification (42, 43). A number of other pharmaceutical ap lications of TLC methods were discussed (44,53). geveral papers dealing with electrochemical techniques in pharmaceutical analysis were published (3, 7,12,25,32). Both theoretical and applied aspects of amperometry, conductometry, ion-selective electrodes, potentiometry, voltammetry, 0 1987 American Chemical Society
PHARMACEUTICALS AND RELATED DRUGS
m- h
Ropn K. cvpk b R o l e ~ o and r Qahnan 01 tha mraem of Chamkby at Kent State Wers(ty. Ha -r h k B.S. db
graa In chemistry lrom Indlana Stale U n m h 1969 and Ms Ph.D. dagree In SnalVacal r n k l l y hm tha unlverany 01 A d z m h 1973. From 1973 to 1975 he was employed as Senior Scientist and hom 1975 10 1978 as 13mp Leader 01 Analytical Chemistry in the Resaarch Dlvision of McNsll LabOTatabs. In 1978 ET. GHph wried me lawny of Kent Slate Universny. BBtween 1981 and 1983 ha also w e e as B Senior Technical Advlsor la IBM Inshumants. His rereardl interests are in I"&&&I and applied gas. liquid. and thin-layer chromatography: ChmmatD 01 chsmicaih modified surlaces: and graphk. ESR. IR, and N M SWS phamceutical analysis. He has numerous publications in lha areas 01 ag a m t a l l i c surface rea&ns: synlhasis 01 aganosiisne and labeled m a g ems: pharmacwkal analysis: lha development of chemically modified surlaces l a TLC. Gc.and HFIC physimmemical studies by chromatographic techniques: electron spin reSOnance and inhared investigalions 01 lmmobillzed liganw and metal and '% NMR. Dr. alpin has cfganired or been Inv0t-j in several Symposia and shwt COVSBS in lha area of chromatographic analysis. Likewise. he has haid OWICBS in bcal chromatography and ACS groups. He is also a member of me Amerbn Chemical Society. Society of Applied Spectroscopy. American Associatl~nlor the Advancement 01 ScC mca. and Sama Xi. and lha Edilorial A d v I S w Board 01 lha Journal Of Chro-
socials (1983-1984), and Senior Ramarch Aasonate (1888-1987) and has lectured a1 Lawenca Insfflute 01 Technology. Hk responsibilHies a1 WLW include Ureenng all snalytlcal. pharmacokinetk. and drug metabolism s w e s lor candidates emanamg born t w h a p % l l c a m s and also charman 01 a drug development team lor an allergy Clinical candidate. His research interests lie in lha areas 01 robotks. eiectroanalytical chemism. liquld chromaand drug metabolism. He has published mwe tography. pharma~oklnelk~. man 30 manuscripts and is a mambar 01 tha American Chemical Soclety. Academy 01 PharmaceuHcal Sciences. American A ~ i e 1 I 0 n01 PharmaUcal ScIBnc~s. and Associalbn 01 OWklal Analytical Chemkts. He IS also On lha EdHorial Board 01 Antimicrobial Agents and ChammSrapy.
and coulometry were considered in one or more of these references. Other citstions of a general nature include an overview of the use of membrane electrodes to analyze pharmacologidy active compounds (10)and the construction (45) and application of (46)*graphite spray electrodes". Other reviews and papers which dealt with particular techniques were concerned with atomic (2,52), UV/visible (51) and multichannel (20) spectroscopy, mass spectrometry (6, 30), flow micro-fluorometry (541, flow-injection analysis (37.48). thermal methods (591,nonaqueous titrations i49J, and automation (15, 27, 47). Reports of a topical nature covered functional group (40)and purity (50) analy&. the u$.e charge transfer complexes in pharmaceutical methods (36), and identity testing (05). Evaluation of method performance was also considered (16. 29).
ALKALOIDS General. Activity in the alkaloid section remains nearly the same as it has in past reviews. The opium alkaloids continue to receive the most attention and chromatography
in the most often used technique for their analysis. A review and a text have appeared which discuss the utilization of multiple-stage mass spectrometry (6A) and gar and liquid chromatography (7A) for the qualitative and quantitative
analysis of alkaloids. GC (3A) and HPLC ( I A , 5 A ) as well as coated-wire electrodes (8A) have been used to determine several different classes of alkaloids. In terms of other techniques an indirect atomic absorption procedure (4A) has been developed for a variety of alkaloida and other classes of compounds. The ion-pair complex which forms between the compound of interest and Co(SCN),*- was extracted into 1,Z-dichloroethaneand measured a t 241 nm. Lastly, circular dichroism spectra have been used to distinguish quinine from quinidine, cinchonine from cinchonidine, hyoscyamine from atropine, and pilocarpine from isopilocarpine @A). Chinchona. Mostly, HPLC has been used to analyze cinchona alkaloids. For example, separations have been obtained on octyl(17A), octadecyl(14A, 18A),and phenyl (16A) columns. An interlaboratory comparison among nine facilities was carried out in an effort to evaluate C-18 columns produced by different manufacturers (13A). Difficulties in establishing standard conditions were discussed. Several other techniques have been utilized to analyze cinchona alkaloids and include the voltammetric determination of colchicine using a rotating disk electrode ( I I A ) and the micro-AA determination of cinchonine, cinchonidine,quinidine, and quinine using KzHgI, (IOA). Membrane electrodes have been developed for both quinidine (IZA) and quinine (SA). Quinine also has been determined a t the picogram level via a radioimmunoassay procedure (15A). The method was highly specific for compounds with 8s configuration. Ergot. Ergot alkaloids have been separated on both LiChrosorb (2lA) and Spherisorb (19A) C-18 columns. In the first instance dihydroergotoxine could be resolved from its hydrolysis and epimerization products. The latter procedure was used to separate four alkaloids in 'Co-dergocrine" tablets, injections, and oral solutions. A normal-phase procedure for ergot alkaloids also has been described with detection limits in the 5-25 ng/pL range (2OA). Opium. During the review period a number of HPLC methods have appeared for codeine (47A) and codeine in combination with other alkaloids such as morphine, papaverine, thebaine, and noscapine (32A, 38A, 50A, 5ZA). In one case on-line coulometric detection was used (38A). Liquid chromatography also has been used to simultaneously determine codeine and acetaminophen in stability samples (&A). Other chromatographicprocedures for codeine samples include the TLC determination of methyldeine in unformulated and formulated samples of codeine (29A) and a combination method involving an initial open column separation followed by gas chromatography to quantitate codeine, noscapine, papaverine, and thebaine (49A). Other methods involving voltammetric, spectropolarimetric, and colorimetric measurements have, respectively, been used to measure codeine in the presence of dihydrocodeine (27A). cocaine (46A),and emetine and pilocarpine (22A). Reversed-phase conditions have been used to simultaneously assay dihydrocodeine, caffeine, aspirin, and promethazine (56A). Each sample could be analyzed in 7 min using a linear gradient. Several gas (%A, 28A, 45A) and liquid (31A, 40A) chromatographic procedures have been reported for diamorphine in illicit preparations. These have been used to determine traces of other alkaloids present (31A, 4OA, 45A) and to study diamorphine samples from different countries (24A). It has been noted that transacetylation occurring in the GC injection port between diamorphine and morphine can produce false results (28A). UV spectrometry also has been used to determine diamorphine (41A). The second-derivative spectra gave improved results. The benzoate complex of 3-0acetylmorphine in illicit diamorphine was determined as the base following treatment with K2C03(52A). Morphine has been quantitated singularly or in combination. In the former case, three of the liquid chromatographic procedures reported for morphine use UV (34A). fluorescence (MA), and electrochemical (39A) methods of detection. Linear electrochemical responses were obtained from 0.1 ng to 1 fig. Structural influences on oxidative amperometric detection of various opiate alkaloids were investigated (55A). Reversedphase procedures have been developed for morphine and possible contaminants in injectables (36A, 42A). Samples containing morphine also have been assayed by spectrophotometric methods and by oxidative and complexometric titration procedures using thiocyanatmhromium(II1) complexes (33A). Rectilinear calibrations in the pg to mg range were ANALYTICAL CHEMISTRY, VOL. 59, NO. 12, JUNE 15. 1987 -175R
PHARMACEUTICALS AND RELATED DRUGS
reported. An extremely sensitive (0.01-0.2 ng) radioimmunoassay method has been used to quantitate morphine in poppy capsules (35A). Binary combinations of morphine and cocaine in the presence of fructose-based syrups have been assayed by spectropolarimetry (25A). This same procedure was used for “Bromption’s cocktails” also prepared from methadone. During the current review period a variety of other papers dealing with opiates were published. General aspects of the MS-MS of the trifluoroacetyl derivatives of 11 bases (57A) and the use of alumina as a HPLC packing for the separation of various opium compounds (26A) were considered. Papaverine was determined by colorimetric methods (37A)and by capillary isotachophoresis (53A). Reversed-phase HPLC was used to measure naloxone in injections (54A) and to study the reaction of acetic anhydride with noscapine, thebaine, and several other opium alkaloids (43A). Additionally, thebaine was determined by colorimetric methods after treatment with HCl and NaNOz (30A) and by IR and TLC-UV methods (23A). In the latter citation both methods were reported to be superior to the K-34(U.N.) procedure. Rauwolfia. Reserpine has been quantitated in various pharmaceutical formulations colorimetrically as the U(V1) complex (59A)as well as by liquid chromatography (58A)and gas chromatography following derivatization (61A, 62A). Fluorometric detection a t 360 nm was used for the HPLC assays. Ajmaline, its chloroacetyl derivative, and pindolol, the internal standard, were separated under reversed-phase conditions (60A). Detection limits of 5 and 10 ng were reported for 244 and 287 nm, respectively. Tropane. Atropine, hyoscyamine, and scopolamine have been the tropane alkaloids studied most often. A variety of chromatographic, spectrometric, and electrochemical methods have been described. Liquid-liquid and poly(viny1 chloride) membrane atropine selective electrodes have been prepared from the compound’s reineckate ion-pair complex (67A). Both electrodes gave results in good agreement with U.S.P. and B.P. methods. A biological method for atropine has been reported which is based on changes in the heart rate of rats (72A). Reversed-phase chromatography in combination with fluorometric detection has been used to analyze commercial preparations containing atropine (63A). Similarly, R P columns have been used to resolve atropine from acidic and basic degradation products (68A)and hyoscyamine and scopolamine fro natural preparations (64A, 73A). Other liquid chromazgraphic procedures have utilized reversed-phase conditions to separate atropine from tropic acid (71A) and from hyoscyamine and scopolamine (69A). Likewise, atropine and scopolamine from plant extracts have been separated under normal-phase conditions (70A). A chiral stationary phase has been used to study several amides and ester derivatives of tropic acid (75A). Tropicamide and related amide derivatives were resolved but atropine and ester derivatives were not. Several tropane derivatives have been quantitated by using a second derivative UV method (66A). Spectra were acquired from the extracted tetraphenylborate salts. Raman spectra have been compiled on crystalline and aqueous solutions of cocaine (65A)for use in toxicological investigations. Similarly, color reactions have been used for identification of cocaine and other benzoyl compounds (74A). Vinca. High-performance liquid chromatography has been used to determine visblastine and vincristine in formulated products (78A). It also has been used for vincristine, vindoline, and catharanthine in extracts of Catharanthus roseus (76A). The oxidation of these same three compounds has been studied by several voltammetric techniques (77A). The mechanisms for electron transfer were suggested. Xanthine. Caffeine has been determined by reversed-phase chromatography (79A, 80A), using a liquid membrane electrode prepared from the picrylsulfonate ion pair complex (81A),and by colorimetry after treatment with KBr03 and HCl (82A). There have been a number of other papers published which deal with the analysis of caffeine. In many cases caffeine has been determined in combination with other classes of compounds. The reader thus is referred to other sections in this review for additional citations. A gas chromatographic procedure has been reported for theophylline and theobromine (83A). Separation was carried out on a 3% OV-17 column following methylation of the alkaloids with trimethylanilinium hydroxide. Several methods 176R
ANALYTICAL CHEMISTRY, VOL. 59,
NO. 12, JUNE 15, 1987
have been used in the identification and quantitation of 3,7-dimethyl-6-(5-oxohexoxy)-W-purin-2-one, an impurity in pentoxifylline (84A). Miscellaneous. One hundred Tabernaemontana alkaloids have been separated by thin-layer chromatography and subsequently identified by using several different visualization techniques (103A). Of the chromogenic reagents employed, ferric chloride was the most effective. Radial TLC has been used to separate protoberberine alkaloids (85A). Berbeine (90A) and cytisine-containing mixtures (106A) have been assayed colorimetrically, and nicergoline (89A)and emetine and cephaeline (93A) have been determined by HPLC. Additionally, spectrofluorometry has been used to simultaneously assay emetine and cephaeline in ipecacuanha preparations (88A). For most samples there was acceptable agreement between results obtained from the cited procedure and the British Pharmacopoeia method. Pilocarpine has been determined in ophthalmic solutions by both liquid chromatographic (94A, 102A) and colorimetric (99A)procedures. One of these methods (102A)was adopted as the official first action for determination of pilocarpine in the presence of isopilocarpine and pilocarpic acid. Although the levels of pilocarpine and physostigmine were unchanged following sterilization, both decreased after 2 months of storage (94A). Other miscellaneous alkaloids studied have been cinchocaine (100A) and brucine and strychnine (91A). In a more general sense, both normal-phase and reversed-phase conditions have been used to resolve Strychnos nux vomica alkaloids (104A). Other classes of compounds studied were carbazole (86A,87A), ephedra (92A,%A), indole (97A),and pyrrolizidine (%A, 98A, 101A) alkaloids. In one instance proton nuclear magnetic resonance spectrometry was used (96A) and in another case isotachophoresis (92A). Isotachophoresis also has been utilized to determine saguinarine and chelerythrine in fractions of Chelidonium majka (105A).
ANTIBIOTICS General. This major section includes drugs that are derived from both natural and synthetic sources. In the past many synthetic compounds may have been included in other sections. These compounds can now be found within this major classification. This section discusses methods for antibacterials, antiinfectives, antifungals, antiparisitics, and antimicrobials. Anticancer drugs will only be included if they were originally discovered in fermentation broths and thought to have antibiotic activity (i.e., mitomycins). As in the past, chromatographic techniques continue to be popular for the analysis of antibiotics. A book has appeared entitled Chromatography of the Antibiotics (9B). Liquid chromatographic methods for the determination of several compounds involved in the biosynthesis of penicillins and cephalosporins (8B),for t,he determination of 17 antibiotics admixed with intravenous infusates ( I B ) ,and for the early characterization of aminoglycosides (SB) have been reported. A review describing the use of mass spectrometry in antibiotic research has appeared (3B). This monograph reviews the literature from 1977 to 1984, contains 197 references, and covers several antibiotic classes. The application of infrared spectrometry in pharmaceutical analysis has also been reviewed (6B). Other general papers include the development of microbiological assays based on the inhibition of ammonia production and employing an ammonia electrode (7B),the flow injection analysis of ,&lactams using an enzyme thermistor detector (2B), and the spectrophotometric estimation of tetracyclines, streptomycins, and chloramphenicol using brucine and sodium metaperiodate (4B). Cephalosporins. A variety of spectrophotometric methods have been described for the general determination of cephalosporins. A visible spectrophotometric method for cephalosporins and penicillins after derivatization with indophenol was described (17B). Colorimetric assays for five cephalosporins were described that involved reacting the drug with acidic ammonium molybdate a t 100 OC and monitoring the absorbance between 684 and 708 nm (IO& 21B).The methods were applicable to both pure drug and pharmaceutical preparations. A rapid method applicable for cephalosporins has been introduced which utilizes an immobilized enzyme reactor and sequential subtractive UV spectrophotometric detection and automated flow injection system (12B).A differential
PHARMACEUTICALS AND RELATED DRUGS
pulse polarographic method was proposed for the sensitive determination of four cephalos rins in pharmaceutical formulations (23B). Two reverse -phase LC procedures applicable to a variety of cephalosporins have been published (%B, 26B). The first method investigated the selectivity of C8 and Clp stationary phases and concluded that no relationship emted between structure and elution order (%B). The second method utilized microbore column technology to determine submicrogram quantities of drug in fermentation broths (25B). A TLC method for cephradine, cephalexin, and cefadroxil in tablets, powders, and capsules that utilizes fluorescamine as the fluorescent visualization/quantification reagent has been proposed (14B). The detection limit was 2 ng which offered a 10- to 20-fold improvement over spectrophotometric or fluorescence detection with o-phthalaldehyde. The selective separation of cephalosporin C has been achieved by use of substituted polystyrene resins (22B). This report evaluated ten ligands enabling the separation of this drug from other fermentation components. The best reproducible separation was achieved with a 1-lysine resin with isocratic elution with 1M NaC1. FDA standards for LC and TLC certification of sterile cefuroxime sodium have been outlined (15B).In addition an LC method for determining cephradine potency and cephalexin content has been recommended (16B). Liquid chromatography has been reported to be a viable technique for process scaleup purification of cefonicid disodium (IIB). A novel electroanalytical procedure has been examined for the determination of cephalexin (18B). Although cephalexin is electroinactive, complexation with Ni(I1) yields a catalytic reversible reduction quantifiable prepeak in differential pulse polarography. Second derivative spectrophotometry was the basis of a method for quantifying cephalexin, cephalothin, and cephradine in the presence of impurities (13B). A rapid microbiolo ical pH assay was proposed for the determination of cep radine in pharmaceutical formulations (19B). A spectrophotometric method utilizing ninhydrin has been reported for the determination of cephradine, cefoxitin, and cephalothin in injectables (20B). An electrochemical method based on cathode ray or differential pulse polarography has been shown to be applicable for the measurement of cephalothin, cephacetrile, cefamandole, and cefoperazone in dosage forms (24B). These authors concluded that the electroactive properties of these compounds were due to the R leaving group located at the 3-position. Cyclosporins. A report has investigated the mechanism of peak broadening of cyclosporin A on an octadecyl stationary phase (27B). A rapid method for quantifying cyclosporins A, B, C, and D in fermentation media was achieved by using a C8 column (28B). Although low theoretical plate counts were obtained, the method was capable of quantifying these antibiotics in 170 samples overnight. Sample preparation consisted of methanol dilution and filtration, followed by detection at 214 nm. The analysis time was 3.5 min and coefficients of variation ranged between 0.8 and 1.5%. Chloramphenicol a n d Isoniazid. A new colorimetric method utilizing a ninhydrin-SnC12 reagent was assessed for determining chloramphenicol in solid and liquid formulations (29B). Several reports have been published characterizing the color reaction for chloramphenicol outlined in the European Pharmacopeia (31B-33B). These reports conclude (a) azoxy derivatives are formed in addition to the hydroxylamines (31B),(b) various 0-benzoylhydroxamic acids are produced if sodium acetate was added to the reaction mixture prior to the benzoylation step (32B),and (c) formation of side products could be minimized if excess benzoyl chloride and no sodium acetate were added to the reaction medium ( B E ) . Differential scanning calorimetry was useful for determining the concentration of enantiomers of chloramphenicol (30B) and a simultaneous polarographic assay for chloramphenicol and nitrofurantoin in pharmaceutical preparations was discussed (34B). A titrimetric method using Mn(1V) for isoniazid determinations has appeared (35B). Penicillins. Two monographs for penicillins have been described in the ref 49B and 50B. A revision of the antibiotic regulations has been issued regarding analysis of ampicillin and amoxycillin (49B). Since iodine is bound to both drugs in solution, the addition of three drops of 1.2 N HC1 to the sample after addition of iodine was recommended in the amended guidelines. The second monograph gives certification requirements for potassium clavulanate and for tablets
8"
K
and oral suspension containing amoxycillin (50B). A variety of LC methodology for penicillin analysis has been reported during this review period. Reversed-phase chromatography on a Chromegabond C18column was the basis of a sensitive and reproducible quantitative method for nine penicillins in commerical dosage forms (42B). Another reversed-phase method relied upon derivatizing several penicillins with acidic imidazole HgC12 followed by separation of the resulting penicillenic acids on a Spherisorb C18 column (60B). Calibration curves were linear up to 50 pg/mL for penicillins K and X, benzylpenicillin, adicillin, methicillin, and phenoxymethylpenicillin in fermentation broths. These authors extended their application to penicillins with a C6 side chain and investigated the use of salts of Ni, Zn, Ag, Cd, Au, and Hg in order to obtain a sensitive method (61B). Separation on a Nucleosil C18 stationary phase formed the basis of a method for quantifying amoxycillin, ampicillin, oxacillin, phenoxymethylpenicillin, benzylpenicillin, flucloxacillin, propicillin, and cloxacillin in the presence of coformulated antibiotics (e. streptomycin) (36B). Liquid chromatography with photofhic degradation and electroanalytical detection has been able to achieve detection limits of 6 , 6 , 8 , and 8 ng for ampicillin, benzylpenicillin, phenoxypenicillin, and cefoperazone, respectively (64B).This unique technology involved reversed-phase separation of the drugs followed by postcolumn photolysis and detection of the photolytic products by amperometry. A TLC identification scheme has been described for 18 penicillins (53B). The chromatographic characteristics of these compounds were elucidated on silica gel and silanized silica gel using 35 mobile phases. Only silanized silica gel proved useful in separating a particular penicillin from all others using an appropriate mobile phase. A rapid, precise, and selective TLC method utilizing spectrodensitometry has been introduced for the determination of several derivatives of penicillin in dosage forms (37B). Enzymatic detection of 0-lactams was the basis of two methods (41B, 52B). Benzylpenicillin and other &lactams were determined in pharmaceutical products via the inactivation of carboxype tidase enzyme, EC 3.4.17.8 (41B). The basis of this methofwas simple. In the absence of the test drug, carboxypeptidase releases D-alanine from the substrate which can act as a substrate for amino acid oxidase, releasing Hz02and pyruvic acid. In the presence of H 2 0 the colorless chromogen was oxidized to a colored compounc! that absorbs a t 513 nm. The enzyme was inhibited by the test penicillin and thus a decrease in absorbance is proportional to concentration. Flow injection analysis of penicillins after incubation with immobilized penicillinase was the basis of a method for benzylpenicillin tablets and injectables and for phenoxymethylpenicillin in fermentation broths (52B). Other general reports include investigating the polarographic behavior of several penicillins after bromometric oxidation (59B) and the determination of water in penicillins using fast Karl Fischer reagents and electronic end-point optimization (57B). A report appeared describing two colorimetric methods for the measurement of amoxycillin in commercial preparations (39B). The first colorimetric assay was based on condensing amoxycillin with 4-aminoantipyrine in the presence of an alkaline oxidizing reagent. The method was linear from 5 to 60 pg/mL and had a precision of 1.3%. The alternative method involved the reduction of Folin reagent. Maximum color development occurred at 20 min and was stable in excess of 6 h. Both procedures gave comparable results to the British Pharmacopeia method. Two liquid chromatographic methods were examined for quantifymg amoxycillin and its degradation products (47B,51B). Both procedures gave acceptable resolutions and were based on reversed-phase chromatography with gradient elution. A stability-indicating method was published for acid-stable penicillins (e.g., amoxycillin, ampicillin, and penicillin V) (48B).The method consisted of alkaline hydrolysis, addition of excess KIOB,and titration with Na2S203. Recoveries were greater than 99% and method specificity was confirmed with TLC. A specific ion pairing LC method was introduced for ampicillin analysis in bulks, injectables, capsules, and oral suspensions (56B). This was proposed as a stability-indicating method since it was specific for ampicillin in the presence of phenoxymethylpenicillin, phenylglycine, 6-aminopenicillanic acid, penicilloic acid, and the analogous acid derived from ampicillin. ANALYTICAL CHEMISTRY, VOL. 59, NO. 12, JUNE 15, 1987
177R
PHARMACEUTICALS AND RELATED DRUGS
Reversed-phase ion pairing chromatography was investigated for quantifying controlled-release penicillin complexes of benzathine-cloxacillin (1:2), benzathine-benzylpenicillin (1:1),benzathinepenicillin V (1:2), procaine-benzylpenicillin (l:l),and benethamine-benzylpenicillin (1:l) (55B).The importance of controlling the pH of the mobile phase was examined for optimal separation of the drugs from their degradation products. The reports also discuss method applicability to stability evaluations. Classical column chromatography on Sephadex G-25 was useful for the determination/identification of polymer degradation products formed from benzylpenicillin solutions that were stored in the dark (67B). A sensitive fluorometric-enzymatic assay was introduced for benzylpenicillin (38B). Sample preparation consisted of incubation with penicillin acylase and the liberated 6-aminopenicillanic acid derivatized with fluorescamine and quantified fluorometricdy with excitation at 390 nm. Proton NMR was examined for quantifying cloxacillin in capsules (54B) and a pBondapak Phenyl column was recommended for quantifying mezlocillin in stability studies (46B). Two reports have appeared for ascertaining the enantiomeric purity of D-peniCillamine (43B, 44B). The first paper relied on converting the drug to 5,5-dimethylthiazolidine-4carboxylic acid followed by treatment with aqueous formaldehyde (43B). Separation was achieved on a LiChrosorb RP-8 column coated with the Cu(I1) complex of (2S,4R,2’RS)-4-hydroxy-l-(2-hydroxydodecyl)proline with
