[9] Gas-liquid chromatography of antibiotics

[9l

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

213

p e n s e r ( B r i n k m a n ) f i t t e d w i t h a 0.1-ml H a m i l t o n s y r i n g e t h a t d e l i v e r s 0.002 m l of s o l u t i o n ; t h e T L C p l a t e w a s p l a c e d 1 m m b e l o w t h e syringe. T h i s p r o c e d u r e p r o v i d e d s p o t s of u n i f o r m size. A s t a n d a r d d e v e l o p i n g t a n k , l i n e d w i t h filter p a p e r , was used for c h r o m a t o g r a p h y , a n d t h e m o b i l e p h a s e w a s e t h e r : a c e t o n e ( 3 : 2 ) . I n t h i s s y s t e m , t h e R I of griseof u l v i n was 0.62 a n d t h a t of g r i s e o f u l v i n - 4 ' - a l c o h o l was 0.50. T h e d r i e d p l a t e s were s c a n n e d a f t e r 12 h r a n d d e t e r m i n a t i o n s were m a d e in duplicate. Q u a n t i t a t i v e e s t i m a t i o n s of t h e a n t i b i o t i c a n d its m e t a b o l i t e were m a d e b y c a l c u l a t i n g t h e a r e a s u n d e r t h e curves p r o d u c e d b y r e c o r d e d s c a n n i n g of t h e fluorescent spots. T h e e s t i m a t e s a g r e e d well w i t h a s s a y s of s i m i l a r l y s p o t t e d T L C p l a t e s t h a t h a d n o t been c h r o m a t o g r a p h e d . T h e p h a r m a c o k i n e t i c conclusion of t h i s s t u d y was t h a t g r i s e o f u l v i n - 4 ' - a l c o h o l , i n a c t i v e in vitro, is c o n v e r t e d r a p i d l y in vivo to b i o l o g i c a l l y a c t i v e griseofulvin.

[9] Gas-Liquid Chromatography of Antibiotics By KIYOSHI T s u J i a n d JOHN H . ROBERTSON I. Introduction . . . . . . . . . . . . . . . . . II. Amino-Cyclitol Antibiotic . . . . . . . . . . . . . Spectinomycin . . . . . . . . . . . . . . . . III. Amino-Glycoside Antibiotics . . . . . . . . . . . . A. Gentamicin . . . . . . . . . . . . . . . . B. Kanamycin . . . . . . . . . . . . . . . . C. Lividomycin . . . . . . . . . . . . . . . . D. Neomycin . . . . . . . . . . . . . . . . . E. Paromomycin . . . . . . . . . . . . . . . IV. Aromatic Antibiotics . . . . . . . . . . . . . . A. Chloramphenicol . . . . . . . . . . . . . . . B. Griseofulvin . . . . . . . . . . . . . . . . V. Glutarimide Antibiotic . . . . . . . . . . . . . . Cycloheximide . . . . . . . . . . . . . . . . VI. Lincomycin-Clindamycin Family . . . . . . . . . . . A. Lincomycin . . . . . . . . . . . . . . . . B. Clindamycin . . . . . . . . . . . . . . . . C. Clindamycin Phosphate . . . . . . . . . . . . . D. Clindamycin Palmitate . . . . . . . . . . . . . VII. Macrolide Antibiotic . . . . . . . . . . . . . . Erythromycin . . . . . . . . . . . . . . . . VIII. Penicillin . . . . . . . . . . . . . . . . . . IX. Tetracycline . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . .

214 215 215 217 217 218 220 220 228 228 228 232 234 234 237 237 239 242 243 245 245 248 251

214

METHODS FOR THE STUDY OF ANTIBIOTICS

X. Miscellaneous . . . . . . . . . . . . . . . . . A. Peptolide Antibiotic--Antimycin A . . . . . . . . . . B. Celestosaminide . . . . . . . . . . . . . . . . C. Phosphonomycin . . . . . . . . . . . . . . . . D. Thiamphenicol . . . . . . . . . . . . . . . . E. Validamycin . . . . . . . . . . . . . . . . . F. Summary of GLC Methods for Antibiotic Analysis . . . . .

[9]

253 253 253 254 254 254 255

I. Introduction Antibiotics in general are complex organic molecules, whose commercial preparations usually contain various amounts of isomers, biosynthetic intermediates, and degradation products, each of which m a y possess a different antimicrobial spectrum from its parent compound. I-4 Microbiological methods of analysis,5 the officiallyaccepted method of analysis for the majority of antibiotics,measure the total antimicrobial activity of the various components against a specificmicroorganism. Since the antimicrobial activity of one antibiotic varies from microbial species to species,~,~ the microbiological responses to a given antibiotic compound are not always constant and frequently are influenced by chemical and physical factors.7 During the past decade, gas-liquid chromatography (GLC) has become one of the most important analytical tools for the investigation of various organic compounds, particularly after the development of numerous trimethylsilyl (TMS) reagents to derivatize a compound to give added volatility and thermostability. Several G L C methods have been developed to separate and quantitate various isomers, process intermediates, and degradation products of antibiotics.Drug potency, as calculated from G L C data, generally agrees well with values obtained by microbiological assay methods. Only a handful of the G L C methods have received approval of the Food and Drug Administration as an alternate assay method ~ (lincomycin, clindamycin, spectinomycin, and neomycin) ; however, the listwill certainly increase in the near future. This paper describes the G L C methods for chromatography of intact 1K. Tsuji, J. H. Robertson, R. Bass, and D. J. McInnis, Appl. Microbiol. 18, 396 (1969). K. Tsuji and J. H. Robertson, Anal. Chem. 43, 818 (1971). 8 O. K. Sebek, J. Bacteriol. 75, 199 (1958). ' A. F. Zak, T. I. Navolotskays, G. I. Loseva, N. I. Shukailo, O. B. Ermolova, and L. M. Yacobson, Antibiotiki (Moscow) 18, 324 (1973). Code of Federal Regulations, Title 21, Food and Drugs U.S. Government Printing Office, Washington, D.C., 1974. W. A. Freyburger and L. E. Johnson, Antibiot. Chemother. 6, 586 (1956). W. T. Sokolski, C. G. Chidester, and D. G. Kaiser, J. Pharm. Sc/. 53, 726 (1964).

[0]

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

215

antibiotic molecules, many by derivatization, and discusses some of the difficulties associated with quantitative derivatization and chromatography and describes precautionary measures for the successful GLC determination of antibiotics. 11. Amino-Cyclitol Antibiotic Spectinomycin 5,s

Spectinomycin is chromatographed intact as the tetrakistrimethylsilyl derivative using an SE-52 column.

Procedure Materials SE-52, 5%, on Diatoport S, 80-100 mesh Column, glass, 3 mm i.d. X 610 mm (2 ft) Vial, glass, 1- or 5-dram size with polyethylene stopper Dimethylformamide (DMF), dry Hexamethyldisilazane (HMDS) Triphenyl antimony Benzyl alcohol Acetone

Solutions Internal standard solution. Prepare a 2 mg/ml solution of triphenylantimony in dry DMF. Apparatus Gas chromatograph: For requirements see the neomycin section. Detector: Flame-ionization detector

Chromatographic Conditions Column: glass, 3 mm i.d. X 610 ram, packed with 5% SE-52 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 190 ° Detector temperature: 220 ° Flash heater: 200 ° Chart speed: 0.64 cm/min Sample size: 1 ~l at attentuation and range setting of 32 X 10 8 L. W. Brown and P. B. Bowman, J. Chromatog. Sci. 12, 373 (1974).

216

METHODS FOR THE STUDY OF ANTIBIOTICS

[9]

Preparation o] Chromatographic Column Follow the procedure described in the neomycin section. Procedures for the conditioning of the column are listed in the erythromycin section.

Preparation o] Bulk Drug and Re]erence Standard Powder Accurately weigh approximately 50 mg of. spectinomycin bulk powder and spectinomycin dihydrochloride pentahydrate reference standard into separate 5-dram vials.

Sample Preparation Sterile Product. Reconstitute a vial with the labeled volume of sterile water for injection with benzyl alcohol and mix. Withdraw 5.0 ml of the suspension, transfer quantitatively to a 50-ml volumetric flask, dissolve, and dilute to volume with deionized water. Transfer 1.0 ml of this solution to a 5-dram vial. Add 15 ml of acetone and evaporate to dryness with a gentle stream of nitrogen. Silylation Procedure Add 10.0 ml of the internal standard solution and 1.0 ml of H M D S to each vial containing sample and reference standard. Intermittently swirl the vials gently to mix the liquid for 1 hr at room temperature. Centrifuge if necessary and chromatograph.

Calculation Calculate the content of spectinomycin base in micrograms per milligram sample using Eq. (1) described in the kanamycin section.

Comments on the Assay Method Silylation of spectinomycin produces several GLC peaks depending on the silylation conditions and reagents. With HMDS, four TMS groups are added to the molecule while with BSA, a fifth group is added. After silylation, the sample must be chromatographed within approximately 2 hr since the silylated samples are not stable. A decrease in peak area of approximately 10% for the spectinomycin GLC peak is observed after a 12-hr period. The degradation product, actinamine, has a shorter retention time than the internal standard while actinospectinoic acid has a longer reten-

[91

GAS-LIQUID

CHROMATOGRAPHY

OF ANTIBIOTICS

I

I

[

I

0

4

8

12

217

TIME (MINUTES)

FIG. 1. Chromatogram of spectinomycm indicating separation of (1) actinamine, (2) internal standaxd, and (3) spectinomycin [J. Chromatog. Sci., 12, 373 (1974)]. tion time than spectinomycin (Fig. i). Both degradation products usually produce two peaks due to incomplete silylation. A single peak can be produced from actinamine if the silylation time is extended; however, actinospectinoic acid is unstable under such a condition.

III. Amino-Glycoside Antibiotics A. Gentamicin 9

Chromatographic Conditions Detector: Flame-ionization Column: Glass, 3 mm i.d. X 610 mm (2 ft) packed with 3% OV-1 K, Tsuji and J. H. Robertson,unpublishedmethod.

218

METHODS FOR THE STUDY OF ANTIBIOTICS

[9]

on Gas Chrom Q, 100-120 mash (Applied Science Lab, Inc., State College, Pennsylvania) Gas flow rate: Hydrogen, 50 ml/min; air, 600 ml/min; and carrier gas (helium), 70 ml/min Oven temperature: 240 ° Detector temperature: 280 ° Flash temperature: 245 °

Silylation Reagent Add 50 ~l of N-trimethylsilyldiethylamine (TMSDEA, Pierce Chemical Co., Rockford, Illinois) per milliliter of TRI-SIL "Z" (Pierce Chemical Company).

Silylation Procedure Add 1 ml of the silylation reagent in a sealed vial containing approximately 5 mg gentamicin. Heat the vial in a 75 ° oil bath for 45 min. Detailed procedure for the preparation of the column, sample preparation, and silylation procedure may be seen in Section III,D (neomycin). B . K a n a m y c i n 1°

Kanamyein is chromatographed intact as the silyl ether-silyl amine derivative using an OV-1 column.

Procedure Unless otherwise specifically stated, materials, solutions, and apparatus required and preparation of chromatographic column may be referred to in Section III,D.

Materials OV-1, 3% on Gas Crom Q, 100-120 mesh (Applied Science Lab) Column, glass 3 mm i.d. X 1830 mm (6 ft)

Solutions Internal standard--silylation reagent: Add 25 t~l of TMSDEA and 8 mg of trilaurin per ml of TRI-SIL "Z". Cap and mix. Place the vial in an airtight container and store under refrigerator temperature. 1~K. Tsuji and J. H. Robertson, Anal. Chem. 42, 1661 (1970).

[9]

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

219

Reference standard:Dry the kanamycin sulfate reference standard at 60 ° in a vacuum oven at less than 5 Inm Hg for 3 hr. Immediately weigh and prepare a water solution containing 10 mg/ml of the standard.

Chromatographic Conditions Column: Glass, 3 mm i.d. X 1830 mm, packed with 3% OV-1 Gas flow rate: Hydrogen 40 ml]min, air 600 ml/min, and carrier gas (helium) 70 ml/min Oven temperature: 300 ° Detector temperature: 315 ° Flash heater: 300 °

Preparation o] the Chromatographic Column Follow the procedure described in Section III,D. The column thus prepared has normally 900 theoretical plates per meter for the silylated kanamycin A.

Sample Preparation Accurately weigh kanamycin bulk sample "as is" and prepare a water solution containing approximately 10 mg/ml of the kanamycin sulfate. Pipette 1.0 ml of the reference standard and sample solution into a vial and freeze dry. After freeze-drying, cap the vial with Teflon closures and metal retainers.

Silylation Procedure Add 1.0 ml of the internal standard-silylation reagent to each vial containing the freeze-dried sample using a 1-ml glass tuberculin syringe. Immerse and heat the vials in a 75 ° silicon oil bath for 35-40 min, swirling occasionally. Store the silylated samples in an airtight container under refrigeration temperature. Warm the sample before chromatography.

Chromatographic Procedure Using a microsyringe remove 1.5-2.0 t~l of sample from the vial and immediately inject it into the chromatograph at an attenuator and range

220

METHODS FOR THE STUDY OF ANTIBIOTICS

[9]

setting of 32 X 10. The sample transferral from the vial to the chromatograph must be made as quickly as possible. Calculation According to Omoto et al. 1~ kanamycin C elutes just prior to the kanamycin B peak. The content of kanamycin in micrograms per milligram of sample is calculated using the following formula: [RI/R~] X [Wr/W~] × F

(1)

where, R1 = (area of sample kanamyein A)/(area of the sample internal standard); R~ = (area of the standard kanamycin A)/(area of the standard internal standard) ; Wr = weight of kanamycin reference standard (mg/ml) ; We = weight of kanamycin sample (mg/ml) ; F = assigned value of kanamycin reference standard expressed in micrograms of anhydrous kanamycin base per milligram of the kanamycin sulfate. Problem Points o] the Assay Method Since the GLC analysis of kanamycin requires silylation of 4 NH~ groups as well as 7-OH groups, problems similar to those of neomycin GLC analysis are commonly encountered. Solutions to some of these problems may be found in Table I. Separation of kanamycin B from kanamycin A requires an extremely efficient column. Strict adherence to the column preconditioning, packing, and conditioning are essential for successful GLC analysis. C. Lividomycin 12 Chromatographic Conditions Column: Glass, 3 mm X 1000 mm, packed with 1% OV-1 on Gas Chrom Q, 100-120 mesh Gas flow rate: Carrier gas (nitrogen), 30 ml/min Oven temperature: 265 ° No silylation condition or other detail in procedure is given. D. Neomycin ~ Neomycin is ehromatographed intact as the silyl ether-silyl amine derivative using an 0V-'I column. ~1S. 0moto, S. Inouye, and T. Miida, J. Antibiol. 24, 430 (1971). 12 T. Mori, Y. Kyotani, I. Watanabe, and T. 0da, J. Antibiot. 25, 149 (1972).

[9]

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

221

Procedure Materials

OV-1, 3% on Gas Chrom Q, 100-120 mesh (Applied Science Laboratory, Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 610 mm (2 ft) Septum, high temperature (No. HT-9, Applied Science Lab.) Ferrule, Teflon front and back ferrules (Swagelok, Crawford Fitting Co., Solon, Ohio) Vial, glass, 2 ml (No. 5080-8712, Hewlett-Packard Co., Avondale, Pennsylvania) with Teflon-coated cap (Disks, No. 5080-8713, Hewlett-Packard) Chloroform, A.R. Hexane Pyridine, A.R. Dimethylchlorosilane iDMCS), (Pierce Chemical Co., Rockford, Illinois) Trimethylsilylimidazole (TSIM) (Pierce Chemical Co.) N-Trimethylsilyldiethylamine (TMSDEA) (Pierce Chemical Co.) TRI-SIL "Z" (Pierce Chemical Co.) or a solution containing approximately 40% TSIM in pyridine may be used Silyl-8 (Pierce Chemical Co.) Trilaurin (Supelco, Inc., Bellefonte, Pennsylvania) Solutions

DMCS 50% in hexane Internal standard--silylation reagent: Add 40 ~1 of TMSDEA and 2 mg of trilaurin per milliliter of TRI-SIL "Z." Cap the vial with a rubber septum and a metal retainer and mix. Place the vial in an airtight container and store under a refrigeration temperature. Reference standard solution: Dry the neomycin reference standard, USP Lot I or equivalent, at 60 ° in a vacuum oven at less than 5 mm Hg pressure for 3 hr. Immediately weigh and prepare a water solution containing 10 mg/ml of the neomycin sulfate standard. The dried neomycin powder is very hygroscopic and, when exposed to an atmosphere of 50% R H on an open surface, the sample can increase in weight by 8% in 3.5 min. In an open weighing bottle, it increases in weight by 1% in the 'first minute and then at a rate of approximately 0.4% per minute. 13 1~W. It. O. International Lab. Biol. Standards, Mill Hill, London January (1970).

222

METHODS FOR THE STUDY OF ANTIBIOTICS

[0]

Apparatus Gas Chromatograph: A Hewlett-Packard 402 or equivalent should be used. The sample injection must be such as to allow on-column sample injection approximately 2 cm into the end of the column. Metal parts and excessive dead space must be excluded from the chromatographic system. 1~,15 Detector: Flame ionization detector Freeze-dryer: A shelf-type freeze-dryer, e.g., No. 10-145MR-BA, VirTis Co., Gardiner, New York, is preferred over a manifold type freeze-dryer. Oil bath: Silicone oil at 75 ° equipped with stirring motor Micropipette: 500-~1 (Eppendorf micropipette, distributed by Brinkman Instruments, Inc., Westbury, New York, or equivalent). Microsyringe: 10 ~l (Hamilton Co., Whittier, California, or equivalent)

Chromatographic Conditions Column: Glass, 3 mm i.d X 610 ram, packed with 3% OV-1 Gas flow rate: Hydrogen, 50 ml/min; air, 600 ml/min; and carrier gas (helium), 70 ml/min. A gas-drying filter trap must be used to remove moisture and organic contaminants, e.g., oil, from the carrier gas, hydrogen, and air. The drying agent, e.g., 4A molecular sieve, must be periodically changed. Oven temperature: 290 ° Detector temperature: 310 ° Flash heater: 290 ° Chart speed: 0.64 cm/min

Column Connection Since the totally silylated neomycin has a molecular weight of 1550, 2 the GLC has to be performed at a high operating temperature (near 300°). Use of Teflon front and back ferrules with a lock nut (Swagelok) and a daily change of the high temperature septum are essential for trouble-free operation.

Preparation o] the Chromatographic Column Precondition an empty glass column, 3 mm i.d. X 610 mm, before packing by filling the column with a 50% solution of DMCS in hexane ~ M. Margosis and K. Tsuji, J. Pharm. Sci. 62, 1836 (1973). ~5G. Belec, W. L. Wilson, and D. W. Hughes, Can. J. Pharm. Sci. 8, 48 (1973).

[91

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

223

and allow it to stand for 5 min. Empty the column and wash with 50 ml of hexane followed by 50 ml of chloroform. Dry the column with a stream of dry, clean air. For best results, pack the column 3-4 cm at a time, while gently tapping the side of the column. The column is packed to within 8-10 mm of the outlet end and 15-20 mm of the inlet end of the column. The height of the column packing material at the inlet end should be adjusted to provide "on-column" injection--the tip of a microsyringe should touch the top of the column packing material and a sample should be injected on the column packing material. Fill the remaining portion of the column with a small piece of silylated glass wool. Glass wool is not really necessary at the inlet side of the column. The silylation of glass wool is performed in a similar manner to the preconditioning of an empty column. The theoretical plates of the column thus prepared showed nearly a 20% increase over a column prepared simply by filling the column with OV-1 material all at one time. Heat the column at 350 ° for 30 min with the carrier gas off. Cool the colmnn to room temperature and inspect the column. If a break or settling of the column packing material is observed, add the packing material as needed. Heat the column to 300 ° with gas flow on. When the oven temperature reaches 200% inject two 50-~1 portions of Silyl-8 and when the oven temperature reaches 300 ° continuously inject 2-~1 quantities of silylated neomycin, 15-20 times, into the column. The neomycin peak will be low for the first couple of injections; however, the peak will progressively increase with an increase in the number of injections and stabilize after 10-15 injections. Adjust the oven temperature and gas flow rates to give optimum separation between neomycins B and C, in 10-15 minutes of chromatography. The column is now ready for quantitative use. Prior to the start of the daily routine neomycin analysis, inject a couple of silylated neomycin samples.

Performance of the Column The column thus prepared has normally about 1050 theoretical plates per meter for the silylated neomycin B. The theoretical plates may be calculated using the following formula: Theoretical plates per meter of column = (5.545/*0 X (U/W~/~) 2 (2) where n = length of column in meters; U = distance between the injection point and the peak of the neomycin B; W1/2 = width at half-height of neomycin B peak.

224

METHODS FOR THE STUDY OF ANTIBIOTICS

[0]

Sample Preparation Procedure Neomycin Bulk Powder. Weigh neomycin sulfate bulk powder "as is" and prepare a water solution containing approximately 10 mg/ml of the neomycin sulfate. Pipette 500 ~l each of the reference standard and sample into glass vials, using a 500-~1 micropipette, and freeze-dry. Neomycin in Petrolatum-Based Ointments. 16 Accurately weigh approximately 5 g of ointment or the equivalent of approximately 25 mg neomycin sulfate into a centrifuge tube. Add 25 ml of chloroform and stopper. Chloroform should be washed with water to remove any ethyl alcohol present. Place in a 60 ° water bath for 5 rain and then shake vigorously until the ointment is well dispersed. Centrifuge at 2000 g for 10 min. Remove the chloroform by aspiration; the neomycin remains in the bottom of the tube. Add 15 ml of chloroform and resuspend neomycin. Use of an ultrasonicator is helpful. Centrifuge and remove the chloroform. Add 5.0 ml of water and 15 ml of n-heptane to the tube and shake or sonicate until the neomycin is completely dissolved in the water. Centrifuge at 220 g for 3 rain, and discard the heptane. Pipette 1.0 ml of the water solution into a serum vial and freeze-dry. Neomycin in Cream and Lotion. Prepare product standard solution as follows: Weigh 25 mg of the dried neomycin reference standard into a 50-ml centrifuge tube. Add approximately 5 g of the ointment base into the tube. Melt the contents of the tube using heat and mix thoroughly. Accurately weigh approximately 5 g of the sample containing about 25 mg of neomycin sulfate into a 50-ml centrifuge tube. Melt the contents of the tube as needed with mild heat. Add 40 ml of 2,2-dimethoxypropane to each tube containing sample and the product standard. Stopper and shake vigorously until the sample is well dispersed. To each tube, add 1 drop of 10 N sulfuric acid. Shake vigorously and place the tubes in a water bath at 60 °. Every 2 rain shake the tubes for approximately 10 sec. After 10 min remove the tubes from the bath. Centrifuge the tubes at 1500 g for 10 min. Remove and discard the solvent with suction, taking care not to lose any of the neomycin which is in the bottom of the tube. Add 3.0 ml of water and shake or ul~rasonicate until the neomycin is dissolved. Add 25 ml of 2,2-dimethoxypropane and shake vigorously. Place the tubes in the water bath at 60 ° and every 2 rain shake for 10 sec. After 10 min remove the tubes from the bath. Add 20 ml of heptane and shake or ultrasonicate until the neomycin is well dispersed. Centrifuge at 1500 g for 10 rain and discard the heptane. Add 5.0 ml of water and shake or ultrasonicate until the neomycin is dissolved. ~eB. v a n Giessen and K . Tsuji, J. Pharm. Sci. 60, 1068 (1971).

19]

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

225

Centrifuge at 1500 g for 5 rain. Pipette 1.0 ml of the water solution into a glass vial and freeze-dry.

Freeze-Dry Procedure Freeze the vials at or below --40 ° , then freeze-dry overnight at less than 10 tLm Hg pressure. The freeze-drying step eliminates lot-to-lot differences in moisture content and in granule size, which significantly influences the solubility characteristics of neomycin powder in the internal standard silylation reagent, hence, greatly affects the quantitation. During high humidity, high temperature Midwestern summer months, we experienced considerable problems from sample melt-back and losses by flaking using a manifold type freeze-dryer. Use of a shelf type freezedryer eliminated these problems. At the end of the freeze-drying cycle, cap the vial immediately with a Teflon-coated septum and seal tightly with a metal retainer. Natural rubber septums interfere with the silylation reaction and lessen the stability of the silylated sample.

Silylation Procedure Add 1.0 ml of the internal standard silylation reagent to each vial containing the freeze-dried sample using a 1-ml glass tuberculin syringe. Immerse and heat the vials in a 75 ° silicone oil bath for 35-40 min swirling occasionally. The silylated neomycin sample thus prepared is extremely sensitive to moisture and temperature. Better stability can be obtained by storing the vial in an air-tight container and keeping it in a refrigerator until analyzed. If a sample is stored at refrigeration temperatures, warm the sample before chromatography. Using a microsyringe remove a 1.5-2.0 ~1 sample from the vial and immediately inject into the chromatograph at an attenuator and range setting of 64 X 10. Since the silylated neomycin rapidly hydrolyzes by moisture in the air, transfer of the sample from the vial to the chromatograph is critical and must be made as quickly as possible. An automatic GLC sample injector, e.g., Hewlett-Packard Model 7671A, may be used satisfactorily provided that the sample-holding tray is kept cool and the syringe is rinsed with pyridine between the sample injection. The entire injection assembly had to be lowered about 15 mm closer to the GLC column than originally designed so that the sample could be iniected on the column.

226

METHODS FOR THE STUDY OF ANTIBIOTICS

[0]

Calculation A typical chromatogram is shown in Fig. 2. Measure peak areas of the internal standard and neomycins B and C. The content of neomycin, in micrograms per milligram of sample, is calculated using the following formula:

[R1/R2] × [Wr/Ws] X F

(3)

where R1 = (area of the sample neomycin B ~ 1/2 area of the samp]e neomycin C)/(area of the sample internal standard peak); R2 = (area of the standard neomycin peak)/(area of the standard internal standard peak); Wr = weight of neomycin reference standard (mg/ml); Ws = weight of neomycin sample (mg/ml); F = assigned value of neomycin reference standard expressed in micrograms of anhydrous neomycin base

3

I 4

I

I

S 12 TIME (MINUTES)

I 16

FIo. 2. Chromatogram of neomycins indicating separation of (1) neamine, (2) neobiosamine; (3) internal standard, (4) neomycin B, (5) neomycin C, (6) LP,, and (7) LPc.

[9]

227

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

HO

~

Neamine

CH2NH2

"" "" "" ". ~

"Zm

~0

R3

)7~--~--~~

"~" "~ .~. 0~/=.= ~ CH2OH I "-~....UH

Neobiosamine

NH2

o,,,4 H

~"

Neomycin B

R1=H

R2=CH2NH2

R3=NH2

Neomycin C

R1 =CH2NH2

R2=H

R3=NH2

Neomycin LPB

RI=H

R2=CH2NH2

R3=NHCOCH3

Neomycin LPC

RI=CH2NH 2

R2=H

R3=NHCOCH 3

FIG. 3. Structure of neomycins [K. Tsuji and J. H. Robertson, Anal. Chem. 42, 1661 (1970). Copyright by the American Chemical Society].

per milligram of the neomycin sulfate (F = 767 pg/mg for the USP Lot I Reference Standard).

Assessment o] Results Mono-N-acetylneomycin C, LPc, (see Fig. 3) elutes after the LPR peak, and paromomycin I, if present, elutes prior to the neomycin B peak. The drug potency of neomycin sample as calculated from the GLC data should be comparable to that obtained by the microbiological cylinder cup agar diffusion assay method using Staphylococcus epidermidis (ATCC 12228) as the assay microorganism. 17 Changes in the assay microorganism require a different response factor for neomycin C. 1

Comments on the Assay Method Each mole of 2,2-dimethoxypropane reacts with 1 mole of water to form 1 mole of acetone and 2 moles of methanol. The reaction requires the use of an acid catalyst. Sulfuric acid was chosen to keep the neomycin in the sulfate form. The concentration of acid is quite important for the ~J. H. Robertson, R. Baas, R. L. Yeager, and K. Tsuji, Appl. Microbiol. 22, 1164 (1971).

228

METHODS FOR THE STUDY OF ANTIBIOTICS

[9]

speed of reaction. When the reaction rate is too fast, the neomycin particles occlude with small amounts of excipient in a product, resulting in interference with the analysis. Melt-back and loss by boiling often occurred during freeze-drying, and interference in silylation and chromatography were also experienced when the extraction was not optimal. When the reaction rate is too slow, the neomycin is not completely dehydrated and tends to stick to the side of the tubes. The technique which gives the best results is to use 1 drop of 10 N sulfuric acid at the initial dehydration and add no acid during the clean-up step. The residual sulfuric acid remaining acts as the catalyst. The neomycin is slightly soluble in 2,2-dimethoxypropane-acetonemethanol solution, thereby resulting in a loss of approximately 5% of neomycin. For this reason, the method calls for the use of the product standard as the reference standard. As the sample ages, the neomycin extracted becomes brownish. These neomycins result in a broader neomycin GLC peak than the standard neomycin peak and are low in assay value. The difficulty is not experienced with fresh samples, however. Because of the difficulty in silylating 6 NH2 groups and 7 OH groups in one large molecule, coupled with the extreme sensitivity of the TMS-NH group to moisture, and the requirement of high chromatographic temperature due to the low volatility of the high molecular weight compound (MW 1550), the GLC determination of neomycin may be easily interfered with by numerous factors. Strict adherence to the procedures described above is essential for the successful analysis of neomycin. Table I is provided to assist in identifying problems frequently encountered during the GLC analysis of neomycin and lists solutions to the problems.

E. Paromomycin The procedure for the GLC analysis of paromomycin is analogous to that of neomycin powder} ° Follow the procedure as described under neomycin powder, using paromomycin in place of neomycin. IV. Aromatic Antibiotics

A. Chloramphenico118 Chloramphenicol is chromatographed intact as the bistrimethylsilyl ether derivative using an OV-1 column. ~mM. Margosis, J. Chromatogr. 47, 341 (1970).

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TABLE I PROBLEMS AND SOLUTIONS FOR THE ANALYSIS OF NEOMYCIN BY GAs-LIQuID CHROMATOGRAPHY

Problem Very small or fat peak

Indication of a shoulder peak

Lack of quantitation

Short column life

Tailing of peak

Excessive darkening of packing material in inlet Stability of silylated sample

Solution a. Too much moisture in sample. Check freeze-dryer for proper operation. b. Column is not properly conditioned. c. Assure on-column injection. d. Eliminate metal parts from the GLC system. e. Check silylation reagent. f. Sample degradation--high storage temperature or leakage in vial. g. Inject immediately after withdrawing sample from vial. h. Excessive organic materials in sample. Sample clean-up may be required. a. Adjust amount of T M S D E A . b. Check freeze-dryer for proper operation. c. Assure on-column injection. d. Eliminate metal part from the GLC system. e. Excess loss of liquid phase. Change or renew column packing material. f. Sample degradation--high storage temperature or leakage in vial. a. Check freeze-dryer for proper operation. b. Inject immediately after withdrawing. c. Assure on-column injection. d. Remove all metal and/or Teflon in inlet system. e. Check silylation temperature for completion of the reaction. f. Keep samples tightly sealed and store in a cool place. g. Condition column with silylated neomycin. a. Reduce column temperature (less than 300 °) and compensate with increase in carrier gas flow. b. Check drying agent for carrier, hydrogen, and air. c. Check silylation temperature and reagent for complete reaction. d. Incomplete silylation or degradation of silylated sample. a. Assure on-column injection. b. Eliminate metal and/or Teflon in inlet system. c. Check packing material for proper loading of liquid phase. d. Replace column. a. Incomplete silylation, check silylation temperature and reagent. Longer silylation time may be needed. b. Replace first 3 cm of packing material in inlet. Use minimum amount of glass wool and replace as needed. a. Use sealed reaction vial; store at refrigeration temperature. b. Presence of metal particles in reaction vial. Vial should be stoppered tightly with a Teflon-coated stopper.

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Procedure Materials OV-1, 3-5% on Gas Chrom Q 100-120 mesh (Applied Science Lab., Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 1220 mm or 1830 mm (4 ft or 6 ft) Vial, glass, 1-dram size with polyethylene or Teflon cap Ethyl acetate, A.R. Diethyl ether, A.R. Pyridine, A.R. Acetonitrile, glass distilled Celite 545 (acid washed) Cyclohexane N,O-Bis(trimethylsilyl)-acetamide (BSA) (Pierce Chemical Company, Rockford, Illinois) m-Phenylene dibenzoate

Solutions HCI solution, 0.01 N Internal standard-silylation reagent. dibenzoate and dissolve in about of BSA and make the solution pyridine. Phosphate buffer, pH 5.8 Ethyl acetate-diethyl ether mixture

Weigh 200 mg of m-phenylene 6 ml of acetonitrile. Add 1 ml to 10 ml with acetonitrile or

(2:1)

Apparatus Gas Chromatograph: See requirements in Section III,D. Detector: Flame ionization detector

Chromatographic Conditions Column: Glass, 3 mm i.d. X 1220 or 1830 mm, packed with 3 or 5% OV-1 Gas flow rate: Hydrogen, 50 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min. Oven temperature: 240 ° Detector temperature: 255 ° Flash heater temperature: 250 °

Preparation o] Chromatographic Column Follow the procedure described in the neomycin section. The column thus prepared normally has 1500 theoretical plates per meter for silylated chloramphenicol.

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Preparation o] Sample and Re]erence Standard Solution Accurately weigh approximately 10 mg of sample powder and reference standard powder into separate, 1-dram vials, or prepare the reference standard solution by accurately weighing about 125 mg of powder into a 25 ml volumetric flask. Dilute to volume with ethyl acetate. Pipette 2.0 ml of this solution into a glass vial and evaporate to dryness under a stream of nitrogen.

Sample Preparation Hard-Filled Capsules. Empty 20 capsules, collecting the contents quantitatively. Weigh the powder and determine the average capsule fill weight. Mix the powder and accurately weigh a portion equivalent to about 125 mg of chloramphenicol into a 25-ml volumetric flask. Add ethyl acetate to volume and shake vigorously. Allow insoluble material to settle or filter if necessary. Pipette 2.0 ml of the supernatant liquid (or filtrate) into a glass vial. Evaporate to dryness under a stream of nitrogen. Tablets. Accurately weigh l0 tablets and then finely grind, using a Wiley mill with a 60-mesh screen. Accurately weigh a portion of the powder to contain approximately 125 mg of chloramphenicol into a 25-ml volumetric flask. Add ethyl acetate to volume and shake vigorously. Allow insoluble material to settle or filter. Pipette 2.0 ml of the supernarant (or filtrate) into a glass vial. Evaporate to dryness under a stream of nitrogen. Solutions. Pipette a suitable portion of the solution into a separatory funnel and dilute with a 5-fold amount of water. Extract chloramphenicol three times with a mixture of ethyl acetate-diethyl ether. Collect the extracts and wash with water. Evaporate the extract to dryness with a stream of nitrogen. Add ethyl acetate to reconstitute and quantitatively transfer into a volumetric flask of suitable size and add ethyl acetate to have a final concentration of 5-10 mg chloramphenicol per milliliter of solution. Pipette a portion of the solution containing 10 mg of chloramphenicol into a glass vial. Evaporate to dryness under a stream of nitrogen. Ointments. A mixture of about 3 g of Celite 545 and 2 ml of a pH 5.8 phosphate buffer is placed directly into a glass chromatographic column and tamped to a uniform mass. Mix 3 g of celite and 2 g of ointment and place on top of the prepared column and again tamp gently. Add 100 ml of cyclohexane to the column and discard the eluate. Add 100 ml of ethyl acetate to elute chloramphenicol. Collect the eluate and evaporate to dryness. Reconstitute and bring to volume with ethyl acetate to a concentration of 5-10 mg chloramphenicol per milliliter of solution.

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Pipette a portion of the solution containing approximately 10 mg of chloramphenicol into a glass vial. Evaporate to dryness under a stream of nitrogen. Silylation Procedure

Using a micropipette add 500 ~l of the internal standard-silylation reagent to each vial containing samples and the reference standard. Swirl the vial gently for a few minutes at room temperature. Inject approximately 1 ~l of the silylated chloramphenieol into the gas chromatograph. Calculation

The micrograms of chloramphenicol per milligram of sample may be calculated using Eq. (1) described in Section III,B. Comments on the Assay Method

According to Janssen and Vanderhaeghe,19 silylation with BSA in acetonitrile may form a mixture of mono-, bis-, and tris-TMS derivatives of chloramphenicol. A single bis-TMS derivative may be obtained when the mixture of TMCS and HMDS in pyridine is used. The meta and erythro isomers may be separated from each other; however, the L isomer may not be separated from the chloramphenicol peak. Ch!oramphenicol appears to be stable in ethyl acetate for about 2 weeks, however, the internal standard-silylation reagent with ethyl acetate may not be stable and should therefore be made fresh just prior to silylation. B. Griseofulvin s° Griseofulvin is ehromatographed intact without derivatization using an OV-1 column.

Procedure

Materials

OV-17, 1% on Gas-Chrom Q, 100-120 mesh (Applied Science Lab, State College, Pennsylvania) Column, glass, 4 mm i.d. X 914 mm (3 ft) lo G. Janssen and H. Vanderhaeghe, J. Chromatogr. 82, 297 (1973). 2, M. Margosis, J. Chromatogr. 70, 73 (1972).

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Chloroform, A.R. Tetraphenylcyclopentadienone

Solutions Internal standard solution: tetraphenylcyclopentadienone dissolved in chloroform at a concentration of 5 mg/ml

Apparatus Perkin-Elmer Model 900GC or equivalent. For requirements see Section III, D. Detector: flame-ionization detector

Chromatographic Conditions Gas flow rate: hydrogen and air at the optimum setting; carrier gas (helium), 60 ml/min Oven temperature: 245 ° Detector temperature: 260 ° Injector temperature: 260 °

Preparation o] Chromatographic Column Pack an empty glass column, 4 mm i.d. X 914 mm, with 1% OV-17 column packing material. No-flow condition the column at 340 ° for 1 hour and then bring the column temperature to 250 ° . Maintain the column temperature at 250 ° with carrier gas until a stable baseline is obtained. The column thus prepared has approximately 1350 theoretical plates per meter for both griseofulvin and the internal standard.

Preparation o] Bulk Drug and Re]erence Standard Solution Accurately weigh approximately 125 mg of bulk drug and the reference standard powder into a 25.0-ml volumetric flask. Add chloroform and shake vigorously to dissolve and dilute to volume. Pipette a 2.0-ml aliquot into a glass vial and evaporate to dryness under a stream of dry nitrogen.

Sample Preparation For solid dosage forms (capsules, tablets, boluses) accurately weigh 10 solid dosage units to obtain an average weight of one dosage form. Grind the dosage form, pool the contents of the capsule, and weigh accu-

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METHODS FOR THE STUDY OF &NTIBIOTICS

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rately a suitable amount of powder into volumetric flasks. Add chloroform and shake with beating if necessary, to dissolve griseofulvin. Dilute to volume with chloroform. Pipette an aliquot of the supernatant containing approximately 10 mg griseofulvin into a glass vial and evaporate to dryness with a stream of dry nitrogen. For suspensions, transfer 5.0 ml of the mixed suspension containing 250 mg of griseofulvin into a separatory funnel and dilute to about 25 ml with water. Extract the mixture with 25 ml of chloroform and collect the chloroform layer. Repeat the extraction twice with 10-ml amounts of chloroform. Pool the chloroform extract and backwash with 5 ml of water, then filter through chloroform-moistened glass wool into a 50 ml volumetric flask and dilute to volume with chloroform. Pipette 2.0 ml of the aliquot into a glass vial and evaporate to dryness with a stream of nitrogen. Chromatographic Procedure Add 1.0 ml of the internal standard solution into each vial containing samples and the reference standard and stir vigorously to obtain a uniform solution. Inject 1 ~l of this solution into the GC. Calculation The content of griseofulvin in micrograms per milligram of sample is calculated by direct comparison of the ratio of the peak area (griseofulvin/tetraphenylcyclopentadienone) with that of the griseofulvin reference standard. Comments on the Assay Method In the preparation of bulk drugs or the reference standard, dissolution in chloroform and evaporation steps are not really necessary but the steps are recommended so that the procedure is consistent with that of sample preparation for the dosage forms. The method resolves dechlorogriseofulvin from griseofulvin but does not resolve dehydrogriseofulvin from isogriseofulvin. V. Glutarimide Antibiotic Cycloheximide ~1 Cycloheximide is derivatised as the monotrimethylsilyl ether by treatment with isopropanol after silylation. =L. W. Brown, Agr. Food Chem. 21, 83 (1973).

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Procedure Materials QF-1, 1% on Gas Chrom Q, 80-100 mesh (Applied Science Lab., Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 610 mm (2 ft) Vial, glass, 1-dram size with polyethylene or Teflon cap Isopropyl alcohol, A.R. Chloroform Benzene Pyridine, A.R. Bis (trimethylsilyl) trifluoroacetamide (Regisil) containing 1% trimethylchlorosilane (TMCS). Available as Regisil TMCS from Regis Chemical Co., Chicago, Illinois. Cholesteryl acetate

Solutions Isopropyl alcohol, 3%, in pyridine Internal standard-silylation reagent. Prepare a pyridine solution containing approximately 6 mg of cholesteryl acetate and 0.1 ml of Regisil TMCS per milliliter of solution.

Apparatus Gas chromatograph. See requirements in Section III, D. Detector: flame-ionization detector

Chromatographic Conditions Column: Glass, 3 mm i.d. X 610 ram, packed with 1% QF-1 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (t~elium), 60 ml/min Oven temperature: 200 ° Detector temperature: 225 ° Flash heater temperature: 200 ° Chart speed: 0.64 cm/min Inject approximately 1 td of sample into the chromatograph.

Preparation o] the Chromatographic Column Follow the procedure described in Section III, D. After no-flow conditioning of the column at 240 ° for 30 min, bring the oven to room temperature. Turn on the oven and carrier gas. Iniect

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METHODS FOR THE STUDY OF ANTIBIOTICS

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a 50-t~l quantity of Silyl-8. Maintain the gas chromatograph under the chromatographic condition overnight. Prior to analysis inject a couple of silylated cycloheximide samples. The column ¢hus prepared normally has 1300 theoretical plates per meter for silylated cycloheximide.

Re]erence Standard Preparation Accurately weigh approximately 5 mg of the cycloheximide reference standard into a 1-dram vial.

Sample Preparation Bulk Drug Sample. Accurately weigh approximately 5 mg of bulk cycloheximide sample in a 1-dram vial. Formulation. For formulations containing materials that are soluble in organic solvents but relatively insoluble in water, a sample equivalent to 10 mg of cycloheximide should be accurately weighed into a 15-ml centrifuge tube and the cycloheximide extracted from the sample with 2 ml of water. A 1.0-ml quantity of this solution is then extracted with chloroform, and the resulting chloroform solution is evaporated to dryness with nitrogen. For formulations containing materials such as ferrous sulfate and sodium alkyl aryl sulfonic acid, which are insoluble in organic solvents, the sample is extracted directly with benzene. A suitable quantity of the benzene solution containing 5 mg of cycloheximide is transferred into a vial and evaporated to dryness. Silylation Procedure Add 1.0 ml of the internal standard-silylation reagent into each vial containing samples and reference standard. Swirl the vial gently to dissolve the solid and place in an oil bath at 50 ° for 2 hr. Remove the vial from the oil bath and add 1.0 ml of the pyridine solution containing 3% isopropyl alcohol. After gently swirling the solution to mix, the vial is allowed to stand for 20 min and then chromatographed.

Calculation The micrograms of cyeloheximide per milligram of sample may be calculated using Eq. (1) of Section III, B.

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Comments on the Assay Method

Normal silylation conditions yield a mixture of mono- and bistrimethylsilyl derivatives of cycloheximide. Since the bistrimethylsilyl derivative could not be formed quantitatively, isopropyl alcohol was added after the silylation reaction to quantitatively convert the bis derivative to the mono derivative, thus yielding a single peak. Addition of alcohol prior to the silylation reaction resulted in no silylation of the cycloheximide. Isocycloheximide and the principal dehydration product, anhydrocycloheximide, two possible impurities in cycloheximide samples, can be determined in one chromatograph. VI. Lincomycin-Clindamycin Family A. Lincomycin 5,2~

Lincomycin is chromatographed intact as the tetrakistrimethylsilyl ether derivative using a 3% SE-30 column. Procedure Materials

SE-30, 3-5% on Gas Chrom Q, 100-120 mesh (Applied Science Lab, Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 1830 mm (6 ft) Vial, glass, suitable size with nonreacting airtight cap Pyridine, A.R. N,O-Bis(trimethylsilyl)acetamide (BSA) (Pierce Chemical Co., Rockford, Illinois) Silyl-8 (Pierce Chemical) Tetraphenylcyclopentadienone (Aldrich Chemical Co., Inc., Milwaukee, Wisconsin) Solutions

Internal standard solution. Prepare a pyridine solution containing approximately 2 mg of tetraphenylcyclopentadienone per milliliter of solution. Apparatus

Gas chromatograph. See requirements in Section III, D. Detector: flame-ionization detector ~ R. L. Houtman, D. G. Kaiser, and A. J. Taraszka, J. Pharm. Sci. 57, 693 (1968)

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METHODS FOR THE STUDY OF ANTIBIOTICS

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Chromatographic Conditions Column: glass, 3 mm i.d. X 1830 mm, packed with 3 or 5% SE-30 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 275° Detector temperature: 300 ° Flash heater temperature: 275 ° Chart speed: 0.64 cm/min

Preparation o] the Chromatographic Column Follow the procedure described in the neomycin section. The column thus prepared has normally 2000 theoretical plates per meter for silylated lincomycin.

Re]erence Standard Preparation Weigh accurately about 20 mg of lincomycin hydrochloride reference standard and transfer into a suitable vial.

Sample Preparation Drug, Tablets, and Capsules. Transfer an accurately weighed sample containing about 20 mg of lincomycin hydrochloride monhydrate into a suitable vial. For tablets, weigh at least 20 tablets to establish the average weight per tablet. For capsules, weigh the contents of at least 10 capsules to establish the average fill weight per capsule. Aqueous Solutions and Syrups. Transfer an accurately measured volume containing 0.9-1.0 g of lincomyein hydrochloride monohydrate to a 100-ml volumetric flask. Dilute to volume with methanol and mix well. Transfer 2.0 ml of this solution to a vial and evaporate to dryness. Silylation Procedure Add 2.0 ml of pyridine into each vial containing lincomycin. Warm the solution on a hot plate for approximately 5 min to completely dissolve lincomycin. Add 1.0 ml of BSA, stopper the vials, and heat at 70-75 ° for 1 hr or allow the vials to stand overnight at room temperature. Pipette 10.0 ml of the internal standard preparation into each vial and mix well. Chromatograph the silylated samples.

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Calculation Calculate the content of lincomycin base "as is" expressed in micrograms per milligram of sample from Eq. (1) described in Section III, B. Comments on the Assay Method The lincomycin tetrakis-TMS derivative is one of the most stable TMS derivatives. For the quantitative derivatization of lincomycin care must be taken to completely dissolve lincomycin in pyridine prior to the addition of BSA. B. Clindamycin T M The clindamycin hydrochloride salt is converted to the free base and extracted with an organic solvent containing the internal standard. The clindamycin base is reacted with trifiuoroacetic anhydride and chromatographed intact using a SE-30 column. Procedure Materials SE-30, 1% on either (a) Gas Chrom Q, 100-120 mesh, (b) Diatoport S, 80-100 mesh, or (c) Chromosorb W, 80-100 mesh Column, glass, 3 mm i.d. X 610 mm (2 ft) Trifluoroacetic anhydride Chloroform A.R. Sodium sulfate, anhydrous Sodium carbonate, anhydrous granules Hexacosane Solutions Sodium carbonate solution, 1% Internal standard solution. Prepare a chloroform solution containing approximately 0.45 mg of hexacosane per milliliter Apparatus Gas chromatograph. See requirements in Section III, D. Detector: flame-ionization detector 23L. W. Brown J. Pharm. Sci. 63, 1597 (1974).

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METHODS FOR THE STUDY OF ANTIBIOTICS

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Chromatographic Conditions Column: Glass, 3 mm i.d. X 610 ram, packed with 1% SE-30 Gas flow rate: Hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 180 ° Detector temperature: 200 ° Flash heater temperature: 180 ° Chart speed: 0.64 cm/min

Preparation o] the Chromatographic Column Follow the procedure described in Section III, D. The SE-30 column thus prepared has normally 1300 theoretical plates per meter for trifluoroacetylated derivative of clindamycin.

Preparation o] Clindamycin Sample and Re]erence Standard Solution Accurately weigh the bulk powder and reference standard powder, equivalent to approximately 13 mg of clindamycin hydrochloride monohydrate into a 15-ml glass-stoppered centrifuge tube. For hard-filled capsules, empty 20 capsules, collecting the contents quantitatively. Weigh the powder and determine the average capsule fill weight. Mix the powder and dccurately weigh a portion equivalent to about 13 mg of clindamycin into a 15-ml stoppered centrifuge tube. Add 6.0 ml of the Internal Standard solution and 6.0 ml of 1% sodium carbonate solution to each tube. Shake vigorously for 30 min and centrifuge.

Derivatization Procedure Transfer a portion of the chloroform solution into a suitable vial conraining 1 g of anhydrous sodium sulfate to dry the solution. Shake vigorously for 1 min then transfer 2 ml of the dried chloroform solution into a 15-ml centrifuge tube and add 0.50 ml of trifluoroacetic anhydride. Place stoppered centrifuge tubes in a heating block or oil bath to an approximate depth of 5 cm so that the upper part of the centrifuge tubes will act as a reflux condenser. Heat at 45 ° for 30 rain. Chromatograph aliquots of the sample and standard preparations. Sample size: 0.5-2 t~l; range and attenuation 15 X10.

Calculation Measure the area under the peak of clindamycin and the internal standard. Calculate the content of clindamyein base in micrograms per

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GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

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milligram of sample from the following formula:

[R1/R2] X [Wr/W.] X [El~F2] X F3

(4)

where R1 = (area of clindamycin sample peak)/(area of the internal standard peak); R2 = (area of the clindamycin standard peak)/(area of the internal standard peak); Wr -- weight of clindamycin hydrochloride hydrate reference standard (rag) ; W8 = sample weight (rag) ; F1 = ml of internal standard solution added to the sample preparation; F2 = ml of internal standard preparation added to the standard preparation; F3 = Assigned potency of clindamycin hydrochloride hydrate reference standard expressed in micrograms of anhydrous clindamycin base per milligram of reference standard.

Comments on the Assay Methods A typical chromatogram may be seen in Fig. 4. The reaction conditions of the trifluoroacetyl procedure are less severe than the acetyl proce-

4

TiM

L 8 (MINUTES)

I 12

FIG. 4. Chromatogram of clindamycin indicating separation of (1) epilincomycin, (2) clindamycin B, (3) clindamycin, and (4) internal standard. (Courtesy of L. W. Brown.)

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METHODS FOR THE STUDY OF ANTIBIOTICS

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dure, 2. and the trifiuoroacetyl procedure is not affected by hydrochloric acid in clindamycin samples, thus eliminating any degradation and shortening the assay time. Also, solvent tailing, a problem with the acetylation is eliminated. Peaks due to incomplete acetylation interfere in the measurement of the impurities, epilincomycin and epiclindamyein, while no interference is experienced with the trifluoroacetyl procedure which separates epilincomycin from epiclindamycin. C. Clindamycin Phosphate 5,23

The GLC method for clindamycin phosphate is based on the hydrolysis of clindamycin phosphate with alkaline phosphatase and chromatography of clindamycin base.

Procedure Materials Intestinal alkaline phosphatase Sodium carbonate, anhydrous

Solutions Borate buffer, pH 9.0. Transfer 3.0 g of boric acid into a l-liter volumetric flask containing 500 ml of water. Mix and add 21 ml of 1.0 N sodium hydroxide and 10 ml of 0.1 M magnesium chloride. Dilute to volume with water and mix well.

Preparation o] Clindamycin Hydrochloride Standard Solution Accurately weigh approximately 9 mg of the clindamycin hydrochloride reference standard into a 35-ml glass-stoppered centrifuge tube and dissolve in 20 ml of pH 9.0 borate buffer.

Preparation o] Sample Solution Accurately weigh approximately 12 mg of the clindamycin phosphate sample into a 50-ml glass-stoppered centrifuge tube. Pipette 25 ml of the pH 9.0 borate buffer into the centrifuge tube. Add 10 ml of chloroform and shake vigorously for 15 rain. Centrifuge the resulting mixture and pipette a 20-ml aliquot of the aqueous phase into a 35-ml centrifuge tube. Add a weighed amount of intestinal alkaline phosphatase equivalent to 24 T. O. Oesterling, ]. Pharm. Sci. 59, 63 (1970).

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50 units of activity and allow the solution to stand until the enzyme has completely dissolved. Place the tube into a water bath at 37 ° for 2.5 hr. After the 2.5-hr hydrolysis, allow the solution to cool.

Extraction Procedure Add 10 ml of the internal standard solution to each sample and standard solution. Shake the centrifuge tubes vigorously for 30 min and centrifuge. Remove the aqueous layer and discard. Shake the tube again and mix in an ultrasonicator for 2 min, then centrifuge. No emulsion should be present at this stage. Remove the remaining aqueous layer by suction and transfer a 3-ml quantity of the chloroform layer to a 1-dram vial containing approximately 1 g of anhydrous sodium sulfate, swirl the vial and allow sodium sulfate to settle. Transfer 1 ml to another 1 dram vial.

Trifluoroacetylation Procedure Add 250 ~l of trifluoroacetic anhydride to each vial and place them into a water bath at 45 ° for 30 rain. Add about 10 granules of anhydrous sodium carbonate to each vial and allow to stand at room temperature for 30 rain. Chromatogram samples under the conditions described in the clindamycin section. D. Clindamycin P a l m i t a t e 5,2~

Procedure Materials SE-54 or UCW-98, 1% on either Diatoport S, 60-80 mesh or Chromosorb WHP 80-100 mesh Column, glass 3 mm i.d. X 610 mm (2 ft) Trilaurin Acetic anhydride Pyridine, A.R. Chloroform, A.R.

Solutions Internal standard solution. Prepare a solution containing 5.0 mg of trilaurin per milliliter in chloroform. ~ K. Tsuji and J. H. Robertson, Anal. Chem. 43, 818 (1971).

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METHODS FOR THE STUDY OF ANTIBIOTICS

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Apparatus Gas chromatograph. For requirements see Section III, D. Detector: flame-ionization detector

Chromatographic Conditions Column: Glass, 3 mm i.d. X 610 mm, packed with 1% SE-54 or UC-W98 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 275 ° Detector temperature: 290 ° Inj ection port: 280 ° Chart speed: 0.64 cm/min

Preparation o] Chromatographic Column Follow the procedure described in Section III, D.

Preparation o] Sample and Re]erence Standard Solutions Accurately weigh approximately 15 n)g of both the sample and the reference standard into separate glass-stoppered, 15-ml centrifuge tubes. Add 1.0 ml of internal standard solution, 1.0 ml of pyridine, and 0.5 ml of acetic anhydride to each tube. Agitate the tubes to ensure dissolution and complete mixing of the liquids.

Acetylation Cover the top of each centrifuge tube with a plastic cap. Punch a small, 18-gauge needle, hole in the top of each cap to allow vapor to escape. Place the tubes in a 100 ° drying oven for 2.5 hr. Remove the tubes from the oven and allow to cool. Take the plastic cap from each tube and replace with the glass stopper. Centrifuge 10-15 min at 2000-2500 rpm to separate the white solid from the liquid in the tube. Inject 1 ~l of the clear solution into the gas chromatograph.

Calculation The internal standard will elute before the clindamycin palmitate peak. Calculate the clindamycin palmitate in microgram per milligram of sample using Eq. (1) described in Section III, B.

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VII. Macrolide Antibiotic E r y t h r o m y c i n25

Erythromycin is chromatographed intact as the pentakistrimethylsilyl ether derivative using an OV-225 column.

Procedure Materials 0V-225, 3% on Gas Chrom Q, 100-120 mesh (Applied Science Lab., Inc. State College, Pennsylvania) Column, glass, 3 mm i.d. X 1830 mm (6 ft) Septum, high temperature-acid resistant (No. 4971, Anspec Co., Ann Arbor, Michigan) Vial, glass 1-dram size with screw cap (No. 60910, Kimble Glass, Owens-Illinois, Toledo, Ohio) with 26 mil polyethylene liner Pyridine, A.R. Trimethylchlorosilane (TMCS) (Pierce Chemical Co., Rockford,Illinois) N,O-Bis(trimethylsilyl) acetamide (BSA) (Pierce Chemical) N-Trimethylsilylimidazole (TSIM), (Pierce Chemical) 1,3-Dimyristin (Applied Science Lab)

Solutions Internal standard-silylation reagent. Add 5 ml of TMCS to 5 ml of BSA, mix, and add 2 ml of TSIM. Add 12 ml of pyridine containing about 50 mg of 1,3-dimyristin. Cap the vial with a polyethylene or Teflon stopper. Place the vial in an airtight container and store under refrigerated temperature.

Apparatus Gas chromatograph. See requirements in Section III, D. Detector: flame-ionization detector

Chromatographic Conditions Column: Glass, 3 mm i.d. X 1830 mm, packed with 3% 0V-225 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 55 ml/min

246

METHODS FOR THE STUDY OF ANTIBIOTICS

[0]

Oven temperature: 275 ° Detector temperature: 290 ° Flash heater temperature: 280 ° Chart speed: 0.64 cm/min Chromatograph a 1-~I sample at an attenuation and range setting of 64 X 10.

Preparation o~ Chromatographic Column Follow the procedure described in Section III, D. After the no-flow conditioning at 300 ° for 45 rain, bring the oven temperature to room ternperature. Turn the oven temperature to 275 ° with carrier gas at 55 ml/min. Inject 50 ~1 of Silyl-8 two or three times. When the oven temperature reaches 275 °, inject 2 ~1 of silylated erythromycin sample. Maintain the GC at chromatographic conditions overnight. Prior to the daily analysis, inject a sample of silylated erythromycin. The column thus prepared normally has 1320 theoretical plates per meter for silylated erythromycin A.

Preparation of Bulk Drug and Reference Standard Powder Accurately weigh approximately l0 mg of erythromycin bulk powder and the reference standard powder into a 1-dram screw-cap vial.

Sample Preparation for Enteric Coated Tablet 26 Materials: Methylene chloride, A.R. Apparatus: Wiley mill with a 60-mesh screen. Platform shaker (Eberbach Corp., Ann Arbor, Michigan). Tube rotator (BBL, Cockeysville, Maryland) or equivalent Accurately weigh 10 erythromycin tablets and finely grind using a Wiley mill with a 60-mesh screen. Accurately weigh a portion of the powder containing approximately 500 mg of erythromycin into a roundbottom ground glass-stoppered centrifuge tube. Add 25.0 ml of methylene chloride and stopper tightly. Shake vigorously or rotate continuously for 45 rain. Centrifuge at 10,000 g for 15 rain. Pipette 500 ~1 of methylene chloride extract into a l-dram screw cap vial and evaporate to dryness under a stream of dry nitrogen. To ensure complete dryness, the samples are then dried for an additional 10 rain in a vacuum oven at 60 ° at less than 5 mm Hg pressure. ~J. H. Robertson and K. Tsuji, J. Pharm. Sci. 61, 1633 (1972)

[91

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

247

Silylation Procedure One milliliter of the internal standard silylation reagent is added to the vial containing erythromyein by means of a glass tuberculin syringe. The cap, lined with a 26-ml polyethylene liner, is tightly sealed and placed in an oil bath (silicone) at 75 ° for 24-36 hr. Approximately 1 tA of the silylated sample is injected into the chromatograph at an attenuation and range setting of 64 X 10.

Calculation Since the microbiological response of erythromycins B and C are 50 and 40%, respectively, that of erythromycin A against Staphylococcus aureus H (ATCC 9144), 2~ the following formula was devised to make the GLC data comparable with that of the microbiological assay method: GLC calculated biopotency (ug/mg) EA+0.5EB+0.4EC = E~-~-0~5E~--~4~CJ

] × [Is~I,] × [ W s / W , I × F

(5)

where EA = erythromycin A peak area of a sample; EB = erythromyein B peak area of a sample; EC = erythromycin C peak area of a sample; ESA = erythromycin A peak area of the reference standard; ESB erythromycin B peak area of the reference standard; ESC = erythromycin C peak area of the reference standard; Is = internal standard peak area of the reference standard; It = internal standard peak aiea of the sample; W8 = weight of the erythromycin reference standard (mg) ; Wt = weight of erythromycin sample (rag); F = assigned value of the erythromycin reference standard expressed in micrograms of erythromycin base per milligram of the standard. A typical chromatogram may be seen in Fig. 5.

Comments on the Assay Method Because of the high operating temperature, an 0V-225 column is stable for approximately 3 weeks. Although the use of the 0V-225 column made it possible to separate erythromycin A from various impurities, the use of the PPE-20 or OV-210 column is required for the separation of erythromycin B and anhydroerythromycin A. The GLC method for erythromycin may be used to analyze monoand diacetyl erythromycin A, propionate esters of erythromycin, and the stearate salts of erythromycin.

248

METHODS FOR THE STUDY OF ANTIBIOTICS

5

I 4

8

I

[9J

6

I

12 16 TiME (MINUTES)

I 20

24

FIG. 5. Chromatogram of erythromycin indicating separation of (1) internal standard, (2) acid-hydrolyzed erythromycin, (3) erythromycin C, (4) erythromycin A, (5) and (7) isomers of erythromycin A, and (6) erythromycin B and/or anhydroerythromycin A. V I I I . Penicillin 27 Penicillin is chromatographed intact as the silylether derivative using an OV-17 column. Procedure Materials

OV-17, 3% on Gas Chrom Q 10(O120 mesh (Applied Science Lab, Inc.) Column, glass, 3 mm i.d. X 610 mm (2 ft) Chloroform, A.R. 2, C. ttishta, D. L. Mays, and M. Garofalo, Anal. Chem. 43, 1530 (1971).

I91

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

249

Pyridine, A. R. Vial, glass 5-a-Cholestane (Mann Research Lab, Inc., New York, New York) Hexamethyldisilazane (HMDS), Pierce Chemical Co.

Solutions pH 2.2 buffer solution. A saturated aqueous solution of ammonium sulfate is adjusted to pH 2.2 with concentrated sulfuric acid. Internal standard-silylation reagent. A 50% (v/v) solution of HMDS in pyridine containing 0.375 mg/ml of 5-a-cholestane was prepared.

Apparatus Gas chromatograph. For requirements, see Section III, D Detector: flame-ionization detector

Chromatographic Conditions Column: glass 3 mm i.d. X 610 mm packed with 3% OV-17 Gas flow rate: hydrogen, 50 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 215 ° Detector temperature: 230 ° Flash heater temperature: 215 ° Chart speed: 0.64 cm/min

Preparation o] Chromatographic Column Follow the method described in Section III, D. The column thus prepared normally has 1450 theoretical plates per meter for silylated penicillin G.

Preparation o] Bulk Drug and Re]erence Standard Solutions Penicillin bulk powder and reference standard powder are accurately weighed and dissolved in water at a concentration of 20 mg/ml. Pipette 2.0 ml of this solution into a glass centrifuge tube with ground-glass stoppers and add 8.0 ml of chloroform and 2 ml of pH 2.2 buffer. Shake the mixture vigorously for 1 min and centrifuge. Remove the aqueous layer and pipette 2.0 ml of the chloroform layer to a serum vial. Because of the poor solubility of procaine penicillin G in water, about 5 mg is accurately weighed into a 5-ml serum vial with 2 ml of chloroform.

250

METHODS FOR THE STUDY OF ANTIBIOTICS

[9]

Silylation Procedure

Add 2 ml of the internal standard-silylation reagent to each vial. Cap the vial tightly, mix, and then allow to stand at room temperature for 10 min for penicillin G, penicillin V, D- and L-phenethicillin, and methicillin. Oxacillin, eloxacillin, and dicloxacillin require up to 60 min for complete silylation. Chromatograph 2 tJ of the silylated penicillin. Calculation

Calculate the content of penicillin in micrograms per milligram of sample using Eq. (1) described in Section III, B. Comments on the Assay Method

Since penicillin is unstable in the pH 2.2 buffer, the extraction step must be done as quickly as possible. During silylation the procaine cleaves off from procaine penicillin G molecule and chromatographs as procaine and TMS-penicillanic acid. No degradation product was seen

I 4 TiME (MINUTES)

I 12

FIQ. 6. Separation of (1) penicillin G and (2) penicillin V.

[9]

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

251

under these chromatographic conditions when penicillin was degraded by either penicillinase or by acid. A typical chromatogram is shown in Fig. 6. The method is not applicable to ampicillin.

I X . T e t r a c y c l i n e 28

Tetracycline is chromatographed intact as the pentatrimethylsilyl ether derivative using a 3% JXR column.

Procedure Materials JXR, 3% on Gas Chrom Q, 100-120 mesh (Applied Science Lab, Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 1830 mm (6 ft) Vial, glass 1-dram screw-cap vial (No. 60910, Kimble Glass, OwensIllinois, Toledo, Ohio) with 26 mil polyethylene liner Pyridine, A.R. Trimethylchlorosilane (TMCS), Pierce Chemical Co. N,O-Bis(trimethylsilyl)acetamide (BSA), Pierce Chemical Co. Trioctanoin (Eastman Organic Chemicals, Rochester, New York)

Solutions Internal standard-silylation reagent. Add 5 ml of BSA and 5 ml of TMCS into 10 ml of pyridine containing about 3 ~i trioctanoin per milliliter. Cap tightly with a polyethylene lined stopper and mix well. Prepare the internal standard silylation reagent just prior to the silylation of samples.

Apparatus Gas chromatograph. For requirements, see Section III, D. Detector: flame-ionization detector

Chromatographic Conditions Column: Glass, 3 mm i.d. X 1830 mm packed with 3% JXR Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium) 55 ml/min Oven temperature: 260 ° K. Tsuji and J. H. Robertson, Anal. Chem. 45, 2136 (1973).

252

METHODS FOR THE STUDY OF ANTIBIOTICS

[9]

Detector temperature: 290 ° Flash heater temperature: 260 ° Chart speed: 0.64 cm/min

Preparation o] Chromatographic Column Follow the procedure described for neomycin powder in Section III, D. After the no-flow conditioning of the column at 310 ° for 30 rain, bring the oven to room temperature. Turn the oven temperature to 260 ° and carrier gas to 55 ml/min. Inject approximately 50 tL1 of Silyl-8 when the oven temperature reaches 200 ° and 2-3 t~l of the silylated tetracycline when the oven temperature reaches 260 °. Maintain the GC under the chromatographic condition overnight. Prior to the analysis, inject two silylated tetracycline samples to stabilize the column. The J X R column thus prepared normally has about 2500 theoretical plates per meter for silylated tetracycline.

Preparation o] Bulk Drug and Re]erence Standard Powder Accurately weigh approximately 10 mg of tetracycline bulk powder and the reference standard powder into a 1-dram vial.

Silylation Procedure One milliliter of the internal standard silylation reagent is added to each vial containing tetracycline by use of a glass tuberculin syringe. Seal the vial tightly and silylate at 20-25 ° for 24 hr. Approximately 2 t~l of the silylated sample is injected into the chromatograph at an attenuation and range setting of 32 X 10.

Calculation Calculate the content of tetracycline in micrograms per milligram of sample using Eq. (1) described in Section III, B.

Comments on the Method Since the separation factors between tetracycline (TC), 4-epitetracycline (ETC), anhydrotetracycline (ATC), and 4-epianhydrotetracycline (EATC) by the J X R column are small, it is essential to have a highly efficient column and operate it at optimum chromatographic conditions. In our hands, the J X R column routinely showed approximately 2500

[9l

GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS

253

theoretical plates per meter for the silylated tetracycline and the JXR column was the only one which made possible the chromatography of TC on a 6 ft-long column. Degradation products of TC may easily be formed during the silylation procedure. Therefore, strict adherence to the silylation procedure described plus the use of fresh silylation reagents for the preparation of the internal standard-silylation reagent and the maintenance of the reaction temperature at 20-25 ° are essential. Even under carefully controlled conditions, a slightly higher ETC in TC samples resulted by the GLC when compared to those obtained by the high-pressure liquid chromatography (HPLC) 29 which requires no derivatization. We prefer the HPLC method for TC analysis because of its superior sensitivity, high speed, and ease of analysis. X. Miscellaneous

The following GLC methods for the analysis of antimyein A, celestosaminide, phosphonomyein, thiamphenicol, and validamycin have not been critically evaluated by the authors. The procedures described in this section are as they appeared in publications. A. Peptolide Antibiotic--Antimyein A 8° Trimethylsilyl ether of antimycin A (blastomycin) was prepared. Five milligrams of antimycin or blastomyein were dissolved in 0.5 ml T H F and heated for 5 min with 0.2 ml HMDS and 0.1 ml TMCS. Then, most of the reagents were removed under a stream of dry nitrogen. A 3 mm i.d. X 1500 mm glass column packed with 1.5% SE-30 on 80-100 mesh diatomaceous earth (Shimalite, Shimazu Seisakusho Ltd., Japan) was used isothermally at 151 ° with a flame ionization detector. He, 57 ml/min. B. C e l e s t o s a m i n i d e 31

Approximately 5 mg of the compound were sealed in a 0.3-ml Reactivial (Pierce Chemical) and was evacuated. Either Supelco silylating reagent or Powersil (DuPont Co.), both having the same composition of BSA, TSIM, and TMCS = 3:3:2, were used for derivatization. The vials wcre heated 5-10 rain at 70 °. A 3 mm i.d. }( 1220 mm glass column packed with 3.8% UCW-98 on 80-100 mesh Diatoports was used isothermally at 280 ° or 290 ° with a flame ionization detector. 59 K. Tsuji, J. H. Robertson and W. F. Beyer, Anal. Chem. 46, 539 (1974). 2oT. Endo and H. Yonehara, J. Antibiot. 23, 91 (1970). 31T. F. Brodasky and A. D. Argoudelis, J. Antibiot. 0.6, 131 (1973).

254

METHODS FOR THE STUDY OF ANTIBIOTICS

[9]

C. P h o s p h o n o m y c i n 32

The sample was dissolved in BSA and heated for 5 min at 60 ° or allowed to stand at room temperature for 30 min to form the TMS derivative. Chromatography was carried out with a 1-2 ~l injection of the silylated derivatives using a glass column, 4 mm i.d. X 1830 mm (6 ft), packed with 4% F-60 (DC-560). coated over 1.5% SE-30 on 100-120 mesh Gas Chrom P. The oven temperature was programmed for 65 ° to 220 ° at 3 ° per minute and then held at 2 2 0 ° for 10 min. A hydrogen flame ionization detector was used. D. Thiamphenico183

Samples of 0.5 or 1.0 ml of biological fluid containing thiamphenicol was accurately added to 5.0 ml of ethyl acetate, and the mixture was vigorously shaken for 5 min. Anhydrous Na~S04 was used to dehydrate the sample. Inject 3 ~l of the ethyl acetate solution directly into a glass column, 4 mm i.d. X 1500 mm, packed with 1.5% DEGS (diethylene glycol succinate polyester) on Chromosorb W, 60-80 mesh, and chromatographed under the following conditions: column and detector temperature, 185°; injector port temperature, 220°; nitrogen (carrier gas), 50 ml/min; for quantitative determinations, nitrogen flow rate was increased to 150 ml/min. When using the TMS ester method, 0.5 or 1.0 ml of the ethyl acetate solution was pipetted into a test tube and evaporated in a vacuum desiccator. Two hundred microliters of TMS reagent were added, and the test tube was stoppered and left :at room temperature for 10 min. The solution was evaporated to dryness in a vacuum desiccator. A suitable volume of pyridine was added to the residue, and 2 ~l was injected into a stainless steel column, 4 mm i.d. X 750 mm, packed with 1.5% OV-17 on Shimalite IV, 80-100 mesh (Shimazu, diatomaceous earth) under the following chromatographic conditions: Column and detector temperature, 215°; injection port temperature, 240°; nitrogen flow rate, 130 ml/min. E. Validamycin 34

Approximately 1 mg of sample was weighed into a rubber-capped small tube and dissolved into 100 ~l pyridine. BSA (100 ~l) and TMCS (50 ~l) were then added. The tube was heated for 30 min at 70-80 °. A 32 H. Shafer, W. J. A. Vanderheuyel, R. Ormand, F. A. Kuehl, and F. J. Wolf, I. Chromatogr. 52, 111 (1970). ~ T. Aoyama and S. Iguchi, J. Chromatogr. 43, 253 (1969). 3, S. Horii, Y. Kameda, and K. Kawahara, J. Antibiot. 25, 48 (1972).

[9]

GAS-LIQUID

CHROMATOGRAPHY

OF

ANTIBIOTICS

255

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256

METHODS FOR THE STUDY OF ANTIBIOTICS

[10]

Hitachi 063GC equipped with flame ionization detector was used with the following chromatographic conditions: 1. For TMS validamycins A to F: Glass column, 3 mm i.d. X 2000 mm packed with 1% OV-1 on Chromosorb W AW DMCS. Temperature: column, 280°; injection, 300 °. Carrier gas (helium), 60 ml/min. 2. For separation of TMS-validoxylamines A and B: Glass column, 3 mm i.d. X 2000 mm packed with 3% OV-17 on Chromosorb W AW DMCS. Temperature: oven, 250c; injection, 300 °. Carrier gas (helium), 30 ml/min. 3. For separation of TMS-derivatives of degradation products: Glass column, 3 mm i.d. X 2000 mm packed with 5% OV-17 on chromosorb W AW DMCS. Column temperature: initial, 150°; final, 280 ° (10°/rain). Injection temperature, 300 °. Carrier gas (helium) : 45 ml/min.

Acknowledgment The authors are indebted to their many colleagueswho provided suggestionsand data for this chapter.

[10] I o n - E x c h a n g e C h r o m a t o g r a p h y o f Streptothricin-like Antibiotics

By

DONALD B . BORDERS

I. Introduction . . . . . . . . . . . . . . . . . . II. Ion-Exchange Chromatography of Intact Antibiotics. . . . . . . III. Ion-Exchange Chromatography of Hydrolysis Fragments . . . . .

256 258 260

I. Introduction Members of the streptothricin family of antibiotics are very watersoluble, strongly basic compounds with broad antimicrobial spectra. One of the most effective means of resolving these antibiotics and their hydrolysis products is by ion-exchange chromatography. It is assumed that the ion-exchange chromatographic techniques which apply to streptothricin-type antibiotics would be of general interest since so'me of the techniques might also apply to a number of other structurally different antibiotics having similar chromatographic and ionic properties.