A Comprehensive Gas Chromatography Procedure for Measurement of Drugs in Biological Materials
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Annals of Clinical and Laboratory Science, Vol. 5, N o. 1 Copyright © 1975, Institute for Clinical Science A Comprehensive Gas Chromatography Procedure for Measurement of Drugs in Biological Materials JAMES R. SHIPE, B.S. AND JOHN SAVORY, Ph .D. Division of Clinical Chemistry, Departments of Medicine, Biochemistry and Pathology and Hospital Laboratories, The University of North Carolina, Chapel Hill, NC 27514 ABSTRACT Procedures have been developed for the determination in serum and urine of 21 drugs which include all of the barbiturates, methyprylon, glutethimide, meprobamate, propoxyphene, methaqualone, primidone, diphenylhydantoin, methadone, codeine, morphine, amphetamine and methamphetamine. The method involves pipeting 1.0 ml of serum or urine into an extraction tube, adding an internal standard together with either a pH 4.9 or pH 8.3 buffer and extracting with chloroform. The drugs in the extract are concentrated by evaporation of the organic layer and identified and quantitated by gas-liquid chromatography (GLC) using a column packed with 3 percent OV 17. GLC analysis times are kept below seven minutes by using temperature program ming which allows good separations of drugs of widely varying volatilities. All calculations are based on peak areas determined by an electronic inte grator. Losses of drugs during extraction, evaporation and chromatography steps are compensated for by the use of internal standards. Actual extraction efficiencies of drugs from serum or urine range from 75 to 100 percent although recoveries, based on standards carried through the entire procedures, are quantitative. Precision (s.d.) based on replicate determinations is approx imately ± 0.2 mg per dl. Introduction the final analytical measurement of drugs. Increasing emphasis is being placed on Resolution of different compounds is ex identification and quantitation of drugs in cellent, low limits of detection are possible patients suspected of overdose. In addition, and the technique affords high precision. the monitoring of therapeutic levels of a Numerous reports have been made of number of drugs, especially those in the the measurement of individual drugs or antiepileptic category, is of considerable groups of drugs using gas chromatographic importance.7’9’13 techniques.1’3’4’6’10’11’12 However, the clin For most clinical laboratories, gas chro ical laboratory scientist has the problem of matography offers the best technique for making a selection from this wide variety 57 5 8 SHIPE AND SAVORY TABLE I chromatographic conditions are outlined in Gas Chromatographic Conditions table I. Carrier gas N2 45 ml per min C o l u m n s Hydrogen flow rate 50 ml per min Air flow rate 1.4 cubic ft per hr Glass U-shaped columns 3 ft X 2 mm in Detector temperature 250° C ternal diameter are packed with 3 percent Infector temperature 230° C OV-17 on Gas-Chrom Q 100-120 mesh. This packing is available commercially. § Column temperature Sedatives 140 to 240° C The columns are conditioned with normal program at 20° per min Amphetamines 150° C isothermal carrier gas flow by heating at 100° for one Alkaloids 240° C isothermal hour and increasing the temperature 2° per Electrometer range 200 on Model 2600 minute up to 240°. This final temperature Chart speed 1 in per min is maintained for 18 hours. Columns condi Recorder Fall scale range - 1 mV tioned in this manner will give excellent resolution for approximately one year. of methods to fulfill the needs of his labo S o l v e n t E v a p o r a t io n B a t h ratory. A fairly comprehensive approach to drug measurements was reported by Bar A water bath maintained at 75° is used rett1 and involved the determination of the to evaporate solvents following extractions. barbiturates, glutethimide, amphetamines, Nitrogen is used to aid the evaporation and alkaloids and the common tranquilizers. is supplied through a glass manifold and The present study uses most of the basic directed into the evaporation tubes through methodology described by Barrett but also Pasteur pipets. involves several modifications which in clude temperature programming for drug R eagen ts screening purposes and for monitoring B arbiturates , N e u t r a l s , || D il a n t i n therapeutic levels of the antiepileptic drugs. Sodium acetate buffer ( 1.0 M, pH 4.8). Precisely 410 ml of 1.0 N acetic acid and Special Apparatus 590 ml of 1.0 N sodium acetate are mixed and the pH adjusted to 4.8 with either G a s C hromatograph acetic acid or sodium acetate. A dual-column instrument* equipped Barbital internal standard. A stock solu with dual hydrogen flame ionization de tion of 100 mg per 100 ml is made up in tectors, dual electrometers and linear tem methanol. perature programming is employed. This Buffer (containing internal standard). system is coupled to two strip chart re Two ml of the barbital internal standard corders f and an electronic digital inte stock solution are diluted to 100 ml with grator equipped with a printer, j The gas acetate buffer. Diphenylhydanoin internal standard. A ° Model 2600 Bendix Corporation, Process In struments Div., Drawer 477, Ronceverte, WV stock solution is prepared by dissolving 100 24970. f Model SRG, Sargent-Welch Scientific Co., § Applied Science Laboratories, Inc., P.O. Box 7300 N. Linder Ave., Skokie, IL 60076. 440, State College, PA 16801. | Model 6230, Vidar Autolab, 77 Ortega Ave., || Neutrals are those drugs which extract at any Mountain View, CA 94040; NOW Div. of Spectra- pH. Those commonly encountered in the clinical Physics, 655 Clyde Ave., Mountain View, CA laboratory are: methyprylon, gluthethimide, and «4040. meprobamate. DRUG MEASUREMENT IN BIOLOGICAL MATERIALS BY GAS CHROMATOGRAPHY 5 9 mg 5-(p-methylphenyl)-5-phenyl hydan- S t a n d a r d S o l u t io n s toinfi in 100 ml of chloroform. Sedative slock standards. The pure drugs Chloroform (containing Dilantin inter were obtained from the appropriate manu nal standard). Two ml of the stock Dilan facturer. A stock standard of each of the tin internal standard are diluted to one following drugs was prepared by dissolv liter with redistilled chloroform. ing 100 mg (if present as sodium salt, Trimethylanilinium hydroxide (TMAH) volume weight for free acid must be in methanol ( 0.2 M ). Thirty ml of N, N- corrected) of each compound in 100 ml dimethyl aniline* are dissolved in 200 ml of methanol: methyprylon, aprobarbital, of ethyl acetate and added to 25 ml of butabarbital, amobarbital, pentobarbital, methyl iodide. The reaction mixture is al secobarbital, glutethimide, propoxyphene, lowed to stand overnight at room tempera methaqualone, phenobarbital, primidone, ture and the product, N,N, N-trimethylani- diphenylhydantoin, flurazepam. line iodide, recovered by filtration followed Sedative working standards. One ml of by recrystallization from hot absolute eth each of the stock sedative standards is anol. Exactly 5.2 g of the iodide are dis combined and diluted to 100 ml with solved in 100 ml of absolute methanol to water. The final concentration of each drug which are added 3.5 g of powdered silver is then one mg per 100 ml. oxide. The slurry is mixed at room temper Stock amphetamine standards (2.5 mg ature for one hour and the silver iodide per 100 ml). Exactly 42.75 mg of ampheta removed by filtration. The TMAH reagent mine sulfate and 31.10 mg of methamphet- is then stored in a 120 ml ground-glass amine HC1 are dissolved in one liter of stoppered bottle. water. Stock alkaloid standards (100 mg per 100 m l). Each of the following compounds is A l k a l o id s a n d A mphetamines dissolved in 100 ml of water: 131.6 mg Borate buffer (pH 8.3). A saturated so codeine phosphate, 133.6 mg morphine sul lution of sodium borate is adjusted to pH fate and 100 mg methadone. 8.3 with 2 N NaOH. Alkaloid and amphetamine working stan Amphetamine internal standard. Five mg dards. One ml of each of the stock alka N-propylamphetamine HClf are dissolved loids is combined with 20 ml of the am in 100 ml of water. phetamine stock standard and diluted to 100 ml with water. The final concentration Alkaloid internal standard. Five hundred of each alkaloid is then one mg per 100 ml mg of nalorphine hydrochloride are dis and 0.5 mg per 100 ml for the ampheta solved in 100 ml of water. mines. 'Nalorphine buffer. One ml of the stock nalorphine internal standard is diluted to Procedures 100 ml with borate buffer. S e d a t iv e a n d A nticonvulsant D r u g s Re-distilled acetic anhydride. Reagent grade acetic anhydride is distilled through One ml of serum or standard, one ml of a 12 inch Vigreaux column. acetate buffer and 10 ml of chloroform are added to a 15 ml ground-glass stoppered If Aldrich Chemical Co., Inc. centrifuge tube. For diphenylhydantoin de * Eastman Kodak. terminations, the chloroform contains the f Generously provided by J. W. Brackett, San Mateo County Coroner Toxicology Laboratory, diphenylhydantoin internal standard. The San Mateo, CA 94403. tubes are shaken for one minute to extract 60 SHIPE AND SAVORY phetamine and 20 ml of chloroform are combined in a 50 ml ground-glass stop pered centrifuge tube. The tubes are shaken vigorously for one minute, centri fuged and the upper aqueous layer re 9 moved by aspiration and discarded. The 14 organic phase is dried by shaking with one g of anhydrous sodium sulfate. A 10 ml aliquot of the chloroform is combined with 0.2 ml acetic anhydride in a 15 ml conical centrifuge tube and the contents evaporated to dryness.