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CLIN. CHEM. 20/2, 307-309 (1974) Technique for Rapidly Separating Drugs from Biological Samples Vijay Aggarwal, Raymond Bath, and Irving Sunshine With use of a sample/solvent ratio of 8.5 ml to 100 zl, certain drugs may be extracted efficiently from urine that has been saturated with a solid buffer. An aliquot of this extract may be injected gas chromatograph. directly into a is of some drugs of abuse. We show how this procedure applied to confirmation Additional urine Keyphrases: #{149} toxicology gas chromatography drug abuse drugs in #{149} #{149} Many commonly used techniques for detecting drugs in biological samples (e.g., gas chromatography, thin-layer chromatography, fluorimetry, and ultraviolet spectrophotometry) require that the drugs first be separated from the biological matrix. They usually are separated by extraction with an immiscible organic solvent or by adsorp- most other procedures, in which relatively large volumes of ether or chlorinated hydrocarbons are used as extractants. However, total sample preparation time is still long, because it is necessary to “clean up” the extract. We have developed a faster and simpler procedure, in which the need for purification of the initial extract is eliminated without, in most cases, a concomitant decrease in the quality of the final extract. Most of our experience with this technique, and the major concern of this note, is with the separation of organic bases from urine samples, but the same principles can be applied to the separation of barbiturates. Materials and Methods Reagents tion onto a solid support. These are time-consuming methods, and frequently yield extracts that contain many normally present substances that may interfere with the subsequent identification procedure. In an effort to avoid these problems, several investigatom have used solid buffers and small volumes of solvent to extract drugs rapidly. Bastos et al. (1) reported that or- All solvents and chemicals are ACS reagent grade. Solid buffer: a 3:2 (by wt) mixture of NaHCO3 K2C03. ganic bases can be isolated from urine by saturating the urine with potassium carbonate and then extracting with 1.0 ml of ethanol. This ethanol extract is then purified by shaking it with a pH 8.5 buffer and ethyl ether. The drugs in question concentrate in the ether-ethanol phase. Horning et al. (2) reported a separation procedure in which diluted plasma is saturated with potassium carbonate and then extracted with two 1.0-ml portions of isopropanol. This isopropanol extract is evaporated, and the residue dissolved in methanol, which is then washed with isooctane. The isooctane, which now contains any lipid material, is discarded and the washed methanol extract is used for the analysis. Use of small volumes of ethanol in these procedures decreases the solvent evaporation time as compared with Procedure County Coroner’s Laboratory, and the Department School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106. Cuyahoga of Pathology, Received Oct. 25, 1973; accepted Nov. 19, 1973. Internal standard: 5 mg of methoxyphenamine and in 100 ml of water. Extraction solvent: a 4:1 (by vol) mixture of chloroform and isopropanol. Add enough solid buffer (about 5 g) to saturate 10 ml of urine. The resulting solution has a pH of 9.5. Shake the mixture well and centrifuge, to separate excess buffer and any other insoluble material. Transfer 8 ml of the supernatant buffered urine to a 20-mi test tube that tapers to a sharp tip designed to contain 10-20 zl (available from Fuchs and Sons, Pittsburgh, Pa. or from Fisher Scientific, 711 Forbes Ave., Pittsburgh, Pa. 15219). Add 0.5 ml of the internal standard solution and, while mixing with use of a vortex-type mixer, add 100 zl of the extraction solvent. Vortex-mix the contents of the tube for 20 s after the solvent is added, then centrifuge for 5 mm. Aspirate and discard most of the supernatant urine and with a 100-zl syringe, remove an aliquot of the solvent layer (usually about 60 ,il). Use of the test tube with a sharp taper best facilitates removal of the solvent layer, but if these specific tubes are unavailable, conventional tapered centrifuge tubes may be used. CLINICAL CHEMISTRY, Vol. 20, No.2, 1974 307 r IS Results and Discussion Elution Al I times for the acetyl derivatives of amphet- amine, methamphetamine, and methoxyphenamine are close to those of the other organic bases; use of acetic anhydride permits a higher initial temperature and thus shortens the time required for gas chromatography. Codeine is not completely derivatized under the given conditions and so gives peaks for both codeine and acetylco- a Ii deine. The principal advantage of this extraction scheme is its part to the use of the very small volof extraction solvent. Because the volume of the speed, owing in large umes final extract is so small, it can be quickly concentrated at room temperature, obviating the need for heating baths, air streams, and the like. Complete evaporation must be 54 54 IS IS 54 TISE IN MINUTES Fig. 1. A: Analysisof a typicaldrug-free urine (IS, internal standard). B: Analysis of the same urine as in A, with drugs added to a final concentration of 2.0 zg/ml of each drug. C: Analysis of a urine sample from a methadonemaintained patient. Note 6) and acetylcodeine were also observed peaks (peak similar to codeine (peak 7). These same two peaks when acetic anhydride was omitted 3, caffeine;4,methadone metabolite; 5, methadone; 6,codeine;7,acetylcodeine; 8. quinine 1, amphetamine; 2, methamphetamine; Table 1. Sensitivity and Extraction Data Percent cxtracted Amphetamine Methamphetamine Methadone metabolite Methadone Codeine Quinine Based #{176} on analysis of Senslvity, CV, %(R)#{176} pg/mt 30 60 55 50 60 60 to which the of 2.5 ,hg/mI. (n of drug standard. 4.4 6.4 5.5 6 1 114 50 urines added to a concentration R = area of drug peak/area were = respective 10). 0.5 0.1 0.3 0 5 05 01 drugs avoided to ensure adequate recovery of volatile bases. Figure 1A shows the gas chromatographic trace obtained on analysis of a typical drug-free ,urine. Figure lB shows the trace for the same urine after drugs and drug metabolites were added to it. There are no major unknown peaks in these two traces, which demonstrates the selective isolation of drugs from the biological sample without the need for a “cleanup” step. In addition, as shown parable in Table to that 1, the amount of drugs achieved by techniques volumes of solvent are used. Analyses of over 1000 urine samples extracted is comin which larger obtained values for methadone and its metabolite were obtained by a procedure similar to that just described. When analyzed as described above, about 80% of the urine samples from patients in methadone maintenance programs yield peaks with retention times identical to those of codeine and acetylcodeine (Figure 1C). They also yield these same two peaks when acetic anhydride is omitted. This behavior is unlike that observed with authentic samples of codeine, so such urine samples presumably do not contain codeine. Because of this problem, another analytical be used if codeine has to be confirmed. The extract can be analyzed by any of several different techniques. After evaporation, an aliquot of the residue may be injected into a gas chromatograph without further treatment or after reaction with any one of several suitable derivatizing reagents such as N,O-bis(trimethylsilyl)acetamide, trimethylsilylimidazole, or acetic anhydride. We routinely transfered 40 ,l of the extract into the small aluminum capsule used in the Perkin-Elmer As-41 Autosampler. This capsule was allowed to stand at room temperature for 15 mm, to permit most of the solvent to evaporate. Before the capsule was sealed, 3 d of acetic anhydride was added. The capsule was then injected into a Perkin-Elmer Model 900 gas chromatograph, and chromatographed under the following conditions: Column: glass, 3 ft x 1/4 inch (o.d.), packed with “3% OV-17 on Chromasorb W, 80/100 mesh” (Supelco, Inc., Bellefonte, Pa. 16823). from pa- tients on a methadone rehabilitation program produced some peaks with retention times identical to those of peaks 1 to 8 in Figure 1. The areas of these peaks were always smaller than those used in calculating the minimal detectable amounts (sensitivity) listed in Table 1. The procedure should However, all other urines can be analyzed for codeine by this procedure, and conversely, the absence of peaks with retention times of 15.5 and 16.3 mm. reliably indicates that codeine is absent. Morphine also cannot be determined by this procedure. We used solid NaOH or NH4OH potassium to adjust carbonate because use of the pH of the urine samples resulted in the formation of emulsions, a tendency decreased by use of the solid buffer. An extraction solvent that is denser than sater must be used, because recovery of the solvent in the tapered tip after centrifugation is much easier than attempting to recover such a small volume from the surface should a low-density solvent be used. If the detection of amphetamine in urine is of primary concern, sensitivity may be improved by substituting 3 g of Na2CO3 for the recommended solid buffer. Although recovery of the other bases is decreased, the recovery of Oven temp: 130 #{176}C for 3 mm, then increased by 12 #{176}C amphetamine is about 60%. per minute to 280 #{176}C, and maintained at 280 #{176}C for 8 mm. When barbiturates are to be extracted from urine, a 3:1 Injection-port temp: 250 #{176}C. (by wt) mixture of NaH2PO4 and Na2HPO4 in place of the NaHCO3 and K2C03 buffer can be used. Plasma, diManifold temp: 300#{176}C. Gas flow rates: helium, 30 ml/min; hydrogen, 30 ml! luted with an equal volume of water, may also be used as mm; air, 300 mi/mm. the sample in this manner. 308 CLINICAL CHEMISTRY, Vol. 20, No.2, 1974 If the extract, prepared as described, is too contaminated for reliable analysis, a double small-volume extraction can be used. In this modification, the buffered urine is extracted with 1.0 ml of extraction solvent. After centrifugation, an aliquot of this extract is transferred to a tube containing 100 il of HC1 (0.4 mol/liter). After the mixture has been shaken and centrifuged, the HC1 layer is removed and made alkaline with 50 jl of NaOH (1 mol/ liter). This tion solvent, matograph. We have mixture is then an aliquot used the extracted with 50 il of extrac- of which is injected described procedure into the chroeffectively CHEM. 20/2, 309-311 (b) Sample manipulation is minimized, since a purification of the initial extract is not generally required. (c) Most importantly, this procedure’s brevity does not affect the quality of the extract, and highly reliable information may be gained from its use. Supported in part by grant 09863-11 the NIGMS, from NIH, USPHS. References 1. Bastos, and reliably for longer than a year as a confirmatory test for organic bases, other than morphine and codeine, in urine. We favor the described procedure over other techniques for several reasons. (a) Evaporation of large volumes of CLIN. solvents is eliminated. M. L., Kannanen, G. L., Young, R. M., Montforte, J., and Sunshine, I., Detection of organic bases and their metabolites in urine. Clin. Chem. 16,931 (1970). 2. Horning, M. E., Boucher, E. A., Stafford, M., and Horning, E. C., A rapid procedure for the isolation of dregs and drug metabolites from urine. Clin. Chim. Acta 37, 381 (1972). (1974) Single-pH Extraction Procedure for Detecting Drugs of Abuse1 Ramon E. Stoner and Connie Parker2 A method is presented for determining certain drugs of urine. The urine samto pH 6.0 with a buffer containing and determined phy (TLC). of abuse in a single extract ple is adjusted bromcresol by (e.g.) use of thin-layer chromatogra- Materials and Methods purple. Basic drugs, such as amphetam- ines and narcotics, form an organic salt with the ionized bromcresol purple, which is extractable with a mixture of chloroform and 2-propanol (3:1 by volume). At pH 6.0, weak acids such as barbiturates and neutral drugs such as glutethimide are also soluble in this solvent. Consequently, the major classes of drugs are extracted simultaneously. The extract is then concentrated and the individual drugs are determined by thin-layer chromatography in a solvent that will separate bromcresol purple from the drugs. Materials Silber-Porter 50-mi centrifuge H. Thomas, Philadelphia, Pa. 2619-F20, respectively). tubes 19105; (A. with stoppers No. 2619-B25 TLC development apparatus (No. 6071; Eastman N.Y. 14650). TLC sheets, Eastman no. 6061 silica gel. and Kodak Co., Rochester, Micropipets, 10-id (Camag Inc., New Berlin, Wis. 53151). Ultraviolet view box, containing and 366 nm. Spectrofluorometer, halla, N.Y. 10595). lamps emitting light at 254 Additional Keyphrases: drug screening thin-layer chromatography We would cedure needed a means save time without #{149}toxicology #{149} screening of screening sacrificing #{149} procedure urine samples proficiency. The that pro- described here was developed to extract acid, neutral, and basic drugs from urine at pH 6, with a buffer containing bromcresol purple. At this pH, acid and neutral drugs this drugs form are soluble an organic in the organic solvent. The salt with bromcresol purple, basic and salt (but not bromcresol purple) is also soluble in the solvent. Consequently, the major classes of drugs are extracted simultaneously, and may then be concentrated Clinical Laboratory Service, West Side VA Hosp., 820 S. Damen Ave., Chicago, Ill. 60612. 1 Presented in part at the 25th National Meeting, AACC, New York, N. Y., July 15-20, 1973. 2 Present address: Dept. of Medicine, University of Illinois College of Medicine, Chicago, Ill. Received Oct. 13, 1973; accepted Nov. 26, 1973. MK I (Farrand Optical Co., Val- Gas chrornatograph, (Hewlett Packard No. 5700A) fitted with 6 ft. x ‘ in. stainless-steel column packed with “3% OV 17 on HP Chromosorb W” (Supelco, Inc., Bellefonte, Pa. 16823) Evaporating bath, To handle a large number of samples per day, one needs a method of evaporating the solvent quickly. Such a device may be constructed by building an aluminum tube rack to fit into a water bath (set at 70 #{176}C). A 12-unit hematocrit tube cleaner is mounted over this rack and a latex rubber tube, 3 mm (i.d.) by 30 cm, is connected to each U-tube of the cleaner. To the other end of each of the rubber tubes is connected a 5-in. “Pharmaseal” connector (Scientific Products, McGaw Park, Ill. 60085; No. T5300). The free end of this connector is loosely inserted into the sample tube. The outlet of the hematocrit tube cleaner is connected to a vacuum line. Such a unit produces tubes. a decreased CLINICAL pressure CHEMISTRY, that is the same for all Vol.20, No. 2, 1974 309