Oeilerich, Külpmann and Haeckel: Drug screening by enzyme immunoassay and .tliin-layer chromatography 275 J. Clin. Chem. Clin. Biochem. Vol. 15, 1977, pp. 275-283 Drug Screening by Enzyme Immunoassay (EMIT) and Thin-Layer Chromatography (Drug Skreen)1) By M. Oellerich, W. R. Külpmann and/?. Haeckel Technical Assistance : F. Behrends,L hberner and K. Petry Institut für Klinische Chemie (Geschäftsführcnder Direktor: Prof. Dr. Dr. J. Büttner) Medizinische Hochschule Hannover (Received June 12/December 16, 1976) Herrn Prof. Dr. Dr. h. c. G. Schettler zum 60. Geburtstag gewidmet Summary: Urine samples (n = 300) were examined for drugs by thin-layer chromatography ("Drug Skreen", Brink- mann Corp.) and by the "Enzyme Multiplied Immunoassay Technique" ("Emit", Syva Corp.). The results of both methods were compared for the detection of opiates, barbiturates and amphetamines. In more than 90% of the determinations identical results were obtained with both methods. About 10% of the results of the Emit barbiturate assay differed from those of thin-layer chromatography and therefore had to be further investigated by gas liquid chromatography. It could be demonstrated that the barbiturate determination by the Emit system correlated better with the results of gas liquid chromatography. From the results of this study it is suggested that thin-layer chromato- graphy is used as a screening test, and to confirm positive results with other methods such as Emit. If the abuse of barbiturates or opiates is suspected the corresponding Emit test should also be performed, even in cases of a negative thin-layer Chromatograph*, -screening. Confirmation with a third method such as gas liquid chromatography is necessary, if thin-layer chromatography and Emit lead to divergent results. Drogenscreening mit Enzymimmuntests (EMIT) und Dünnschichtchromatographie (Drug Skreen) Zusammenfassung: Urinproben (n = 300) wurden dünnschichtchromatographisch („Drug Skreen", Brinkmann Corp.) und mit Enzymimmuntests („Emit", Syva Corp.) auf Drogen untersucht. Die Ergebnisse beider Methoden wurden für den Nachweis von Opiaten, Barbituraten und Amphetaminen miteinander verglichen. In über 90% der Bestimmungen fanden sich identische Resultate mit beiden Methoden. Etwa 10% der Ergebnisse des Emit Barbiturattests divergierten mit denen der Dünnschichtchromatographie und mußten daher zusätzlich gaschromatographisch untersucht werden. Es konnte gezeigt werden, daß die Barbituratbestimmung mit dem Emit System besser mit den Resultaten der Gas- chromatographie korrelierte. Auf Grund der Ergebnisse dieser Studie wird vorgeschlagen, die Dünnschichtchromato- graphie als Screenings-Test zu verwenden und positive Resultate mit anderen Methoden wie z. B. Emit zu bestätigen. Bei Verdacht auf Barbiturat- und Opiatmißbrauch ist die zusätzliche Durchführung des entsprechenden Emit-Testes auch in Fällen eines negativen Dünnschichtchromatographie-Screenings empfehlenswert. Die Bestätigung mit einem dritten Verfahren wie z. B. Gaschromatographie ist erforderlich, wenn Dünnschichtchromatographie und Emit zu divergenten Resultaten führen. The treatment of dtug overdosage and the detection of The identification of drugs with thin-layer chromato- drug abuse require rapid analytical methods for the graphic techniques alone is difficult. Therefore drugs identification of drugs in biological material. have been eluted from the thin-layer plate and subse- t ,. quently examined by various methods such as color, In the past few years many thiiulayer Chromatographie H^ ^ ^ hotometric tests (13) techniques for the detection of drugs have been described ^ ^ sensitive methods (1-7). Instead of the time-consuming extraction of drugs for ^ verification of ^.^ chromatographic from unne with organic solvents Amberhte XAD-2 ne geveral ^^ columns were proposed (8-12). Combinations of drug lca rr / extraction by XAD-2 resin with a thin-layer ehromato- graphic screening technique (3) are commercially avail- i) p^ of this study have been reported at Anaiytica 76 in Munich 1976 (abstract: Z. Anal. Chem. 279, 132 (1976)). J. Clin. Chem. Clin, Biochem. / Vol. 15, 1977 / No. 5 276 Ocllerich, Külpmann and Haeckel: Drug screening by enzyme immunoassa'y and thin-layer chromatography metliods such as radioimmunoassay, spin immuno- Tab. 1. Detection criteria for hypnotics and sedatives. assay, hemaglutination inhibition and enzyme immuno- Compounds (trade name) Rf valuea Color reaction assay have also been applied for the detection of drugs with dip hen yl- (14-23). carbazone/mer- curie sulfate In the present study an enzyme immunoassay system (Emit, Syva Corp.) was evaluated for its use in the Phenobarbital (Luminal) 0.54 violet Brallobarbital 0.70 violet confirmation of opiates, barbiturates and amphetamines Barbital (Veronal) 0.70 violet detected by thin layer chromatography. Cyclobarbital (Phanodorm) 0.72 violet Allobarbital (Dial) 0.76 white Propylallylonal 0.79 violet Heptabarbital (Medomin) 0.80 violet Aprobarbital (Numal) 0.89 white Materials and Methods Vinylbital 0.89 violet Methylphenobarbital (Prominal) 0.91 violet Crotylbarbital 0.92 violet Origin of specimens Butalbital (Sandoptal) 0.93 white Urine samples (n = 300) were received from a drug abuse Cyclopal 0.94 white rehabilitation center, from the State Health Department Amobarbital (Amytal) . 1.00 violet Hannover, and from our hospital. Drug free control urine Butabarbital 1.03 violet was obtained from laboratory personal. Lyophilized human Pentobarbital (Neodorm) 1.06 violet urine spiked with various drugs (urine toxicology control pro- Amylallylbarbital 1,06 violet ficiency and urine drug check kit) was purchased from Cyana- Secobarbital (Seconal) 1.09 white mide GmbH (D-8000 Munich). Thiogenal 1.16 white Hexobarbital (Evipan) 1.21 violet Materials Enibomal (Euriarcori) 1.28 violet The reagents for the enzyme immunoassay (Emit) were purchased Glutethimide (Doriden) 1.64 violet from Syva Corp., Palo Alto, Calif, and materials for thin-layer Methaqualone (Mandrax)b 1,77 NRC chromatography (Drug Skreen) from Macherey & Nagel Carbromal (Adalin) - NR (D-5160 Diiren). Materials for gas liquid chromatography were ? obtained from Supelco (Bellfonte, Pa, USA) and all other - ) Relative to amobarbital. Solvent system: ethyl acetate/ chemicals from Merck AG (D-6100 Darmstadt). methano 1/330 g/1 ammonium hydroxide, 17 ml + 2 ml + 1 ml. k) Positive reaction with iodoplatinate and Dragendorffs reagent. Thin-layer chromatography (Drug Skreen) °) No reaction. Thin-layer Chromatographie determinations were performed according to the manufacturer's instructions. The drugs were Tab. 2. Detection criteria for various drugs. extracted by disposable Amberlite XAD-2 columns and separated by thin-layer chromatography on silica gel coated glass plates Compound (trade name) Rf Nin- iodo- (solvent system: ethyl acetate/methanol/330 g/1 ammonium value3 hydrin platinate hydroxide, 17 ml + 2 ml + 1 ml). For visualization of the drugs UV radiation, heat and group specific reagents were applied to Narcotics the plates. With each plate a control urine was processed con- Morphine 0.41 dark blue taining morphine, codeine, meperidine, methadone, ampheta- Codeine 0.72 brown mine, pheno-, seco- and amorbarbital at levels of 1-3 mg/L The Ketobemidone (Cliradon) 1.09 brown Rf-values of drugs were calculated relative to amobarbital (1). Levorphanol (Dromorari) 1.19 brown-violet Drugs were identified by their relative Rf-value, color reaction Pethidine (Dolantin) 1.28 violet and the comparison with the reference substances on the same Normethadone (Ticarda) 1.28 brown plate. Detection criteria of various drugs are given in table 1 Levallorphan (Lorfan) 1.59 brown^Violet and 2. Blind controls were performed with toxicological control Methado'ne ( ,-Polamidon) 1.64 (pink) brown urine (urine drug check kit, Lederie Diagnostics). Methadone Metabolite 1.70 (pink) brown Enzyme immunoassay (Emit) Stimulants The original procedure of the enzyme immunoassay for the Methamphetamine (Pervitin) 0.68 brown detection of drugs in urine (18) was slightly modified to in- Amphetamine (Benzedrin) 0.96 pink corporate the Eppendorf system 5085 (tab. 3). The further Strychnine 0.68 brown-violet evaluation of the results was performed according to the Fenethylline (Captagon) 1.32 brown-violet manufacturer's instructions. At the beginning and end of Methylphenidate (Ritalin) 1.43 brown-violet b each series a urine sample spiked with the drug tested was Bemegride (Eukraton) .·=· NR analyzed as precision control. The quantitative determination Miscellaneous of phenobarbital in serum by Emit was performed with an 0 Eppendorf analyzer 5010 (tab. 4). Quinine 0.72 brown 4~Aminophenazone 1.04 pink (pink) Propylhexediine (Eventin) 1.05 brown Gas-liquid chromatography Nicotine 1.34 green After the addition of 5 jug hexobarbital as internal standard the Fenfluramine 1.43 brown urinary samples were purified by chromatography on Amberlite Thioridazine (Melleril) 1.58 brown-yellow XAD-2. The eluates were evaporated under vacuum. The residues Diphenhydramine 1.58 brown were dissolved in a small volume of ethyl acetate/acetic acid Chlorpromazine (Megaphen) 1.59 blue-violet (100 ml + 1 ml) and applied to an aluminium sheet coated with Trifluopromazine (Psyquil) 1.61 browri^violet silica gel F2S4 (thickness of the layer 0.25 mm). The chromato- Amitryptiline (Laroxyl) 1.65 brown gram was developed with ethyl acetate/methanol/330 g/1 ammonium hydroxide (17 ml+2 ml+1 ml) and chloroform/ a) Relative to amobarbital. Solvent system: ethyl