Screening for Basic Drugs in 2-mL Urine Samples by Dual-Plate Overpressured Layer Chromatography and Comparison with Gas Chromatography-Mass Spectrometry

Journal of Analytical Toxicology, Vol. 27, May/June 2003 Screeningfor BasicDrugsin 2-mt Urine Samplesby Dual-Plate OverpressuredLayerChromatographyand Comparisonwith Gas Chromatography-Mass Spectrometry Anna Pelander*, Ilkka Ojanperli, Johanna Sistonen, Ilpo Rasanen, and Erkki Vuori Department of ForensicMedicine, P.O. Box 40, F/N-O0014 University of He/sin~d, Finland Abstract I A dual-plate overpressuredlayer chromatography (OPLC) method was evaluated for broad-scale screeningof basic drugs in 2-mL autopsy urine samples. Extraction was carried out by mixed.mode solid.phase extraction, and identification was based on automated comparison of corrected Rf values (hRtc) and in situ UV spectra with library values by dedicated software. The day-to-day precision of hRfcvalues was good in both OPLC1 and OPLC2 systemswith median relative standard deviations of 2.4% and 3.4%, respectively. Both Rf and hRfc values were independent of the amount of analyte (0.5-10 pg) applied to the plate. Detection limits were determined for 47 drug substancesin 2-mL urine samples, and they varied between 0.05 and 3.5 mg/L with a median of 1.0 mg/L. The performance of OPLC was evaluated by analyzing 30 autopsy urine samplesby both OPLC and gas chromatography-massspectrometry (GC-MS). The majority of findings by OPLC were in agreement with GC-MS. Some substanceswith low concentrations were not detected by OPLC, whereas GC-MS failed to detect a few polar substances.The OPLC method thus provides an alternative for current planar and column liquid chromatographic drug screening methods with the possibility of lowering detection limits by usinga larger sample size. Introduction Broad-scale screening analysis for drugs and poisons continues to be a major challenge in analytical toxicology. Thinlayer chromatography (TLC) in instrumental mode, utilizing corrected Rfvalues (hR~:)(1) and in situ ultraviolet (UV)spectra as identification parameters, has proved to be a reliable and efficient technique in drug screening (2--4). However,a drawback of free-flowTLC is limited separation power, and with the complex matrices encountered in forensic and clinical toxicology, 9 Author to whom correspondenceshould be addressed.E-mail:. 226 insufficient separation may severely affect the analysis. Overpressured layer chromatography (OPLC)is a forced-flow planar chromatographic technique originally developed by 'I~ih~ and Mincsovics(5). In OPLC,the separationis performed in a pressurized chamber and the flow rate of the mobile phase is kept constant by an external pump. As diffusion is diminished with constant flowrate, longer separation distances can be used, resulting in higher separation numbers than in TLC (6,7). In addition, OPLC is less vulnerable to external conditions than free-flowTLC because the separation system is closed. A broad-scale drug-screening approach based on OPLC with two independent separation systems (6) and improved UV library search (8) has been described earlier. The present study validates the method for postmortem toxicology in terms of chromatographic precision and limits of identification in autopsy urine. Experimental Materials and apparatus The chromatographic plates were 20- • 20-cm aluminum sheets coated with a 140- to 180-1Jm layer of silica gel F2s4of 5-1Jmparticle size. The plates were original Merck (Darmstadt, Germany) products, sealed for OPLC purposes, and marketed under the trademark HTSorbTM by Bionisis OPLC (Le PlessisRobinson, France). Drug standards were obtained from various pharmaceutical companies and were of pharmaceutical purity. Standard stock solutions and correction standard mixtures were made in methanol at a concentration of 1 or 2 mg/mL, and dilutions were made in methanol as needed. The solid-phase extraction (SPE) of urine samples was performed with IST mixed-mode (C8 and cation exchange) HCX 130-rag cartridges (International Sorbent Technology,Hengoed, U.K.). All solvents used were of analytical reagent grade. Fast Black K Reproduction (photocopying) of editorial content of thisjournal is prohibited without publisher's permission. Journal of Analytical Toxicology, Vol. 27, May/June 2003 salt (FBK)was from Aldrich (Milwaukee,WI). ~Glucuronidase was from Roche Diagnostics (Mannheim, Germany). Urine samples were collected at autopsy. Sample application to the plates was performed with an ATS III instrument (Camag, Muttenz, Switzerland).The OPLC instrument was a Personal OPLC Basic System 50 (Bionisis). A TLC Scanner 3 (Camag) operated with Cats 4.03 software was used for scanning densitometry. Gas chromatography-mass spectrometry (GC-MS) was performed with a 5973 mass selective detector coupled to a 6890 Plus GC, equipped with a 7683 injector and an HP-5MS (12 m • 0.20mm i.d., 0.33-pm film) capillary column (Agilent Technologies, Palo Alto, CA). GC--MS was operated by Chemstation software. Sample pretreatment Urine samples of 2 mL were hydrolyzed for 2 h at 57~ after the addition of 10 mL of 13-glucuronidaseenzyme solution. SPE was performed according to the manufacturer's application note with minor modifications (9). The pH of the samples was adjusted between 5 and 7 by adding 2 mL of 0.1M pH 6 phosphate buffer. The SPE cartridges were solrated and equilibrated with 2 mL of methanol, 2 mL of water, and 3 mL of 0.1M pH 6 phosphate buffer. After sample addition, the cartridges were rinsed with 1 mL of 0.1M pH 6 phosphate buffer and dried under full vacuum for 5 min. The cartridges were further rinsed with I mL of 1M acetic acid, and again dried for 5 rain. The acidic-neutral fraction was eluted with 3 mL of ethyl acetate/hexane (25:75, v/v) and discarded. The cartridges were dried for 2 min and rinsed with 6 mL of methanol, and again dried for 2 rain. Basic drugs were eluted with 3 mL of ethyl acetate/ammonium hydroxide (98:2, v/v). The extracts were evaporated to dryness at 40~ and reconstituted with 50 pL of methanol. An aliquot of 10 pL was applied to each of the two OPLC plates as a narrow band using the spray mode, and art aliquot of 2 pL was injected to GC--MS. Planarchromatographyand detection For OPLC1, the correction standard components were codeine (hP~c= 16), promazine (hR~:= 38), nortriptyline (hP~ = 58), moperone (hRic = 76), and theophyiline (hl~ = 86). The amount of each component applied to the plate was 1 pg. The mobile-phase composition was trichloroethylene/ methylethylketone/n-butanol/acetic acid/water (17:8:25:6:4, v/v), and the platewas developedwithout presaturation. The external pressure was 50 bar, the flow rate 450 laUmin, the volume of rapid delivery 300 pL, and the mobile phase volume 5500 pL (development time 12 min 19 s) (6). For OPLC2, the correction standard components were codeine (hRfc = 9), promazine (hR~: = 19), amitriptyline (hRtc = 40), levomepromazine (hRtc = 60), and dextropropoxyphene (hRfc = 94). The amount of each component applied to the plate was I pg. The mobile-phase composition was butyl acetate/et (...truncated)