Journal of Chromatography B (v.842, #2)

The phenethylamine-derived designer drug 4-ethyl-2,5-dimethoxy-β-phenethylamine (2C-E) was found to be mainly metabolized in rats by O-demethylation, N-acetylation, hydroxylation of the ethyl side chain at C2′ or at C1′ followed by oxidation at C1′ to the corresponding ketone, by deamination followed by reduction to the corresponding alcohols or by oxidation to the corresponding acids, and finally combinations of these steps. Most of the metabolites were excreted in conjugated form. The authors’ systematic toxicological analysis (STA) procedure using full-scan GC–MS allowed the detection of an intake of a dose of 2C-E in rat urine that corresponds to a common drug users’ dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-E in human urine.
Keywords: 4-Ethyl-2,5-dimethoxy-β-phenethylamine; 2C-E; Designer drug; Metabolism; GC–MS;

LC/MS/MS analysis of quaternary ammonium drugs and herbicides in whole blood by Marinah M. Ariffin; Robert A. Anderson (91-97).
Quaternary ammonium drugs (atracurium, bretylium, edrophonium, ipratropium, mivacurium, neostigmine, pancuronium and rocuronium) and herbicides (difenzoquat, diquat and paraquat) in human whole blood were analysed by LC/MS/MS with positive electrospray ionisation (ESI), following extraction with Bond Elut® LRC-CBA cartridges. Internal standards were benzyldimethylphenylammonium chloride monohydrate and ethyl viologen for drug and herbicide analysis, respectively. Ion-pair chromatography used heptafluorobutyric acid (15 mM)–ammonium formate (20 mM) buffer adjusted to pH 3.30 with formic acid and a linear gradient from 5 to 90% methanol run over 18 min. Recoveries ranged from 79.7 to 105.1%, detection limits were between 3.6 and 20.4 ng/ml and the intra- and inter-day precisions were less than 18.6% at a concentration of 10 ng/ml. The method was applied to a case of accidental paraquat poisoning in which the concentration of paraquat in blood was 0.64 mg/l, which is within the range associated with fatal paraquat poisoning.
Keywords: Quaternary ammonium compounds; Liquid chromatography–mass spectrometry;

A novel chiral derivatisation agent, (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, was used for the indirect resolution of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers using gas chromatography coupled to mass spectrometry operating in the negative-ion chemical ionization mode (GC/MS-NICI). This new chiral derivatisation reagent was readily obtained in optically pure form after a simple two-step synthesis. Optimal derivatisation was accomplished in 15 min at room temperature in a carbonate buffer and the resulting diastereoisomers were base line separated by GC in 12 min only. No racemization was observed during the derivatisation. The method was applied and fully validated for the enantiomeric quantification of amphetamines and methylenedioxylated amphetamines in hair. The analyses of 24 hair specimens from suspected ATS abusers showed that 24 cases were positive for MA and/or AM enantiomers and that in most cases the concentrations of (S)-MA and (S)-AM exceeded those of the corresponding (R)-enantiomers. One hair specimen was tested positive for both enantiomers of MDMA and MDA.
Keywords: Amphetamine; Amphetamine-type stimulants; (2S,4R)-N-Heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride; Enantiomeric ratios; Hair analyses;

A sensitive method for detection of methamphetamine (MA) and amphetamine (AP) in human hair was developed using solid phase microextraction (SPME) and one-pot derivatization. MA and AP were directly derivatized to N-propoxycarbonyl derivatives in an aqueous solution by propylchloroformate in a one-pot reaction before extraction by SPME. The derivatives were extracted to a coating of SPME from a headspace of the vial. The adsorbed derivatives were thermally desorbed in the injection port of a gas chromatograph. Pentadeuterated MA was used as an internal standard. The absolute recoveries of MA and AP from the spiked hair were 2.80–17.5%, respectively. The calibration curves showed linearity in the range of 0.05–20 ng/0.08 mg/vial for MA and 0.1–20 ng/0.08 mg/vial for AP in hair. Detection limits (S/N = 3) of MA and AP were 0.02 and 0.05 ng/0.08 mg/vial. The coefficients of variation of intraday were 1.04–26.4%. Additionally, this proposed method was applied to segmental analysis in clinical and medico-legal cases of MA intoxication.
Keywords: Amphetamine; Methamphetamine; Hair; Segmental analysis; Solid phase microextraction;

Hair to document exposure to glibenclamide by Marion Villain; Christine Tournoud; Françoise Flesch; Vincent Cirimele; Pascal Kintz (111-115).
Among the drugs that are used to incapacitate victims such as kids or elderly for sedation or for criminal gain such as sexual offences or robberies, glibenclamide, an antidiabetic was never mentioned. To document the interest of hair testing in such forensic situations, we have developed an original method to test for glibenclamide. A 30-year-old man was admitted to the Emergency Unit for coma and seizures after a party with some members of his family. Blood glucose was 0.40 g/l. A hair specimen was collected several weeks after the event and divided into two segments of 2 cm. Twenty milligrams of each segment cut into small pieces were incubated overnight in a phosphate buffer (pH 5.5), in presence of gliclazide used as internal standard (IS). A liquid/liquid extraction was realized with a mixture of diethyl ether/methylene chloride, and hair extract was separated on a XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection of glibenclamide was achieved using two transitions: m/z 493.9 to 168.9 and 493.9 to 368.8. Linearity was observed from 5 to 1000 pg/mg (r 2  = 0.956) with a limit of quantification at 5 pg/mg and a clean-up recovery of about 61%. Within-batch precision and bias were 9.0 and 9.5%, respectively. Ion suppression tested on drug-free hair was about 50%. Glibenclamide tested positive in the two consecutive segments (root to 2 cm: 23 pg/mg and 2–4 cm: 31 pg/mg). These findings were in accordance with a repetitive exposure to the drug. The concentrations were compared with those obtained after a single and a daily dose administration. In the hair of a subject receiving a single 5 mg dose and collected 4 weeks later, glibenclamide was detected in the proximal segment at 5 pg/mg. After a 20 mg/day dose, the hair concentration of a subject under glibenclamide therapy was 650 pg/mg.
Keywords: Drug facilitated crime; Glibenclamide; Antidiabetic; Hair; LC–MS/MS;

Rapid GC–MS analysis of methamphetamine and its metabolites in urine—application of a short narrow-bore capillary column to GC–MS by Hiroshi Fujii; Kenji Hara; Seiichi Kashimura; Mitsuyoshi Kageura; Masayuki Kashiwagi; Aya Miyoshi; Sachiko Ikeda (116-120).
A rapid analysis of methamphetamine and its metabolites in urine was performed by gas chromatography–mass spectrometry (GC–MS) using a short narrow-bore capillary column (NBC) (5 m × 0.1 mm I.D.). For detection, selected ion monitoring (SIM) was performed for the characteristic ions of each of the compounds. The analytes were independently detected within 2 min. Linearity was demonstrated over a range from 25–2500 ng/ml. As an application of this study, a urine sample from a drug-abuse suspect was analyzed. The analytes from the actual sample were detected with reasonable reproducibility. The results indicate the possibility of rapid analysis using a conventional GC–MS with a short NBC at a relatively low inlet pressure.
Keywords: GC–MS; Short narrow-bore capillary column; Amphetamines;

Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid–liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05–20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.
Keywords: Bovine serum albumin; LC–MS/MS; Urine; Stabilization of acylglucuronide metabolite; Automated liquid–liquid extraction;

Tiletamine and zolazepam injection (Telazol®) is used in veterinary surgical practice to induce short-term anesthesia and also to immobilize wild animals. The present work describes a sensitive method to measure tiletamine and zolazepam concentrations in plasma by means of GC/EI–MS on a 5% phenyl/95% methylpolysiloxane column. A simple liquid extraction procedure with ethyl acetate was used to isolate the two compounds and the same were separated and analyzed by GC/MS without derivatization. A formal validation of the assay demonstrated good accuracy and precision for both tiletamine (98–100.8%; C.V.total  < 6.7%) and zolazepam (98.3–103.4; C.V.total  < 13.2%). With 500 μl of plasma, the limits of quantification for both tiletamine and zolazepam were found to be 10 ng/ml. Both compounds were stable after three freeze-thaw cycles. The assay was used to analyze plasma samples collected from a pig after intramuscular administration of 10 mg/kg of Telazol®. The plasma concentration–time profile of tiletamine and zolazepam from this representative pig is also provided.
Keywords: GC/MS; Pharmacokinetics; Pig; Telazol®; Tiletamine; Zolazepam;

The enantioselective composition of the amphetamines is of interest, as the enantiomers show differences in their pharmacological effects and several methods for chiral separation of amphetamines have been described. Only a few methods have used whole blood as matrix and none of these separates both classic amphetamines (amphetamine and methamphetamine) and designer amphetamines (MDA, MDMA and MDEA). The aim of this study was, therefore, to develop a method for enantioselective analysis of AM, MA, MDA, MDMA, and MDEA in whole blood. The amphetamines were extracted from 0.5 g of whole blood by liquid–liquid extraction. After derivatization with R-MTPCl, the resulting diastereomers were separated by GC on a HP-5MS column and detected by SIM-MS. R-MTPCl was used as derivatization reagent because of the stability of this reagent and good separation of these analytes. Through the method, development time and temperature of the derivatization were optimized, and by admixture of 0.02% triethylamine it became possible to detect the amphetamines in adequately low concentrations as more analytes were derivatized. The method was validated and it was linear from 0.004 to 3 μg/g per enantiomer. The accuracy was within 91–115%, while the repeatability and reproducibility were ≤15% R.S.D. A method suitable for enantioselective separation and analysis of the amphetamines has been achieved, and the method was applied to analysis of whole blood samples originating from traffic and criminal cases and post mortem cases.
Keywords: Chiral separation; Amphetamines MDMA; Whole blood; GC/MS;