Journal of Chromatography B (v.825, #1)

Preface by Marc LeBeau; Madeline Montgomery (ix).

Determination of urinary steroid sulfate metabolites using ion paired extraction by Adam T. Cawley; Rymantas Kazlauskas; Graham J. Trout; Adrian V. George (1-10).
The need for laboratories accredited by the World Anti-Doping Agency (WADA) to develop methods of analysis for steroids excreted primarily as their sulfate conjugates has faced significant analytical challenges. One of the issues relates to the extraction of these metabolites from urine in a relatively pure state. The use of (−)-N,N-dimethylephedrinium bromide as an ion pairing reagent was optimised to produce a method that is selective for the extraction of steroid sulfates prior to GC-MS or LC-MS analysis, with minimal contributions from the urine matrix. The recovery of androsterone from its sulfate conjugate was determined to be 67% with a relative quantitative uncertainty of ±14% (k  = 2).
Keywords: Doping control; Steroid sulfates; Ion pairing;

Methodology using MAE/SPME/GC–MS is being pursued for the analysis of organic pollutants in sebum. The microwave-assisted extraction (MAE) of standards of semi volatile organic pollutants from sebum was optimized. All compounds were extracted from sebum with recoveries analyzed by GC/MS ranging from 94% to 100% under the optimum MAE conditions: 10 mL acetone–hexane (2:1), 60 °C, and 10 min microwave heating. To improve the detection limits a SPME procedure was optimized. Linearity ranged from 0.70 ppb to 25 ppb. R.S.D. were in the range of 1–23% for the SPME step. Preliminary real samples were analyzed and a range of compounds was detected. The optimized MAE/SPME/GC–MS methodology promises to be useful for different applications.
Keywords: Sebum; Organic pollutants; Microwave-assisted extraction; Solid phase microextraction; GC–MS;

A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for the determination of LSD, iso-LSD and the metabolite 2-oxo-3-hydroxy-LSD in forensic applications. The procedure involves liquid–liquid extraction of the analytes and LSD-D3 (internal standard) from 1.0 g whole blood or 1.0 ml urine with butyl acetate at pH 9.8. Confirmation and quantification were done by positive electrospray ionisation with a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each compound were established and identification criteria were set up based on the retention time and the ion ratio. The curves of extracted standards were linear over a working range of 0.01–50 μg/kg for all transitions of LSD and iso-LSD. The limit of quantification was 0.01 μg/kg for LSD and iso-LSD. The method was applied to a case investigation involving a 26-year-old male suspected for having attempted homicide, where blood concentrations of LSD and iso-LSD were determined to 0.27 and 0.44 μg/kg, respectively. 2-Oxo-3-hydroxy-LSD was detected in the urine and confirmed the LSD abuse. The case illustrated the importance of analyte separation before MRM detection of a sample due to identical fragmentation ions of the isomers.
Keywords: LSD; ISO-LSD; Whole blood; LC/MS/MS;

To investigate the trend of non-controlled drugs of abuse, simultaneous analytical methods were developed using GC–MS and LC–ESI-MS for 8 tryptamines/β-carbolines, 6 phenethylamines of typically non-controlled substances in Japan, and, additionally, five legally controlled tryptamines and phenethylamines originally found in fungi or plants. Moreover, the proposed methods were applied to analyses of these drugs in 99 kinds of products (a total number of 123 products purchased at adult shops or via the Internet over the past 2 years in Japan), which potentially advertised psychotropic/psychoactive effects. The samples were extracted with methanol under ultrasonication. After centrifugation, the extracts were filtered prior to injections. GC–MS analysis was performed using a DB-5MS capillary column. Regarding the LC–ESI-MS analysis; the separation of the target drugs was optimized on an ODS column in acetonitrile/MeOH (7:3)–10 mM ammonium formate buffer (pH 3.5)/acetonitrile (95:5) by a linear gradient program and a quantitative analysis was carried out by the monitoring of each [M + H]+ in the positive ion mode of ESI-MS. As a result of the analyses using GC–MS and LC–ESI-MS, 5-MeO-DIPT (the synthetic substance known by the street name “Foxy”) was found in 8 out of the 99 kinds of products. Additionally, AMT (from brown powder), DMT (from dried plant), harmine and harmaline (from dried plant) were also found in some of the 99 products. These analytical methods could be useful for the investigation of the distribution of the non-controlled psychotropic tryptamines/β-carbolines and phenethylamines in the market.
Keywords: Tryptamines; Phenethylamines; Non-controlled substances; GC–MS; LC–ESI-MS;

A typical use of hair analysis in forensic toxicology is the documentation of previous drug administration. This is illustrated in a suicidal death of a 58-year-old epileptic patient who was treated with oxcarbazepine and probably with levomepromazine. The toxicological analysis carried out by HPLC/APCI/MS included also the hair (6 cm length) besides postmortem blood. The method was validated for levomepromazine, oxcarbazepine (OXCBZ) and its two metabolites, 10-hydroxycarbazepine (CBZ-10OH) and trans-diol-carbazepine (CBZ-diOH) in various biological matrices. The analysis of the postmortem blood indicated oxcarbazepine and its two main metabolites were present at therapeutic concentrations; levomepromazine was detected at a fatal concentration. In three 2-cm segments of hair, oxcarbazepine and its two metabolites were detected; however, levomepromazine was not detected in this specimen. As a result of complex chemical-toxicological investigation it was confirmed the information that the decedent. was an epileptic patient and was treated with oxcarbazepine for at least 6 months before death. In addition, he took a toxic dose of levomepromazine in order to commit suicide. The analysis revealed differences between the concentration levels of oxcarbazepine and its active metabolite CBZ-10OH in postmortem specimens and hair, suggesting different mechanisms of penetration of metabolites and their precursors into this matrix.
Keywords: Trileptal; Oxcarbazepine; Hair; LC/APCI/MS;

High-throughput screening of corticosteroids and basic drugs in horse urine by liquid chromatography-tandem mass spectrometry by Gary N.W. Leung; Evonne W. Chung; Emmie N.M. Ho; W.H. Kwok; David K.K. Leung; Francis P.W. Tang; Terence S.M. Wan; Nola H. Yu (47-56).
This paper describes two high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the screening of two important classes of drugs in equine sports, namely corticosteroids and basic drugs, at low ppb levels in horse urine. The method utilized a high efficiency reversed-phase LC column (3.3 cm L  × 2.1 mm i.d. with 3 μm particles) to provide fast turnaround times. The overall turnaround time for the corticosteroid screen was 5 min and that for the basic drug screen was 8 min, inclusive of post-run and equilibration times. Method specificity was assessed by analysing a total of 35 negative post-race horse urine samples. No interference from the matrices at the expected retention times of the targeted masses was observed. Inter-day precision for the screening of 19 corticosteroids and 48 basic drugs were evaluated by replicate analyses (n  = 10) of a spiked sample on 4 consecutive days. The results demonstrated that both methods have acceptable precision to be used on a routine basis. The performance of these two methods on real samples was demonstrated by their applications to drug administration and positive post-race urine samples.
Keywords: Basic drugs; Corticosteroids; Horse urine;

Enantioselective quantification of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers in hair using gas chromatography–mass spectrometry (GC–MS) is described. Hair specimens were digested with 1 M sodium hydroxide at 100 °C for 30 min and extracted by a solid phase procedure using Cleanscreen ZSDAU020. Extracted analytes were derivatised with (S)-heptafluorobutyrylprolyl chloride and the resulting diastereoisomers were quantified by GC–MS operating in the negative chemical ionization mode. Extraction yields were between 73.0 and 97.9%. Limits of detection varied in the range of 2.1–45.9 pg/mg hair, whereas the lowest limits of quantification varied between 4.3 and 91.8 pg/mg hair. Intra- and inter-assay precision and respective accuracy were acceptable. The enantiomeric ratios (R versus S) of AM, MA, MDA, MDMA and MDEA were determined in hair from suspected amphetamine abusers. Only MA and AM enantiomers were detectable in this collective and the quantification data showed in most cases higher concentrations of (R)-MA and (R)-AM than those of the corresponding (S)-enantiomers.
Keywords: Hair analysis; Amphetamine; Amphetamine derivatives; Enantiomeric ratio;

A new simple and rapid liquid chromatographic–mass spectrometric technique was designed for the determination of nine benzodiazepines in plasma and oral fluid. Benzodiazepines were extracted from alkalinised spiked and clinical plasma and oral fluid samples using a single step, liquid–liquid extraction procedure with diethyl ether. The chromatographic separation was performed with a Xterra® RP18, 5 μm (150 × 2.1 mm i.d.) reversed-phase column using deuterated analogues of the analytes as internal standard. The recovery ranged from 70.3 to 86.9% for plasma and 63.9 to 77.2% for oral fluid. The limits of detection ranged from 0.5 to 1 ng/ml in plasma and 0.1 to 0.2 ng/ml for oral fluid. The method was validated for all the compounds, including linearity and the main precision parameters. The procedure, showed to be sensitive and specific, was applied to real plasma and oral fluid samples. The method is especially useful to analyse saliva samples from drivers undergoing roadside drug controls.
Keywords: Liquid chromatography; Benzodiazepines; Plasma; Oral fluid; Saliva; Liquid-chromatography-mass spectrometry;

A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC–MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid–liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography–tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r 2  > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.
Keywords: Benzodiazepines; Hypnotics; Hair; LC–MS/MS;

The recent advances in fiber manufacturing technology, solid-phase microextraction (SPME) is now widely studied for its effectiveness for the pretreatment of various categories of samples. This study explores a novel SPME approach for enantiomeric analysis of amphetamines, in which absorption/derivatization are accomplished in one step. Specifically, (S)-(−)-N-(Trifluoroacetyl)-prolyl chloride was adopted as the chiral derivatizing reagent and added directly into the sample matrix. Analytical parameters, such as temperature, absorption/desorption duration, and the amount of derivatizing reagent, were studied to determine their effects on the yield of analytes. The derivatization products resulting from this study show excellent desorption characteristics on the polydimethylsiloxane-coated fiber adopted in this study. Optimal operational parameters (absorption: 70 °C for 10 min; injection: 250 °C for 5 min) cause minimal negative impact on the fiber, allowing repeated use of the fiber for more than 30 times. For quantitation, data were collected under selected ion monitoring (SIM) mode using m/z 237 and 251 to designate derivatized amphetamine and methamphetamine. This method was evaluated and proved to be effective in (a) quantitative determination of the enantiomeric pairs of amphetamine and methamphetamine – in terms of repeatability, linearity, and limits of detection and quantitation; and (b) generating case-specimen data comparable to those derived from a conventional Liquid–liquid extraction approach. Good linearity for the calibration curves were established in the range of 1000–50 ng/mL for both analytes. The limits of detection for these analytes were 30 ng/mL.
Keywords: Amphetamine; Methamphetamine; Enantiomer separation; Derivatization; GC–MS;

Enantiomeric determination of ephedrines and norephedrines by chiral derivatization gas chromatography–mass spectrometry approaches by Sheng-Meng Wang; Russell J. Lewis; Dennis Canfield; Tien-Lai Li; Chang-Yu Chen; Ray H. Liu (88-95).
Concerned with variations in abuse potential and control status among various isomers of ephedrines and norephedrines, this study was conducted to develop an effective method for the simultaneous analysis of eight ephedrine-related compounds along with structurally similar cathinones. Among various approaches studied, a 60-m HP-5MS (0.25 mm i.d., 0.25 μm film thickness) was successfully used to characterize the following compounds that were derivatized with (−)-α-methoxy-α-trifloromethylphenylacetic acid (MTPA): (+)-cathinone, (−)-cathinone, (+)-norephedrine, (−)-norephedrine, (+)-norpseudoephedrine, (+)-ephedrine, (−)-ephedrine, (−)-pseudoephedrine, (+)-pseudoephedrine. (−)-Cathine standard was not available, but should also be resolvable under this analytical procedure. This method was successfully applied to the analysis of selected cold remedies for characterizing the enantiomeric compositions of the compounds present in these samples.
Keywords: Enantiomer separation; Ephedrine; Norephedrine; Derivatization; GC–MS;