Journal of Chromatography B (v.811, #1)

Preface by Jim Gerostamoulos; Olaf H. Drummer (1).

Although substance identification is a key factor in analytical toxicology, it is amazing that the subject is receiving very limited and often inappropriate attention. With regard to the latter, a “confirmation” approach is usually chosen, which does not yield unambiguous identification. Moreover, the criteria for establishing a “positive match” leave much to be desired. These observations are corroborated when comparing some recent guidelines for qualitative analysis (issued for various forensic areas by SOFT/AAFS, NCCLS, NLCP, WADA and EU). Apart from showing substantial differences between them on pivotal issues, the guidelines contain various elements that appear scientifically incorrect and/or legally untenable. Also, the guidelines focus primarily on mass spectrometry (MS) and pay little or no attention to other identification possibilities (such as chromatographic techniques, either in combination with MS or as stand-alone techniques. Moreover, they do not offer alternatives in situations where access to MS is not available. One must conclude, therefore, that substance identification is a neglected and misunderstood domain in analytical toxicology. Rapid and concerted actions are needed to: (1) improve the general knowledge; (2) to define uniform strategies in the analytical approach and in the interpretation of the results; and (3) to set up and maintain suitable banks of reference substances and computerized data bases to allow unambiguous identification.
Keywords: Substance identification;

Quantitative analysis of 33 benzodiazepines, metabolites and benzodiazepine-like substances in whole blood by liquid chromatography–(tandem) mass spectrometry by B.E. Smink; J.E. Brandsma; A. Dijkhuizen; K.J. Lusthof; J.J. de Gier; A.C.G. Egberts; D.R.A. Uges (13-20).
A quantitative method using high-performance liquid chromatography–mass spectrometry (LC–MS, ion trap) after matrix supported liquid–liquid extraction is described for the simultaneous determination in whole blood of 33 benzodiazepines including metabolites and benzodiazepine-like substances. The limits of detection (LOD) range from 0.0001 to 0.0126 mg/l. Linearity is satisfactory for all compounds. The extraction recoveries for the benzodiazepines in whole blood are between 60 and 91%, desmethyldiazepam, OH-bromazepam and brotizolam excepted. Selectivity, accuracy and precision are satisfactory for clinical and forensic purposes.
Keywords: Benzodiazepines; Whole blood analysis; Drugs and driving;

Recently, high-performance liquid chromatography–tandem mass spectrometry (LC/MS/MS) has become a powerful tool for quantitative confirmatory analysis of drugs of abuse and has begun to spread in the field of forensic toxicology. Guidelines for confirmatory analysis by GC/MS and LC/MS/MS have been published recently by several organizations (WADA, IOC, SOFT, GTFCh, EU). However, these guidelines have not yet been included in procedures for drug analysis with LC/MS/MS. The prerequisites for forensic confirmatory analysis by LC/MS/MS with respect to EU guidelines are chromatographic separation, a minimum number of two MS/MS transitions to obtain the required identification points and predefined thresholds for the variability of the relative intensities of the MS/MS transitions (MRM transitions) in samples and reference standards. LC/MS/MS methods for determination of several classes of drugs of abuse including some basic drugs (opiates, stimulants), cannabinoids and some of their phase-I- and phase-II-metabolites (especially glucuronides) in urine and serum of drug abusers and/or crime offenders or victims have been developed and validated following current recommendations and are presented in this paper. At least two MRM transitions for each substance were monitored to provide sufficient identification of drugs, deuterated analogues of analytes were used as internal standards for quantitation where possible and chromatographic separation has been performed on reversed-phase columns with gradient elution. Validation data obtained and the application to real samples show that the requested criteria for confirmatory analysis of drugs of abuse by EU guidelines can be fulfilled with a total number of four identification points by LC/MS/MS methods using a triple-quadrupole mass spectrometer. Furthermore, the methods are sufficiently sensitive to meet current requirements for confirmatory analysis of drugs of abuse in driving under the influence of drugs (DUID) cases established by the Society of Toxicological and Forensic Chemistry (GTFCh).
Keywords: Drugs of abuse;

A liquid chromatographic mass spectrometric (LC/MS/MS) method has been developed for the determination of loperamide in whole blood and other biological specimens. The procedure involves liquid–liquid extraction of loperamide, desmethylloperamide and methadone-D3 (internal standard) with butyl acetate. Confirmation and quantification was 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 ratio of the responses between the two MRM transitions of each compound. The standard curves were linear over a working range of 0.1–500 μg/kg for all transitions. The limit of quantification was 0.1 μg/kg in whole blood. The repeatability and reproducibility within the laboratory expressed by relative standard deviation were less than 5 and 11%, respectively, and the accuracy was better than 9%. The method was developed to examine a feces sample from a child whose mother was suspected of Münchausen syndrome by proxy and it proved to be suitable for forensic cases being simple, selective and reproducible. The method was also applied for a case investigation involving a overdose of loperamide.
Keywords: Loperamide; Desmethylloperamide;

Detection of cocaine and cocaethylene in sweat by solid-phase microextraction and gas chromatography/mass spectrometry by Maria José Damas Follador; Mauricio Yonamine; Regina Lucia de Moraes Moreau; Ovandir Alves Silva (37-40).
In the present work, a semi-quantitative method was developed to detect simultaneously cocaine (COC) and cocaethylene (CE) (transesterification product of the coingestion of COC with ethanol) in sweat. Sweat samples were collected by means of a non-occlusive sweat patch device supplied by PharmChek™. The method was based on the dissolution of COC and CE incorporated into the patch, with 0.2 M sodium acetate buffer (pH 5.0) and the extraction of the analytes by solid-phase microextraction (SPME). Gas chromatography/mass spectrometry (GC–MS) was used to detect the analytes in selected ion monitoring mode (SIM). The method showed to be very simple, rapid and sensitive. The limits of detection were 5 ng/ml for COC and CE (12.5 ng/patch). Good inter and intra-assay precision was also observed (coefficient of variation <8%) with the use of deuterated internal standards.
Keywords: Cocaine; Cocaethylene; Sweat samples;

This paper describes the development and validation of analytical methodology for the determination of the use of MDMA, MDEA and MDA in urine. After a simple liquid extraction, the analyses were carried out on a high performance liquid chromatography (HPLC) in an octadecyl column, with fluorescence detection. The mobile phase using a sodium dodecyl sulfate ion-pairing reagent allows good separation and efficiency. The method showed good linearity and precision. Recovery was between 85 and 102% and detection limits were 10, 15 and 20 ng/ml for MDA, MDMA and MDEA, respectively. No interfering substances were detected with fluorescence detection.
Keywords: Drug of abuse; MDMA; MDEA;

A fully validated procedure for the simultaneous determination of morphine (MOR), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), 6-acetylmorphine (6AM), codeine (COD), codeine-6-glucuronide (C6G), acetylcodeine (AC), noscapine (NOS) and papaverine (PAP) based on liquid chromatography followed by electrospray mass spectrometry applying multiple reaction monitoring (LC-ESI-MS/MS) in urine samples is described. The extraction was carried out on a Zymark Rapid Trace Workstation using C18 solid-phase extraction cartridges. The separation was performed in 19 min on an Agilent 1100 HPLC system, using a Phenomenex C18 AQUA column (4 μm, 150 mm × 2 mm), which is coupled with an Applied Biosystems API 2000 mass spectrometer. Deuterated analogues were used as internal standards. The limits of detection were in the range of 0.1 ng/ml (PAP) to 7.4 ng/ml (M6G), the coefficients of correlation were higher than 0.996, the precisions ranged from 3% to 12% and the absolute recoveries were between 45% (M3G) and 98% (MOR). Analyses of samples from patients of a heroin prescription program demonstrated the usefulness of the procedure for the analytical differentiation between prescribed synthetic heroin (diamorphine) use and non-prescription heroin abuse on the basis of urine analysis. After the ingestion of pharmaceutical heroin only general markers for heroin use were detected, which are MOR, M3G, M6G and 6AM, respectively. When illicit heroin was abused, additionally to further general markers (COD, C6G) specific markers for non-prescription heroin abuse (AC, NOS, PAP) were found. However, it must be kept in mind that only AC may be regarded as absolute specific marker of non-prescription heroin, because all other compounds may appear in urine after ingestion of opiate alkaloids containing medicines or foods (e.g. poppy seeds). Therefore, patients of a heroin prescription program should be advised not to ingest such products.
Keywords: Morphine; Morphine glucuronides; Codeine; Acetylcodeine; Noscapine; Papaverine; Heroin;

Kinetic characteristics and toxic effects of benzalkonium chloride following intravascular and oral administration in rats by Yuying Xue; Yoko Hieda; Kojiro Kimura; Koji Takayama; Junko Fujihara; Yoshio Tsujino (53-58).
Kinetic characteristics and toxic effects of benzalkonium chloride (BZK) following injection via jugular vein (JV), femoral artery (FA) and oral administration (PO) were experimentally investigated using rats. The BZK concentrations in blood and tissues (lung, liver and kidney) were determined by high-performance liquid chromatography with solid phase extraction. Toxic doses of 15 and 250 mg/kg of BZK were used for intravascular (JV and FA) and PO administration, respectively. The fatal effects appeared soon after the dose in JV-rats, while delayed in FA- or PO-rats. The blood BZK concentrations and the elimination half-lives were similar between JV- and FA-rats, while the distribution of BZK in tissues was slightly different. In PO administration, the rats that aspirated BZK into their lungs had some symptoms, while the rats that did not aspirate BZK appeared to be normal. The BZK concentrations in blood and tissues were significantly higher in the aspirated PO-rats. The toxic degree of BZK was correlated with the BZK concentration in orally dosed rats. Lung and kidney had higher BZK concentrations compared to blood or liver, and they could be the target organs of BZK.Keyword: Benzalkonium chloride

Testing for zolpidem in oral fluid by liquid chromatography–tandem mass spectrometry by Pascal Kintz; Marion Villain; Bertrand Ludes (59-63).
The purported enhancement of sexuality, coupled with a possible abrupt coma-inducing effect and ease of administration in spiked drinks have resulted in the use of hypnotics to facilitated a sex offence. Among these compounds, zolpidem possesses amnesic properties, is short-acting and can rapidly impair an individual. In order to document zolpidem exposure, we have developed an original analytical method in oral fluid. 500 μl of oral fluid was added to 500 μl pH 7.6 Soerensen buffer was extracted by 2 ml dichloromethane in presence of 5 ng diazepam-d5, used as internal standard. An aliquot of the extract was injected into a 5 μm Novapak C18 column (150 mm × 2.1 mm). Reversed-phase separation was achieved in 6 min, under isocratic conditions (90% acetonitrile, 10% 2 mM ammonium formate pH 3.6) at a flow rate of 150 μl/min. Detection was achieved by tandem mass spectrometry. Molecular ions (m/z 308 and 290 for zolpidem and the IS, respectively) were selected in Q1 and the corresponding daughter ions (m/z 235 and 263 for zolpidem and m/z 154 and 198 for the IS) were detected in Q3 after collision with argon. Linearity was observed for zolpidem concentrations ranging from 0.2 to 100 ng/ml, and the assay was capable of detecting 0.05 ng/ml. Oral fluid was collected for 480 min after oral zolpidem administration of 10 mg to 2 subjects. In both cases, zolpidem was detectable (0.4 ng/ml) after 15 min intake. Peak zolpidem concentrations were obtained at 150 min (53.5 ng/ml) and 180 min (75.7 ng/ml), respectively. Oral fluid tested positive for zolpidem for over 8 h (9–15 ng/ml).
Keywords: Zolpidem; Sexual assault;

Detection of anti-diabetics in equine plasma and urine by liquid chromatography–tandem mass spectrometry by Emmie N.M. Ho; Kenneth C.H. Yiu; Terence S.M. Wan; Brian D. Stewart; Keith L. Watkins (65-73).
Anti-diabetics such as sulfonylurea and thiazolidinedione derivatives are hypoglycemic drugs used for the treatment of diabetes. However, they can also be used as a stopper in horseracing. This paper describes a convenient method for the separation and simultaneous detection of 10 anti-diabetic drugs (namely glipizide, glibenclamide, glimepiride, gliclazide, tolazamide, tolbutamide, nateglinide, repaglinide, rosiglitazone and pioglitazone) in equine plasma and urine by LC–MS-MS. Method: The anti-diabetics were isolated from equine plasma and urine by liquid–liquid extraction with 1,2-dichloroethane at acidic pH, and analysed by LC–MS-MS in the positive electrospray ionisation mode. Separation of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a mixture of aqueous ammonium formate (pH 3.0, 10 mM) and methanol as the mobile phase. Results: Detection and confirmation of the 10 anti-diabetic drugs at 10 ng/mL each in equine plasma and equine urine were achieved by full-scan MS-MS. All of these drugs were detected consistently at this concentration in spiked samples of different plasma and urine (n = 15 each). No significant matrix interferences were observed at the expected retention times of the targeted ions in blank urine samples (n = 30). This method has been used successfully in the analysis of drug-administration samples as well as official racing samples. Conclusion: An LC–MS-MS method has been developed for the simultaneous detection of 10 anti-diabetics in equine plasma and urine. This method can be used to detect the abuse of anti-diabetic drugs in racehorses.
Keywords: Anti-diabetics;

Detection of stanozolol and its metabolites in equine urine by liquid chromatography–electrospray ionization ion trap mass spectrometry by Andrew R. McKinney; Craig J. Suann; Anthony J. Dunstan; Stephen L. Mulley; Damon D. Ridley; Allen M. Stenhouse (75-83).
The equine phase I and phase II metabolism of the synthetic anabolic steroid stanozolol was investigated following its administration by intramuscular injection to a thoroughbred gelding. The major phase I biotransformations were hydroxylation at C16 and one other site, while phase II metabolism in the form of sulfate and β-glucuronide conjugation was extensive. An analytical procedure was developed for the detection of stanozolol and its metabolites in equine urine using solid phase extraction, acid solvolysis of phase II conjugates and analysis by positive ion electrospray ionization ion trap LC–MS.
Keywords: Stanozolol;

Protopine alkaloids in horse urine by Paul M Wynne; John H Vine; R Gary Amiet (85-91).
Protopine was extracted from Fumaria officinalis and purified by column chromatography. Urine samples were collected from horses and a human volunteer that had been administered either F. officinalis or protopine free base. Plant and urine samples were acetylated and analysed by GCMS after solid-phase extraction (SPE). The urinary metabolites of protopine were identified as 4,6,7,13-tetrahydro-9,10-dihydroxy-5-methyl-benzo[e]-l,3-benzodioxolo [4,5-1][2] benzazecin-12(5H)-one, 4,6,7,13-tetrahydro-10-hydroxy-9-methoxy-5-methyl-benzo[e]-1,3-benzodioxolo[4,5-1][2] benzazecin-12(5H)-one and 4,6,7,13-tetrahydro-9-hydroxy-10-methoxy–5-methyl-benzo[e]-1,3-benzodioxolo[4,5-l][2] benzazecin-12(5H)-one, chelianthifoline, isochelianthifoline and 2-O-desmethylchelianthifoline. The metabolic formation of the tetrahydroprotoberberines by closure of the bridge across N5 and C13 is rate limited and protopine-like metabolites accumulate only when the route is overloaded. Metabolism was qualitatively similar in the horse and human.
Keywords: Protopine; Alkaloids; Opiates; Chemotaxonomy;

3-Methoxytyramine as an indicator of dopaminergic manipulation in the equine athlete by Paul M. Wynne; John H. Vine; R. Gary Amiet (93-101).
The influence of sampling variables on the concentration of the dopamine metabolites 3-methoxytyramine (3MT), dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was examined in equine urine. A logarithmic transformation of the data for all horses gave distribution which approximated the normal distributions for each metabolite. The mean urinary concentration of 3MT in horses was 214 ng/mL and the application of a threshold with a probability of 1 in 10,000 gave an actionable level of 4 μg/mL. Environmental variables were not forensically significant in determining the population distribution. HVA was not found to be a reliable indicator of dopamine or levodopa administration.
Keywords: 3-Methoxytyramine; Dopamine manipulation; Regulatory threshold;