Screening procedure for detection of antidepressants of the selective serotonin reuptake inhibitor type and their metabolites in urine as part of a modified systematic toxicological analysis procedure using gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric (GC-MS) screening procedure was developed for detection of selective serotonin reuptake inhibitors (SSRIs) in urine as part of a systematic toxicological analysis procedure. After acid hydrolysis of one aliquot of urine, another aliquot was added. The mixture was then liquid-liquid extracted at pH 8-9, acetylated, and GC separated. Using mass chromatography with the ions m/z 58, 72, 86, 173, 176, 234, 238, and 290, the possible presence of SSRIs and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. The overall recoveries of citalopram, sertraline, and paroxetine ranged between 60 and 80%, and those of fluoxetine and fluvoxamine, which were destroyed during acid hydrolysis, were between 40 and 45%. The coefficients of variation were less than 10-20%, and the limit of detection was at least 100 ng/mL (signal-to-noise ratio = 3). This method allowed the detection of therapeutic concentrations of citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline in human urine samples.     

Piperazine-derived designer drug 1-(3-chlorophenyl)piperazine (mCPP): GC-MS studies on its metabolism and its toxicological detection in rat urine including analytical differentiation from its precursor drugs trazodone and nefazodone

Studies on the metabolism and the toxicological analysis of the piperazine-derived designer drug 1-(3-chlorophenyl)piperazine (mCPP) in rat urine using gas chromatography-mass spectrometry (GC-MS) are described. mCPP was extensively metabolized, mainly by hydroxylation of the aromatic ring and by degradation of the piperazine moiety to the following metabolites: two hydroxy-mCPP isomers, N-(3-chlorophenyl)ethylenediamine, 3-chloroaniline, and two hydroxy-3-chloroaniline isomers. The hydroxy-mCPP metabolites were partially excreted as the corresponding glucuronides and/or sulfates, and the aniline derivatives were partially acetylated to N-acetyl-hydroxy-3-chloroaniline isomers and N-acetyl-3-chloroaniline. Our systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction, and microwave-assisted acetylation allowed the detection of mCPP and its previously mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. The hydroxy-mCPP metabolites should be used as target analytes being the major metabolites of mCPP. Assuming similar metabolism, our STA procedure should be suitable for detection of an intake of mCPP in human urine. Furthermore, possibilities for differentiating an intake of mCPP from that of its precursor drugs trazodone or nefazodone, two common antidepressants, are described. Within the context of these studies, N-(3-chlorophenyl)ethylenediamine was identified as a new metabolite of these two antidepressants.     

Screening procedure for detection of stimulant laxatives and/or their metabolites in human urine using gas chromatography-mass spectrometry after enzymatic cleavage of conjugates and extractive methylation

A gas chromatography-mass spectrometry (GC-MS)-based screening procedure was developed for the detection of stimulant laxatives and/or their metabolites in human urine after enzymatic cleavage of conjugates followed by extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. By use of mass chromatography with the ions m/z 305, 290, 335, 320, 365, 350, 311, 326, 271, and 346, the possible presence of stimulant laxatives and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra. This method allowed the detection of the diphenol laxatives bisacodyl, picosulfate, and phenolphthalein and of the anthraquinone laxatives contained in plant extracts and/or their metabolites in human urine samples. The overall recoveries of the stimulant laxatives and/or their metabolites ranged between 33% and 89% with a coefficient of variation of less than 15%, and the limits of detection ranged between 10 and 25 ng/mL (S/N 3) in the full scan mode. After ingestion of the lowest therapeutic dose of sodium picosulfate, its main metabolite, bisacodyl diphenol, was detectable in urine samples for 72 hours. After ingestion of the lowest therapeutic dose of a senna extract, the main metabolite of sennosides, rhein, was detectable in urine samples for 24 hours. This procedure is part of a systematic toxicological analysis procedure for acidic drugs and poisons with the modification of enzymatic cleavage of conjugates.     

Screening procedure for detection of dihydropyridine calcium channel blocker metabolites in urine as part of a systematic toxicological analysis procedure for acidic compounds by gas chromatography-mass spectrometry after extractive methylation

A gas chromatographic-mass spectrometric (GC-MS) screening procedure was developed for the detection of dihydropyridine calcium channel blocker ("calcium antagonist") metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. Using mass chromatography with the ions m/z 139, 284, 297, 298, 310, 312, 313, 318, 324, and 332, the possible presence of calcium channel blocker metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of amlodipine, felodipine, isradipine, nifedipine, nilvadipine, nimodipine, nisoldipine, and nitrendipine in human urine samples. Because urine samples from patients treated with nicardipine were not available, the detection of nicardipine in rat urine was studied. The overall recovery ranged between 67 and 77% with a coefficient of variation of less than 10%, and the limit of detection was at least 10 ng/mL (signal-to-noise ratio = 3) in the full-scan mode.     

Screening procedure for detection of non-steroidal anti-inflammatory drugs and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography-mass spectrometry after extractive methylation

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used as analgesic and anti-rheumatic drugs, and they are often misused. A gas chromatographic-mass spectrometric (GC-MS) screening procedure was developed for their detection in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The compounds were separated by capillary GC and identified by computerized MS in the full-scan mode. Using mass chromatography with the ions m/z 119, 135, 139, 152, 165, 229, 244, 266, 272, and 326, the possible presence of NSAIDs and their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of acemetacin, acetaminophen (paracetamol), acetylsalicylic acid, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flufenamic acid, flurbiprofen, ibuprofen, indometacin, kebuzone, ketoprofen, lonazolac, meclofenamic acid, mefenamic acid, mofebutazone, naproxen, niflumic acid, phenylbutazone, suxibuzone, tiaprofenic acid, tolfenamic acid, and tolmetin in urine samples. The overall recoveries of the different NSAIDs ranged between 50 and 80% with coefficients of variation of less than 15% (n = 5), and the limits of detection of the different NSAIDs were between 10 and 50 ng/mL (S/N = 3) in the full-scan mode. Extractive methylation has proved to be a versatile method for STA of various acidic drugs, poisons, and their metabolites in urine. It has also successfully been used for plasma analysis.     

Screening procedure for detection of diuretics and uricosurics and/or their metabolites in human urine using gas chromatography-mass spectrometry after extractive methylation

A gas chromatography-mass spectrometry (GC-MS)-based screening procedure was developed for the detection of diuretics, uricosurics, and/or their metabolites in human urine after extractive methylation. Phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by GC and identified by MS in the full-scan mode. The possible presence of the following drugs and/or their metabolites could be indicated using mass chromatography with the given ions: m/z 267, 352, 353, 355, 386, and 392 for thiazide diuretics bemetizide, bendroflumethiazide, butizide, chlorothiazide, cyclopenthiazide, cyclothiazide, hydrochlorothiazide, metolazone, polythiazide, and for canrenoic acid and spironolactone; m/z 77, 81, 181, 261, 270, 295, 406, and 438 for loop diuretics bumetanide, ethacrynic acid, furosemide, piretanide, torasemide, as well as the uricosurics benzbromarone, probenecid, and sulfinpyrazone; m/z 84, 85, 111, 112, 135, 161, 249, 253, 289, and 363 for the other diuretics acetazolamide, carzenide, chlorthalidone, clopamide, diclofenamide, etozoline, indapamide, mefruside, tienilic acid, and xipamide. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with reference spectra. This method allowed the detection of the abovementioned drugs and/or their metabolites in human urine samples, except torasemide. The limits of detection ranged from 0.001 to 5 mg/L in the full-scan mode. Recoveries of selected diuretics and uricosurics, representing the different chemical classes, ranged from 46% to 99% with coefficients of variation of less than 21%. After ingestion of the lowest therapeutic doses, furosemide was detectable in urine samples for 67 hours, hydrochlorothiazide for 48 hours, and spironolactone for 52 hours (via its target analyte canrenone). The procedure described here is part of a systematic toxicological analysis procedure for acidic drugs and poisons.     

Target urinary analytes for the gas chromatographic- mass spectrometric detection of procyclidine and benzhexol in drug abuse cases

The two antiparkinsonian drugs procyclidine and benzhexol are presently finding considerable favor for their euphoric hallucinogenic effects among drug abusers in some countries. In anticipation of their possible scheduling in national drug laws, gas chromatography-mass spectrometry (GC-MS) methods for their detection in urine will be required. However, because of uncertainty of the metabolic fate of the two drugs in humans, the urinary target analytes for GC-MS detection were not well defined. The problem was addressed in the present study in which it was found that mono-hydroxy metabolites, where hydroxylation took place at the cyclohexane ring in both drugs, could be endorsed as the major target analytes. The metabolites could only be detected as the mono- and/or di-trimethylsilyl (TMS) derivatives. The predominance of either derivative depended on the temperature and time of heating with the derivatizing reagent. Because of the basic properties of the hydroxy metabolites, analytic method optimization was needed for their detection in urine included extraction under basic pH conditions. Urine hydrolysis with β-glucuronidase did not have an effect on the recovery of the metabolites, but was usually performed in search for other drugs. Because of the relative abundance of ions, the electron impact mass spectra of the mono-TMS derivatives and the chemical ionization (CI) mass spectra of the mono- and di-TMS derivatives of the hydroxy metabolites of both drugs were found to be more structurally informative. The CI mass spectra of the di- TMS derivatives have the additive advantage of being potentially useful for quantitative analysis.     

The effect of neuroleptics, tranquillizers, narcotics, antidepressants and anticonvulsive drugs on the alterations of mouse behaviour caused by acetaldehyde

In a study of the effect of 26 psychoactive drugs on the complex of mouse behaviour changes caused by acetaldehyde it was found that tranquillizers, hypnotics, sodium oxybutyrate and ethanol are the most efficient. The differences in teh drug effects were determined by a tan alpha criterion. Antidepressants and neuroleptics have equal tan alpha, whereas narcotics, anti-convulsive drugs and tranquillizers all have similar tan alpha, but greater than that of the antidepressants and neuroleptics. A direct dose/effect relationship was found to exist for all the drugs investigated except for neuroleptics and antidepressants. For the latter drugs an increase in dose is followed by an increased effect, which, however, decreases on further increase of the dose. The convulsive syndrome is completely abolished only by tranquillizers and narcotics. Our results are quite in agreement with clinical data about the effectiveness of psychoactive drugs in the treatment of the alcohol abstinence syndrome. The method can be used not only for screening purposes, but also to define the specificity in the action of antialcoholic drugs.     

Rapid detection of benzoylecgonine in vitreous humor by enzyme immunoassay

The usual specimens submitted by a medical examiner for toxicological analysis include blood, urine, bile, vitreous humor, stomach contents, and solid-organ tissue. The detection of drugs in these specimens typically involves a combination of techniques including colorimetry, immunoassay, and gas chromatography. Although many laboratories rely principally on urine for the detection of drugs of abuse by immunoassay, these assays may be applied to other specimen types. An evaluation of Microgenics Corporation's cloned enzyme donor immunoassay (CEDIA) was conducted in order to evaluate its use in the detection of cocaine/cocaine metabolites in vitreous humor specimens. During a 14-month period, 392 vitreous humor specimens were analyzed by the CEDIA DAU Cocaine assay. Instrument parameters were set according to published manufacturer's guidelines. All presumptive positive immunoassay results prompted confirmatory testing and quantitation by gas chromatography-mass spectrometry (GC-MS) of other specimens including blood. Vitreous humor specimens were not tested by GC-MS. Using a approximately 100-ng/mL cutoff, the CEDIA assay produced 23 presumptive positive results, 22 of which were confirmed by GC-MS. The only specimen which could not be confirmed, elicited an immunoassay screen value near the cutoff limit. Routine analysis of blood, urine, bile, and/or bladder wash specimens by gas chromatography-nitrogen phosphorus detection revealed the presence of cocaine/cocaine metabolites in only 7 (31.8%) of the 22 confirmed cases. The concentration ranges of cocaine and benzoylecgonine in the blood specimens were none detected to 337 ng/mL and 17 to 8598 ng/mL, respectively. Cocaethylene was not detected in these cases. Analysis of vitreous humor specimens by CEDIA improved the detection rate of cocaine/cocaine metabolites by 0.7% in the cases submitted to our laboratory during the 14-month period.     

The role of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to test blood and urine samples for the toxicological investigation of drug-facilitated crimes

The authors present an overview of the drug-facilitated crime (DFC) phenomenon, especially in France. Recently, there has been an increase in reports of incidents (mainly sexual assaults and robbery) as well as in scientific publications and congress presentations on the topic. From enquiries conducted nationally, a list of drugs reportedly associated with DFC was established and includes benzodiazepines and benzodiazepine-like drugs (zolpidem, zopiclone), minor tranquilizers and neuroleptics, barbiturates, narcotics, hallucinogens, and anaesthetics. Some of these molecules are specific to France in DFC cases. A study using healthy volunteers who had taken benzodiazepines (lorazepam, bromazepam, flunitrazepam, clonazepam), zolpidem and zopiclone, showed that the only way to increase the duration of detection of these drugs is to use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to test blood and urine samples. The very high sensitivity of this method appears to be an essential condition to document the cases, because the drugs tested were still detectable in urine at least 6 days after the ingestion of one therapeutic dose. Limits of detection were always lower than 0.5 ng/mL in urine. The actual list of molecules and metabolites the authors screened for in urine and blood by LC-MS/MS, in every DFC, is given in detail: 25 benzodiazepines and benzodiazepine-like drugs, 11 minor tranquilizers and neuroleptics, 2 barbiturates, 12 narcotics, 4 hallucinogens, and 1 anaesthetic. However, the distinction between continual therapeutic use of a psychotropic drug or illegal narcotic and a single ingestion has to be documented by sequential analysis of hair, again with LC-MS/MS.     

Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches

4‐methyl‐N‐ethcathinone (4‐MEC), the N‐ethyl homologue of mephedrone, is a novel psychoactive substance of the beta‐keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4‐MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N‐deethylation, hydroxylation of the 4‐methyl group followed by further oxidation to the corresponding 4‐carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N‐methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N‐deethyl‐dihydro isomers and the 4‐carboxy‐dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4‐MEC should be detectable in urine by the GC‐MS and LC‐MSⁿ standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of zotepine and various drugs on apomorphine- and methamphetamine-induced rotational behaviour in rats with unilateral lesions of the substantia nigra

The effect of zotepine (2-chloro-11-(2-dimethyl-aminoethoxy) dibenzo [b, f] thiepin), a new neuroleptic, and other neuroleptics, minor tranquilizers, antidepressants, anticholinergic drugs, antihistamine drugs, antiparkinsonian drugs, alpha-, beta-adrenergic blocking agents, hypnotics and central muscle relaxants on apomorphine (APM)- and methamphetamine (MAP)-induced rotational behaviour was investigated in rats with unilateral lesions of the substantia nigra. Only the neuroleptics dose-dependently depressed both rotational behaviours. The minor tranquilizers also depressed both behaviours, but their effect was non-specific. The neuroleptics could be divided into two groups according to their relative effect on the APM- and MAP-induced rotational behaviour. The high-ratio group (fluphenazine, haloperidol, perphenazine, pimozide and zotepine) depressed the MAP-induced rotational behaviour more strongly than the APM-induced one, whereas the depressive effect of the low-ratio group (chlorpromazine, levomepromazine, thioridazine and clozapine) was the reverse. Zotepine, while belonging to the high-ratio group, was markedly lower in ratio than the other neuroleptics in this group. The correlation between the different effect of the two groups of neuroleptics and their clinical effect is discussed.     

Replacing immunoassays for mephedrone, ketamines and six amphetamine-type stimulants with flow injection analysis tandem mass spectrometry

A screening procedure was developed for the simultaneous detection of mephedrone, six amphetamine-type stimulants (ATS), ketamine and its two metabolites with electrospray ionization flow injection analysis tandem mass spectrometry (FIA-MS-MS). Urine samples were fortified with deuterated analogues as internal standards, extracted with ethyl acetate and analyzed with FIA-MS-MS. The mass analyzer was operated in multiple reaction monitoring mode. Two product ions were monitored for each drug and internal standards. For each analyte, the limit of detection was less than 4 μg/L, within-day and between-day precisions (percent coefficient of variation) at three different concentrations were less than 7.3% and bias was between -17.3 and 11.8%. Total analysis time with FIA-MS-MS is 1.8 min per sample. A group of 215 urine samples were screened with immunoassay for ATS and analyzed with FIA-MS-MS and gas chromatography-mass spectrometry (GC-MS) for ketamines and ATS. The analysis of ATS by immunoassay and GC-MS was 96.7% concordant. The analysis of three ketamines and seven ATS by FIA-MS-MS and GC-MS was 97.2% concordant. The FIA-MS-MS procedure is efficient, accurate, flexible and capable of detecting analytes of different chemical groups. It can replace immunoassays for the screening of new designer drugs when commercial immunoassays are unavailable.     

Serum and urine concentrations of flunitrazepam and metabolites, after a single oral dose, by immunoassay and GC-MS.

A clinical study was conducted to assess the ability of commercially available immunoassays to detect flunitrazepam (FNP) in plasma and urine samples and to compare the results with those obtained by gas chromatography-mass spectrometry (GC-MS). The clinical study consisted of four individuals (two male and two female) who had taken a single 2-mg dose of FNP. Serum was collected over a 48-h period and urine was collected over a 72-h period. The serum and urine samples were analyzed by the COBAS INTEGRA Serum Benzodiazepines assay (SBENZ), the TDx serum and urine Benzodiazepines assay, and GC-MS. The GC-MS procedure was developed for analysis of FNP and metabolites in plasma and urine using an acid hydrolysis step resulting in the formation of specific benzophenones corresponding to FNP and its metabolites. The relative sensitivities of the assays for the detection of FNP and metabolites in serum and urine were GC-MS > SBENZ > TDx. The immunoassay results for serum samples showed peak concentrations of FNP metabolites at 8 h after FNP ingestion for three individuals and at about 1 h for the fourth individual. The GC-MS, SBENZ, and TDx urine immunoassays detected drug above the stated limit of detection (LOD) in 44, 41, and 35 serial FNP urine samples, respectively. FNP metabolites were detected in urine samples with all three assays for up to 72 h after a 2-mg dose. The improved detection rate with the SBENZ assay as compared to the TDx assay is likely explained by its higher cross-reactivity with the major metabolite, 7-amino-flunitrazepam (7-amino-FNP), and its lower LOD.     

Development and validation of a multi‐analyte LC‐MS/MS approach for quantification of neuroleptics in whole blood,plasma, and serum

Based on a similar approach for quantification of antidepressants, benzodiazepines, and z‐drugs, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) multi‐analyte approach with simple liquid‐liquid extraction was extended for fast target screening and quantification of neuroleptics in whole blood, plasma, and serum. As this method is part of a multi‐analyte procedure for over 100 analytes from different drug classes and as the extracts were additionally used in the authors' laboratory for gas chromatography‐mass spectrometry (GC‐MS) analysis, one universal stable‐isotope‐labelled internal standard (SIL‐IS) was used to save time and resource. The method was validated with respect to international guidelines. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. Selectivity problems could not be observed, but matrix effects ranged from 68 to 211% in all samples. For the low quality control (QC), recovery ranged from 32 to 112%, process efficiency from 31 to 165% and for the high QC recovery from 42 to 141%, process efficiency from 29 to 154%. In addition statistical data evaluation of the variances of the recovery, matrix effects, and process efficiency data between whole blood vs. plasma, whole blood vs. serum, and plasma vs. serum were done. The presented LC‐MS/MS approach was applicable for selective detection of 33 neuroleptics as well as accurate and precise quantification of 25 neuroleptics in whole blood, 19 in plasma, and 17 in serum. More significant matrix effects (ME) for neuropletic drugs overall in plasma and serum as compared with whole blood were detected. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid chromatography–tandem mass spectrometry screening method using information‐dependent acquisition of enhanced product ion mass spectra for synthetic cannabinoids including metabolites in urine

The total number of synthetic cannabinoids (SCs) – a group of new psychoactive substances (NPS) – is increasing every year. The rapidly changing market demands the latest analytical methods to detect the consumption of SCs in clinical or forensic toxicology. In addition, SC metabolites must also be included in a screening procedure, if detection in urine is asked for. For that purpose, an easy and fast qualitative liquid chromatography—tandem mass spectrometry (LC?MS/MS) urine screening method for the detection of 75 SCs and their metabolites was developed and validated in terms of matrix effects, recovery, and limits of identification for a selection of analytes. SC metabolites were generated using in vitro human liver microsome assays, identified by liquid chromatography?high resolution tandem mass spectrometry (LC?HRMS/MS) and finally included to the MS/MS spectra in‐house library. Sample preparation was performed using a cheap‐and‐easy salting‐out liquid–liquid extraction (SALLE) after enzymatic hydrolysis. Method validation showed good selectivity, limits of identification down to 0.05 ng/mL, recoveries above 80%, and matrix effects within ±25% for the selected analytes. Applicability of the method was demonstrated by detection of SC metabolites in authentic urine samples.     

Large-volume injection gas chromatography-mass spectrometry for automated broad-spectrum drug screening in horse urine

A rapid, sensitive, and rugged method for detecting drugs and drug metabolites in extracts of horse urine is described. The use of large-volume injection (LVI) gas chromatography-mass spectrometry (GC-MS) for analysis of horse urine extracts allowed automation of the derivatization procedure and reduction of the sample volume from 5 mL to 1 mL of urine. An autosampler and temperature-programmable inlet were used to automatically dissolve the sample extract and form trimethylsilyl derivatives of over 200 analytes. The suitability of this procedure for routine GC-MS detection of approximately 80 basic analytes in extracts of racehorse urine was investigated. The formation of derivatives using LVI with in-liner derivatization was compared to a manual procedure involving the dissolution of sample extracts in N,O-bis(trimethylsily)trifluoroacetamide, heating the resulting mixture, and injecting 1 or 2 microL of the mixture through a splitless injector into the GC-MS instrument. In all cases, the in-liner derivatization reactions were found to be as complete as conventional heating block procedures. Ruggedness testing of the method demonstrated that peak resolution, shape, and area were maintained through 40 consecutive injections of sample extracts. No evidence of the accumulation of interfering substances was observed. The limits of detection using LVI GC-MS for routine screening of basic drugs in urine were generally in the range of 5-25 ng/mL. The method is currently being used to detect basic analytes in horse urine extracts with a throughput of approximately 50 urine sample extracts per instrument per day.     

Detection of beta-blockers in human urine by GC-MS-MS-EI: perspectives for the antidoping control

We have developed a general method for the detection of beta-blockers and/or of their metabolites in human urine. The method comprises a pretreatment procedure (enzymatic hydrolysis, liquid/liquid extraction and derivatization by pentafluoropropionic anhydride, PFPA), carried out on an initial aliquot of 2.5-5.0 ml of urine, and the instrumental analysis of the derivatives, performed by GC-MS-MS (ion trap) with electronic impact ionization (EI). The GC-MS-MS analysis allows to isolate and to characterize specific fragments of the original molecular structure, and particularly the fragments originating from parent ion clusters specific for all beta blocking drugs, giving rise to m/z = 366 and 202 ions respectively. MS-MS analysis of the parent ion allows checking for the presence of the above-mentioned peaks in the GC-MS chromatogram. The proposed method is capable of detecting a great variety of known (and possibly also of newly synthesized) beta-blockers, with an average sensitivity limit of 20 ng/ml of drug/metabolite in urine. The method is presently being evaluated as a general screening protocol to be followed by an antidoping laboratory to detect illicit beta-blockers administration to the athletes.     

The use of nuclear magnetic resonance spectroscopy in the detection of drug intoxication

The use of nuclear magnetic resonance (NMR) spectroscopy as a method for drug analysis has the advantages of reduced pre-analytical preparation time and the potential to detect and quantitate drug conjugates and metabolites simultaneously. NMR was investigated as a method to screen for organic substances (and metabolites) in 25 patients who presented to the Emergency Department with clinical indications of a drug overdose. Urine specimens were examined by 1H NMR spectroscopy at 300 MHz and the results compared with gas chromatography-mass spectrometry (GC-MS) results. There was a 56% concordance (14 of 25 samples) between NMR and GC-MS. NMR identified acetaminophen, ibuprofen, aspirin, valproate, carbamazepine, and pseudoephedrine as parent compounds or metabolites. For a patient for whom GC-MS results were negative, NMR strongly suggested the presence of erythromycin. NMR was most successful in identifying analgesics and antiepileptic drugs (sensitivity 83-100%). In 10 patients, signals from 1,2-propanediol, a common vehicle for some pediatric medications, were observed by NMR spectroscopy. NMR had 0% sensitivity in identifying tricyclic antidepressants and antipsychotic drugs. In these samples, GC-MS detected a variety of compounds, including tricyclic antidepressants and their metabolites and chlorpromazine. In addition, other substances that had not been disclosed as having been ingested, such as caffeine, diphenhydramine, and nicotine, were detected by GC-MS. NMR spectroscopy represents an emerging supplementary analytical technique that is applicable to a wide range of possible intoxicants and to the evaluation of the intoxicated patient, particularly when larger amounts of the intoxicant (> 200 mg) are ingested.