Characterization of URB Series Synthetic Cannabinoids by HRMS and UHPLC-MS/MS.

A large number of synthetic cannabinoids are included in new psychoactive substances (NPS) and constitute an open research area in analytical pharmaceutical and toxicology when methods are needed to unambiguously identify these substances and their metabolites in biological fluids. A full molecular characterization of five synthetic molecules of the URB series that is able to interact with the endocannabinoid system was achieved with a high-resolution mass spectrometry (HRMS) in positive ion electrospray ionization and collisional experiments on the protonated parent ions, obtaining characteristic fragmentation patterns. Ultra-high-performance liquid chromatography coupled with a triple quadrupole (UHPLC-MS/MS) has also been used, which can help develop methods for screening and confirming synthetic cannabinoids in biological fluids.

Does Eumelanin Oxidation Play a Role on the Photostability of Ethyl Glucuronide in Hair Exposed to Simulated Solar Radiation?

The action of solar radiation on the concentration of the ethanol metabolite ethyl glucuronide (EtG) in 40 hair samples of nonabstinent subjects was investigated. Hair samples of different colors were analyzed before and after irradiation with artificial sunlight under a light dose corresponding to 3 months of sun exposure. After irradiation, an increase of EtG concentration was detected in 55% of the samples ranging from 5% to 141%. In 16 cases, a concentration reduction ranging from -2% to -74% was observed. The measure of the level of pyrrole-2,3,5-tricarboxylic acid (PTCA), a marker of eumelanin oxidation, demonstrated the largest increase in oxidation in light brown hair where the greatest degradation of EtG was observed after irradiation. However, the rise of PTCA in all hair tested was accompanied by increase in EtG concentration in 8/10 samples and by decrease in 2/10, suggesting no correlation between the two markers. To verify if hair structure was modified by light, scanning electron microscopy (SEM) analysis was performed on irradiated hair of different colors and compared with the dark samples. SEM revealed modification of hair structure in all samples showing partial shaft exfoliation and reduction of hair thickness under the treatment with solar radiation.

A Study on Photostability of Ethyl Glucuronide in Hair Irradiated under Artificial Sunlight.

Drugs incorporated into hair are exposed to the environment, and cosmetic and chemical treatments, with possible decreases in their content. Knowledge concerning the effect of sunlight on drug content in hair can be helpful to forensic toxicologists, in particular, when investigating drug concentrations above or below pre-determined cut-offs. Twenty-eight authentic positive hair samples were selected which had previously tested positive for ethyl glucuronide (EtG). Washed hair were divided into two identical tufts, with the former exposed at 13,219 J/cm2 (300-800 nm spectrum of irradiance) for 48 h in a solar simulator, and the latter kept in the dark. Hair samples were extracted and analyzed by ultra performance liquid chromatography-tandem mass spectrometry. The percentage of photodegradation was calculated for EtG. In parallel, photodegradation processes of standard molecule dissolved in aqueous and organic solutions were studied. In 28 hair samples, positive for the targeted analyte, exposure to artificial sunlight induced an appreciable increase in EtG concentrations. The concentration range in the non-irradiated hair samples was 6.0-772.0 pg/mg, and 64.3% of samples exhibited an increase in post-irradiation samples, ranging from 7% to 255%. In seven cases, a decrease was observed ranging from -5.0% to -36.0%. Thus, either a decrease or an increase of EtG may be observed post-irradiation, depending on hair color and/or hair thickness. Because the denaturation status of hair fibers and the thickness of hair before irradiation could play a role, a scanning electron microscope study should be envisaged.

Multiple incidence of the prescription diuretic hydrochlorothiazide in compounded nutritional supplements.

Diuretic agents are prohibited in sports in- and out-of-competition according to the regulations of the World Anti-Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (µg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA-accredited laboratories. We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10 ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analyzed revealing HCTZ presence in different concentrations, at the ppm level (µg/g and ng/mL). With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with three healthy volunteers was performed. HCTZ-contaminated powder (6.4 µg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24 hours, and underwent liquid-liquid extraction and liquid chromatography-tandem mass spectrometry determination. Post-administration specimens were found to contain HCTZ at concentrations of 5-230 ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete. This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products.

Hair analysis to discriminate voluntary doping vs inadvertent ingestion of the aromatase inhibitor letrozole.

Letrozole is an aromatase inhibitor, used to treat postmenopausal women with hormone receptor-positive or unknown advanced breast cancer. It is prohibited in sport because it is used together with androgen anabolizing steroids to avoid their adverse effects. In the case of an adverse analytical finding, it may be important to distinguish between repetitive use due to voluntary administration and occasional use, possibly due to involuntary intake. With the objective to identify the dose capable of producing a positive hair test, and to apply these results to the scenarios of inadvertent letrozole ingestion by an athlete, this study investigates the urinary excretion and incorporation into hair of single doses of letrozole. Seven subjects were recruited for an excretion study of letrozole and its metabolite bis(4-cyanophenyl) methanol (M1) in urine, after the consumption of 0.62 mg, 1.25 mg, and 2.5 mg of letrozole, and to investigate the incorporation in hair after ingestion of 0.62 mg and 2.5 mg of letrozole. Urine and hair samples were also obtained from two women in chronic therapy. Urinary concentrations of letrozole and its metabolite M1 were lower in subjects administered once with 0.62 mg, 1.25 mg, or 2.5 mg letrozole than in women in regular therapy with 2.5 mg/day. In hair collected after a single dosage, concentrations of 16-60 pg/mg were detected while in women in chronic therapy concentrations were higher than 160 pg/mg all along the hair shaft. Hair analysis turned to be a promising possibility for the discrimination of letrozole repetitive use vs occasional/inadvertent administration.

Non-fatal Overdose with U-47700: Identification in Biological Matrices.

BACKGROUND/OBJECTIVE: We report on a case of severe intoxication after insufflation of U-47700, a synthetic opioid that acts as a selective agonist of the µ-opioid receptor, and is several times more potent than morphine. A man in his 30s was found irresponsive in his apartment and was brought to the emergency department of a local hospital. A comatose state and severe respiratory depression were present. Hetero anamnesis revealed that the patient could have taken the substance named "U-47700", bought on the Internet. After supportive care, the patient fully recovered. METHOD: Urine, blood and a white powder found at his home were collected during his hospital stay and sent for testing using liquid chromatography-high resolution mass spectrometry (LC-HRMS) on an Orbitrap instrument. Later, his pubic hair was also collected. A standard comprehensive toxicology screening was performed. RESULTS: U-47700 was identified in all biological samples and in the seized white powder. Using liquid chromatography-high resolution mass spectrometry (LC-HRMS) the presence of U-47700 and its phase I and phase II metabolites in blood, urine and pubic hair was confirmed. U-47700 was determined at 94 ng/mL and 5.2 ng/mL in blood at the admission and the day after, respectively, and 3.02 ng/mg in pubic hair, together with its metabolites. No other opioid nor designer drug could be detected in blood and urine, while in pubic hair Cocaine, Benzoylecgonine, Norcocaine, Mephedrone, Ketamine, Norketamine, 3,4-Methylenedioxymethamphetamine, Tetrahydrocannabinol and Cannabinol were also detected. CONCLUSION: The toxicological findings confirmed the use of U-47700 in the intoxicated patient and also revealed a history of a poly-drug use. The use of LC-HRMS allowed the easy identification of the NPS and its metabolites in fluids and hair.

A Study on Photostability of Amphetamines and Ketamine in Hair Irradiated under Artificial Sunlight.

Drugs incorporated into hair are exposed to the environment, and cosmetic and chemical treatments, with possible decreases in their content. Knowledge concerning the effect of sunlight on drug content in hair can be helpful to forensic toxicologists, in particular, when investigating drug concentrations above or below pre-determined cut-offs. Twenty authentic positive hair samples were selected which had previously tested positive for amphetamines and/or ketamine. Washed hair were divided into two identical strands, with the former exposed at 765 W/m² (300⁻800 nm spectrum of irradiance) for 48 h in a solar simulator, and the latter kept in the dark. Hair samples were extracted and analyzed by liquid chromatography high-resolution mass spectrometry detection. The percentage of photodegradation was calculated for each analyte (i.e., amphetamine, methamphetamine, methylendioxyamphetamine, ketamine, and norketamine). In parallel, photodegradation processes of standard molecules dissolved in aqueous and organic solutions were studied. In 20 hair samples positive for the targeted analytes, exposure to artificial sunlight induced an appreciable decrease in drug concentrations. The concentration ranges in the non-irradiated hair samples were 0.01⁻24 ng/mg, and 65% of samples exhibited a decrease in post-irradiation samples, with reduction from 3% to 100%. When more drugs were present in the same hair sample (i.e., MDMA and ketamine) the degradation yields were compound dependent. A degradation product induced by irradiation of ketamine in aqueous and methanol solutions was identified; it was also found to be present in a true positive hair sample after irradiation. Ketamine, amphetamines, and their metabolites incorporated in the hair of drug users undergo degradation when irradiated by artificial sunlight. Only for ketamine was a photoproduct identified in irradiated standard solutions and in true positive irradiated hair. When decisional cut-offs are applied to hair analysis, photodegradation must be taken into account since sunlight may produce false negative results. Moreover, new markers could be investigated as evidence of illicit drug use.

Sample preparation methods for determination of drugs of abuse in hair samples: A review.

Hair analysis has assumed increasing importance in the determination of substances of abuse, both in clinical and forensic toxicology investigations. Hair analysis offers particular advantages over other biological matrices (blood and urine), including a larger window of detection, ease of collection and sample stability. In the present work, an overview of sample preparation techniques for the determination of substances of abuse in hair is provided, specifically regarding the principal steps in hair sample treatment-decontamination, extraction and purification. For this purpose, a survey of publications found in the MEDLINE database from 2000 to date was conducted. The most widely consumed substances of abuse and psychotropic drugs were considered. Trends in simplification of hair sample preparation, washing procedures and cleanup methods are discussed. Alternative sample extraction techniques, such as head-space solid phase microextraction (HS-SPDE), supercritical fluid extraction (SFE) and molecularly imprinted polymers (MIP) are also reported.

Monitoring haloperidol exposure in body fluids and hair of children by liquid chromatography-high-resolution mass spectrometry.

BACKGROUND: Haloperidol, 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone (HP), one of the most widely used antipsychotics in the treatment of schizophrenia, mania, and other psychiatric disorders, is frequently encountered in cases of unintentional pediatric intoxication because the ingestion of a small amount can cause significant toxic effects in children. For monitoring HP in suspected ingestions, a liquid chromatography-high-resolution mass spectrometry method has been developed and validated in urine, blood, and hair samples. METHODS: The analyte was extracted from 1 mL blood or urine by liquid/liquid extraction and from 5 mg of hair by micropulverized extraction; gradient elution on an Atlantis T3 column was realized using HP-d4 as an internal standard. Positive ion electrospray ionization and high-resolution mass spectrometry determination were performed in an Orbitrap mass spectrometer. RESULTS: The method exhibited a r > 0.999 in the studied ranges (0.1-50 ng/mL in urine and blood and 0.1-50 ng/mg in hair) and a limit of quantification of 0.1 ng/mL for urine and blood and 0.1 ng/mg for hair; intra-assay and interassay relative SDs were always more than 18%. The method was applied to determine haloperidol in 3 children who were admitted to emergency departments. HP concentrations ranged from 2 to 21 ng/mL in urine, from not detected to 4.9 ng/mL in blood, and from 0.37 to 0.73 ng/mg in hair samples. CONCLUSIONS: The utilization of high-resolution/high-accuracy mass spectrometry in full scan mode allowed the identification of HP metabolites in urine and blood, thus unequivocally documenting the exposure to the drug. HP metabolites were structurally characterized by high-resolution multiple mass spectrometry. For the first time, a HP metabolite was detected in hair.

Determination of ketamine and norketamine in hair by micropulverized extraction and liquid chromatography-high resolution mass spectrometry.

Ketamine (KT), primarily used as a general anaesthetic agent in clinical practice, has become very popular in recent years as a recreational drug, due to its dissociative and hallucinogenic effects. A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method has been developed and validated for the quantification of KT and its main metabolite norketamine (NK) in 2.0mg of hair. Sample preparation consisted of a rapid, simultaneous pulverization and extraction step in acidic solution, followed by centrifugation and filtration. Gradient elution was performed by an Atlantis T3 analytical column, and deuterated KT was used as the internal standard. Positive ion electrospray ionization and HRMS determination in full-scan mode were achieved with an Orbitrap mass spectrometer. The method has a linear range of 0.05-50 ng/mg, a limit of quantisation of 0.05 ng/mg and a limit of detection of 0.02 ng/mg for both KT and NK. The validated method was applied for the determination of KT and NK in two authentic hair samples from subjects suspected of taking psychoactive substances. The detection of the metabolite at low concentration gave proof for systemic drug origin and an investigation into the possible presence of further metabolites was performed by means of retrospective screening.

Journals publishing bio-medicolegal research in Europe.

Fragmentation of bio-medicolegal knowledge has led to a proliferation of ultra-specialised sub-disciplines and branches, often published in 'field-oriented' scientific journals.The aim of this work is to provide an in-depth analytical picture of bio-medicolegal sources of publication, within and outside the traditional conception of legal medicine. An extensive search of bio-medicolegal articles published in the last five and a half years was performed on the MEDLINE database according to MeSH terms combined with free-text protocols. We performed a systematic analysis of targeted journals after merging, selecting and categorising all retrieved records, taking into account data from the 2009 JCR Science Edition (released on June 2010); 1,037 different journals were identified, of which only 48 (4.6%) focus specifically on bio-medicolegal matters, and of which only seven (14.6%) have an impact factor (IF). Despite this apparent dispersion, 47% of articles were published in bio-medicolegal journals (BML), of which 70.2% were in journals with IF (BML-IF). Articles published in BML-IF journals (33% of total papers) reach almost 50%, mainly in "Forensic Science International", "International Journal of Legal Medicine" and "Journal of Forensic Sciences". Instead, publications in not specifically bio-medicolegal journals (Not BML-IF) are greatly scattered and even fragmented in about 650 journals.The sub-disciplines that appear most frequently in Not BML-IF rather than BML-IF journals are Forensic Psychiatry (48.2% vs. 5.1%), Criminology (37.1% vs. 8.3%), Malpractice (50.7% vs. 4.0%), Medical Law and Ethics (46.4% vs. 6.9%) and Clinical Forensic Medicine (39.5% vs. 21.3%). The proposed bibliometric analysis revealed the preference of Forensic Pathology, Criminalistics (Biological), Forensic Genetics, Forensic Anthropology and Forensic Entomology for journals traditionally considered pertinent to the medico-legal discipline, with a considerable dispersion involving Toxicology, Psychiatry, Criminology and Malpractice, which were published in less well-known journals. This dispersion could be reduced adapting specialised forensic sections and increasing the IF of forensic journals, in order to respond suitably to the present demand for visibility by bio-medicolegal scientists, clearly oriented towards enhancing the objective impact of their curricula and attempting to attract funding to their research projects.

High performance liquid chromatography-high resolution mass spectrometry and micropulverized extraction for the quantification of amphetamines, cocaine, opioids, benzodiazepines, antidepressants and hallucinogens in 2.5 mg hair samples.

A high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method for simultaneous screening and quantification of 28 drugs was developed and validated for 2.5 mg hair samples. Target drugs and their metabolites included amphetamines, cocaine, opioids, benzodiazepines, antidepressants, and hallucinogens. After decontamination, hair samples were extracted with 200 µL of a mixture of water: acetonitrile:1 M trifluoroacetic acid (80:10:10, v/v) using a 5 min simultaneous pulverization/extraction step. The extracts were analysed by HPLC-HRMS in an Orbitrap at a nominal resolution of 60,000, with concomitant in source collisional experiments (in source CID). Gradient elution on an Atlantis T3 column resolved 28 target compounds and 5 internal standards. Total chromatographic run time was 26 min. Calibration was achieved by linear regression analysis utilizing six calibration points; R2 ranged from 0.9964 to 0.9999, the limits of quantification were 0.1 ng/mg for 8 compounds, 0.2 ng/mg for 16 compounds and 0.5 ng/mg for 4 compounds; mean relative errors from -21% to +23% were obtained; relative standard deviation, used to estimate repeatability and intermediate reproducibility at three concentrations, was always less than 20%. Process efficiency and recoveries for all analytes were better than 65 and 73%, respectively, at any concentration. The method was applied to hair samples from forensic investigations that contained a broad assortment of drugs of abuse and pharmaceuticals. The use of concomitant HRMS full scan and CID afforded the possibility of retrospective analysis for discovering untargeted drugs.

Simultaneous LC-HRMS determination of 28 benzodiazepines and metabolites in hair.

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the simultaneous identification and quantification of 28 benzodiazepines, including 6 metabolites, in 50 mg of hair has been validated. Positive ion electrospray ionization and HRMS determination in full-scan mode were realized on an Orbitrap mass spectrometer at a nominal resolving power of 60,000. In-source collisional experiments were conducted to obtain additional information for a more reliable identification of the investigated drugs. HRMS in full-scan mode allowed the exact determination of molecular masses of all analytes eluting in the HPLC run, so that both the immediate and retrospective screening of results for drugs and their metabolites were available. Sample preparation consisted of an overnight incubation in phosphate buffer pH 8.4 and a subsequent liquid/liquid extraction with methylene chloride/diethyl ether (90:10). Gradient elution was performed on a Luna C18 analytical column and four deuterated analogues were used as internal standards (IS). Validation was performed using both spiked hair samples and hair samples from subjects treated with benzodiazepines. Selectivity was evaluated by analysis of 20 certified blank hair samples. Extraction efficiency and matrix effects were evaluated by analysis of true positive samples. The lowest limits of quantification (LLOQs) ranged from 1 to 10 pg/mg. Linearity was investigated in the range from LLOQ to 1,000 pg/mg, for each compound (R(2) 0.998-0.999). Mean relative errors, calculated at three concentration levels, ranged from 1 to 20% (absolute value). Precision, at concentrations higher than the LLOQs, was always less than 15% expressed as percentage relative standard deviation. After validation, the procedure was applied to real samples collected for clinical and forensic toxicology purposes from subjects who were assumed to have taken benzodiazepines.

Validation of a fast screening method for the detection of cocaine in hair by MALDI-MS.

The sensitivity and specificity of a novel method of screening for cocaine in hair, based on matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS), have been evaluated. The method entails a rapid extraction procedure consisting of shaking 2.5 mg pulverised hair at high frequency in the presence of an acidic solution (160 microL of water, 20 microL of acetonitrile and 20 microL of 1 M trifluoroacetic acid) and a stainless-steel bullet. Following centrifugation, the supernatant is dried under a nitrogen stream, and the residue is reconstituted in 10 microL of methanol/trifluoroacetic acid (7:3; v/v). One microlitre of the extract is deposed on a MALDI sample holder previously scrubbed with graphite; an alpha-cyano-4-hydroxycinnamic acid (matrix) solution is electrosprayed over the dried sample surface to achieve a uniform distribution of matrix crystals. The identification of cocaine is obtained by post-source decay experiments performed on its MH(+) ion (m/z 304), with a limit of detection of 0.1 ng/mg of cocaine. A total of 304 hair samples were analysed in parallel by MALDI-MS and a reference gas chromatography-MS method. The obtained results demonstrate specificity and sensitivity of 100% for MALDI-MS. Evidence of cocaine presence was easily obtained even when hair samples exhibiting particularly low cocaine levels (<0.5 ng/mg) were analysed.

Neonatal hair analysis by liquid chromatography-high-resolution mass spectrometry to reveal gestational exposure to venlafaxine.

Venlafaxine (VEN) is a second generation antidepressant drug, belonging to the class of selective serotonine and norepinephrine reuptake inhibitors, widely used in the treatment of depression and anxiety disorders. Though its pharmacological profile is considered safe, treatment with VEN can cause several nervous, gastrointestinal, cardiovascular and genitourinary adverse effects. Therapeutic drug monitoring for VEN could be useful in specific situations, including exposure to the drug during pregnancy. A liquid chromatography-high-resolution mass spectrometry method was developed and validated for the assay of VEN in 2.5-mg hair samples from 2 newborn identical twin sisters. The analyte was extracted by a rapid, simultaneous pulverization and extraction step, allowing analysis when tiny amounts of hair are available, such as in the case of a newborn. Gradient elution on an Atlantis T3 column was performed using nordiazepam-d5 as an internal standard. Positive ion electrospray ionization and high-resolution full scan determination were performed in an Orbitrap mass spectrometer. The method was linear range in the range 0.2-25 ng/mg, and had a quantification limit of 0.2 ng/mg, a relative standard deviation in the range 0.7%-1.4% (intra-assay) and 2.9%-5.9% (interassay), and was accurate (as % relative error) in the range -9% to + 2%, using a hair sample size as low as 2.5 mg. The utilization of high-resolution/high accuracy mass spectrometry in full-scan mode allowed both the quantitative determination of VEN in the hair of the 2 newborns and the straightforward identification of 4 VEN metabolites, namely O-desmethylvenlafaxine, N-desmethylvenlafaxine, N,N-didesmethylvenlafaxine, and N,O-didesmethylvenlafaxine, by means of retrospective screening, thus unequivocally documenting in utero exposure.

A fast screening MALDI method for the detection of cocaine and its metabolites in hair.

Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry was used for the rapid detection of cocaine, benzoylecgonine and cocaethylene in hair. Different MALDI sample preparation procedures have been tested and the employment of a multi-layer 'graphite-sample-electrosprayed alpha-cyano-4-hydroxycinnamic acid (HCCA)' yielded the best results for standard solutions of the target analytes. The same approach was subsequently applied to hair samples that were known to contain cocaine, benzoylecgonine and cocaethylene, as determined by a classical GC-MS method. It was however necessary to extract hair samples by incubating them in methanol/trifluoroacetic acid for a short time (15 min) at 45 degrees C; 1 microl of the obtained supernatant was deposed on a metal surface treated with graphite, and HCCA was electrosprayed on it. This procedure successfully suppressed matrix peaks and was effective in detecting all the target analytes as their protonated species. The results obtained give further confirmation of the effectiveness of the MALDI for detecting drugs and their metabolites in complex biological matrices. The method can be useful as a fast screening procedure to detect the presence of cocaine and metabolites in hair samples.

Quantification of citalopram or escitalopram and their demethylated metabolites in neonatal hair samples by liquid chromatography-tandem mass spectrometry.

Citalopram and escitalopram are highly selective serotonin reuptake inhibitors widely used in the treatment of depression. They exhibit adverse drug reactions and side effects, however, and the development of specific methods for their determination is of great interest in clinical and forensic toxicology. A liquid chromatography-tandem mass spectrometry method has been developed and validated for the assay of citalopram, escitalopram, and their demethylated metabolites in 10-mg hair samples. The analytes were extracted by incubation in methanol and liquid/liquid extraction with diethyl ether/dichloromethane. Gradient elution on a narrow bore C18 column was realized using clomipramine-d3 as an internal standard. Positive ion electrospray ionization and tandem mass spectrometry determination by collision-induced dissociation were performed in an ion trap mass spectrometer. The method exhibited a linear range of 25 to 2000 pg/mg, a quantification limit of 25 pg/mg for all analytes, relative standard deviations in the range of 12.10 to 9.80 (intraassay), and 13.80 to 11.78 (interassay), and accuracies (as percent recovery of the spiked standards) in the range of 90% to 110%; it was applied to the determination of citalopram and escitalopram and their metabolites in hair samples of two newborns to document their in utero exposure to the drugs. The method proved suitable for neonatal hair analysis of citalopram or escitalopram and was applied to two real cases of gestational exposure.

Simultaneous determination of atracurium and its metabolite laudanosine in post-mortem fluids by liquid chromatography/multiple-stage mass spectrometry on an ion trap.

A method was developed to accurately quantify atracurium (a non-depolarizing skeletal muscle relaxant) and its metabolite laudanosine in post-mortem specimens. Analytes were isolated from blood and tissues by liquid/liquid extraction after adding vecuronium as an internal standard. Chromatographic separation was accomplished by gradient elution in a Synergy Max RP 150 x 2.1 mm column. Positive ion electrospray ionization and mass spectrometric analyses were carried out in an ion trap mass spectrometer under collision-induced dissociation conditions. The method proved selective and sensitive, and was validated in post-mortem blood, heart, lung and liver in the range of 1-2000 ng/mL (blood) and 5-5000 ng/g (tissues); the limits of quantification obtained were 1 ng/mL in blood and 5 ng/g in tissues.

Highly specific quantification of ergotamine in urine, blood, and hair samples by liquid chromatography-tandem mass spectrometry.

Ergotamine has been used for therapeutic purposes since the 1950s, usually to treat vascular headache. It is highly toxic and in large, repeated doses can produce all the symptoms of ergot poisoning. A selective and sensitive method, based on liquid chromatography-tandem mass spectrometry (LC-MS2), has been developed for quantifying ergotamine in biological fluids with use of a quick and easy sample preparation. Ergotamine and the internal standard, trideuterated lysergic acid diethylamide, were extracted from human urine, blood, and hair by means of liquid-liquid extraction at alkaline pH. Gradient elution on a cyanopropyl column was used for chromatographic separation. Positive ion electrospray ionization and tandem mass spectrometry determination by collision-induced dissociation were performed in an ion trap mass spectrometer. The method was validated and successfully applied to a case of iatrogenic ergotism resulting from the intake of ergotamine tartrate for treating headache. For the first time, ergotamine was identified and quantified in hair. The ergotamine concentrations measured were 320 pg/mL in blood, 100 pg/mL in urine, 24 pg/mg in proximal hair, and 15 pg/mg in distal hair.

Potentials of ion trap collisional spectrometry for liquid chromatography/electrospray ionization tandem mass spectrometry determination of buprenorphine and nor-buprenorphine in urine, blood and hair samples.

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the analysis of buprenorphine (BUP) and nor-buprenorphine (NBUP) in biological fluids. Analytes are isolated from urine and blood, after addition of d4-buprenorphine (d4-BUP) as internal standard, by solid-phase extraction. Preparation of hair involves external decontamination, mechanical pulverization, overnight incubation in acidic medium, and neutralization prior to extraction. Enzymatic hydrolysis with beta-glucuronidase may be performed to distinguish between free and total BUP. Chromatographic separation is accomplished by gradient elution on a cyanopropyl 2.1 x 150 mm column. Positive ion ESI and MS analyses are carried out in an ion trap mass spectrometer. The use of this mass analyzer allows effective collisional experiments to be performed on ESI-generated MH+ species. Abundant product ions are produced, which can be monitored together with precursor ions without losing sensitivity. Thus, assay selectivity is definitely increased with respect to LC/ESI-MS/MS methods in which only precursor ions are monitored. The method has good linearity (calibration curves were linear in the range 0.1-10 ng/mL in urine and blood, in the range 10-160 pg/mg in hair) and limits of detection of 0.05 ng/mL for both BUP and NBUP in blood and urine samples, of 4 pg/mg for both analytes in hair. Both intra- and inter-assay precision and accuracy were satisfactory at three concentrations studied: relative standard deviations were <13.7% in urine, <17.3% in blood, <17.8% in hair; percent deviation of the mean from the true value was always <10.5% in urine and blood, <16.1% in hair. The method can be used to determine both analytes in the urine and hair of drug addicts on replacement therapy, and in post-mortem blood specimens when there is suspicion of drug-related death.