Ana de Castro

Target screening and confirmation of 35 licit and illicit drugs and metabolites in hair by LC–MSMS

A liquid chromatography-tandem mass spectrometry (LC-MSMS) target screening in 50mg hair was developed and fully validated for 35 analytes (Δ9-tetrahidrocannabinol (THC), morphine, 6-acetylmorphine, codeine, methadone, fentanyl, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, benzoylecgonine, cocaine, lysergic acid diethylamide, ketamine, scopolamine, alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, 7-aminoflunitrazepam, lorazepam, lormetazepam, nordiazepam, oxazepam, tetrazepam, triazolam, zolpidem, zopiclone, amitriptyline, citalopram, clomipramine, fluoxetine, paroxetine and venlafaxine). Hair decontamination was performed with dichloromethane, and incubation in 2 mL of acetonitrile at 50°C overnight. Extraction procedure was performed in 2 steps, first liquid-liquid extraction, hexane:ethyl acetate (55:45, v:v) at pH 9, followed by solid-phase extraction (Strata-X cartridges). Chromatographic separation was performed in AtlantisT3 (2.1 mm × 100 mm, 3 μm) column, acetonitrile and ammonium formate pH 3 as mobile phase, and 32 min total run time. One transition per analyte was monitored in MRM mode. To confirm a positive result, a second injection monitoring 2 transitions was performed. The method was specific (no endogenous interferences, n=9); LOD was 0.2-50 pg/mg and LOQ 0.5-100 pg/mg; linearity ranged from 0.5-100 to 2000-20,000 pg/mg; imprecision <15%; analytical recovery 85-115%; extraction efficiency 4.1-85.6%; and process efficiency 2.5-207.7%; 27 analytes showed ion suppression (up to -86.2%), 4 ion enhancement (up to 647.1%), and 4 no matrix effect; compounds showed good stability 24-48 h in autosampler. The method was applied to 17 forensic cases. In conclusion, a sensitive and specific target screening of 35 analytes in 50mg hair, including drugs of abuse (THC, cocaine, opiates, amphetamines) and medicines (benzodiazepines, antidepressants) was developed and validated, achieving lower cut-offs than Society of Hair Testing recommendations.

Liquid chromatography tandem mass spectrometry determination of selected synthetic cathinones and two piperazines in oral fluid. Cross reactivity study with an on-site immunoassay device

Since the past few years, several synthetic cathinones and piperazines have been introduced into the drug market to substitute illegal stimulant drugs such as amphetamine and derivatives or cocaine due to their unregulated situation. These emerging drugs are not usually included in routine toxicological analysis. We developed and validated a LC-MS/MS method for the determination of methedrone, methylone, mephedrone, 3,4-methylenedioxypyrovalerone (MDPV), fluoromethcathinone, fluoromethamphetamine, 1-(3-chlorophenyl)piperazine (mCPP) and 3-trifluoromethylphenylpiperazine (TFMPP) in oral fluid. Sample extraction was performed using Strata X cartridges. Chromatographic separation was achieved in 10min using an Atlantis(®) T3 column (100mm×2.1mm, 3μm), and formic acid 0.1% and acetonitrile as mobile phase. The method was satisfactorily validated, including selectivity, linearity (0.2-0.5 to 200ng/mL), limits of detection (0.025-0.1ng/mL) and quantification (0.2-0.5ng/mL), imprecision and accuracy in neat oral fluid (%CV=0.0-12.7% and 84.8-103.6% of target concentration, respectively) and in oral fluid mixed with Quantisal™ buffer (%CV=7.2-10.3% and 80.2-106.5% of target concentration, respectively), matrix effect in neat oral fluid (-11.6 to 399.7%) and in oral fluid with Quantisal™ buffer (-69.9 to 131.2%), extraction recovery (87.9-134.3%) and recovery from the Quantisal™ (79.6-107.7%), dilution integrity (75-99% of target concentration) and stability at different conditions (-14.8 to 30.8% loss). In addition, cross reactivity produced by the studied synthetic cathinones in oral fluid using the Dräger DrugTest 5000 was assessed. All the analytes produced a methamphetamine positive result at high concentrations (100 or 10μg/mL), and fluoromethamphetamine also at low concentration (0.075μg/mL).

Methadone, cocaine, opiates, and metabolite disposition in umbilical cord and correlations to maternal methadone dose and neonatal outcomes.

Objectives: The purpose was to explore methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) umbilical cord disposition, correlate with maternal methadone dose and neonatal outcomes, and evaluate the window of drug detection in umbilical cord of in utero illicit drug exposure. Methods: Subjects comprised 19 opioid-dependent pregnant women from 2 clinical studies, one comparing methadone and buprenorphine pharmacotherapy for opioid-dependence treatment and the second examining monetary reinforcement schedules to maintain drug abstinence. Correlations were calculated for methadone and EDDP umbilical cord concentrations and maternal methadone dose, and neonatal outcomes. Cocaine- and opiate-positive umbilical cord concentrations were compared with those in placenta and meconium, and urine specimens collected throughout gestation. Results: Significant positive correlations were found for umbilical cord methadone concentrations and methadone mean daily dose, mean dose during the third trimester, and methadone cumulative daily dose. Umbilical cord EDDP concentrations and EDDP/methadone concentration ratios were positively correlated to newborn length, peak neonatal abstinence syndrome (NAS) score, and time-to-peak NAS score. Methadone concentrations and EDDP/methadone ratios in umbilical cord and placenta were positively correlated. Meconium identified many more cocaine- and opiate-positive specimens than did umbilical cord. Conclusions: Umbilical cord methadone concentrations were correlated to methadone doses. Also, our results indicate that methadone and EDDP concentrations might help to predict the NAS severity. Meconium proved to be more suitable than umbilical cord to detect in utero exposure to cocaine and opiates; however, umbilical cord could be useful when meconium is unavailable due to in utero or delayed expulsion.

Quantification of selected synthetic cannabinoids and Δ9-tetrahydrocannabinol in oral fluid by liquid chromatography-tandem mass spectrometry.

An LC-MS/MS method for the quantification of the synthetic cannabinoids JWH-200, JWH-250, JWH-073, JWH-018, HU-211, CP 47,497 and CP 47,497-C8, and THC in oral fluid was developed and validated. Samples (0.5 mL) were extracted using Strata X cartridges (Phenomenex). Chromatographic separation was achieved with a Sunfire™ IS column (20×2.1 mm, 3.5 μm) (Waters Corp.), with formic acid 0.1% and acetonitrile as mobile phase. A different chromatographic gradient was applied for the separation of the analytes depending on the ionization mode employed, with a total chromatographic run of 14 min. Detection was performed in a Quattro Micro™ API ESCI (Waters Corp.), using electrospray in the positive mode (ESI+) for JWH-200, JWH-250, JWH-073, JWH-018 and THC, and ESI- for HU-211, CP 47,497, and CP 47,497-C8. Validation of the method included the assessment of selectivity, linearity (0.1-2.5 to 200 ng/mL), limits of detection (0.025-1 ng/mL) and quantification (0.1-2.5 ng/mL), imprecision (%CV≤14.4%), accuracy (91.8-109.7% of target concentration), extraction recovery (65.4-105.6%) and Quantisal recovery (56.1-66.7%), and matrix effect (neat oral fluid: -56.0% to 38.5%; oral fluid in Quantisal buffer: -15.1% to -71.7%). The application of this method to oral fluid samples from roadside testing will provide unique information on the use of these new synthetic drugs by Spanish drivers.

An LC-MS/MS methodological approach to the analysis of hair for amphetamine-type-stimulant (ATS) drugs, including selected synthetic cathinones and piperazines

Amphetamine-type-stimulants (ATS) are the second most commonly used group of illicit drugs worldwide. However, in the last few years, new psychoactive substances (NPS) with stimulant effects have appeared on the illegal market, which are not detected with traditional analytical methods. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination in hair of classic ATS (amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine), synthetic cathinones (methylone, methedrone, mephedrone, 3,4-methylenedioxypyrovalerone, (±)-4-fluoromethamphetamine and 4-fluoromethcathinone), synthetic piperazines (1-(3-chlorophenyl)piperazine (mCPP) and 3-trifluoromethylphenylpiperazine), and medicines (trazodone and phenazone) that produce mCPP as a metabolite, was developed and fully validated. Hair samples (30 mg) were incubated in acid methanol (0.1% HCl) and extracted by a mixed-mode solid-phase extraction. Chromatographic separation was performed using an Atlantis T3 (3 µm; 2.1x50 mm) analytical column, and ammonium formate 2 mM pH 3 and acetonitrile as mobile phase. The method was validated, including selectivity (no endogenous or exogenous interferences); linearity (2-20 to 2000-4000 pg/mg); limits of detection (0.2 to 5 pg/mg) and quantification (2 to 20 pg/mg); accuracy (93.4-109.4% of target concentration); imprecision (%CV<11.6%); extraction recovery (40.5-92.1%); matrix effect (24.1-227.2%); process efficiency (9.8-165.7%) and stability in the autosampler (-14.5% of loss). The method was applied to the analysis of 16 hair samples. Amphetamine (n=7; 69.1-777.1 pg/mg), methamphetamine (n=3; 120.4-1,538.9 pg/mg), MDA (n=2; 27.8-135.4 pg/mg) and MDMA (n=8; 73.4-3,654.5 pg/mg) were found. Moreover, 10 positive results for mCPP were detected (341.7->4000 pg/mg); however, in all cases trazodone identification (2085.3->4000 pg/mg) probed a licit origin of mCPP. Copyright

Water-compatible imprinted pills for sensitive determination of cannabinoids in urine and oral fluid

A novel molecularly imprinted solid phase extraction (MISPE) methodology followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed using cylindrical shaped molecularly imprinted pills for detection of Δ(9)-tetrahydrocannabinol (THC), 11-nor-Δ(9)-tetrahydrocannabinol carboxylic acid (THC-COOH), cannabinol (CBN) and cannabidiol (CBD) in urine and oral fluid (OF). The composition of the molecular imprinted polymer (MIP) was optimized based on the screening results of a non-imprinted polymer library (NIP-library). Thus, acrylamide as functional monomer and ethylene glycol dimethacrylate as cross-linker were selected for the preparation of the MIP, using catechin as a mimic template. MISPE pills were incubated with 0.5 mL urine or OF sample for adsorption of analytes. For desorption, the pills were transferred to a vial with 2 mL of methanol:acetic acid (4:1) and sonicated for 15 min. The elution solvent was evaporated and reconstituted in methanol:formic acid (0.1%) 50:50 to inject in LC-MS/MS. The developed method was linear over the range from 1 to 500 ng mL(-1) in urine and from 0.75 to 500 ng mL(-1) in OF for all four analytes. Intra- and inter-day imprecision were <15%. Extraction recovery was 50-111%, process efficiency 15.4-54.5% and matrix effect ranged from -78.0 to -6.1%. Finally, the optimized and validated method was applied to 4 urine and 5 OF specimens. This is the first method for the determination of THC, THC-COOH, CBN and CBD in urine and OF using MISPE technology.

Development and validation of a liquid chromatography tandem mass spectrometry method for the determination of cannabinoids and phase I and II metabolites in meconium

A liquid chromatography-tandem mass spectrometry (LC-MSMS) method was developed and fully validated for the determination of Δ9-tetrahydrocannabinol (THC), 11-hydroxyTHC (OHTHC), 11-nor-9-carboxyTHC (THCCOOH), 8-β-11-dihydroxyTHC (diOHTHC), cannabinol, cannabidiol, and THC and THCCOOH glucuronides in 0.25±0.02g meconium. Samples were homogenized in methanol and subjected to cation exchange solid-phase extraction. Chromatographic separation was performed on a Kinetex C18 column (50 mm×2.1mm, 2.6μm) at 35°C, with a gradient of 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min; total run time was 10min. Two transitions per analyte were monitored in MRM mode. The method was specific and sensitive; LOD was from 1 to 2ng/g, and LOQ from 4 to 10ng/g; linearity ranged from 4 to 400 ng/g for all the analytes, except for THC glucuronide (10-400ng/g); intra-assay, inter-assay and total imprecision were <11.2%, <13.45% and <15.6%, respectively; accuracy ranged from 93.9% to 109.0% of the target concentration; matrix effect, extraction and process efficiency ranged from -26.4% to -71.4%, 49.9% to 69.5% and 14.3% to 45.0%, respectively. The inclusion of THC and THCCOOH glucuronides avoided the need for the hydrolysis process, thus facilitating sample pretreatment. Application of the method to 19 authentic meconium specimens from uncontrolled pregnancies or women suspicious of drug consumption revealed fetal cannabis exposure in 4 newborns. THCCOOH (24.1-288.8ng/g), diOHTHC (53.2-332.4ng/g), THC (4.2-7.7ng/g), CBN (30.7-93.3ng/g) and CBD (7.1-251.5ng/g) were detected in all cases; THCCOOH glucuronide (190.2-306.8ng/g) in 3 cases; and OHTHC (11.9ng/g) in the remaining one; however, THC glucuronide was not identified in any specimen.

Bioanalysis for cocaine, opiates, methadone and amphetamines exposure detection during pregnancy.

Drug exposure during pregnancy constitutes a major legal issue and a public health concern. Drug and metabolite determination in biological matrices from mother and newborn is an objective indication of prenatal drug exposure. However, limited data are available regarding the interpretation of these analytical results in terms of window of detection and degree of exposure. We collected paired maternal hair, meconium, placenta, and umbilical cord from 727 mother-newborn dyads. We analyzed these specimens by liquid chromatography-tandem mass spectrometry for the determination of cocaine, opioids, methadone, and amphetamines, and compared the analytical results from the four different matrices. The cases were divided in non-exposure, low, and frequent exposure, based on maternal hair concentrations and segmental analysis by trimesters. For cocaine, 62 cases tested positive in hair, 9 in meconium, 6 in placenta and 7 in umbilical cord. In the case of opioids, 14 maternal hair cases were positive, 11 meconium and umbilical cord and 9 placenta samples. For methadone, 11 cases were positive in hair, 9 in meconium and 6 in placenta and umbilical cord. For amphetamines, 18 cases were positive according to maternal hair, but all meconium, placenta, and umbilical cord tested negative. Maternal hair was the most sensitive specimen to detect drug exposure during pregnancy. Meconium, placenta, and umbilical cord tested positive if hair concentrations showed frequent drug use during the whole pregnancy, especially during the third trimester. Meconium, placenta, and umbilical cord also tested positive for morphine and metabolites, if this drug was administered during labour and delivery. Copyright

Liquid Chromatography-Mass Spectrometry for the Determination of Antidepressants and Some of their Major Metabolites in Human Biological Matrices

Due to the high prescription of antidepressants and their frequent involvement in clinical and forensic intoxications, reliable analytical techniques for identification and quantification of these therapeutic drugs should be available in clinical and toxicological laboratories. Improvements in liquid chromatography-mass spectrometry LC-MS(MS) technology over the last two decades have favored this technique to be one of the most commonly employed for this purpose, as it combines the high selectivity and sensitivity of the mass spectrometer with the great versatility of the liquid chromatographic separation. In this chapter, LC-MS(MS) applications for antidepressants determination are reviewed, detailing typical sample preparation techniques used for these therapeutic drugs, as well as common chromatographic and mass spectrometric characteristics. In addition, an LC-MS/MS method for the most common antidepressants used in clinical practice is described as an example of a whole method development and validation.