Elena Lendoiro

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.

Methamphetamine transiently increases the blood–brain barrier permeability in the hippocampus: Role of tight junction proteins and matrix metalloproteinase-9

Methamphetamine (METH) is a powerful stimulant drug of abuse that has steadily gained popularity worldwide. It is known that METH is highly neurotoxic and causes irreversible damage of brain cells leading to neurological and psychiatric abnormalities. Recent studies suggested that METH-induced neurotoxicity might also result from its ability to compromise blood-brain barrier (BBB) function. Due to the crucial role of BBB in the maintenance of brain homeostasis and protection against toxic molecules and pathogenic organisms, its dysfunction could have severe consequences. In this study, we investigated the effect of an acute high dose of METH (30mg/kg) on BBB permeability after different time points and in different brain regions. For that, young adult mice were sacrificed 1h, 24h or 72h post-METH administration. METH increased BBB permeability, but this effect was detected only at 24h after administration, being therefore a transitory effect. Interestingly, we also found that the hippocampus was the most susceptible brain region to METH, comparing to frontal cortex and striatum. Moreover, in an attempt to identify the key players in METH-induced BBB dysfunction we further investigated potential alterations in tight junction (TJ) proteins and matrix metalloproteinase-9 (MMP-9). METH was able to decrease the protein levels of zonula occludens (ZO)-1, claudin-5 and occludin in the hippocampus 24h post-injection, and increased the activity and immunoreactivity of MMP-9. The pre-treatment with BB-94 (30mg/kg), a matrix metalloproteinase inhibitor, prevented the METH-induced increase in MMP-9 immunoreactivity in the hippocampus. Overall, the present data demonstrate that METH transiently increases the BBB permeability in the hippocampus, which can be explained by alterations on TJ proteins and MMP-9.

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).

Simultaneous quantification of Δ9-tetrahydrocannabinol, 11-nor-9-carboxy-tetrahydrocannabinol, cannabidiol and cannabinol in oral fluid by microflow-liquid chromatography–high resolution mass spectrometry

Δ(9)-Tetrahydrocannabinol (THC) is the primary target in oral fluid (OF) for detecting cannabis intake. However, additional biomarkers are needed to solve interpretation issues, such as the possibility of passive inhalation by identifying 11-nor-9-carboxy-THC (THCCOOH), and determining recent cannabis smoking by identifying cannabidiol (CBD) and/or cannabinol (CBN). We developed and comprehensively validated a microflow liquid chromatography (LC)-high resolution mass spectrometry method for simultaneous quantification of THC, THCCOOH, CBD and CBN in OF collected with the Oral-Eze(®) and Quantisal™ devices. One milliliter OF-buffer solution (0.25mL OF and 0.5mL of Oral-Eze buffer, 1:3 dilution, or 0.75mL Quantisal buffer, 1:4 dilution) had proteins precipitated, and the supernatant subjected to CEREX™ Polycrom™ THC solid-phase extraction (SPE). Microflow LC reverse-phase separation was achieved with a gradient mobile phase of 10mM ammonium acetate pH 6 and acetonitrile over 10min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by targeted-MSMS experiments. The assay was linear 0.5-50ng/mL for THC, CBD and CBN, and 15-500pg/mL for THCCOOH. Intra- and inter-day and total imprecision were <10.8%CV and bias 86.5-104.9%. Extraction efficiency was 52.4-109.2%, process efficiency 12.2-88.9% and matrix effect ranged from -86 to -6.9%. All analytes were stable for 24h at 5°C on the autosampler. The method was applied to authentic OF specimens collected with Quantisal and Oral-Eze devices. This method provides a rapid simultaneous quantification of THCCOOH and THC, CBD, CBN, with good selectivity and sensitivity, providing the opportunity to improve interpretation of cannabinoid OF results by eliminating the possibility of passive inhalation and providing markers of recent cannabis smoking.

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

Applications of Tandem Mass Spectrometry (LC–MSMS) in estimating the post-mortem interval using the biochemistry of the vitreous humour

It is widely accepted that the quantification of potassium concentrations ([K+]) and hypoxanthine ([Hx]) in the vitreous humour is useful in estimating the time of death within a recent time interval. Despite all the advances made in this area, it is well recognized that difficulties in calibration, validation and the use of different methodologies and instrumentation from different laboratories may lead to obtaining different concentrations from the same sample. The extraction of the vitreous humour itself should also be carried out with a precise technique, taking care during the procedure to avoid causing any vascular injury that might lead to haematic contamination. Any fluid that is not clear could lead to erroneous results and should be discarded. We present a new LC-MSMS method developed for quantitative and qualitative analysis of [Hx] (valid also for guanine and xanthine), and qualitative determination of uric acid in vitreous humour. We also introduce a methodology to assess haematic contamination in order to improve the estimation of time since death. The method was fully validated in terms of linearity, sensitivity, imprecision, analytical recovery, extraction and process efficiency and matrix effect.

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.

The Application of Flow Cytometry as a Rapid and Sensitive Screening Method to Detect Contamination of Vitreous Humor Samples and Avoid Miscalculation of the Postmortem Interval

Research into maximizing the speed, precision, and reliability of estimating the postmortem interval (PMI) has been a recurring object of investigation and methodologies based on the vitreous humor (VH) have provided good results. However, contamination from causes not readily apparent, such as blood, can occur, and thus lead not only to an erroneous estimation of PMI, but also interfere with the correct identification of other substances in the VH. We have developed a flow cytometry method which quantifies blood contamination and is able to detect erythrocytes in 1:750,000 dilution of contaminated VH which affects the results of hypoxanthine. It is an improvement on the previous more complex mass spectrometry method, being faster, more sensitive, and readily available. As such, it could be proposed for the rapid screening of appropriate samples by detecting and eliminating blood contaminated samples from PMI estimation.

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.