LC-MS-MS Determination of Cytostatic Drugs on Surfaces and in Urine to Assess Occupational Exposure.

The ever-increased usage of cytostatic drugs leads to high risk of exposure among healthcare workers. Moreover, workers are exposed to multiple compounds throughout their lives, leading to cumulative and chronic exposure. Therefore, multianalyte methods are the most suitable for exposure assessment, which minimizes the risks from handling cytostatic drugs and ensures adequate contamination containment. This study describes the development and full validation of two liquid chromatography-tandem mass spectrometry methods for the detection of gemcitabine, dacarbazine, methotrexate, irinotecan, cyclophosphamide, doxorubicinol, doxorubicin, epirubicin, etoposide, vinorelbine, docetaxel and paclitaxel in working surfaces and urine samples. The urine method is the first to measure vinorelbine and doxorubicinol. For surfaces, limits of detection (LOD) and limits of quantification (LOQ) were 5-100 pg/cm2, and linearity was achieved up to 500 pg/cm2. Inaccuracy was between -11.0 and 8.4%. Intra-day, inter-day and total imprecision were <20%, except for etoposide and irinotecan (<22.1%). In urine, LOD and LOQ were 5-250 pg/mL, with a linear range up to 1,000-5,000 pg/mL. Inaccuracy was between -3.8 and 14.9%. Imprecision was <12.4%. Matrix effect was from -58.3 to 1,268.9% and from -66.7 to 1,636% in surface and urine samples, respectively, and extraction efficiency from 10.8 to 75% and 47.1 to 130.4%, respectively. All the analytes showed autosampler (6°C/72 h), freezer (-22°C/2 months) and freeze/thaw (three cycles) stability. The feasibility of the methods was demonstrated by analyzing real working surfaces and patients' urine samples. Contamination with gemcitabine, irinotecan, cyclophosphamide, epirubicin and paclitaxel (5-4,641.9 pg/cm2) was found on biological safety cabinets and outpatients' bathrooms. Analysis of urine from patients under chemotherapy identified the infused drugs at concentrations higher than the upper LOQ. These validated methods will allow a comprehensive evaluation of both environmental and biological contamination in hospital settings and healthcare workers.

Comparison of conventional dried blood spots and volumetric absorptive microsampling for tacrolimus and mycophenolic acid determination.

Therapeutic drug monitoring (TDM) of immunosuppressants is essential to avoid either rejection or toxicity after solid organ transplantations. Capillary microsampling approaches are an outstanding alternative to conventional venous sampling for TDM (easy and non-invasive collection, enabling self-sampling, and cost-saving shipment, processing and storage). Volumetric absorptive microsampling (VAMS) has gained importance in the last years, as it was meant to overcome the hematocrit (Hct) related issues commonly associated to DBS analysis. Despite all the benefits, microsampling techniques performance (including a thorough clinical validation) should be set up before their implementation in clinical practice. The aim of this study was to perform a clinical validation for both tacrolimus (TAC) and mycophenolic acid (MPA) in both DBS and Mitra™ VAMS. For the clinical validations, two different requirements were set up: analytical (following EMA and FDA guidelines) and clinical (following the Royal College of Pathologists of Australasia -RCPA- recommendations) acceptance criteria. For DBS, both analytical and clinical acceptance criteria were fulfilled for TAC, with 98.7% and 95% of the paired samples within the preset limits, respectively. For MPA, the analytical criterion was met (70.6% of paired specimens), although only half of the pairs were within the clinical limits. For VAMS, the clinical validation for both TAC and MPA showed good correlations but significant lower concentrations in VAMS compared to the routine matrices. After VAMS concentrations correction, the analytical requirement was fulfilled for both analytes (71.1% for TAC, 75% for MPA), although the more restrictive criteria recommended by the RCPA were not met for any analyte (half of the samples fell within the acceptance area). In addition, no significant Hct impact on the quantification was found in any case. Also, a preliminary home-sampling trial was set up, showing promising results. Moreover, a comparison between VAMS vs. DBS analytical and clinical performances was carried out, including a home-sampling trial, sample quality results and costs. Although the analytical performance for both VAMS and DBS was similar, DBS were superior regarding clinical criteria, sampling quality and cost.

Determination of antidepressants and benzodiazepines in paired hair and nail samples.

Hair and nails are keratinized matrices that can be used in Toxicology as matrices for the long-term detection of substances. Whereas hair is an established matrix with decades of use in this field, nails have been less studied, especially including a comparison to hair samples. Specifically in the case of antidepressant and benzodiazepine drugs, very few publications analyzing these drugs in nail samples exist as of yet. For this reason, in the present study a method for the detection of 12 antidepressant and benzodiazepine drugs in hair and nail samples was developed. Samples were decontaminated with 3 washes of dichloromethane, and 25 or 30 mg of hair and nails, respectively, were pulverized. Then, the samples were incubated with 1.5 mL water:ACN (50:50, v/v) with horizontal agitation for 90 min. The supernatant was evaporated and reconstituted in 200 µL of methanol and 2 mL of 2% FA in water, submitted to solid phase extraction (SPE) using Oasis MCX cartridges and analyzed by LC-MS/MS. The method was satisfactorily validated in nail and hair samples for the following parameters: linearity, LOD (0.005-0.02 ng/mg), LOQ (0.01-0.02 ng/mg), selectivity, carryover, accuracy, imprecision, matrix effect, extraction efficiency, process efficiency and autosampler stability. Matched fingernail, toenail and hair samples were obtained from 21 patients under treatment with any of the studied drugs and analyzed with the developed method. The most frequently detected drugs were venlafaxine (n = 11), trazodone (n = 6), zolpidem (n = 5), alprazolam (n = 5) and nordiazepam (n = 5). Concentrations in hair, fingernails and toenails, respectively, were 44.31 ng/mg, 8.05-43.35 ng/mg and 7.02-22.69 ng/mg for venlafaxine; 5.40-19.08 ng/mg, 0.13-1.00 ng/mg and 0.42-1.04 ng/mg for trazodone; 13.86 ng/mg, 5.19 ng/mg and 9.11 ng/mg for fluoxetine; 7.42 ng/mg, 1.85 ng/mg and 0.03-2.81 ng/mg for sertraline; 0.40-1.42 ng/mg, 0.12 ng/mg and 0.16 ng/mg for zolpidem; and 0.02-0.11 ng/mg, 0.07-1.07 ng/mg and 0.05 ng/mg for alprazolam for the patients under active treatment. Hair concentrations were higher than nail concentrations for most drugs in patients under active treatment, with the exception of diazepam (n = 1; 0.12 ng/mg in hair and 0.41 ng/mg in fingernails). Fingernail concentrations were lower than toenail concentrations in patients under active treatment in most compared cases. Comparison of fingernails and toenails of a patient with antifungal treatment did not show an observable effect in concentrations.

Determination and Distribution of Cannabinoids in Nail and Hair Samples.

Hair has been used for decades in toxicology as a biological matrix for long-term detection of substances. Nails are another keratinized matrix that is being studied as an alternative when hair cannot be obtained. Although cannabis is the most prevalent illicit drug in the world, cannabinoid distribution in nails compared with hair has been scarcely studied. In this work, we described two methods for the determination of cannabidiol (CBD), cannabinol (CBN) and Δ9-tetrahydrocannabinol (THC), and main metabolites of THC [11-nor-9-carboxy-THC (THCCOOH), 11-hydroxy-THC (OHTHC) and 8-ß-11-dihydroxyTHC (diOHTHC)] in nail and hair samples. After an alkaline hydrolysis, samples were submitted to solid-phase extraction and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS-MS). The methods were fully validated, with good linearity (r2 > 0.99) in the range of 20 to 100 to 20,000 pg/mg. No endogenous or exogenous interferences were found. Accuracy was from 99.5% to 109.8%, and imprecision was <6.9%. Ion suppression (up to -74.4%) was observed for all the analytes, except for diOHTHC at low concentrations in hair (46.1%). Extraction efficiency ranged from 21.5% to 84.5%. The methods were applied to matched nail and hair specimens from 23 cannabis users to study the incorporation and distribution of the cannabinoids into these matrices. Only CBD, CBN and THC were detected in the samples, with much higher concentrations in fingernails than in toenails and hair. Correlations between analyte concentrations in the different matrices and with reported drug consumption were studied. A preliminary cut-off for THC in toenails was calculated using the cut-off proposed by the Society of Hair Testing in hair for the identification of chronic cannabis use.

Determination of antipsychotic drugs in nails and hair by liquid chromatography tandem mass spectrometry and evaluation of their incorporation into keratinized matrices.

Nail samples are an alternative to hair for long-term monitoring of drug use, although there are a limited number of studies about its applicability. This study presents the development and validation of a LC-MS/MS method for the determination of five antipsychotic drugs (clozapine, haloperidol, levomepromazine, olanzapine and quetiapine) in nail and in hair samples. Samples were washed with dichloromethane, pulverized with a ball mill, and incubated in water:acetonitrile (50:50 v/v) with horizontal agitation for 90 min. Then, samples were purified by solid phase extraction with OASIS MCX cartridges and analysed by LC-MS/MS. The analytical method was fully validated in nails and in hair, including: limits of detection (2.5 pg/mg and 2.5-10 pg/mg, respectively), limits of quantification (LOQ) (10 pg/mg and 10-20 pg/mg, respectively), linearity (LOQ to 10,000 pg/mg), selectivity (no endogenous or exogenous interferences), accuracy (97.4-106.9% and 97.3-108.2%, respectively), imprecision (<7.9% and <8.6%, respectively), extraction efficiency (62.3%-109.8% and 45.1%-83.6%, respectively), matrix effect (-35.6% to 654.4% and -71.0% to -10.8%, respectively) and autosampler stability after 72 h (%loss <12.4%). Moreover, paired fingernail, toenail and hair samples from 13 patients under chronic treatment were analysed, and concentrations in the different matrices were compared. Concentrations in real samples ranged from 11.3-8306 pg/mg in fingernails, from 12.7-1755.4 pg/mg in toenails and from 17.6-24045.4 pg/mg in hair. Hair concentrations were generally higher than nail concentrations; however, a variable pattern was found in fingernails and toenails depending on the case. In addition, concentrations in paired hair and nail samples from a patient under chronic treatment with quetiapine at different doses were studied for 1-year.

Volumetric Absorptive Microsampling (VAMS) for assaying immunosuppressants from venous whole blood by LC-MS/MS using a novel atmospheric pressure ionization probe (UniSpray™).

Therapeutic drug monitoring (TDM) of immunosuppressants (IMS) is crucial to prevent rejection or toxicity after solid organ transplantation. Microsampling techniques (sampling <50 µL of blood) can be a good alternative to conventional venous sampling for TDM, due to their numerous advantages, including its easy and low-invasive sampling, enabling self-collection, and cost-saving shipment and storage. Furthermore, volumetric absorptive microsampling (VAMS) enables the collection of precise and accurate blood volumes, overcoming the hematocrit (Hct) effect related to dried blood spots, while offering the same benefits. In this work, an LC-MS/MS method for the determination of the 5 most common IMS (mycophenolic acid -MPA-, tacrolimus -TAC-, sirolimus -SIR-, everolimus -EVE- and cyclosporin A -CsA-) in venous blood collected with Mitra™ VAMS devices was developed and validated, employing a novel LC-MS/MS interface, Unispray™. The method was fully validated including linearity, limits of detection (LOD) and quantification (LLOQ), accuracy, precision, selectivity, carry-over, matrix effect, recovery, impact of Hct on recovery and autosampler and short-/long-term stability, satisfying acceptance criteria in all cases. LLOQs were 0.5 ng/mL for TAC, SIR and EVE, 20 ng/mL for CsA and 75 ng/mL for MPA. No impact of the Hct (range: 0.2 to 0.62 L/L) on recovery was found for any analyte. All compounds were stable in VAMS for at least 8 months at -20 °C. In addition, as part of the VAMS analytical method validation, we performed for the first time a broad statistical study to compare liquid venous blood concentrations from patients under TAC (n = 53) and MPA (n = 20) treatment to those observed when the same specimens were absorbed into VAMS. Our results showed that venous blood VAMS concentrations were correlated to those found in the original liquid venous blood, proving that the VAMS material itself will not bias blood drug concentrations. Therefore, the present method could be applied to evaluate possible correlations between venous blood and capillary blood collected with VAMS.

Assessment of Tobacco Exposure During Pregnancy by Meconium Analysis and Maternal Interview.

Smoking during pregnancy can have serious obstetric and fetal complications. Therefore, it is essential to identify in utero exposure to tobacco, being meconium the matrix of choice for this purpose. Meconium (n = 565) was analyzed for nicotine, cotinine and hydroxycotinine by LC-MS-MS. Then, tobacco meconium results were compared with smoking habits during pregnancy and neonatal outcomes measures (birth weight, length, head circumference, gestational age and Apgar scores). Although meconium analysis increased identification of in-utero exposure to tobacco (17.7% meconium positive specimens vs 13.5% mothers admitting tobacco use during pregnancy), there was a statistically significant relationship between meconium results and interview answers (P < 0.001). Birth weight was significantly lower for newborns with meconium positive results in males (P = 0.023) and females (P = 0.001), while for length significance was only observed in females (P = 0.001); however, when excluding meconium specimens positive for other drugs, a statistically significant difference was only found for female weight (P = 0.045). Meconium analysis proved to be more reliable for tobacco prenatal exposure detection than maternal interview. In addition, positive meconium results increased the probability for low birth weight, especially in females.

A multidrug LC-MS/MS method for the determination of five immunosuppressants in oral fluid.

Aim: This study aimed: to develop and validate an LC-MS/MS method for mycophenolic acid, tacrolimus, sirolimus, everolimus and cyclosporin A in oral fluid (OF), as an essential tool to study the usefulness of OF as an alternative matrix for immunossuppressants' therapeutic drug monitoring; and to find the best OF collector for these analytes. Materials & Methods: Chromatographic separation was achieved using an XBridge® Shield RP18 analytical column maintained at 65ºC, using 2 mM ammonium formate and 0.1% formic acid in water (A) and acetonitrile (B) as mobile phase. OF sample was extracted with solid phase extraction after sonication and protein precipitation. Results & Conclusions: Method validation met all the acceptance criteria. LODs were 0.05-1 ng/ml, and LOQs 0.1-5 ng/ml. Silanized tubes offered the best recoveries. The method was successfully applied to 31 OF specimens, describing everolimus detection in OF for the first time. Conclusion: The proposed method is sensitive enough for the detection of OF trough concentrations in patients receiving immunosuppressants when using an appropriate OF collector.

A LC-MS/MS method for the determination of common synthetic cathinones in meconium.

New psychoactive substances have been introduced into the market in the last years due to their unregulated status. Synthetic cathinones are one of their main representatives, and they have shown to produce neonatal complications. It is important to have objective tools to identify in utero exposure to drugs that have shown to produce neonatal complications. An analytical method was developed and fully validated for the determination of common synthetic cathinones, including methylone, methedrone, mephedrone, 3,4-methylenedioxypyrovalerone (MDPV), (±)-4-fluoromethamphetamine and 4-fluoromethcathinone in meconium. Meconium (0.25 ±â€¯0.02 g) was homogenized with methanol by sonication for 30 min. After centrifugation, the sample was extracted with Oasis MCX columns. The analysis was performed by LC-MS/MS using an Atlantis T3 column (3 µm, 2.1 × 50 mm) and a gradient with acetonitrile and 0.1% formic acid in water. Method validation included the following parameters: selectivity (no endogenous or exogenous interferences), limits of detection (n = 3, 0.5-1 ng/g) and quantification (n = 3, 1-2 ng/g), linearity (n = 5, LOQ-200 ng/g), imprecision (n = 15, 0% to 10%), accuracy (n = 15, 87.3% to 97.8%), matrix effect (n = 10, -76% to -28.1%), extraction efficiency (n = 6, 63.7% to 91.3%), total process efficiency (n = 6, 16% to 60.2%) and stability for 72 h in the autosampler (n = 3, %loss = -6.7% to 5.1%). The method was applied to 28 meconium specimens.

Toxicological oral fluid results among Spanish drivers testing positive on on-site drug controls from 2013 to 2015.

BACKGROUND: Driving under the influence of drugs (DUID) increases the risk of serious injury or death in traffic accidents. The aim of this study was to provide information about DUID in Spanish drivers. METHODS: 10,064 oral fluid samples were collected from Spanish drivers that tested positive on the roadside using the Dräger DrugTest 5000 (DDT5000) between 2013 and 2015. Samples were collected using Quantisal™ and analysed by LC-MS/MS at the Toxicology Laboratory of the Institute of Forensic Science of the University of Santiago de Compostela. RESULTS: Drivers were mainly young men (85.1% male, 29.7 ± 8.1 years old). In 98.5% of cases, LC-MS/MS results confirmed at least one of the positive results detected on the roadside. Cannabis (82.4%) and cocaine (42.1%) were the most commonly detected drugs. Poly-drug use was observed in 42.7% of drivers, mostly for all illicit drugs (>80%) except for cannabis (42.6%). Illicit drug and single-drug use was more frequent among drivers under 35 years old, and medicines and poly-drug use more common among drivers older than 35 years old. The on-site device performance was calculated using both the DDT5000 cut-offs and the LC-MS/MS method LOQs. Sensitivity (>73% vs >58%), specificity [>94% for all the compounds regardless the cut-offs used, except for cannabis (71%)] and accuracy (>87.5% with both cut-offs) fulfilled the DRUID Project requirements in all cases. CONCLUSION: LC-MS/MS confirmation result was negative in only 1.5% of the cases. The DUID driver profile was a young man, consuming cannabis or a combination of cannabis and cocaine.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of nicotine and its metabolites in placenta and umbilical cord.

Tobacco exposure during pregnancy is associated with obstetric and fetal complications. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine nicotine, cotinine, and hydroxycotinine (OH-cotinine) in placenta (PL) and umbilical cord (UC). Specimens were homogenized in water, followed by solid-phase extraction. Chromatographic separation was performed using an Atlantis® HILIC Silica column. Detection was accomplished in electrospray in positive mode. Method validation included: linearity (5 to 1000 ng/g), accuracy (86.9 to 105.2% of target concentration in PL, and 89.1 to 105.0% in UC), imprecision (6.8 to 11.8% in PL, and 7.6 to 12.2% in UC), limits of detection (2 ng/g for cotinine and OH-cotinine, and 5 ng/g for nicotine) and quantification (5 ng/g), selectivity (no endogenous or exogenous interferences), matrix effect (-34.1 to -84.5% in PL, %CV = 9.1-24.0%; -18.9 to -84.7% in UC, %CV = 10.2-23.9%), extraction efficiency (60.7 to 131.5% in PL, and 64.1 to 134.2% in UC), and stability 72 h in the autosampler (<11.5% loss in PL, and < 13% loss in UC). The method was applied to 14 PL and UC specimens from tobacco users during pregnancy. Cotinine (6.8-312.2 ng/g in PL; 6.7-342.3 ng/g in UC) was the predominant analyte, followed by OH-cotinine (

Usefulness of hair analysis and psychological tests for identification of alcohol and drugs of abuse consumption in driving license regranting.

The implementation of the points-based driving license helps to change the drivers' behavior and is related to a reduction of traffic accidents and fatalities. In Spain, when a driver loses all points, the driving license is revoked, so the driver must enroll on a Driver Awareness and Re-education (DARE) course. However, at the moment offenders are not submitted to any test to confirm absence of alcohol or drugs of abuse consumption, even when 9% of Spanish drivers lose their driving license for driving under the influence (DUI). The objective of this pilot study was the comparison of the usefulness of psychological tests and hair analysis to identify those individuals with a chronic consumption of alcohol and drugs of abuse among drivers performing DARE courses. Volunteers were submitted to the AUDIT and DAST-10 tests. Also a hair sample was collected and analyzed for ethylglucuronide (EtG) (LOQ 5pg/mg) and 35 licit and illicit drugs (LOQ 5-50pg/mg) by LC-MS/MS. Sixty-one participants with a mean age of 37.2±11.6years, and mainly men (90.2%), were recruited and performed AUDIT and DAST-10 tests. All hair samples were analyzed for EtG and 17 samples for licit and illicit drugs. Mean AUDIT score was 9.6 (SD=7.5), showing a value ≥8 (indicator of hazardous and harmful alcohol use) in 52.4% of cases. Mean DAST-10 score was 2.9 (SD=3.3), but a score ≥6 was detected in 21.3% of cases (indicating drug abuse or dependence). Twenty-two samples were positive for EtG, 8 for drugs of abuse (8 cocaine, 2 opioids, 1 amphetamines, 1 cannabis), and 3 for medicines. EtG concentration (20.7-1254.1pg/mg) was higher than the Society of Hair Testing (SoHT) cut-off for chronic alcohol consumption (≥30pg/mg) in 21 cases. All positive cases for methadone and cannabis, and half of positive cases for opioids and cocaine presented higher concentrations than SoHT cut-offs for chronic consumption. Higher AUDIT score and higher EtG concentration in hair were statistically associated with declaration of alcohol consumption ≥4 times/month and with previous fine for DUI of alcohol. In addition, AUDIT scores and EtG concentration in hair had a moderate but significant Spearman correlation (r=0.331, p<0.05). The combination of psychological tests and hair analysis seems to be a promising tool to identify individuals with chronic and problematic consumption of alcohol and drugs of abuse. Moreover, their application during driving license regranting procedures could increase the effectiveness of DARE courses, reduce recidivism and improve road safety.

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-ß-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.

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 © 2016 John Wiley & Sons, Ltd.

ABCC3 Polymorphisms and mRNA Expression Influence the Concentration of a Carboxylic Acid Metabolite in Patients on Clopidogrel and Aspirin Therapy.

Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism. These results suggest that up-regulation of ABCC3 mRNA expression leads to increased plasma CAM levels through MRP3-mediated cell efflux. The ABCC3 rs757421 polymorphism may contribute to gene expression. Therefore, ABCC3 may be a potential biomarker for the response to clopidogrel.

ABCC3 Polymorphisms and mRNA Expression Influence the / Concentration of a Carboxylic Acid Metabolite in Patients on / Clopidogrel and Aspirin Therapy

Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism. These results suggest that up-regulation of ABCC3 mRNA expression leads to increased plasma CAM levels through MRP3-mediated cell efflux. The ABCC3 rs757421 polymorphism may contribute to gene expression. Therefore, ABCC3 may be a potential biomarker for the response to clopidogrel.

ABCC3 Polymorphisms and mRNA expression influence the concentration of a carboxylic acid metabolite in patients on clopidogrel and aspirin therapy

Acetylsalicylic acid (ASA) and clopidogrel combined therapy has been reported to be beneficial in patients with acute coronary syndrome (ACS). Antiplatelet drug resistance, especially to clopidogrel, is a multifactorial phenomenon that affects a large number of ACS patients. The genetic contribution to this drug response is not fully elucidated. We investigated the relationship of ABC-type efflux subfamily C member 3 (ABCC3) polymorphisms and mRNA expression with plasma concentrations of clopidogrel, salicylic acid (SA) and a carboxylic acid metabolite (CAM). Clopidogrel, CAM and SA plasma concentrations were measured simultaneously by liquid chromatography-tandem mass spectrometry (LCMS/MS) from 83 ACS patients undergoing percutaneous coronary intervention. ABCC3 (rs757421, rs733392 and rs739923) and CYP2C19*2 (rs4244285) polymorphisms as well as mRNA expression were evaluated. A positive correlation was found between CAM concentrations and ABCC3 mRNA expression (r = 0.494, p < 0.0001). Patients carrying genotype AA (rs757421 variant) had higher CAM concentrations and ABCC3 mRNA expression as compared to those of GG + GA carriers (p = 0.017). A multiple linear regression analysis revealed that ABCC3 mRNA expression (p = 0.017), rs757421 AA genotype (p = 0.001), blood collection time (p = 0.018) and clopidogrel dose (p = 0.001) contributed to the concentration of CAM. No associations were observed for the CYP2C19*2 polymorphism...

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.

Determination of ketamine and its major metabolite, norketamine, in urine and plasma samples using microextraction by packed sorbent and gas chromatography-tandem mass spectrometry.

Ketamine is a club drug widely abused for its hallucinogenic effects, being also used as a "date-rape" drug in recent years. We have developed an analytical method using gas chromatography-tandem mass spectrometry (GC-MS/MS) for the identification and quantification of ketamine and its major metabolite in urine and plasma. No derivatization step is needed to accomplish analysis. The compounds were extracted from 0.25mL of sample using microextraction by packed sorbent on mixed mode (M1) cartridges. Calibration curves were linear in the range of 10-250ng/mL for urine and 10-500ng/mL for plasma, with determination coefficients higher than 0.99. The limit of detection (LOD) was 5ng/mL for both compounds in both specimens. Recoveries ranged from 63 to 101%, while precision and accuracy were below 14% and 15%, respectively. These low limits of detection and the quite high recoveries obtained, in very low sample amounts, allow detecting small quantities of the compounds, making this procedure suitable for those laboratories performing routine analysis in the field of forensic toxicology. Compared with existing methods, the herein described procedure is fast, since no derivatization step is required, and cost effective for the quantification of ketamine and norketamine in biological specimens by gas chromatography.

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).