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.

Detection of unsuspected cathinone and piperazine-type drugs in urine samples positive for methamphetamine and amphetamine collected in emergency departments. / Detección no sospechada de catinonas y piperacinas en pacientes consumidores de metanfetamina y anfetamina atendidos en servicios de urgencias hospitalarios.

OBJECTIVES: To detect the presence of unsuspected and/or undeclared cathinone and piperazine-type designer drugs in methamphetamine (METH) and amphetamine users treated in emergency departments, and to compare clinical and toxicologic profiles. MATERIAL AND METHODS: Retrospective observational study of emergency department patients treated for confirmed acute intoxication by recreational drugs (METH and amphetamines) between March 2019 and December 2020. We ordered high-performance liquid chromatography with tandem mass spectrometry to detect cathinones (methylone, fluoromethcathinone, mexedrone, fluoromethamphetamine, mephedrone, methylenedioxypyrovalerone) and synthetic piperazines (meta-chlorophenylpiperazine and trifluoromethylphenylpiperazine). Demographic, clinical, and toxicologic variables were analyzed with SPSS software (version 23). RESULTS: Thirty-nine patients were included: 24 (61.5%) had used METH and 15 (38.5%) an amphetamine. Synthetic cathinones were detected in samples from 11 patients (28.2%), 10 (90.9%) in the METH group and 1 (9.1%) in the amphetamine group (P = .028). The METH users had taken mephedrone (8 patients) or methylone (2 patients); the amphetamine user had taken mephedrone. None of the patients had declared use of a cathinone; nor was use suspected. The mean (SD) number of substances involved was higher among users of cathinones (3.5 [1.13] vs 2.5 [1.40] in those who took no cathinones; P = .036). Among the cathinone users, 90.9% were men, 90.9% had used METH, and 45.5% had practiced chemsex. HIV positivity was significantly associated with cathinone use (in 45.5% vs 10.7% of those not using cathinones; P = .028). All 5 of the patients who had taken cathinones and also practiced chemsex were HIV positive. Significantly more patients who had taken cathinones presented with anxiety (72.7% vs 21.43%; P = .007). No differences in clinical management were found. CONCLUSION: Detection of METH in intoxicated patients should raise suspicion of probable use of a synthetic cathinone. Patients in whom new psychoactive substances are detected should be kept under observation, and clinical protocols should include referring them to addiction treatment centers.

OBJETIVO: Determinar la incidencia de catinonas y piperazinas, no sospechadas y/o declaradas en consumidores de metanfetamina (MANF) y anfetamina (ANF) atendidos en servicios de urgencias hospitalarios (SUH) y comparar los perfiles clínicos y toxicológicos. METODO: Estudio retrospectivo de pacientes con intoxicación aguda por drogas recreativas con MANF y ANF confirmadas analíticamente atendidos en 3 SUH entre marzo de 2019 y diciembre de 2020. Se detectaron por HPLC-MS/MS las catinonas [metilona, fluorometcatinona, mecedrona, fluorometanfetamina, mefedrona, metilendioxipirovalerona (MDPV)] y las piperazinas sintéticas [meta-clorofenilpiperazina (mCPP), trifluorometilfenilpiperazina (TFMPP)]. RESULTADOS: Se incluyeron 39 pacientes: 24 (61,5%) en el grupo MANF y 15 (38,5%) en el ANF. En 11 (28,2%), se detectaron catinonas sintéticas (grupo CAT), 10 en el grupo MANF (8 mefedrona, 2 metilona) y 1 en el grupo ANF (1 mefedrona) (90,9% vs 9,1%; p = 0,028). Ninguno de los pacientes declaró consumo de catinonas. El número de drogas implicadas en la intoxicación fue superior en el grupo CAT (3,5 [1,13] vs 2,5 [1,40]; p = 0,036). El perfil clínico del grupo CAT fue: varón (90,9%), consumidor de MANF (90,9%) y usuario de chemsex (45,5%). El diagnóstico de VIH se asoció significativamente al grupo CAT (45,5% vs 10,7%; p = 0,028). Los pacientes del grupo CAT presentaron mayor ansiedad (72,7% vs 21,4%; p = 0,007). No se hallaron diferencias en su manejo clínico. CONCLUSIONES: La detección de MANF debería considerarse un dato de sospecha de consumo de catinonas sintéticas, y en esos casos debería contemplarse la detección de nuevas sustancias psicoactivas de abuso.

Fast and Sensitive Method for the Determination of 17 Designer Benzodiazepines in Hair by Liquid Chromatography-Tandem Mass Spectrometry.

In recent years, identification and analysis of designer benzodiazepines have become a challenge in forensic toxicology. These substances are analogs of the classic benzodiazepines, but their pharmacology is not well known, and many of them have been associated with overdoses and deaths. As a result, there has been a surge in efforts to develop analytical methods to determine these compounds in different biological samples. Our aim was to develop and validate a fast, sensitive and specific method for determining 17 designer benzodiazepines (adinazolam, clobazam, clonazolam, delorazepam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, flunitrazolam, N-desmethylclobazam, nifoxipam, nitrazolam, meclonazepam, pyrazolam and zolazepam) in hair by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples were decontaminated and pulverized, and a 20 mg aliquot was incubated in methanol in an ultrasound bath (1 h, 25°C). The supernatant was evaporated and reconstituted in 200 µL of mobile phase, and the extracts were filtered (nano-filter vials) before injection into LC-MS-MS. All analytes were eluted from the chromatographic column in 8 min, and two multiple-reaction monitoring (MRM) transitions were used to identify each compound. The limits of quantification were 5 or 25 pg/mg depending on the analyte, and the calibration functions were linear to 200 pg/mg. Imprecision was <19.2% (n = 15), and bias was from -13.7 to 18.3% (n = 15). All the analytes yielded high extraction efficiencies >70% and displayed ion suppression between -62.8% and -23.9% (n = 10). The method was applied to 19 authentic cases. Five samples were positive for flualprazolam ( 200 pg/mg) and/or etizolam (47.4-88.5 pg/mg). In conclusion, the present validated method has proven to be fast, sensitive, specific and capable of determining 17 designer benzodiazepines in hair using LC-MS-MS.

Detección no sospechada de catinonas y piperacinas en pacientes consumidores de metanfetamina y anfetamina atendidos en servicios de urgencias hospitalarios / Detection of unsuspected cathinone and piperazine-type drugs in urine samples positive for methamphetamine and amphetamine collected in emergency departments

Objetivos. Determinar la incidencia de catinonas y piperazinas, no sospechadas y/o declaradas en consumidores de metanfetamina (MANF) y anfetamina (ANF) atendidos en servicios de urgencias hospitalarios (SUH) y comparar los perfiles clínicos y toxicológicos. Método. Estudio retrospectivo de pacientes con intoxicación aguda por drogas recreativas con MANF y ANF confirmadas analíticamente atendidos en 3 SUH entre marzo de 2019 y diciembre de 2020. Se detectaron por HPLC-MS/MS las catinonas [metilona, fluorometcatinona, mecedrona, fluorometanfetamina, mefedrona, metilendioxipirovalerona (MDPV)] y las piperazinas sintéticas [meta-clorofenilpiperazina (mCPP), trifluorometilfenilpiperazina (TFMPP)]. Resultados. Se incluyeron 39 pacientes: 24 (61,5%) en el grupo MANF y 15 (38,5%) en el ANF. En 11 (28,2%), se detectaron catinonas sintéticas (grupo CAT), 10 en el grupo MANF (8 mefedrona, 2 metilona) y 1 en el grupo ANF (1 mefedrona) (90,9% vs 9,1%; p = 0,028). Ninguno de los pacientes declaró consumo de catinonas. El nú- mero de drogas implicadas en la intoxicación fue superior en el grupo CAT (3,5 [1,13] vs 2,5 [1,40]; p = 0,036). El perfil clínico del grupo CAT fue: varón (90,9%), consumidor de MANF (90,9%) y usuario de chemsex (45,5%). El diagnóstico de VIH se asoció significativamente al grupo CAT (45,5% vs 10,7%; p = 0,028). Los pacientes del grupo CAT presentaron mayor ansiedad (72,7% vs 21,4%; p = 0,007). No se hallaron diferencias en su manejo clínico. Conclusiones. La detección de MANF debería considerarse un dato de sospecha de consumo de catinonas sintéticas, y en esos casos debería contemplarse la detección de nuevas sustancias psicoactivas de abuso.

Objectives. To detect the presence of unsuspected and/or undeclared cathinone and piperazine-type designer drugs in methamphetamine (METH) and amphetamine users treated in emergency departments, and to compare clinical and toxicologic profiles. Method. Retrospective observational study of emergency department patients treated for confirmed acute intoxication by recreational drugs (METH and amphetamines) between March 2019 and December 2020. We ordered high-performance liquid chromatography with tandem mass spectrometry to detect cathinones (methylone, fluoromethcathinone, mexedrone, fluoromethamphetamine, mephedrone, methylenedioxypyrovalerone) and synthetic piperazines (meta-chlorophenylpiperazine and trifluoromethylphenylpiperazine). Demographic, clinical, and toxicologic variables were analyzed with SPSS software (version 23). Results. Thirty-nine patients were included: 24 (61.5%) had used METH and 15 (38.5%) an amphetamine. Synthetic cathinones were detected in samples from 11 patients (28.2%), 10 (90.9%) in the METH group and 1 (9.1%) in the amphetamine group (P = .028). The METH users had taken mephedrone (8 patients) or methylone (2 patients); the amphetamine user had taken mephedrone. None of the patients had declared use of a cathinone; nor was use suspected. The mean (SD) number of substances involved was higher among users of cathinones (3.5 [1.13] vs 2.5 [1.40] in those who took no cathinones; P = .036). Among the cathinone users, 90.9% were men, 90.9% had used METH, and 45.5% had practiced chemsex. HIV positivity was significantly associated with cathinone use (in 45.5% vs 10.7% of those not using cathinones; P = .028). All 5 of the patients who had taken cathinones and also practiced chemsex were HIV positive. Significantly more patients who had taken cathinones presented with anxiety (72.7% vs 21.43%; P = .007). No differences in clinical management were found. [...]

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.

Assessment of biological matrices for the detection of in utero cannabis exposure.

Cannabis consumption has been increasing worldwide among pregnant women. Due to the negative effects of prenatal cannabis exposure, it is necessary to develop an objective, sensitive, and specific method to determine cannabinoids use during pregnancy. In this study, we compared four different biological samples, maternal hair, meconium, umbilical cord, and placenta, for the detection of in utero cannabis exposure. The biological samples were collected from 627 mother-newborn dyads. All hair and meconium samples were analyzed, and umbilical cord and placenta if hair and/or meconium were positive for cannabinoids. Meconium and hair showed to complement each other, with an agreement between hair and meconium results of 96.7% but only 34.3% if just positive results were considered. Umbilical cord and placenta results showed a better agreement with meconium (91.3% and 92.6%, respectively) than with hair (39.1% and 34.6%, respectively). The predominant metabolites in meconium were 11-nor-carboxy-THC (THCCOOH) and 8,11-dihydroxy-THC (diOHTHC), and in umbilical cord and placenta was THCCOOH-glucuronide. Cannabidiol (CBD) and cannabinol (CBN) were detected in meconium but not in any umbilical cord or placenta. For the first time, prenatal marijuana exposure was analyzed and compared in paired hair, meconium, umbilical cord, and placental samples. Hair and meconium positivity rate was similar, but a more sensitive and specific analytical method for the hair may resolve discrepancies between the matrices. Umbilical cord and placenta may be considered suitable alternative matrices to meconium through the determination of THCCOOH-glucuronide as a biomarker of cannabis exposure.

Diminished Consciousness in a Woman Following an Unsuspected Scopolamine Overdose.

Scopolamine is used clinically, but it is also used as a recreational drug and as an incapacitating drug, in sexual crimes and robberies. In this paper, the authors report the case of a woman with a diminished consciousness following an unsuspected overdose with scopolamine and review published articles on scopolamine poisoning that included concentrations in biological samples. Scopolamine was identified in the patient's serum and urine samples collected 1 h post-admission to intensive care unit at concentrations of 8.4 ng/mL and 62,560 ng/mL (169,539 ng/mg creatinine), respectively. In non-fatal cases, the median [interquartile range] of serum scopolamine levels was 1.9 [2.1] ng/mL. The serum concentration found in our case would explain the abrupt clinical presentation suffered by the patient. Scopolamine in urine could be detected up to 48 h after admission. This report illustrates that broad toxicology screening, including scopolamine, should be considered when patients with diminished consciousness are attended after ruling out infection or cerebrovascular disease. This can play an important role in identifying this potentially life-threatening etiology.

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.

Drug testing in biological samples vs. maternal surveys for the detection of substance use during whole pregnancy.

BACKGROUND: Early diagnosis of nicotine, ethanol and drug use during pregnancy is critical in order to provide adequate care. Current screening procedures show limitations in terms of reliability and short windows of detection. OBJECTIVES: To investigate the prevalence and identify biomarkers of substance use and changes in substance use during pregnancy. To compare drug testing results in different types of biological samples (maternal hair, meconium, placenta, umbilical-cord) with self-reported data. PARTICIPANTS AND SETTING: Prospective cohort study using data from pregnant women and their newborns. METHODS: Biological matrices were collected at birth and analyzed by liquid chromatography tandem mass spectrometry. A paper survey was provided to determine substance use habits. RESULTS: 867 mother-newborn pairs were included. According to the analysis of biological samples, 29.1% cases were positive for one or more substances (13.6% nicotine, 8.4% ethanol, 8.3% cocaine, 6.4% cannabis, 5.7% opioids). The profile of the substance-using mother was a single woman, <28 years-old, with no higher education and unemployed. Segmental maternal hair analysis showed a decrease in tobacco, cannabis and cocaine use throughout pregnancy (p < 0.001). The level of concordance between results from interviews and from biological analyses was weak for opioids, cocaine, and cannabis (kappa coefficient < 0.40). Maternal hair detected the highest number of cases, followed by meconium and by placenta and/or umbilical-cord. CONCLUSIONS: Maternal survey was not a reliable screening technique. Analysis of maternal hair detected the highest number of cases with the broadest detection window (whole pregnancy).

Quantification of Classic, Prescription and Synthetic Opioids in Hair by LC-MS-MS.

The current use and misuse of synthetic and prescription opioids in the USA has reached epidemic status. According to the US Department of Health and Human Services, every day more than 130 people in the USA die after overdosing on opioids, and 2.1 million had an opioid use disorder in 2018. Hair is becoming an alternative matrix of increasing interest in forensic toxicology to investigate drug use and abuse patterns due to its long window of detection. The focus of this project was to develop and validate a method that simultaneously detects and quantifies 27 classic, prescription and synthetic opioids in hair by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples were decontaminated and pulverized in a bead mill. Twenty-five milligrams of hair powder were incubated in a buffer overnight. Mixed mode cation exchange solid phase extraction was carried out before undergoing reversed-phase chromatographic separation, successfully resolving isobaric opioids. We used two multiple reaction monitoring transitions in positive mode to identify each analyte. The linearity range was 1-500 pg/mg for fentanyl and synthetic opioids and 10-500 pg/mg for prescription and classic opioids. Imprecision was <17.5% and bias ranged from -13.6 to 12.0%. Majority of compounds showed extraction efficiency >50%, and ion suppression from -89.2 to -26.6% (CV < 19%, n = 10). This method was applied to 64 authentic cases, identifying 13 compounds from our panel. A sensitive and specific method was developed for the identification and quantification of 27 classic, prescription and synthetic opioids in hair by LC-MS-MS.

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.

Folate, genomic stability and colon cancer: The use of single cell gel electrophoresis in assessing the impact of folate in vitro, in vivo and in human biomonitoring.

Intake of folate (vitamin B9) is strongly inversely linked with human cancer risk, particularly colon cancer. In general, people with the highest dietary intake of folate or with high blood folate levels are at a reduced risk (approx. 25%) of developing colon cancer. Folate acts in normal cellular metabolism to maintain genomic stability through the provision of nucleotides for DNA replication and DNA repair and by regulating DNA methylation and gene expression. Folate deficiency can accelerate carcinogenesis by inducing misincorporation of uracil into DNA, by increasing DNA strand breakage, by inhibiting DNA base excision repair capacity and by inducing DNA hypomethylation and consequently aberrant gene and protein expression. Conversely, increasing folate intake may improve genomic stability. This review describes key applications of single cell gel electrophoresis (the comet assay) in assessing genomic instability (misincorporated uracil, DNA single strand breakage and DNA repair capacity) in response to folate status (deficient or supplemented) in human cells in vitro, in rodent models and in human case-control and intervention studies. It highlights an adaptation of the SCGE comet assay for measuring genome-wide and gene-specific DNA methylation in human cells and colon tissue.

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.

A reliable method for estimating the postmortem interval from the biochemistry of the vitreous humor, temperature and body weight.

The estimation of the time elapsed since death is of paramount importance in the field of forensic sciences and criminal investigation, owing, among other factors, to the possible legal repercussions. Over the past few years various formulae have been developed to calculate this interval using a combination of different statistical methods and the concentrations of substances found in the vitreous humor. Corrective factors, such as ambient temperature, cause of death or age, which can modify the concentration of these substances and therefore the estimation of the postmortem interval, have been incorporated into models. In this paper five simple and reliable models to estimate PMI based the on the analysis of potassium, hypoxanthine and urea in the vitreous humor are presented. Corrective factors, such as body weight, rectal temperature and ambient temperature, which can influence the estimation of this interval have been incorporated into the formulae. Finally, the R2 and the mean squared error have been calculated for each model in order to select the best of the five. A free software program which calculates the PMI from the model and parameters used is available from the authors. It provides quick and reliable results as well as the error committed and R2 for each case.