Establishing effective interpretation criteria in hair analysis to distinguish between passive and active cocaine exposure: Insights from authentic hair samples collected from professional individuals exposed to cocaine.

Interpretation results of hair analysis, particularly for cocaine, can be challenging due to the need to differentiate between active use or passive contamination. Our study aimed to assess the impact of varying degrees of passive cocaine exposure hair analysis results and their interpretation. Hair samples (n = 25) were categorized based on the declared cocaine exposure of volunteers: (a) high, involving handling up to several kilograms of cocaine powder from dismantling illegal distribution sites; (b) medium, where staff dealt with cocaine blocks (up to kilograms); and (c) low, with staff in contact with up to grams of cocaine for laboratory analysis. Hair samples were decontaminated using dichloromethane, water, and methanol. The samples and final wash were analyzed for cocaine, benzoylecgonine, norcocaine, cocaethylene, m-OH-benzoylecgonine, and ecgonine methyl ester using a validated UPLC-MS/MS method. Cocaine hair concentrations ranges were as follows (pg/mg): high (n = 53 segments) < LLOQ(32)-7046; medium (n = 91) < LLOQ-939; and low (n = 54) < LLOQ-292. All hair samples had concentrations below the LLOQ for cocaethylene, ecgonine methyl ester, and m-OH-benzoylecgonine. Applying the SoHT cocaine cut-off in combination with a hair/wash ratio criterion identified 97% of the samples as contaminated. This study advocates for a comprehensive approach in evaluating cocaine hair concentrations. This involves integrating the 500 pg/mg decision limit for cocaine with a criterion comparing wash and hair concentration. Additionally, confirming the presence of specific metabolites is crucial. This multifaceted method effectively distinguishes between environmental contamination and active cocaine usage. The research contributes significantly to refining cocaine exposure assessment in professional contexts.

Incorporation of doxylamine and N-doxylamine-oxide in human hair and the impact of a permanent oxidative hair dye.

Knowledge of the drug incorporation in hair and impact of cosmetic treatments remains essential to correctly interpret forensic cases. The study shows the analysis of doxylamine and doxylamine-N-oxide and the evaluation of the relationship between dose and hair concentration and the impact of hair treatment (oxidative dying). The study included (A) three subjects participated to the study: a regular user (Subject 1) and two single-dose users (Subject 2, 1 single dose; and Subject 3, 2 single doses spaced 5 months apart). Subject 3 applied a permanent oxidative hair dying monthly. (B) A permanent oxidative hair dying was applied twice to the hair collected from Subject 2. (A) The average concentrations in head hair for doxylamine and its N-doxylamine-oxide, respectively, were as follows: Subject 1, 1825 pg/mg and 16 pg/mg; Subject 2, 182 and

Evaluation of decontamination procedures for drug testing in undamaged versus damaged hair.

Although substances incorporated by ingestion are strongly bound to hair, their loss may occur if aggressive decontamination procedures are applied, especially in highly damaged/porous hair. Evaluation of cleaning procedures using hair samples with different porosity obtained from ethanol or drug users (cocaine, heroin, methamphetamine, methadone, fentanyl, tramadol, diazepam, buprenorphine, dihydrocodeine, citalopram and trazodone). The effect of washing time and multiple wash steps with water and methanol were evaluated. Hair samples (n = 16) were selected and evaluated according to (a) the drug pattern consumption, (b) available amount, and (c) hair porosity (c1 'cosmetic treatment', c2: storage time). Six of them were soaked with an aqueous deuterated analogue solution. The samples were cut in 1-cm segments and homogenized. All hair samples were then decontaminated one or six times with 1.5 ml of water or methanol during 1, 5, 15, 30, 60 and/or 90 min (n = 1 to 3/sample, depending on the available amount of hair). Hair extracts were then cleaned up via a solid-phase extraction (SPE) or liquid-liquid extraction (LLE), while the washes were evaporated to dryness. All were thereafter reconstituted and analysed with routine ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods. Although concentrations of parent drugs and/or metabolites presented a negative trend along the washing time with methanol (up to 80%), the compounds were relatively well retained in hair even after a 90 min wash (with methanol or water) in most samples, and their retention would depend mostly on the hair nature rather than their physicochemical properties (whether incorporated by ingestion and/or from external contamination). Moreover, parent drugs and/or metabolites were detected in the washes in most samples, and the ratio between hair and washes decreased along the washing time. More than 50% of the deuterated analogues soaked into hair were still present after the different washing steps. Losses were observed more frequently for long-term stored hair samples, after decontamination with methanol for more than 30 min. Therefore, prolonged or repeated cleaning with methanol should be avoided in general procedures.

The Interest of a Systematic Toxicological Analysis Combined with Forensic Advice to Improve the Judicial Investigation and Final Judgment in Drug Facilitated Sexual Assault Cases.

The conviction rate in drug facilitated sexual assault (DFSA) cases is known to be very low. In addition, the potential impact of toxicological results on the case is often not well understood by the judicial authorities. The aims of this study were (1) to obtain more knowledge concerning the prevalence of incapacitating substances in DFSA cases, (2) to create a more efficient DFSA analysis strategy taking background information into account, and (3) to evaluate the potential impact of systematic toxicological analysis (STA) on the final judicial outcome. This small-scale epidemiological study (n = 79) demonstrates that 'commonly-used' illicit drugs, psychoactive medicines and ethanol are more prevalent in DFSA cases in contrast to the highly mediatized date rape drugs. Additionally, via case examples, the interest of performing STA-to prove incapacitation of the victim-in judicial procedures with mutual-consent discussions has been demonstrated as it led to increased convictions. However, more attention has to be paid to ensure a short sampling delay and to get more accurate information from the medical treatment of the alleged victim. This will improve the interpretation of the toxicological analysis and thus its applicability in a DFSA case. The future is multi-disciplinary and will certainly lead to an efficient and more cost-effective DFSA approach in which STA can impact the final judgment.

Quantitation of phosphatidylethanol in dried blood after volumetric absorptive microsampling.

BACKGROUND: Stimulated by the increased recognition of phosphatidylethanol (PEth) as sensitive direct marker of alcohol intake, the Ghent University's Laboratory of Toxicology and the National Institute of Criminalistics and Criminology combined their efforts to develop a quantitative method. To facilitate implementation the focus was on the use of a sampling technique which allows quick and easy blood collection, without the need of dedicated personnel at any place/any time. In the meantime the cooperation of the two labs should also allow to initiate a Belgian network of laboratories capable of quantifying PEth. METHODS: Dried blood microsamples were collected via volumetric absorptive microsampling (VAMS). PEth 16:0/18:1 was quantified after liquid-liquid extraction using two independent isotope dilution - liquid chromatography - tandem mass spectrometry methods. A systematic review of the entire process at both sites was performed before the final method comparison using samples from 59 routine toxicology cases collected within a one-year time interval. RESULTS: Initial differences between both laboratories were solved by focusing on important methodological aspects: (i) trueness verification of the calibration protocol focusing on the primary material, preparation of the stock solutions and adequate equilibration of calibrators and QCs, and (ii) verification of comparability of results obtained with different m/z transitions. Several of these aspects could only be verified by critically assessing spiked and native samples. After a final validation good average comparability of the two methods was observed. The average bias was -0.4%, with 85% of the differences within 20%. Moreover, the methods proved to be reproducible and robust within a one-year time interval. CONCLUSION: This study is the first to develop a quantitative volumetric absorptive microsampling based method for PEth measurements, in addition it is the first to perform a systematic comparison of PEth measurements between two laboratories. From the discussion on the encountered pitfalls it is clear that also on a global scale, more efforts are needed to improve interlaboratory agreement.

A different insight in hair analysis: Simultaneous measurement of antipsychotic drugs and metabolites in the protein and melanin fraction of hair from criminal justice patients.

BACKGROUND: Previous studies have postulated that four structural compartments may be differentiated in hair: surface protein domain, water-accessible protein domain, water-inaccessible protein domain, and melanin. Drugs contained in blood, sweat, sebum, and environment would be deposited in the first two domains, with primarily drugs in blood being incorporated in the latter two domains during hair synthesis. Drugs in the first two domains would be removed by washing procedures. Use of enzymatic extraction procedures and evaluation of hair for damage from harsh cosmetic treatments might help to separately identify and quantify the drugs incorporated in the second two domains. AIMS: a) Development of an UPLC-MS/MS method for the simultaneous quantification of the following 19 antipsychotic drugs and metabolites in hair: amisulpride, aripiprazole, chlorpromazine, clotiapine, clozapine, desmethylclozapine, desmethylolanzapine, haloperidol, norchlorpromazine, 7-OH-quetiapine, 9-OH-risperidone, olanzapine, pimozine, pimpamperone, quetiapine, risperidone, sertindole, sulpride, and tiapride; b) evaluation of measurement of patient adherence to prescribed medication use, c) determination of the influence of biochemical individuality effects on hair drug content, d) evaluation of relative binding of antipsychotic drugs to protein and to melanin hair structures. METHOD: Approximately 10 mg of intact hair were decontaminated with isopropanol and phosphate buffer, and then enzymatically digested overnight with dithiothreitol. After centrifugation, the supernatant digest (protein fraction) was separated from the remaining melanin hair pellet (melanin fraction). Melanin fraction was washed with water, and the drugs were extracted with dimethyl sulfoxide with ball-mill pulverization. Both fractions were purified with solid-phase cation exchange cartridges and injected in the UHPLC-MS/MS system. RESULTS AND DISCUSSION: Validation of the method was carried out on the protein fraction following international guidelines. The limits of quantification ranged from 1.6-40 pg/mg. The method was applied to 59 head hair samples from prisoners from an antipsychotic compliance study in the criminal justice system in US. The patients were under chlorpromazine, haloperidol, risperidone, olanzapine, or quetiapine multiple antipsychotic treatment, during incarceration. The first head hair centimeter, closest to the scalp, was analyzed. The results were evaluated in relation to the type of hair, colour, hair damage, drug melanin affinity, and prescribed dose. In general, no good correlation between the prescribed dose/concentration in hair was obtained. A wide range of antipsychotic concentrations were observed 'dose mg/day (d); pg/mg protein fraction (A)': chlorpromazine (d:50-400;A:1600) and its metabolite norchlorpromazine (A: 1600), haloperidol (d:4-20;A: 1600) and its metabolite 9-OH-quetiapine (A:

Semiquantitative Activity-Based Detection of JWH-018, a Synthetic Cannabinoid Receptor Agonist, in Oral Fluid after Vaping.

The rapid proliferation of new synthetic cannabinoid receptor agonists (SCRAs) has initiated considerable interest in the development of so-called "untargeted" screening strategies. One of these new screening technologies involves the activity-based detection of SCRAs. In this study, we evaluated whether (synthetic) cannabinoid activity can be detected in oral fluid (OF) and, if so, whether it correlates with SCRA concentrations. OF was collected at several time points in a placebo-controlled JWH-018 administration study. The outcome of the cell-based cannabinoid reporter system, which monitored the cannabinoid receptor activation, was compared to the quantitative data for JWH-018, obtained via a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. A total of 175 OF samples were collected and analyzed via both methods. The cannabinoid reporter assay correctly classified the vast majority of the samples as either negative (<0.25 ng/mL; 74/75 = 99%) or having low (0.25-1.5 ng/mL; 16/16 = 100% and 1.5-10 ng/mL; 37/41 = 90%), mid (10-100 ng/mL; 23/25 = 92%) or high (>100 ng/mL; 16/18 = 89%) JWH-018 concentrations. Passing-Bablok regression analysis yielded a good linear correlation, with no proportional difference between both methods (slope 0.97; 95% confidence interval 0.86-1.14) and only a small systematic difference. This is the first study to demonstrate the applicability of an untargeted, activity-based approach for SCRA detection in OF. Additionally, the outcome of the cannabinoid reporter assay was compared to the gold standard (LC-MS/MS), showing a good correlation between both methods, indicating that the cannabinoid reporter assay can be used for an estimation of drug concentrations.

Development of an UPLC-MS/MS method for the analysis of 16 synthetic opioids in segmented hair, and evaluation of the polydrug history in fentanyl analogue users.

Seizures of synthetic opioids have increased since 2012, with a 45 % increase in synthetic opioid related deaths between 2016 and 2017 in US. Recently, concerns have arisen around these substances and their illicit use also in several European countries. Our aim was to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of 16 synthetic opioids in segmented hair, including fentanyl, norfentanyl, acetylfentanyl, U-47700, AH-7921, acrylfentanyl, crotonylfentanyl, butyrylfentanyl, methoxacetylfentanyl, U-49900, valeryfentanyl, 4-fluoro-iso-butyrylfentanyl, ocfentanyl, furanylfentanyl, tetrahydrofuranylfentanyl, and alfetanyl. Sample preparation involved washing the hair in dichloromethane, water and methanol, and extraction in methanol, followed by solid phase extraction clean-up. This method was validated for linearity, limit of quantification (LLOQ), precision and bias, selectivity, stability, matrix effects, extraction efficiency of the clean up procedure, and carryover. LLOQs ranged from 0.15-1pg/mg, and the calibration ranged from the LLOQ up to 500pg/mg. Intra and inter-day precision were evaluated at low and high concentrations, with spiked QCs, during 8 days and the results were satisfactory with RSD<15 % for all the compounds except for norfentanyl (22 %) and alfentanyl (19 %). Two external certified QCs containing fentanyl at 11 and 105pg/mg were also analysed within each batch and the RSD and bias were lower than 16 % and 10 %, respectively. Matrix effects compensated by internal standard fentanyl-d5 (MEIS), were between 77-115 % (RSD<10 %) and extraction efficiency of the clean-up procedure was between 66-93 % (RSD<21 %). Processed sample stability and carryover were acceptable for all of the compounds. The method was applied to 17 authentic hair samples (body or head hair) from US fentanyl analogue users. When head hair was available, the hair strands were analysed in 1cm/segment. Concentrations ranges were as follows: fentanyl (n=16) 2->ULOQ (500) pg/mg, norfentanyl (n=14) 1-38pg/mg, acetylfentanyl (n=7) 0.6->ULOQ (250) pg/mg, furanylfentanyl (n=5) 2-123pg/mg, tetrahydrofuranylfentanyl (n=1) 0.5-63pg/mg and valerylfentanyl (n=1) 2.1->ULOQ (50) pg/mg, along the hair strands. To our knowledge, this is the first time where concentrations of tetrahydrofuranylfentanyl, and valerylfentanyl in hair are reported. The same samples were also analysed for the determination of other drugs of abuse using our routine method (also in 1cm/segment for head hair when available). The results demonstrated poly-drug use in these fentanyl-analogue users population (mean drugs: n=5): amphetamine and/or methamphetamine (n=10), buprenorphine (n=5), cocaine (n=8), methadone (n=8), 6-MAM (n=17), meperidine (n=1), oxycodone (n=11), tramadol (n=3). Evaluation of the concentrations of these drugs, together with the fentanyl analogues is discussed in the present paper. Two authentic samples from two Belgian post-mortem cases, were also analysed showing fentanyl use and in one case polydrug use. The results demonstrated multi-analyte quantitative methods, including fentanyl analogues, are becoming useful in forensic laboratories involved in hair analysis, and in particular when polydrug use is suspected.

Time course detection of dihydrocodeine in body hair after a single dose.

BACKGROUND: When head hair is not suitable or not available, body hair, such as leg or beard hair might be the most suitable sample for drug hair analysis. Information about the time course of drugs in hair, from the different anatomical body sites, should still be well documented. AIM: The aim of this study was to determine and compare (a) the detection window of dihydrocodeine in frequently shaved legs and beard, (b) in unshaved hair from head hair, chest hair, leg hair, and/or arm hair, and (c) the distribution concentrations over the scalp, after a single dihydrocodeine intake. METHOD: Before a single intake of 12 mg dihydrocodeine by subject 1 (woman), both legs hair were shaved in the morning. The subject 2 (man) shaved his beard in the morning and 30 min later he had a dose of 10 mg of dihydrocodeine. The samples were washed with water and shampoo, dried and collected as follows: Subject 1: every 3-days shaved leg hair (n = 9) and 1-month-later head hair (n = 1). Subject 2: daily shaved beard hair (n = 15), 2 months later head hair (n = 145), and every 20 days unshaved arm, leg and chest hair (from different areas) (n = 4/area). The samples were analysed for dihydrocodeine using a validated liquid chromatography-tandem mass spectrometry method with a limit of quantification (LOQ) of 15.6 pg/mg for dihydrocodeine. About 20 mg of hair samples were weighted, washed with dichloromethane, centrifuged, dried, and pulverized in the same disposable tubes. Then the samples were incubated with methanol (under sonication at 45 °C) during 4 h. After centrifugation, the supernatant was evaporated and a cation exchange solid phase extraction followed by separation and quantification using ultra performance liquid chromatography-tandem mass spectrometry (ULC-MS/MS) was carried out. Chromatographic separation was achieved using a BEH phenyl column eluted with 0.1% formic acid: methanol (0.1% formic acid). The UPLC-MS/MS method was validated and used in routine for drug hair analysis for already several years. RESULTS AND DISCUSSION: In the present study leg hair was collected every 3 days, as an average of frequent shaved hair in western woman population. Shaved leg hair was very limited and only one hair sample was available per analysis. Beard was collected daily and in a higher amount. Dihydrocodeine was detected in leg hair from the first sample (3 days after the intake). Maximum concentration at 68 pg/mg for the single intake was obtained after 15 days (±2 days), decreasing later to the LOQ from the 21th day. Beard hair was positive from the first day sample, and the maximum concentration was observed at 66 pg/mg, 6 days after the intake, decreasing later to the LOQ from day 13. This may be explained by growth rate and the amount of growing hairs, in anagen phase. In other body hair samples, dihydrocodeine was negative or detected from 1 month after the intake. No significant differences in dihydrocodeine concentrations over the scalp in the different regions were observed (p > 0.05). CONCLUSION: Body hair presents different time course window detection due to the different growth rates. Frequently shaved leg and beard hair may be suitable samples for recent single dihydrocodeine dose detection from the first days up to 2-3 weeks after the intake, respectively, when a LOQ of 15.6 pg/mg is applied.

Influence of bleaching and thermal straightening on endogenous GHB concentrations in hair: An in vitro experiment.

Since gamma-hydroxybutyric acid (GHB) is present in hair of the general population under physiological concentrations, special attention has to be given to the hair analysis of GHB and its interpretation. Normal levels of endogenous GHB can vary in each individual. As a result, strands of hair from a subject have to be cut in small segments (0.3-0.5 cm long) with analysis of each segment. As such, each subject can be used as its own control with a continuous endogenous signal. If one segment has a GHB concentration 10 times higher than the others, this suggests possible administration of exogenous GHB according to the UNODC guideline for Drug Facilitated Assault Cases. AIM: As cosmetic treatments were found to decrease drug concentrations in hair, the aim of the study was to develop an UPLC®-MS/MS method for the analysis of GHB in hair. An in vitro study was then carried out in order to evaluate the impact of a hair straightener or a bleaching treatment on endogenous GHB concentrations. METHOD: Hair samples (10 mg) were washed with dichloromethane and water. After drying overnight in an oven at 35 °C the samples were pulverized in disposable plastic tubes. Methanol/acetonitrile/ammonium formate buffer 1 mM (25:25:50, v/v/v) was used to extract the drug from the hair matrix in a water bath for 1.5 h at 37 °C. Thereafter, the samples were filtered and evaporated to dryness. The dried extracts were then reconstituted in mobile phase and injected in a UPLC®-MS/MS (Waters, Winslow, UK) with a BEH C18 column. RESULTS: The method was validated using untreated hair samples from three healthy volunteers. The calibration curve ranged from 0.06 to 25 ng/mg and the repeatability and intra-batch precision was lower than 20% evaluated in 8 different batches. Processed samples were stable for 3 days in the auto-sampler. To demonstrate the method applicability, 54 hair samples from healthy volunteers were analysed for endogenous GHB resulting in a concentration range from 0.2 to 6 ng/mg. Three different hair treatments experiments were carried out, in which a hair straightener and/or a bleaching treatment were applied. These experiments demonstrated that hair treatments decreased up to 80% of the GHB endogenous concentrations. CONCLUSION: This in vitro study showed that hair bleaching or a heat source treatment influences GHB concentrations in hair. For a correct interpretation of GHB results in hair, cosmetic treatments should be considered, certainly in cases where only a part of the hair is treated.

Determination of Antidepressants in Hair via UHPLC-MS/MS as a Complementary Informative Tool for Clinical and Forensic Toxicological Assessments.

BACKGROUND: Hair analysis is a complementary approach for the detection of antidepressants (ADs) in clinical and forensic schemes because it yields a picture of long-term exposure over a time window depending on the length of the hair. METHODS: A fast and sensitive ultra-high performance liquid chromatography tandem mass spectrometry method using a BEH C18 column with a mobile phase consisting of ammonium acetate/acetonitrile was developed and validated according to international guidelines for the simultaneous analysis of 24 ADs in hair. Methanol/acetonitrile/ammonium formate buffer 1 mmol/L (25:25:50, vol/vol/vol) was used to extract the drugs from the hair matrix before a solid-phase extraction using cation exchange cartridges was applied. Hair samples (n = 18) obtained from a US workplace drug testing center were analyzed to demonstrate the method applicability. RESULTS: The limit of quantification values ranged from 0.006 to 0.05 ng/mg hair, and the calibration curves ranged from the LOQ up to 10 ng/mg hair. The bias and imprecision were <15% for all the compounds except maprotiline (17%). This was evaluated with 2 "in-house" QCs and 1 authentic hair sample from an amitriptyline user. No significant matrix effects for most of the compounds were observed, and the extraction efficiency of the sample cleanup procedure ranged from 40% to 80% (relative standard deviation <15%) [except for demethylcitalopram, didemethylcitalopram, and trazodone (relative standard deviation <33%)]. The method was then successfully applied to the analysis of hair samples from workplace drug testing. The samples were analyzed in 1-cm segments to determine the medication history of the patient. When a sample was reported positive, information concerning the prescription was obtained anonymously for several samples. Concentrations of (minimum-maximum value in ng/mg) citalopram (0.01-132: extrapolated), trazodone (0.01-5.3), sertraline (0.05-0.1), paroxetine (0.02-1.0), bupropion (0.05-0.6), fluoxetine (0.5-8), and amitriptyline (0.2-4.8), including metabolites, are reported. CONCLUSIONS: This study may be of interest to clinical and forensic laboratories for interpretation because it demonstrates the AD concentration windows in hair and the link to the prescribed drugs.

Detection of Benzodiazepines and z-Drugs in Hair Using an UHPLC-MS/MS Validated Method: Application to Workplace Drug Testing.

BACKGROUND: A sensitive and reproducible Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry method has been developed for the simultaneous quantification of the 29 commonly prescribed benzodiazepines and z-drugs in hair. The method was validated according to international guidelines. METHODS: After decontamination (with dichloromethane and water), compounds were extracted from 20 mg of pulverized hair samples using methanol at 45°C and sonication for 2 hours. The drugs were recovered by liquid-liquid extraction using 1-chlorobutane, evaporated to dryness, and reconstituted with 100 µL of methanol before injection in the UPLC-MS/MS. RESULTS: The applied gradient ensured the elution of all the compounds within 7 minutes using 0.1% formic acid in water and methanol as mobile phase. The lower limit of quantification values ranged from 0.5 to 5 pg/mg of hair. Calibration curves were linear for almost all the compounds and ranged from the limit of quantification to 620 pg/mg hair. The bias and relative standard deviation of the intraday and interday imprecision were lower than 15% in 3 fortified "in-house" quality control samples, 1 external quality control sample, and 1 authentic hair sample (from a diazepam user). No significant matrix effects were observed for most of the compounds, and the extraction efficiency of the sample cleanup procedure ranged from 19% to 82% with a relative standard deviation <15% [except for clobazam (16%), loprazolam (20%), brotizolam (18%), and 7-aminoclonazepam (20%)]. The method was then successfully applied to the analysis of 40 hair samples from the workplace drug testing, containing alprazolam, estazolam, clonazepam, diazepam, zolpidem, and desalkylflurazepam (and metabolites). CONCLUSIONS: The method was completely validated and can be of interest to clinical and forensic laboratories.

Impact of the grinding process on the quantification of ethyl glucuronide in hair using a validated UPLC-ESI-MS-MS method.

The Society of Hair Testing (SoHT) has provided cutoffs for the quantification of ethyl glucuronide (EtG) in hair to indicate occasional or chronic/excessive alcohol consumption. Although several sensitive methods have been reported, past proficiency test results show a lack of reproducibility. An ultra-performance liquid chromatographic mass spectrometric method (LLOQ of 10 pg EtG/mg hair) has been validated according to the international guidelines, including the successful participation in five proficiency tests. This method was subsequently used to evaluate the impact of different grinding conditions (cut, weakly or extensively pulverized hair samples) on the final measured EtG concentration. Hair from alcohol consumers (n = 2) and commercially available quality control samples (QCs) (n = 2) was used. For the QCs, extensive pulverization led to a significantly higher amount of measured EtG. In the hair samples obtained from volunteers, cut or weakly pulverized hair resulted in EtG concentrations below the LLOQ, while the mean concentrations of 14 and 40 pg EtG/mg hair were determined after extensive pulverization. Differences in sample preparation could partially explain inter-laboratory variability. As the differences in results can lead to a different interpretation even when applying the SoHT cutoffs, it is of interest to standardize sample preparation techniques in the field of EtG hair testing.

Validation of an automated solid-phase extraction method for the analysis of 23 opioids, cocaine, and metabolites in urine with ultra-performance liquid chromatography-tandem mass spectrometry.

The aim of this work was to automate a sample preparation procedure extracting morphine, hydromorphone, oxymorphone, norcodeine, codeine, dihydrocodeine, oxycodone, 6-monoacetyl-morphine, hydrocodone, ethylmorphine, benzoylecgonine, cocaine, cocaethylene, tramadol, meperidine, pentazocine, fentanyl, norfentanyl, buprenorphine, norbuprenorphine, propoxyphene, methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine from urine samples. Samples were extracted by solid-phase extraction (SPE) with cation exchange cartridges using a TECAN Freedom Evo 100 base robotic system, including a hydrolysis step previous extraction when required. Block modules were carefully selected in order to use the same consumable material as in manual procedures to reduce cost and/or manual sample transfers. Moreover, the present configuration included pressure monitoring pipetting increasing pipetting accuracy and detecting sampling errors. The compounds were then separated in a chromatographic run of 9 min using a BEH Phenyl analytical column on a ultra-performance liquid chromatography-tandem mass spectrometry system. Optimization of the SPE was performed with different wash conditions and elution solvents. Intra- and inter-day relative standard deviations (RSDs) were within ±15% and bias was within ±15% for most of the compounds. Recovery was >69% (RSD < 11%) and matrix effects ranged from 1 to 26% when compensated with the internal standard. The limits of quantification ranged from 3 to 25 ng/mL depending on the compound. No cross-contamination in the automated SPE system was observed. The extracted samples were stable for 72 h in the autosampler (4°C). This method was applied to authentic samples (from forensic and toxicology cases) and to proficiency testing schemes containing cocaine, heroin, buprenorphine and methadone, offering fast and reliable results. Automation resulted in improved precision and accuracy, and a minimum operator intervention, leading to safer sample handling and less time-consuming procedures.

Driving under the influence of cannabis: pitfalls, validation, and quality control of a UPLC-MS/MS method for the quantification of tetrahydrocannabinol in oral fluid collected with StatSure, Quantisal, or Certus collector.

BACKGROUND: "Driving under the influence of drugs" (DUID) has a large impact on the worldwide mortality risk. Therefore, DUID legislations based on impairment or analytical limits are adopted. Drug detection in oral fluid is of interest due to the ease of sampling during roadside controls. The prevalence of Δ9-tetrahydrocannabinol (THC) in seriously injured drivers ranges from 0.5% to 7.6% in Europe. For these reasons, the quantification of THC in oral fluid collected with 3 alternative on-site collectors is presented and discussed in this publication. METHODS: An ultra-performance liquid chromatography-mass spectrometric quantification method for THC in oral fluid samples collected with the StatSure (Diagnostic Systems), Quantisal (Immunalysis), and Certus (Concateno) devices was validated according to the international guidelines. Small sample volumes of 100-200 µL were extracted using hexane. Special attention was paid to factors such as matrix effects, THC adsorption onto the collector, and stability in the collection fluid. RESULTS: A relatively high-throughput analysis was developed and validated according to ISO 17025 requirements. Although the effects of the matrix on the quantification could be minimized using a deuterated internal standard, and stability was acceptable according the validation data, adsorption of THC onto the collectors was a problem. For the StatSure device, THC was totally recovered from the collector pad after storage for 24 hours at room temperature or 7 days at 4°C. A loss of 15%-25% was observed for the Quantisal collector, whereas the recovery from the Certus device was irreproducible (relative standard deviation, 44%-85%) and low (29%-80%). During the roadside setting, a practical problem arose: small volumes of oral fluid (eg, 300 µL) were collected. However, THC was easily detected and concentrations ranged from 8 to 922 ng/mL in neat oral fluid. CONCLUSION: A relatively high-throughput analysis (40 samples in 4 hours) adapted for routine DUID analysis was developed and validated for THC quantification in oral fluid samples collected from drivers under the influence of cannabis.

Conventional and alternative matrices for driving under the influence of cannabis: recent progress and remaining challenges.

In the past decade much research concerning the impact of cannabis use on road safety has been conducted. More specifically, studies on effects of cannabis smoking on driving performance, as well as epidemiological studies and cannabis-detection techniques have been published. As a result, several countries have adopted driving under the influence of drugs (DUID) legislations, with varying approaches worldwide. A wide variety of bodily fluids have been utilized to determine the presence of cannabis. Urine and blood are the most widely used matrices for DUID legislations. However, more and more publications focus on the usability of oral fluid testing for this purpose. Each matrix provides different information about time and extent of use and likelihood of impairment. This review will focus on the practical aspects of implying a DUID legislation. The pros and cons of the different biological matrices used for Δ(9)-tetrahydrocannabinol screening and quantification will be discussed. In addition, a literature overview concerning (roadside) cannabinoid detection, as well as laboratory confirmation techniques is given. Finally, we will discuss important issues influencing interpretation of these data, such as oral fluid collection, choice of cut-offs, stability and proficiency testing.

Quantification of methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in third instar larvae of Lucilia sericata (Diptera: Calliphoridae) using liquid chromatography-tandem mass spectrometry.

Entomotoxicology studies the application of toxicological analysis on necrophageous insects present on human remains. This paper describes the development and validation of a sensitive liquid chromatography tandem mass spectrometry method for quantification of methadone and its main metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in developmental stages of Lucilia sericata. One single larva was pulverized in a disposable vial and then extracted with 1-chlorobutane. After evaporation of the organic layer, samples were reconstituted in the mobile phase. Chromatographic separation was achieved on a NUCLEODUR Sphinx RP column with a liquid chromatographic gradient (0.1% formic acid and methanol), ensuring the elution of methadone and EDDP within 15 min. The method was fully validated according to international guidelines. The use of liquid liquid extraction was demonstrated to be effective (matrix effect < 27% and recovery > 66%). The method was linear over the dynamic range (10-400 pg/mg larva) with excellent within- and between-run precision and bias (CV% < 5%). The lower limit of quantification was fixed at 10 pg/mg larva. No instability of the extracted samples was observed in the autosampler after three freeze/thaw cycles and after two months at -20 degrees C. The validated method was applied to third instar larvae of Lucilia sericata reared on beef heart spiked with 4 microg/g methadone and on a postmortem methadone overdose case. The validation and actual sample analysis showed that the method is sensitive, rugged, precise, accurate, and well-suited for routine analysis of methadone and EDDP in a single larva obtained from forensic cases.

Analysis of amphetamines and metabolites in urine with ultra performance liquid chromatography tandem mass spectrometry.

A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid-liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C18 column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (<10.7%) at each measured concentration of two external quality controls (QC) and three "in house" QC. No matrix effects were observed and good recoveries (>70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.

Evaluation of on-site oral fluid screening using Drugwipe-5(+), RapidSTAT and Drug Test 5000 for the detection of drugs of abuse in drivers.

Driving under the influence of drugs is a major problem worldwide. At the moment, several countries have adopted a 'per se' legislation to address this problem. One of the key elements in the enforcement process is the possibility of rapid on-site screening tests to take immediate administrative measures. In this study, the reliability of three oral fluid screening devices (Mavand RapidSTAT, Securetec Drugwipe-5(+), and Dräger DrugTest 5000) was assessed by comparing their on-site results with confirmatory GC-MS plasma analysis. Our results demonstrate that for amphetamine screening, the oral fluid on-site devices on the market today are certainly sensitive enough. RapidSTAT, Drugwipe-5(+), and DrugTest 5000 demonstrated respectively a sensitivity of 93%, 100% and 92% for amphetamine/MDMA. For cocaine screening, sensitivities of 75%, 78% and 67% were obtained for the RapidSTAT, Drugwipe-5(+), and DrugTest 5000 devices, respectively. The studied devices were able to detect about 70% of all cannabis users in a roadside setting. However, a newer version of the DrugTest 5000 test cassette demonstrated a sensitivity of 93%, indicating an increased detection of Delta(9)-tetrahydrocannabinol using 'new generation' oral fluid screening tests with lowered cut-offs. Due to these promising results police officers and judicial experts are keen to use oral fluid screening devices. They believe that their ease of use and diminished amount of false positive results in comparison with urine screening will lead to more roadside tests and more appropriate juridical measures.

High-throughput on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of 14 antidepressants and their metabolites in plasma.

A rapid, sensitive and fully automated on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method was developed and validated for the direct analysis of 14 antidepressants and their metabolites in plasma. Integration of the sample extraction and LC separation into a single system permitted direct injection of the plasma without prior sample pre-treatment. The applied gradient ensured the elution of all the examined drugs within 14 min and produced chromatographic peaks of acceptable symmetry. The total process time was 20 min and only 50 microL of plasma was required. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated compounds. The use of SPE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Extraction was found to be both reproducible and efficient with recoveries >99% for all the analytes. The method showed excellent intra-assay and inter-assay precision (relative standard deviation (RSD) and bias <20%) for quality control (QC) samples spiked at a concentration of 40, 200 and 800 microg/L and the r2>0.99 over the range investigated (10-1000 microg/L). Limits of quantification (LOQs) were estimated to be 10 microg/L. Furthermore, the processed samples were demonstrated to be stable for at least 48 h, except for clomipramine and norclomipramine, where a slight negative trend was observed, but did not compromise the quantification. The method was subsequently applied to authentic samples previously screened by a routine HPLC method with diode array detection (DAD).