Quantitative determination of R/S-methadone in human serum using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry: A method for routine use.

Methadone has two enantiomers, which exhibit differences in pharmacological effects, with R-methadone being the active and S-methadone the inactive enantiomer. A robust, simple and rapid method for chiral separation of the two enantiomers in serum samples using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MSMS) has been developed and validated. Enantiomeric separation was achieved using a Chiralpak IH-3 column with a mobile phase consisting of CO2 and 30mM ammonium acetate in methanol/water (98/2, v/v). Runtime was 4 minutes. Sample preparation was semi-automated using a Hamilton ML Star robot with protein precipitation, and phospholipid removal was carried out using a Waters OSTRO™ 96-well plate. The calibration range was 50.0-1,500 nM for each enantiomer. The between-assay relative standard deviations were in the range of 1.2-3.6%. Matrix effects ranged from 99% to 115% corrected with internal standard. The method has been implemented in our laboratory and has proven to be a robust and reliable method for determining the ratio of R/S-methadone in authentic patient samples.

Conductive vial electromembrane extraction of opioids from oral fluid.

The use of oral fluid as sample matrix has gained significance in the analysis of drugs of abuse due to its non-invasive nature. In this study, the 13 opioids morphine, oxycodone, codeine, O-desmethyl tramadol, ethylmorphine, tramadol, pethidine, ketobemidone, buprenorphine, fentanyl, cyclopropylfentanyl, etonitazepyne, and methadone were extracted from oral fluid using electromembrane extraction based on conductive vials prior to analysis with ultra-high performance liquid chromatography-tandem mass spectrometry. Oral fluid was collected using Quantisal collection kits. By applying voltage, target analytes were extracted from oral fluid samples diluted with 0.1% formic acid, across a liquid membrane and into a 300 µL 0.1% (v/v) formic acid solution. The liquid membrane comprised 8 µL membrane solvent immobilized in the pores of a flat porous polypropylene membrane. The membrane solvent was a mixture of 6-methylcoumarin, thymol, and 2-nitrophenyloctyl ether. The composition of the membrane solvent was found to be the most important parameter to achieve simultaneous extraction of all target opioids, which had predicted log P values in the range from 0.7 to 5.0. The method was validated in accordance to the guidelines by the European Medical Agency with satisfactory results. Intra- and inter-day precision and bias were within guideline limits of ± 15% for 12 of 13 compounds. Extraction recoveries ranged from 39 to 104% (CV ≤ 23%). Internal standard normalized matrix effects were in the range from 88 to 103% (CV ≤ 5%). Quantitative results of authentic oral fluid samples were in accordance with a routine screening method, and external quality control samples for both hydrophilic and lipophilic compounds were within acceptable limits.

Determination of amphetamine enantiomers in urine by conductive vial electromembrane extraction and ultra-high performance supercritical fluid chromatography tandem mass spectrometry.

Separation and quantification of amphetamine enantiomers are commonly used to distinguish between consumption of prescription amphetamine (mostly S-amphetamine) and illicit forms of the drug (racemate). In this study, electromembrane extraction with prototype conductive vials was combined with ultra-high performance supercritical fluid chromatography (UHPSFC-MS/MS) to quantify R- and S-amphetamine in urine. Amphetamine was extracted from 100 µL urine, diluted with 25 µL internal standard solution and 175 µL 130 mM formic acid, across a supported liquid membrane (SLM) consisting of 9 µL of a 1:1(w/w) mixture of 2-nitrophenyloctyl ether (NPOE) and bis(2-ethylhexyl)phosphite (DEHPi) into an acceptor phase containing 300 µL 130 mM formic acid. The extraction was facilitated by the application of 30 V for 15 min. Enantiomeric separation was achieved using UHPSFC-MS/MS with a chiral stationary phase. The calibration range was 50-10,000 ng/mL for each enantiomer. The between-assay CV was ≤5%, within-assay CV ≤ 1.5%, and bias within ±2%. Recoveries were 83%-90% (CV ≤ 6%), and internal standard corrected matrix effects were 99-105 (CV ≤ 2%). The matrix effects ranged from 96% to 98% (CV ≤ 8%) when not corrected by the internal standard. The EME method was compared with a chiral routine method that employed liquid-liquid extraction (LLE) for sample preparation. Assay results were in agreement with the routine method, and the mean deviation between methods was 3%, ranging from -21% to 31%. Finally, sample preparation greenness was assessed using the AGREEprep tool, which resulted in a greenness score of 0.54 for conductive vial EME, opposed to 0.47 for semi-automated 96-well LLE.

Serum or Plasma for Quantification of Direct Oral Anticoagulants?

BACKGROUND: Direct oral anticoagulants are increasingly replacing vitamin K antagonists for prevention of stroke in patients with atrial fibrillation, partly owing to the lack of a need for routine monitoring. Therapeutic drug monitoring may still be warranted under certain circumstances. It is generally assumed that serum and plasma can be interchangeably used for this purpose. The aim of this study was to investigate possible differences between the serum, citrate-plasma, and ethylenediaminetetraacetic acid (EDTA)-plasma concentrations of apixaban and rivaroxaban in a larger patient group and their relation to factor X measurements. METHODS: Plasma and serum samples were drawn during the same venipuncture from patients treated with apixaban or rivaroxaban. Drug levels were measured using ultrahigh-performance liquid chromatography combined with tandem mass spectrometry. Three sample matrices were obtained from 8 healthy volunteers for measurement of factor X antigen and activity. RESULTS: Mean concentrations of apixaban and rivaroxaban were 16.8% and 36.6% higher in serum than in citrate-plasma, respectively (both P < 0.001). The corresponding differences in serum versus EDTA-plasma were 4.5% for apixaban and 13.1% for rivaroxaban (both P < 0.001). Factor X antigen measurements in citrate-plasma, EDTA-plasma, serum with clot activator, and serum without additives yielded comparable results, and factor X activity was significantly higher in serum than in plasma. CONCLUSIONS: Apixaban and rivaroxaban concentrations were significantly higher in serum than in plasma. The difference was more pronounced with rivaroxaban and was larger between serum and citrate-plasma than between serum and EDTA-plasma. Higher factor X activity in serum may explain the observed concentration differences. The choice of matrix is, thus, important when interpreting therapeutic drug monitoring results and in research involving analyses of direct oral anticoagulants. The authors recommend citrate-plasma as the preferred matrix.

Stability of Direct Oral Anticoagulants and Antiarrhythmic Drugs in Serum Collected in Standard (Nongel) Serum Tubes Versus Tubes Containing Gel Separators.

BACKGROUND: Separation gels are often used in collection tubes, but adsorption of drugs onto the gel may cause falsely low concentrations in therapeutic drug monitoring. In this study, the stability of apixaban, edoxaban, rivaroxaban, flecainide, amiodarone, and desethylamiodarone was assessed in tubes, with and without gel separators. METHODS: Drug-free blood was spiked and stored for up to 7 days in nongel tubes and gel tubes from 2 manufacturers (Vacuette and Vacutainer). The samples were analyzed in triplicates using ultra-high-pressure liquid chromatography-tandem mass spectrometry. RESULTS: At ambient temperature conditions, the serum concentrations of apixaban, edoxaban, and rivaroxaban in a tube with acrylic-based gel had already decreased at baseline, whereas it took 6 hours to observe the same result in a tube with olefin-based gel. At 4°C, the reduction in serum concentration was considerably slower. For flecainide, the gel tube concentrations were stable at ambient temperature for 3 days, but decreased after 7 days in acrylic-based gel tubes. Amiodarone and desethylamiodarone stored in gel tubes at 4°C showed decrease in concentrations after 24 hours and 6 hours, respectively. CONCLUSIONS: Acrylic-based gel tubes should not be used for any of the tested drugs. Although olefin-based gel tubes may be used for anticoagulants and flecainide, it is advisable to prefer nongel tubes as a general precaution.

Determination of psychoactive drugs in serum using conductive vial electromembrane extraction combined with UHPLC-MS/MS.

Conductive vial electromembrane extraction (EME) with prototype equipment was applied for the first time to extract lipophilic basic drugs from serum. With this equipment, traditional platinum electrodes were replaced with sample and acceptor vials made from a conductive polymer, making the electrodes fully integrated and disposable. EME was combined with UHPLC-MS/MS, and a method to determine selected psychoactive drugs (alimemazine, amitriptyline, atomoxetine, clomipramine, doxepin, duloxetine, fluvoxamine, levomepromazine, nortriptyline and trimipramine) and metabolites (desmethyl clomipramine and desmethyl doxepin) in serum was developed, optimized, and validated. Extractions were carried out with 50 V for 15 min from serum samples (100 µL) diluted 1:3 with formic acid (0.1% v/v), using 2-nitrophenyl octyl ether as the supported liquid membrane (SLM), and formic acid (0.1% v/v, 300 µL) as acceptor phase. Using conductive vial EME, the extraction of lipophilic drugs reached exhaustive or near-exhaustive conditions, with recoveries in the range 75-117%. The method demonstrated excellent accuracy and precision, with bias within ± 6%, and intra- and inter-day CVs ranging 0.9 - 6% and 2 - 6%, respectively. In addition, acceptor phases were completely free of glycerophosphocholines. EME-UHPLC-MS/MS was successfully applied in determination of psychoactive drugs in 30 patient samples, and the results were in agreement with the current hospital routine method at St. Olav University Hospital (Trondheim, Norway). Obtaining comparable results to well-established routine methods is highly important for future implementation of EME into routine laboratories. These results thus serve as motivation for further advancing the EME technology. Until now, EME has been carried out with laboratory-build equipment, and the introduction of commercially available standardized equipment is expected to have a positive impact on future research activity.

Enantiomeric separation and quantification of R/S-amphetamine in serum using semi-automated liquid-liquid extraction and ultra-high performance supercritical fluid chromatography-tandem mass spectrometry.

The amphetamine molecule contains a chiral center and its enantiomers exhibit differences in pharmacological effects, with the S-enantiomer mediating most of the central nervous system stimulating activity. The majority of prescribed amphetamine consists of the pure S-enantiomer, but therapeutic formulations containing the R-enantiomer in various proportions are also available. Illegal amphetamine remains available mainly as a racemic mixture of the R- and S-enantiomers. To distinguish between legal and illegal consumption of amphetamine a method for enantiomeric separation and quantification of R/S-amphetamine in serum was developed and validated using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS). Sample preparation prior to UHPSFC-MS/MS analysis was performed by a semi-automated liquid-liquid extraction method. The UHPSFC-MS/MS method used a Chiralpak AD-3 column with a mobile phase consisting of CO2 and 0.1% ammonium hydroxide in 2-propanol/methanol (50/50, v/v). The injection volume was 2 µL and run time was 4 minutes. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 136.1 > 119.0 and m/z 136.1 > 91.0). The calibration range was 12.5-1,000 nM for each analyte. The between-assay relative standard deviations were in the range of 1.3-3.0%. Recovery was 73% and matrix effects ranged from 95 to 100% when corrected with internal standard. After development and validation, the method has been successfully implemented in our laboratory for both separation and quantification of R/S-amphetamine and has proved to be a reliable and useful tool for distinguishing intake of R- and S-amphetamine in authentic patient samples.

Association between low body weight and cytochrome P-450 enzyme activity in patients with anorexia nervosa.

Very little is known to which extent severe underweight could affect cytochrome P-450 (CYP) enzyme activity. In this study, 24 patients with anorexia nervosa at two occasions ingested single oral doses of five test drugs known to be metabolized by CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. A mixed model analysis was used to evaluate the effect of changes in body mass index (BMI) on the metabolic activities of these enzymes. The primary end point was the change in drug/metabolite ratio of each of the test drugs per kg/m2 change in BMI. With increasing BMI, the metabolic activity of CYP3A4 decreased (change in the CYP3A4 drug/metabolite ratio per unit change in BMI = 0.056; 95% confidence interval [CI] 0.011 to 0.102; P = .017). For CYP1A2, increasing BMI increased the metabolic activity with borderline significance (change in the CYP1A2 drug/metabolite ratio per unit change in BMI = -0.107; CI -0.220 to 0.005; P = .059). For CYP2C9, CYP2C19, and CYP2D6, no significant changes were seen. The clinical impact of these findings for drug treatment in patients with anorexia nervosa and other severely underweight patients needs to be further studied by examining the pharmacokinetics of specific drugs. This might be particularly relevant for drugs metabolized by CYP1A2 and/or CYP3A4.

Stability of 21 Antihypertensive Drugs in Serum Collected in Standard (Nongel) Serum Tubes Versus Tubes Containing a Gel Separator.

BACKGROUND: Therapeutic drug monitoring of antihypertensive drugs is being increasingly used to optimize treatment and to assess nonadherence. Separator gels are often used in blood collection tubes to facilitate serum or plasma separation from other blood constituents before analyses. Drug adsorption into the separator gel presents a possible pre-analytical cause of falsely low concentrations or false negative results. METHODS: Drug-free blood from blood donors was spiked with therapeutic concentrations of 21 antihypertensive drugs, transferred to serum tubes with and without separator gel (Vacuette gel plastic tubes and plain serum plastic tubes, respectively), and centrifuged. Serum was collected immediately after centrifugation and after 24 and 72 hours of room temperature storage, samples were analyzed in triplicates using liquid chromatography-mass spectrometry. RESULTS: Serum samples collected immediately after centrifugation or 24 hours later, had the same drug concentrations in the gel and nongel tubes. After 72 hours of room temperature storage, verapamil and lercanidipine serum concentrations were 43% and 29%, respectively, lower in gel tubes than nongel tubes. Canrenone, diltiazem, and bendroflumethiazide showed between 10% and 20% concentration loss in gel tubes, compared with nongel tubes, with the 2 latter observed as unstable also in nongel tubes. CONCLUSIONS: Except for verapamil, lercanidipine, and canrenone, which showed substantial concentration loss in gel tubes, gel tubes may be used for therapeutic drug monitoring purposes for the most commonly used antihypertensive drugs. Transferring serum to gel-free containers immediately after centrifugation minimizes concentration loss; however, bendroflumethiazide and diltiazem are generally unstable at room temperature.

Serum Concentrations and Pharmacokinetics of Tranexamic Acid after Two Means of Topical Administration in Massive Weight Loss Skin-Reducing Surgery.

BACKGROUND: Topical administration of tranexamic acid to reduce bleeding is receiving increasing attention, as it is inexpensive, simple, and possibly beneficial in most surgery. Concerns regarding potential systemic adverse effects such as thromboembolic events and seizures may prevent general use of tranexamic acid. Although serum concentrations after topical application are assumed to be low, proper pharmacokinetic studies of tranexamic acid after topical application are lacking. METHODS: The authors have investigated systemic absorption of tranexamic acid after two means of topical administration in patients undergoing abdominoplasty after massive weight loss: a bolus of 200 ml of 5 mg/ml into the wound cavity versus moistening the wound surface with 20 ml of 25 mg/ml. Twelve patients were recruited in each group. Serum concentrations achieved were compared with those after administration of 1 g as an intravenous bolus to arthroplasty patients. Serial blood samples for tranexamic acid analysis were obtained for up to 24 hours. RESULTS: After intravenous administration, the peak serum concentration was 66.1 ± 13.0 µg/ml after 6 ± 2 minutes. Peak serum concentration after topical moistening was 5.2 ± 2.6 µg/ml after 80 ± 33 minutes, and in the topical bolus group, it was 4.9 ± 1.8 µg/ml after 359 ± 70 minutes. Topical moistening resulted in homogenous and predictable absorption across the individuals included, whereas topical bolus administration caused variable and unpredictable serum concentrations. CONCLUSION: Topical administration of tranexamic acid in patients undergoing abdominoplasty results in low serum concentrations, which are highly unlikely to cause systemic effects.

Quantification of 21 antihypertensive drugs in serum using UHPLC-MS/MS.

BACKGROUND: Poor drug adherence in hypertensive patients can lead to treatment failure and increased cardiovascular morbidity, as well as increased costs to society. An analytical method based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MSMS) was developed and validated for use in routine therapeutic drug monitoring (TDM). The method includes 21 antihypertensive drugs or active metabolites from the groups beta blockers (n=5), calcium antagonists (n=5), angiotensin II receptor antagonists (n=4), angiotensin converting enzyme (ACE) inhibitors (n=3) and diuretics (n = 3), in addition to one α1-selective alpha blocker. METHOD: A 200 µL serum sample was handled automatically using a pipetting robot. Protein precipitation was performed with 600 µL of 1% formic acid in acetonitrile (v:v) and phospholipid removal was carried out using a Waters OSTRO™ 96-well plate. After evaporation and reconstitution the eluent was injected thrice with different inlet and mass spectrometric methods to cover the different physico-chemical properties of the drugs and the variations in therapeutic concentration ranges between drugs. Acquity UPLC BEH C18 (2.1x50mm, 1.7 µm) column equipped with a corresponding pre-column was used for chromatographic separation. For every analyte an isotopically labelled analogue served as internal standard, except for lisinopril where enalaprilat-d5 was used. RESULTS: Accuracies were in the range of -13.7 to 13.2% and intra-day and inter-day precisions in the range of 1.1 to 10.5%. The linearity within the calibration ranges expressed as coefficient of determination was higher than 0.995 for all compounds. Matrix effects and recovery efficiencies were within acceptable limits. The limits of quantitation varied from 0.02 to 10.7 µg/L. The stability of the drugs in serum at different conditions was tested. Diltiazem was not stable at 4-8 °C with up to 23.5 % loss after six days. Degradation of atenolol, irbesartan, bendroflumethiazide, hydrochlorothiazide and diltiazem was observed when stored at 30 °C. The suitability of the method was demonstrated in a routine TDM setting, analysing samples from 127 patients undergoing antihypertensive drug treatment.

Quantification of Apixaban, Dabigatran, Edoxaban, and Rivaroxaban in Human Serum by UHPLC-MS/MS-Method Development, Validation, and Application.

BACKGROUND: Direct oral anticoagulants (DOACs) are prescribed for anticoagulation in patients with atrial fibrillation and venous thromboembolic disease. Fixed doses are recommended, but measuring their serum drug concentrations as a basis for dose adjustments may be useful in some clinical settings. METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method for the analysis of the DOACs apixaban, dabigatran, edoxaban, and rivaroxaban in human serum was developed and validated. A 100-µL serum sample was handled using a pipetting robot. Protein precipitation was performed with 375 µL of 1% formic acid in acetonitrile (vol/vol), and phospholipid removal was performed using a Waters Ostro 96-well plate. The injection volume was 1 µL, and run time was 3.0 minutes. RESULTS: The calibration range was 5-800 nmol/L. The between-day precision relative SDs were in the range of 3.3%-10%. Recoveries ranged from 85% to 105%, and matrix effects from 88% to 102%, when corrected with internal standard. Edoxaban was, in contrast to the other DOACs, unstable when stored for more than 6 hours at 30°C. The suitability of the method was demonstrated by analyzing routine samples from 345 patients undergoing anticoagulation treatment. CONCLUSIONS: The developed method fulfilled the set validation criteria, and its suitability was demonstrated in a routine setting. The instability of edoxaban may complicate the transport of routine samples to the laboratory.

Enantiomeric separation and quantification of citalopram in serum by ultra-high performance supercritical fluid chromatography-tandem mass spectrometry.

A method for enantiomeric separation and quantification of R/S-citalopram in serum was developed and validated using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS). Sample preparation prior to UHPSFC-MS/MS analysis consisted of protein precipitation with acidic acetonitrile and filtration through a phospholipid removal plate. The UHPSFC-MS/MS method used an UPC2 Trefoil CEL2 column with a mobile phase consisting of CO2 and methanol/acetonitrile (70:30, v/v) with 10mM ammonium acetate. The injection volume was 1µL and run time was 4min. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 325.1>262.0 and m/z 325.1>109.0). The calibration range was 5-500nM for each analyte. The between-assay relative standard deviations were in the range of 3.4-4.5%. Recovery was 81-91% and matrix effects ranged from 96 to 101% (corrected with internal standard). After development and initial testing, the method has been successfully implemented in routine use in our laboratory for both separation and quantification of R/S-citalopram in more than 250 serum samples for therapeutic drug monitoring.

EtG/EtS in Serum by UHPLC-MS-MS in Suspected Sexual Assault Cases.

A method including semi-automated extraction of ethyl glucuronide (EtG) and ethyl sulfate (EtS) from serum followed by ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) has been developed and validated. Sample preparation prior to UHPLC-MS-MS analysis consisted of protein precipitation and filtration through a phospholipid removal plate. Chromatography was achieved using an HSS T3 column and gradient elution with formic acid in water in combination with methanol. The mass spectrometer was monitored in the negative mode with multiple reaction monitoring. Two transitions were monitored for the analytes and one for the deuterated internal standards (ISs). The limits of quantification were 0.025 mg/L for EtG and 0.009 mg/L for EtS. The between-assay relative standard deviations were in the range of 3.8-9.1%, the recovery was 66-102% and matrix effects ranged from 88 to 97% (corrected with IS). Compared to previously published studies, the method presented is semi-automated, uses a simple method for phospholipid removal and has short run times and low limit of quantifications. We analyzed serum samples from 49 female patients presenting to the Sexual Assault Centre at St. Olav University Hospital in Trondheim, Norway, for ethanol, EtG and EtS. EtG and EtS were detected longer than ethanol itself after intake of ethanol, with estimated maximum detection times of >24 h. The ethanol, EtG and EtS concentrations were highly correlated (P < 0.001), but with large inter-individual variations. This study suggests that analysis of EtG and EtS in serum or blood may complement ethanol analysis and shed light on the patient's recent ethanol intake after ethanol itself is no longer detectable.

EtG and EtS in Autopsy Blood Samples With and Without Putrefaction Using UPLC-MS-MS.

Analytical challenges related to postmortem specimens are well known. The degree of putrefaction of the corpse will influence the quality of the blood samples, and both the efficiency of sample preparation and the subsequent chromatographic performance can be affected. An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed and validated for the determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in postmortem whole blood. Sample preparation prior to UPLC-MS-MS analysis consisted of protein precipitation and filtration through a phospholipid removal plate. Chromatography was achieved using an HSS T3 column and gradient elution with formic acid in water in combination with methanol. The injection volume was 0.5 µL. Negative electrospray ionization was performed in the multiple reaction monitoring mode. Two transitions were monitored for the analytes and one for the internal standards. The between-assay relative standard deviations were in the range of 1.7-7.0% and the limits of quantification were 0.025 and 0.009 mg/L for EtG and EtS, respectively. Recovery was 51-55% and matrix effects ranged from 98% to 106% (corrected with internal standard). Blood samples from nine autopsy cases with various extents of putrefaction were analyzed. The sample preparation efficiently removed the phospholipids from the blood specimens. The samples were clean and the analytical quality of the chromatographic performance was satisfactory for both analytes irrespective of the degree of putrefaction.

Detection Times of Carboxylic Acid Metabolites of the Synthetic Cannabinoids JWH-018 and JWH-073 in Human Urine.

Over the past years, use of synthetic cannabinoids has become increasingly popular. To draw the right conclusions regarding new intake of these substances in situations of repeated urinary drug testing, knowledge of their elimination rate in urine is essential. We report data from consecutive urine specimens from five subjects after ingestion of synthetic cannabinoids. Urinary concentrations of the carboxylic acid metabolites JWH-018-COOH and JWH-073-COOH were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) with a limit of quantification of 0.1 ng/mL. In these subjects, specimens remained positive over a period of 20-43 (mean 27) days for JWH-018-COOH and over a period of 11-25 (mean 19) days for JWH-073-COOH. Detection times were shorter for subjects that appeared to have ingested only one, or a few, doses prior to urine collection in the study. Creatinine-normalized concentrations (CN-concentrations) slowly declined throughout the follow-up period in all subjects, suggesting that no new intake had taken place during this period. Mean elimination half-lives in urine were 14.0 (range 4.4-23.8) days for CN-JWH-018-COOH and 9.3 (range 3.6-16.8) days for CN-JWH-073-COOH. These data show that urine specimens could be positive for JWH-018-COOH for more than 6 weeks and JWH-073-COOH for more than 3 weeks after ingestion. However, such long detection periods require a low limit of quantification.

A validated method for simultaneous determination of codeine, codeine-6-glucuronide, norcodeine, morphine, morphine-3-glucuronide and morphine-6-glucuronide in post-mortem blood, vitreous fluid, muscle, fat and brain tissue by LC-MS.

The toxicodynamics and, to a lesser degree, toxicokinetics of the widely used opiate codeine remain a matter of controversy. To address this issue, analytical methods capable of providing reliable quantification of codeine metabolites alongside codeine concentrations are required. This article presents a validated method for simultaneous determination of codeine, codeine metabolites codeine-6-glucuronide (C6G), norcodeine and morphine, and morphine metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in post-mortem whole blood, vitreous fluid, muscle, fat and brain tissue by high-performance liquid chromatography mass spectrometry. Samples were prepared by solid-phase extraction. The validated ranges were 1.5-300 ng/mL for codeine, norcodeine and morphine, and 23-4,600 ng/mL for C6G, M3G and M6G, with exceptions for norcodeine in muscle (3-300 ng/mL), morphine in muscle, fat and brain (3-300 ng/mL) and M6G in fat (46-4,600 ng/mL). Within-run and between-run accuracy (88.1-114.1%) and precision (CV 0.6-12.7%), matrix effects (CV 0.3-13.5%) and recovery (57.8-94.1%) were validated at two concentration levels; 3 and 150 ng/mL for codeine, norcodeine and morphine, and 46 and 2,300 ng/mL for C6G, M3G and M6G. Freeze-thaw and long-term stability (6 months at -80°C) was assessed, showing no significant changes in analyte concentrations (-12 to +8%). The method was applied in two authentic forensic autopsy cases implicating codeine in both therapeutic and presumably lethal concentration levels.

Screening and quantitative determination of drugs of abuse in diluted urine by UPLC-MS/MS.

The purpose of this work was to develop and evaluate a fast, robust and specific UPLC-MS/MS screening platform for the determination and quantification of a variety of commonly used drugs of abuse in urine, i.e. a high-throughput quantitative analysis. Substances in the drug classes opioids, central nervous system stimulants and benzodiazepines and related agents were included in addition to cannabis and pregabalin, a total of 35 different analytes. Based on the concentrations and the physico-chemical properties of the substances, three UPLC-MS/MS methods were developed in parallel. Prior to analysis, sample preparation consisted of two different simple dilutions with 60 and 100 µL urine, respectively, using a Tecan Freedom Evo pipetting robot platform. A Waters Xevo TQ-S tandem quadrupole mass spectrometer coupled to a Waters I-class UPLC was used for quantitative analysis of one quantitative and one qualifying MRM transition for each analyte, except for tramadol for which the metabolite O-desmethyl-tramadol was included in the MRM method to confirm tramadol identity. Deuterated analogs were included as internal standards. The between-assay relative standard deviations varied from 2% to 11% and the limits of quantification were in the range 1-200 ng/mL for the various analytes. After development and initial testing, the method has been successfully implemented and routinely used at our hospital for quantitative screening of drugs of abuse in more than 35,000 urinary samples.

EtG/EtS in Urine from sexual assault victims determined by UPLC-MS-MS.

In cases of sexual assault, victims often present too late for the detection of ethanol in biological samples. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed and validated for the determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in urine. Sample preparation prior to UPLC-MS-MS analysis was a simple sample dilution. The calibration ranges were 0.2-20 mg/L, and between-assay relative standard deviations were in the range of 0.7-7.0% at concentrations of 0.3, 3.0 and 7.0 mg/L. Urine samples were analyzed from 59 female patients presenting to the Sexual Assault Centre at St. Olav University Hospital in Trondheim, Norway between November 2010 and October 2011. EtG and EtS results were fully concordant, and positive in 45 of the 48 cases with self-reported alcohol intake. In contrast, ethanol was detectable in only 20 of these cases, corresponding to sensitivities of 94 and 42%, respectively. Of the patients reporting no alcohol intake, none had positive EtG/EtS findings. These data show that analysis of EtG and EtS greatly increases the detection window of alcohol ingestion in cases of sexual assault, and may shed additional light on the involvement of ethanol in such cases. The victims' self-reported intake of alcohol seems to be reliable in this study, according to the EtG/EtS findings.

Challenges to antagonist blockade during sustained-release naltrexone treatment.

AIMS: Naltrexone is a competitive opioid antagonist that effectively blocks the action of heroin and other opioid agonists. Sustained-release naltrexone formulations are now available that provide long-acting opioid blockade. This study investigates the use of heroin and other opioids among opioid-dependent patients receiving treatment with long-acting naltrexone implants, their subjective experience of drug 'high' after opioid use, and factors associated with opioid use. METHODS: Participants (n = 60) were opioid-dependent patients receiving treatment with naltrexone implants. Outcome data on substance use, drug 'high', depression and criminal activity were collected over a 6-month period. Blood samples were taken to monitor naltrexone plasma levels, and hair samples to verify self-reported opioid use. FINDINGS: More than half [n = 34 or 56%; 95% confidence interval (CI) 44-68%)] the patients challenged the blockade with illicit opioids during the 6-month treatment period; 44% (n = 26; 95% CI 32-56%) were abstinent from opioids. Mean opioid use was reduced from 18 [standard deviation (SD)13] days during the month preceding treatment to 6 days (SD 11) after 6 months. Of the respondents questioned on opioid 'high' (n = 31), nine patients (30%; 95% CI 16-47%) reported partial drug 'high' following illicit opioid use, and three (12%; 95% CI 3-26%) reported full 'high'. Opioid use was associated with use of non-opioid drugs and criminal behaviour. CONCLUSIONS: Challenging naltrexone blockade with heroin on at least one occasion is common among sustained-release naltrexone patients, but only a minority of patients use opioids regularly. Challenges represent a warning sign for poor outcomes and often occur in the context of polydrug use and social adjustment problems.