Solfrid Hegstad

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.

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.

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.

Enantiomeric separation and quantification of R/S-amphetamine in urine by ultra-high performance supercritical fluid chromatography tandem mass spectrometry

To distinguish between legal and illegal consumption of amphetamine reliable analytical methods for chiral separation of the R- and S-enantiomers of amphetamine in biological specimens are required. In this regard, supercritical fluid chromatography (SFC) has several potential advantages over liquid chromatography, including rapid separation of enantiomers due to low viscosity and high diffusivity of supercritical carbon dioxide, the main component in the SFC mobile phase. A method for enantiomeric separation and quantification of R- and S-amphetamine in urine 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 a semi-automatic solid phase extraction method. The UHPSFC-MS/MS method used a Chiralpak AD-3 column with a mobile phase consisting of CO2 and 0.2% cyclohexylamine in 2-propanol. The injection volume was 2 μL and run-time was 6 min. 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 50-10,000 ng/mL for each enantiomer. The between-assay relative standard deviations were in the range of 3.7-7.6%. Recovery was 92-93% and matrix effects ranged from 100 to 104% corrected with internal standard. After development and validation, the method has been successfully implemented in routine use at 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.

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.

Bestemmelse av legemidler og rusmidler i hår

2. What drugs can VIFM test for with hair? VIFM can test for at least 60 drugs in hair. This includes common illicit drugs such as methylamphetamine, heroin, cocaine and cannabis. VIFM can also test for a number of prescription drugs including an extensive range of stimulants, benzodiazepines (sedatives), opioids, barbiturates, antidepressants, anaesthetics, antipsychotics and new synthetic drugs.

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.

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 patients recent ethanol intake after ethanol itself is no longer detectable.