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.

Quantifying Alcohol Consumption in the General Population by Analysing Phosphatidylethanol Concentrations in Whole Blood: Results from 24,574 Subjects Included in the HUNT4 Study.

AIMS: To evaluate the association between self-reported alcohol consumption and phosphatidylethanol (PEth) concentrations in blood in a large general population study, and discuss optimal cut-off PEth concentrations for defined levels of alcohol consumption. METHODS: Population based, longitudinal cohort study including 24,574 adults from The Trøndelag Health Study 4 (HUNT4) conducted in Trøndelag County, Norway. Data included PEth concentration, self-reported alcohol consumption and CAGE score. RESULTS: PEth levels in whole blood increased with the number of alcohol units consumed, the frequency of alcohol consumption, the frequency of binge drinking and the CAGE score (lifetime, i.e. 'have you ever'). The cut-off concentrations with highest combined sensitivity and specificity were 0.057 µmol/l (40 ng/ml) for identification of those consuming >1 alcohol unit per day (sensitivity 86%, specificity 76%), 0.087 µmol/l (61 ng/ml) for consuming >2 units per day (sensitivity 87%, specificity 81%) and 0.122 µmol/l (86 ng/ml) for consuming >3 alcohol units per day (sensitivity 80%, specificity 86%). By defining a CAGE score ≥ 2 as potentially harmful consumption, a cut-off of 0.100 µmol/l (70 ng/ml) identified 52% of all those subjects. CONCLUSIONS: Cut-off limits of PEth concentrations should take into account the indication for sampling. Using cut-offs for the PEth concentrations of about 0.05 µmol/l (35 ng/ml) and 0.08 µmol/l (56 ng/ml) would identify about 90% of the subjects consuming more than 1 and 2 alcohol units per day, respectively. Concentrations above these cut-offs should lead to a more detailed interview related to alcohol use.

Identification of unhealthy alcohol use by self-report and phosphatidylethanol (PEth) blood concentrations in an acute psychiatric department.

BACKGROUND: The use of standard screening methods could improve the detection rate of unhealthy alcohol use in patients admitted to psychiatric acute and emergency departments. The aim of the present study was to investigate the ability of the alcohol biomarker phosphatidylethanol (PEth) to identify patients with high levels of alcohol consumption prior to admission. METHODS: The data were prospectively collected at admittance to an acute psychiatric department in the period January 2016 to June 2017. A blood sample for the analysis of PEth was available from 177 patients. We compared the PEth concentrations with the Alcohol Use Disorders Identification Test (AUDIT) scores during the hospital stay, and psychiatric diagnoses at discharge. RESULTS: A total of 45.8% of the patients had a PEth concentration ≥ 0.03 µmol/L, indicating significant alcohol consumption. AUDIT scores consistent with unhealthy alcohol use were present in 51.7%. There was a significant positive correlation between PEth concentrations and AUDIT scores (r = 0.631, p < 0.001). PEth was above the detection limit of 0.03 µmol/L in 19% of those reporting an average daily intake of zero alcohol units per day during the last week before admission. PEth concentrations were significantly higher among those with an alcohol diagnosis than among those without such a diagnosis (0.82 µmol/L vs. 0.09 µmol/L, p = 0.001). CONCLUSION: PEth provides supplementary information on recent alcohol consumption in a psychiatric population and would be particularly helpful in patients unable or unwilling to give such information at admission.

Severe Neurological Sequelae after a Recreational Dose of LSD.

A young man with an unremarkable medical history suffered a seizure with subsequent cardiorespiratory arrest and severe neurological sequelae after ingesting a blotter. Analysis of a similar blotter and a serum sample obtained 3 h after the event detected lysergic acid diethylamide (LSD) at an amount of 300 µg in the blotter and at a concentration of 4.0 ng/mL (12.4 nmol/L) in the serum. No other drugs were present in concentrations which may confer significant effects. In addition, no individual traits which would make the patient particularly susceptible to adverse LSD effects have subsequently been identified. This suggests that LSD may confer toxic effects in previously healthy individuals.

Stability of Phosphatidylethanol 16:0/18:1 in Freshly Drawn, Authentic Samples from Healthy Volunteers.

Due to its specificity, phosphatidylethanol (PEth) 16:0/18:1 has gained increased popularity as a marker for high alcohol consumption in recent years. As conflicting results regarding the stability of PEth 16:0/18:1 in whole blood have been published, there are still uncertainties related to optimum handling, transport and storage of blood samples for the analysis of PEth 16:0/18:1. A stability study where whole blood samples were drawn from healthy volunteers, who had ingested alcohol, is presented. The samples were collected in tubes with ethylenediamine tetraacetic acid (EDTA) and heparin as additives and stored under standardized conditions within 1 h of blood sampling. Storage times were 28 days in ambient temperature and at 4-8°C, and 90 days at -20°C and -80°C. All samples were analyzed regularly during the storage periods. PEth 16:0/18:1 concentrations were stable (defined as < 15% decrease compared with baseline values) at all temperatures up to 28 days, independent of additive. After 90 days of storage at -20°C, the mean concentrations had decreased by 18.8% in EDTA tubes and by 13.8% in heparin tubes. At -80°C, the concentrations were stable throughout the 90-day period. The present study shows that in samples containing PEth formed in vivo, PEth 16:0/18:1 is stable for 28 days irrespective of storage temperature. During long-term storage, samples should be stored at -80°C.

High Throughput UPLC®-MSMS Method for the Analysis of Phosphatidylethanol (PEth) 16:0/18:1, a Specific Biomarker for Alcohol Consumption, in Whole Blood.

Phosphatidylethanol (PEth) is an alcohol biomarker formed in the presence of ethanol in the body. Both due to its specificity and because it has a detection window of up to several weeks after alcohol intake, its application potential is broader than for other ethanol biomarkers. The aim of this study was to develop and validate a robust method for PEth in whole blood with fast and efficient sample extraction and a short analytical runtime, suitable for high throughput routine purposes. A validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC®-MSMS) method for quantification of PEth 16:0/18:1 in the range 0.05-4.00 µM (R2 ≥ 0.999) is presented. PEth 16:0/18:1 and the internal standard (IS) PEth-d5 (0.55 µM), were extracted from whole blood (150 µL) by simple protein precipitation with 2-propanol (450 µL). Chromatography was achieved using a BEH-phenyl (2.1 × 30 mm, 1.7 µm) column and a gradient elution combining ammonium formate (5 mM, pH 10.1) and acetonitrile at a flow rate of 0.5 mL/min. Runtime was 2.3 min. The mass spectrometer was monitored in negative mode with multiple reaction monitoring (MRM). The m/z 701.7 > 255.2 and 701.7 > 281.3 transitions were monitored for PEth 16:0/18:1 and the m/z 706.7 > 255.3 for PEth-d5. Limit of quantification was 0.03 µM (coefficient of variation, CV = 6.7%, accuracy = 99.3%). Within-assay and between-assay imprecision were 0.4-3.3% (CV ≤ 7.1%). Recoveries were 95-102% (CV ≤ 4.9%). Matrix effects after IS correction ranged from 107% to 112%. PEth 16:0/18:1 in patient samples were stable for several days at 30°C. Repeated freezing (-80°C) and thawing did not affect the concentration. After thawing and analysis patient samples were stable at 4-8°C for at least 4 weeks. Results from a proficiency test program, showing |Z| values ≤1.2, confirm the validity of the method. Analysis of the first 3,169 samples sent to our laboratory for routine use has demonstrated its properties as a robust method suitable for high throughput purposes.

A UPLC-MSMS method for the analysis of olanzapine in serum-with particular emphasis on drug stability testing.

A method including a rapid and automated extraction of olanzapine from serum followed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been developed and validated. Serum aliquots (100µL) and internal standard (olanzapine-d3, 25µL) were pipetted onto an Ostro(TM) 96-well filtration plate and protein precipitated with acidic acetonitrile (300µL) before removal of endogenous phospholipids by filtration followed by analysis. Chromatography was achieved using an HSST 3 (2.1×100mm, 1.8µm) column and gradient elution with acidic water in combination with methanol at a flow rate of 0.5mL/min. The runtime was 1.5min. The mass spectrometer was monitored in positive mode and multiple reaction monitoring (MRM). The m/z 313.1>256.1 and 313.1>198.0 transitions were monitored for olanzapine (m/z 316.1>256.1 for olanzapine-d3). The quadratic calibration curves ranged from 5 to 500nM (R(2)≥0.999). Limit of quantification was 0.5nM (CV 9.6%, accuracy 110%). Within-assay and between-assay inaccuracies were 2.6-11.9% (CV≤4.8%). Recovery was 84-95% (CV≤1.4%) and matrix effects ranged from 100 to 103% (CV≤2.6%). Extensive stability testing showed that at ambient temperature, olanzapine in patient serum samples were stable for at least seven hours on the laboratory bench and for at least 48h in darkness. When exposed to 3000lux, however, significant degradation had occurred after 48h. Notably, olanzapine in spiked serum was unstable already after four hours when exposed to 3000 lux. At 4-8°C and exposure to 550lux, both patient serum and spiked serum were stable for more than 48h but less than a week, whereas in darkness, the samples were stable for at least 14 days. The cumulative light exposure causing significant degradation of olanzapine in patient serum was 50,000-100,000lux-h. In some individual samples, however, the effect of light exposure was more pronounced. Therefore, it seems pertinent to recommend protecting all samples from light, although we found no indication that a few hours of exposure to standard indoor illumination will affect the olanzapine concentration to any significant degree.

Is oxycodone efficacy reflected in serum concentrations? A multicenter, cross-sectional study in 456 adult cancer patients.

CONTEXT: The relationship between oxycodone and metabolite serum concentrations and clinical effects has not previously been investigated in cancer pain patients. OBJECTIVES: The aim of this study was to assess whether there is a relationship between oxycodone concentrations and pain intensity, cognitive functioning, nausea, or tiredness in cancer patients. Also, oxymorphone and noroxymorphone contributions to analgesia and the adverse effects of oxycodone were assessed. METHODS: Four hundred fifty-six cancer patients receiving oxycodone for cancer pain were included. Pain was assessed using the Brief Pain Inventory. The European Organisation for Research and Treatment of Cancer Quality-of-Life Questionnaire-C30 was used to assess the symptoms of tiredness, nausea, constipation, and depression. Cognitive function was assessed using the Mini-Mental State Examination. Associations were examined by multiple linear or ordinal logistic regressions. Whether patients classified as being a "treatment success" or a "treatment failure" had different serum concentrations of oxycodone or metabolites was assessed using Mann-Whitney U-tests. RESULTS: Serum concentrations of oxycodone and metabolites were not associated with pain intensity, nausea, tiredness, or cognitive function, with the exception that increased pain intensity was associated with higher oxymorphone concentrations. Patients with poor pain control and side effects had higher serum concentrations of the oxycodone metabolites, noroxycodone and noroxymorphone, compared with those with good pain relief and without side effects. CONCLUSION: This study of patients receiving oxycodone for cancer pain confirms previous observations that there is most likely no association between serum concentrations of opioid analgesics and clinical effects.

Do CYP2D6 genotypes reflect oxycodone requirements for cancer patients treated for cancer pain? A cross-sectional multicentre study.

OBJECTIVE: Opioids are recommended by the World Health Organization for moderate to severe cancer pain. Oxycodone is one of the most commonly used opioids and is metabolized in the liver by CYP3A4 and CYP2D6 enzymes. The aim of this cross-sectional study was to assess the relationship between oxycodone pharmacokinetics, pharmacodynamics and the CYP2D6 genotypes "poor metaboliser" (PM), "extensive metaboliser" (EM) and "ultra-rapid metaboliser" (URM) in a cohort of patients with cancer pain. METHODS: The patients were genotyped for the most common CYP2D6 variants and serum concentrations of oxycodone and metabolites were determined. Pain was assessed using the Brief Pain Inventory (BPI). The EORTC QLQ-C30 was used to assess the symptoms of tiredness and nausea. Cognitive function was assessed by the Mini Mental State (MMS) examination. Associations were examined by analyses of variance (ANOVA) and covariance (ANCOVA), or ordinal logistic regressions with and without covariates. RESULTS: The sample consisted of 27 PM, 413 EM (including heterozygotes) and 10 URM. PM had lower oxymorphone and noroxymorphone serum concentrations and oxymorphone to oxycodone ratios than EM and URM. No differences between PM, EM and URM in pain intensity, nausea, tiredness or cognitive function was found. CONCLUSION: CYP2D6 genotypes caused expected differences in pharmacokinetics, but they had no pharmacodynamic consequence. CYP2D6 genotypes did not influence pain control, the adverse symptoms nausea and sedation or the risk for cognitive failure in this study of patients treated with oxycodone for cancer pain.

Influences on the pharmacokinetics of oxycodone: a multicentre cross-sectional study in 439 adult cancer patients.

OBJECTIVE: Oxycodone is widely used for the treatment of cancer pain, but little is known of its pharmacokinetics in cancer pain patients. The aim of this study was to explore the relationships between ordinary patient characteristics and serum concentrations of oxycodone and the ratios noroxycodone or oxymorphone/oxycodone in cancer patients. METHODS: Four hundred and thirty-nine patients using oral oxycodone for cancer pain were included. The patients' characteristics (sex, age, body mass index [BMI], Karnofsky performance status, "time since starting opioids", "oxycodone total daily dose", "time from last oxycodone dose", use of CYP3A4 inducer/inhibitor, "use of systemic steroids", "number of medications taken in the last 24 h", glomerular filtration rate (GFR) and albumin serum concentrations) influence on oxycodone serum concentrations or metabolite/oxycodone ratios were explored by multiple regression analyses. RESULTS: Sex, CYP3A4 inducers/inhibitors, total daily dose, and "time from last oxycodone dose" predicted oxycodone concentrations. CYP3A4 inducers, total daily dose, and "number of medications taken in the last 24 h" predicted the oxymorphone/oxycodone ratio. Total daily dose, "time from last dose to blood sample", albumin, sex, CYP3A4 inducers/inhibitors, steroids, BMI and GFR predicted the noroxycodone/oxycodone ratio. CONCLUSION: Women had lower oxycodone serum concentrations than men. CYP3A4 inducers/inhibitors should be used with caution as these are predicted to have a significant impact on oxycodone pharmacokinetics. Other characteristics explained only minor parts of the variability of the outcomes.