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.

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.

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.

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.