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.

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.

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.