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

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 10 mM ammonium acetate. The injection volume was 1 μL and run time was 4 min. 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 5–500 nM 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

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

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

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.publishedVersio