Measurement of the alcohol biomarker phosphatidylethanol (PEth) in dried blood spots and venous blood-importance of inhibition of post-sampling formation from ethanol.

Phosphatidylethanol (PEth) is a group of phospholipids formed in cell membranes following alcohol consumption by action of the enzyme phospholipase D (PLD). PEth measurement in whole blood samples is established as a specific alcohol biomarker with clinical and forensic applications. However, in blood specimens containing ethanol, formation of PEth may continue after sampling leading to falsely elevated concentrations. This study evaluated the use of dried blood spot (DBS) and microsampling specimens to avoid post-sampling formation of PEth. Filter paper cards and three commercial devices for volumetric microsampling of finger-pricked blood were assessed, using PEth-negative and PEth-positive whole blood fortified with 2 g/L ethanol. PEth (16:0/18:1) was measured by LC-MS/MS. Post-sampling formation of PEth occurred in wet blood and in the volumetric devices, but not filter paper cards, when stored at room temperature for 48 h. Addition of an inhibitor of PLD, sodium metavanadate (NaVO3), eliminated post-sampling formation during storage and drying. In conclusion, the present study confirmed previous observations that PEth can be formed in blood samples after collection, if the specimen contains ethanol. The results further demonstrated that post-sampling formation of PEth from ethanol also occurred with commercial devices for volumetric dried blood microsampling. In order for a PEth result not to be questioned, it is recommended to use a PLD inhibitor, whether venous blood is collected in a vacutainer tube or finger-pricked blood is obtained using devices for dried blood microsampling.

Evaluation of a new simple collection device for sampling of microparticles in exhaled breath.

The microparticle fraction of exhaled breath is of interest for developing clinical biomarkers. Exhaled particles may contain non-volatile components from all parts of the airway system, formed during normal breathing. This study aimed to evaluate a new, simple sampling device, based on impaction, for collecting microparticles from exhaled breath. Performance of the new device was compared with that of the existing SensAbues membrane filter device. The analytical work used liquid chromatography-tandem mass spectrometry methods. The new device collected three subsamples and these were separately analysed from eight individuals. No difference was observed between the centre position (0.91 ng/sample) and the side positions (1.01 ng/sample) using major phosphatidylcholine (PC) 16:0/16:0 as the analyte. Exhaled breath was collected from eight patients on methadone maintenance treatment. The intra-individual variability in measured methadone concentration between the three collectors was 8.7%. In another experiment using patients on methadone maintenance treatment, the sampling efficiency was compared with an established filter device. Compared to the existing device, the efficiency of the new device was 121% greater for methadone and 1450% greater for DPPC. The data from lipid analysis also indicated that a larger fraction of the collected material was from the distal parts. Finally, a study using an optical particle counter indicated that the device preferentially collects the larger particle fraction. In conclusion, this study demonstrates the usefulness of the new device for collecting non-volatile components from exhaled breath. The performance of the device was superior to the filter device in several aspects.

Study of measurement of the alcohol biomarker phosphatidylethanol (PEth) in dried blood spot (DBS) samples and application of a volumetric DBS device.

Phosphatidylethanol (PEth) is a group of phospholipids formed in cell membranes following alcohol consumption. PEth measurement in whole blood samples is established as a specific alcohol biomarker with clinical and medico-legal applications. This study further evaluated the usefulness of dried blood spot (DBS) samples collected on filter paper for PEth measurement. Specimens used were surplus volumes of venous whole blood sent for routine LC-MS/MS quantification of PEth 16:0/18:1, the major PEth homolog. DBS samples were prepared by pipetting blood on Whatman 903 Protein Saver Cards and onto a volumetric DBS device (Capitainer). The imprecision (CV) of the DBS sample amount based on area and weight measurements of spot punches were 23-28%. Investigation of the relationship between blood hematocrit and PEth concentration yielded a linear, positive correlation, and at around 1.0-1.5µmol/L PEth 16:0/18:1, the PEth concentration increased by ~0.1µmol/L for every 5% increase in hematocrit. There was a close agreement between the PEth concentrations obtained with whole blood samples and the corresponding results using Whatman 903 (PEthDBS=1.026 PEthWB+0.013) and volumetric device (PEthDBS=1.045 PEthWB+0.016) DBS samples. The CV of PEth quantification in DBS samples at concentrations≥0.05µmol/L were ≤15%. The present results further confirmed the usefulness of DBS samples for PEth measurement.