Dried blood spot (DBS) for ethyl glucuronide (EtG) and ethyl sulfate (EtS) determination in blood with a new design of “volumetric” DBS filter paper card

Abstract

Determination of EtG in blood can be performed from capillary blood with high sensitivity and detection limits of down to 1 ng/mL for EtG–and in addition to phosphatidyl ethanol (PEth) determination, EtG concentrations can be used for interpretation of consumption habits of patients in maintenance therapy. Sampling of capillary blood is a crucial preanalytical step–and different methods have been suggested for collection of a defined volume (VAMS, Capitainer, HemaXis, and capillaries for collection of samples). We used and validated a new volumetric capillary blood sampling card (DBSv cards, Protzek, Lörrach/Germany) which can be used either for self-sampling of dried blood spots (up to 4 times 20 microliter of blood) on filter paper with convenient handling by the patients or also in laboratory work. In contrast to standard DBS cards with printed circles on a filter paper, a newly designed card with a paper comb with four teeth per card, which allows a volumetric collection of 20 microlitres of blood per tooth, was used. Venous blood was pipetted on the teeth of DBSv cards, dried, and one paper tooth was ripped off and extracted by adding 490 microliter of methanol and 10 microliter of ISTD (400 ng/ml D5-EtG and 400 ng/ml D5-EtS). The samples were then vortexed for 5 minutes and centrifuged for 10 minutes. After transferring them into vials, the solutions were evaporated under a stream of nitrogen and reconstituted in 80 microliter of a water/0.1% formic acid. The samples were shaken and 10 μL were injected onto the LC column. A previously published LC-MS/MS method was adapted for a QTrap 5500 triple-quadrupole ion trap mass spectrometer (Sciex, Toronto, Canada) with an Acquity C18 HSS T3 column (Waters, Wexford, Ireland) and detection of EtG, D5-EtG, EtS and D5-EtS was performed according to forensic guidelines using one precursor and two product ions. Lower limits of quantification were 10 ng/mL for EtG and 20 ng/mL for EtS–with linear ranges of 10 to 5000 ng/mL for EtG and (50 to 2500 ng/mL for EtS. Traces of EtS were found in HPLC grade solvent, and a solvent selection between products of different manufacturers had to be performed. One brand methanol showed the lowest baseline level of EtS, which was in the range of 20 ng/mL. Thus, for determination of EtS, a lower limit of quantitation of 50 ng/mL was defined, whereas EtG could be quantified at concentrations above 10 ng/mL. A validation with spiked blood samples was performed. Samples from forensic cases and from drinking studies, where DBS were collected for the analysis of alcohol biomarkers (PEth and EtG/EtS) have been analyzed. The dried blood spot sample collection in laboratory or by self-sampling (of capillary blood) can be used to detect EtG and EtS at very low levels. The results show the potential of DBS analysis for alcohol markers EtG and EtS–which can be used for abstinence monitoring by very low limits of detection, especially for EtG, or for the detection of a recent alcohol consumption, when ethanol has already been eliminated from the body. A contamination of pure HPLC-grade solvent methanol was found for EtS determination, this has to be kept in mind, and needs further optimization for detection of EtS at lower concentrations in biological samples.