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Abstract Introduction
Reliable biomarkers have great significance in the investigation of alcohol consumption patterns and in the treatment of alcohol-induced diseases. Conventional alcohol tests are based on the measurement of indirect biomarkers (GGT,CDT etc.) to determine the toxic effect caused by alcohol consumption. However, these indirect biomarkers are not specific enough since they are also affected by physiological and pathological conditions. Direct biomarkers to detect alcohol intake are non-oxidative products of ethanol metabolism. EtG and EtS are conjugated metabolites of ethanol and have narrow detection windows in biofluids. Additionally, severe clinical errors in results may occur stemmed from post-sampling process, either due to storage circumstances or medical conditions of the person being sampled. Phosphatidylethanol (PEth), blood-based direct alcohol biomarker, represents a group of unnatural phospholipids formed on leukocytes and predominantly erythrocyte membranes. Studies demonstrate that the most dominant of these possible forms is PEth-16:0 18:1 consisting of a palmitic and an oleic acid. Moreover, detecting drugs of abuse (DOA) including opiates, amphetamines, cocaine metabolites, benzodiazepines, THC-metabolites etc. is important to establish patterns of illicit drug usage. In recent years, DBS has been gaining interest in plenty of research areas owing to ease of specimen collection and storage. To overcome challenges coming from blood viscosity, volumetric DBS devices have begun powerful alternative for specific volume of blood collection.
Objectives
The main objective of this study was to determine the concentration of PEth-16:0 18:1 in DBS samples collected using a volumetric DBS device (HemaxisTM DB) and to identify/quantify drugs of abuse (including 119 parameters-Jasem Clinical Toxicology mixture) using another blood spot punch of the same DBS sample.
Methods
According to Jasem method, DBS samples were prepared by sampling from spiked 20 µL of blood drop located onto paraffin film. PEth-16:0 18:1 and DOA were extracted from entire blood spot of DBS specimens implementing two different extraction reagents at room temperature for 10 min then subjected to HPLC system equipped with Agilent 6470 triple quadrupole mass spectrometer. The total run times from injection to injection for PEth and DOA were 4.0 min and 12.0 min respectively.
Results
The linearity and accuracy of the methods were evaluated using 5 DBS calibrators levels for PEth and 6 for DOA. Linearity was confirmed in the range 10 to 1000 ng/mL (r²>0.995) for PEth 16:0/18:1 and in the range 2 to 100 ng/mL for DOA (r²>0.99). According to our results, PEth RSD was %5.61 and DOA RSD values were between %1.34-11.73.
Conclusion
Both of Jasem LC-MS/MS approaches are centered on simple sample preparation following non-invasive sample collection method. Prior to rapid extraction, applying volumetric DBS device assures to collect fixed volume of blood. |
= Discovery stage.
= Translation stage.
= Clinically available.
