Defense Date
2023
Document Type
Directed Research Project
First Advisor
Michelle Peace
Second Advisor
Justin Poklis
Third Advisor
Kim Samano
Fourth Advisor
Matthew Halquist
Abstract
Alcohol use can be indicated by breath alcohol analysis, blood alcohol analysis, and
analysis of several ethanol biomarkers. These biomarkers include ethyl-ß-d-glucuronide (EtG)
and ethyl sulfate (EtS), 5-hydroxy tryptophol (HTOL), 5-hydroxy indole acetic acid (HIAA), 5-hydroxy tryptophol beta-D-glucuronide (GTOL), and phosphatidyl ethanol (PEth). The analysis of multiple biomarkers can help indicate a time frame of consumption. Urinary EtG and EtS concentrations and HTOL/HIAA to GTOL/HIAA ratios are often used as an indicator of recent consumption, while PEth analysis can be useful for indicating use of up to four weeks after ethanol consumption. There are currently very few studies that combine more than three ethanol biomarkers into one assay. This study aims to create a unified extraction and analysis method for six ethanol biomarkers in blood, oral fluid, and urine to provide an all-in-one analysis for detection of ethanol consumption.
A method was developed for the analysis of six ethanol biomarkers. In brief, fortified pig’s blood at 1000 ng/mL (n=3) was extracted using Biotage Supported Liquid Extraction cartridges and eluted with 30:65:5 ethyl acetate: acetonitrile (ACN): 2-propanol, dried down and reconstituted in 90:10 Mobile Phase B: Mobile Phase A (MPB:MPA). MPA consists of 25 mM ammonium formate (pH=3.3), MPB consists of 50:50 ACN: 2-propanol with 0.1% formic acid. All samples were analyzed on a Shimadzu 8050 LC-MS/MS. Chromatographic separation was performed on a Luna Omega 3 µm Sugar 100Å Column (150 x 4.6 mm) with a total run time of 25.01 min. The gradient began with holding 90% MPB for 2.5 min, then decreased to 60% MPB over 11.5 min. MPB was then decreased to 25% over 0.5 min and was held for 8 min, then MPB was increased to 90% MPB over 0.01 min and held for 2.5 min. The flow rate was 1 mL/min, and the column temperature was 50 ºC.
All analytes were resolved in a single 25-minute analytical run. A unified extraction
method was successfully developed and evaluated for efficiency and ion suppression at 1000 ng/mL.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
5-3-2023