THE DETECTION AND QUANTIFICATION OF ETHYL GLUCURONIDE IN PLACENTAL TISSUE AND PLACENTAL PERFUSATE BY HEADSPACE SOLID-PHASE MICROEXTRACTION COUPLED WITH GAS CHROMATOGRAPHY-MASS SPECTROMETRY

Background Ethyl glucuronide (EtG) is arising as a promising biomarker of heavy prenatal alcohol exposure, however its transfer across the human placenta is still unclear and is currently being investigated using the ex vivo placental perfusion model. This model allows for sampling from placental tissue and placental perfusate, which is a surrogate to plasma. Objective To develop a method for detecting and quantifying EtG in placental perfusate and tissue using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Methods A method was optimized by manipulation of the following components to attain the highest peak counts for the quantifying ions of EtG and its deuterated internal standard on the mass spectrum: cartridges used for solid phase extraction, injection method, derivatizing agent, pre-injection parameters, SPME fiber, GC ramp speed, and GC column flow. Results The final method utilized involved solid phase extraction of standards via UCT CleanScreen Cartridges, derivatization with heptafluorobutyric acid, and introduction into the GC via HS-SPME with adsorption to a polydimethylsiloxane fiber. The method has improved sensitivity over other methods that quantify EtG in blood using GC-MS, with detection limits of 1.6 ng/mL and 13.7 ng/g for placental perfusate and tissue, respectively. The method was applied to samples collected from the fetal reservoir during the ex vivo placental perfusion model and EtG was detected in the fetal circulation after 20 minutes of perfusion, indicating transfer of EtG. Conclusions The present method is sensitive and can be used to quantify EtG transfer during ex vivo placental perfusion experiments.