What LC Column Should I Use for EtG and EtS Detection?

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) biomarkers are formed by the liver after ethanol ingestion. They can be detected in urine approximately 2-80 hours after alcohol is consumed. Due to the polar nature of these metabolites, they are difficult to retain on a typical reversed-phase column. So in a recent study by Agilent, a hybrid end-capped charged surface C18 column was used to separate and analyze these compounds by LC/MS/MS.

Experimental

A simple ‘dilute and shoot’ sample preparation method was used. Urine was spiked with standards made from a working stock solution. Calibrators, controls, and samples were spiked with the internal standard and diluted 1:50 in mobile phase A. Then, samples were injected onto an Agilent InfinityLab Poroshell 120 CS-C18 column.

Results and Discussion

Excellent reproducibility was observed for EtG and EtS analyses for various concentrations. Over 1,000 injections were completed – see figure 1, below.

CS-C18 column for detection of ETG and ETS

• Simple sample preparation: dilute and shoot method
• High dilution and low injection volume for best peak shape
• Preliminary data suggests method works well for high-throughput environments due to short run times and easy sample prep

Conclusion

The data indicates that the CS-C18 HPLC columns adequately retain EtG and EtS. A high dilution and low injection volume produce the best peak shape. Retention times and peak areas were stable across hundreds of injections. While further experiments are needed to determine column lifetime and method robustness, preliminary data indicate this method would work well in a high-throughput environment due to its short run time and simple sample preparation.