Influence of preservatives on the stability of ethyl glucuronide and ethyl sulphate in urine.

Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are specific and sensitive markers of ethanol consumption well established in monitoring withdrawal treatment in patients with chronic alcoholism. Recently, bacterial decomposition as well as in vitro and post-mortem formation of EtG was reported. The aim of this study was to investigate the influence of different preservatives on the stability of EtG and EtS concentrations in urine samples. Urine samples were doped with glucuronidase-positive Escherichia coli after sterile filtration. The preservatives used were thymol, chlorhexidine, boric acid and the combination of chlorhexidine, ethylparabene and sodium propionate. Different aliquots of urine samples were stored refrigerated (4-8 degrees C), at room temperature (18+/-1 degrees C) and in an incubator (36+/-1 degrees C) for a period of 9 days with daily sampling. EtG and EtS analyses were performed by LC-ESI-MS/MS. The number of bacteria was detected by counting the colony forming units on Columbia blood agar plates. Chlorhexidine on its own as well as in the aforementioned combination, and boric acid proved useful preservatives, while EtG degraded in samples doped with thymol. Addition of these preservatives did not interfere with the LC-MS/MS analysis.