Stability of volatile organic compound metabolites in urine at various storage temperatures and freeze-thaw cycles for 8 months

Abstract

The urinary concentrations of mercapturic acid metabolites of volatile organic compounds (VOCs) have been used as biomarkers of human exposure to this class of chemicals. However, long-term stability of these VOC metabolites (VOCMs) in urine at various storage conditions such as temperature, duration, and freeze-thaw cycles is not known. In this study, spot urine samples collected from three volunteers, stored at 22 °C (room temperature: RT), 4 °C (refrigerator) and −20 °C (freezer) for up to 240 days were analyzed at weekly to monthly interval for a total of 19 time points. Samples stored at 4 °C and −20 °C underwent 18 freeze-thaw cycles at RT for 30 min at each of the time points. Among 38 VOCMs analyzed, up to 18 metabolites were detected at concentrations above their respective detection limits on Day 0 (baseline concentration), and the concentrations of several VOCMs declined with the storage duration. Eight to ten VOCMs were lost completely within 240 days of storage at RT, compared to between two and five at 4 °C and between one and seven at −20 °C. The loss rate varied depending on the sample, storage temperature, VOCM, and number of freeze-thaw cycles. Storage of urine at RT led to a rapid loss of VOCMs in comparison to that stored at 4 °C or −20 °C. Among VOCMs measured, CEMA, SBMA, GAMA, DHBMA, AMCC, TCVMA, and HPMMA were lost more rapidly than the other metabolites. CMEMA, a major VOCM found in all three urines at baseline, exhibited a rapid loss in those of two volunteers but not of the other volunteer, suggesting sample to sample variation in lose rates. Freeze-thaw cycles considerably affected VOCM concentrations in urines stored at 4 °C or −20 °C. It is recommended that urine samples are analyzed for VOCMs within a couple of months of collection and stored at temperatures below −20 °C, with minimal or no freeze-thaw cycles. This study highlights the need for appropriate storage conditions to maintain the integrity of samples for biomonitoring studies.