Liquid chromatography and ultra-performance liquid chromatography–mass spectrometry fingerprinting of human urine: Sample stability under different handling and storage conditions for metabonomics studies

Typically following collection biological samples are kept in a freezer for periods ranging from a few days to several months before analysis. Experience has shown that in LC–MS-based metabonomics research the best analytical practice is to store samples as these are collected, complete the sample set and analyse it in a single run. However, this approach is prudent only if the samples stored in the refrigerator or in the freezer are stable. Another important issue is the stability of the samples following the freeze–thaw process. To investigate these matters urine samples were collected from 6 male volunteers and analysed by LC–MS and ultra-performance liquid chromatography (UPLC)–MS [in both positive and negative electrospray ionization (ESI)] on the day of collection or at intervals of up to 6 months storage at −20 °C and −80 °C. Other sets of these samples underwent a series of up to nine freeze–thaw cycles. The stability of samples kept at 4 °C in an autosampler for up to 6 days was also assessed, with clear differences appearing after 48 h. Data was analysed using multivariate statistical analysis (principal component analysis). The results show that sample storage at both −20 and −80 °C appeared to ensure sample stability. Similarly up to nine freeze thaw cycles were without any apparent effect on the profile.