Sensitive profiling of phenols, bile acids, sterols, and eicosanoids in mammalian urine by large volume direct injection-online solid phase extraction-ultra high performance liquid chromatography-polarity switching tandem mass spectrometry
Bile acids, sterols, and eicosanoids serve as important chemical categories in mammalian urine and, sometimes, as primary diagnostic markers for diverse diseases, whereas phenols, usually derived from the diet, are usually observed as annoying interferents in pharmacokinetic and metabolic profiling of phenolic derivatives. Precise analysis of these substances widely suffers from instability and trace distributions, as well as errors and uncertainties resulting from tedious sample preparation procedures. Herein, directly simultaneous determination of phenols, bile acids, sterols, and eicosanoids, in particular those trace ones, in mammalian urinary matrices was attempted using large volume direct injection-online solid phase extraction-ultra high performance liquid chromatography-polarity switching tandem mass spectrometry (LVDI-online SPE-UHPLC-psMS/MS). Large volumes (500 μL) of liquid-state samples were directly loaded onto an online SPE column via ten consecutive injections. The SPE column was responsible for accumulating targeted components at the loading phase (−4.5 to 5.0 min), while transmitting those trapped analytes into a Waters HSS T3 column at the elution phase (5.0–27.0 min). Phase switching was accomplished using an electronic 6-port/2-channel valve. Analyte detection of all analytes, 28 ones in total, was performed using both positive and negative multiple reaction monitoring modes. Various method validation assays demonstrated the developed method to be extremely sensitive (most limits of quantitation lower than 0.3 ng mL−1), precise (all RSDs of intra- and inter-day variations lower than 15%) and accurate (recoveries ramped from 80.4% to 120.0% with RSDs lower than 20%). Significant variations occurred for urine samples from different species, as well as amongst urinary matrices from different individuals within the same group. The findings proved that the developed LVDI-online SPE-UHPLC-psMS/MS method takes advantage of detecting the trace components and preserving those mutable substances from degradation, thus offering a meaningful tool for widely targeted monitoring substances in biofluids.