Method development and validation for simultaneous quantitation of endogenous hippuric acid and phenylacetylglycine in rat urine using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

- A simultaneous quantitation method for HA and PAG was developed for potential biomarker of drug-induced phospholipidosis.
- Effective sample dilution reduced matrix effect of urine to be negligible level.
- Intra- and inter day validation were successfully accomplished.
- The method was applied to rat urine after PLD-inducing drugs treatment, and the changes in HA and PAG concentrations were successfully detected.

Urinary hippuric acid (HA) and phenylacetylglycine (PAG) are biomarker candidates for drug-induced phospholipidosis (PLD). To confirm their utility in preclinical and clinical settings, it is essential to develop and validate their quantification method in advance. In this study, we have applied liquid chromatography-tandem mass spectrometry (LC/MS/MS) for simultaneous quantification of HA and PAG in rat urine, and matrix based ion suppression was assessed by post-column infusion assay. Effective sample dilution reduced matrix effect of urine to be negligible level and calibration curves showed good correlation between those in urine diluent and buffer alone. Reliability of this assay was confirmed by the assessments for intra- and inter-day precisions and accuracies of quality control samples. The method was applied to rat urine after multiple oral administrations of PLD-inducing drugs, and the changes in HA and PAG concentrations and their ratio were successfully detected as rat plasma in previous report. This is the first report to quantify HA and PAG easily and accurately as potential biomarkers to monitor PLD status. This assay would be useful tool for monitoring PLD in toxicological studies by non-invasive sampling.