UPLC-MS/MS Method for Simultaneous Quantification of Cyclosporine A and Urolithin A in Plasma and Interspecies Analysis Across Mammals Including Humans

In the past decade, liquid chromatography–mass spectrometry (LC-MS/MS) has become pivotal in clinical diagnosis, drug discovery, and bioanalytical science due to its high sensitivity and rapid analysis. We have developed an ultrasensitive and robust LC-MS/MS method for the simultaneous detection and quantification of cyclosporine A (CsA) and urolithin A (UA) employing ascomycin (ASC) and naringenin (NAR) as internal standards (ISTDs). The method was validated for clinical use, revealing interspecies differences between human plasma and other mammals (e.g., mouse, rat, feline, canine, and bovine serum). Validation parameters, including accuracy, precision, limits of quantification, specificity, selectivity, carryover, linearity, stability, and recovery, met acceptable ICH standards. Linear regression across the full calibration range (1–250 ng/mL for CsA and 0.5–125 ng/mL for UA) yielded an average R² ≥ 0.999 in all mammal models. The method achieved a limit of quantification (LOQ) of 1–2.5 ng/mL across all model plasma samples with negligible carryover and demonstrated sample stability up to 96 h intra- and interday and 48 h for bovine serum. The method was successfully applied to quantify CsA and UA in canine samples following oral administration from a previous study. With a rapid run time of 6 min, this method offers high selectivity and precision, making it ideal for analyzing limited sample sizes and addressing regulatory challenges. The ability to simultaneously quantify CsA and UA has significant clinical potential for managing complex immuno-inflammatory diseases, enabling precise dose adjustments, and optimizing treatment outcomes.