SIL-IS LC-MS/MS method for rapid quantification of phloroglucinol in human plasma: development, validation and application to a pharmacokinetic study.

Abstract

Phloroglucinol is a smooth muscle relaxant widely used for the symptomatic treatment of gastrointestinal, biliary, and genitourinary spasms. A sensitive, rapid, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method incorporating a stable isotope-labeled internal standard (SIL-IS, phloroglucinol-13C6) was developed and validated for the quantification of phloroglucinol in human plasma. Plasma samples were prepared using a simple liquid-liquid extraction (LLE) procedure in a 96-well format and analyzed on a reversed-phase phenyl column with a total run time of 4.5 min. The analyte and the stable isotope-labeled internal standard were monitored by multiple reaction monitoring (MRM) in negative electrospray ionization (ESI) mode on a triple quadrupole mass spectrometer, with transitions of m/z 125.0 → 57.0 and m/z 131.0 → 60.1, respectively. The method demonstrated excellent linearity over the concentration range of 0.750-750 ng/mL with a lower limit of quantification (LLOQ) of 0.750 ng/mL. The validation was conducted in strict accordance with the requirements of bioanalytical guidelines, encompassing assessments of selectivity, linearity, accuracy and precision, recovery, matrix effect, stability, and dilution integrity. The results demonstrated that all validation parameters met the predefined acceptance criteria. It was successfully applied to a pharmacokinetic and bioequivalence study of phloroglucinol orally disintegrating tablets in healthy Chinese volunteers under fasting and fed conditions, enabling reliable characterization of pharmacokinetic profiles.