PII-115 - A SIMPLE AND SENSITIVE ULTRA-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (UHPLC-MS/MS) ASSAY TO QUANTIFY TIZANIDINE AND ITS METABOLITES IN HUMAN PLASMA

Y. Wu¹, J. Lu¹, W. Osei^2,3, A. Rashidian¹, A. Colwell¹, Z. Desta⁴; ¹Division of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis, IN, United States, ²Department of Pharmacy Practice, School of Pharmacy, Purdue University, Indianapolis, IN, USA, ³Purdue University, West Lafayette, IN, USA, ⁴Division of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis,IN, US.

Background: Tizanidine, a muscle relaxant metabolized by CYP1A2, exhibits large intersubject variability, especially with CYP1A2 inhibitors. Current assays quantify only tizanidine and require complex extraction, prompting the need for a simple and sensitive method to quantify tizanidine and its metabolites, to better understand tizanidine disposition and drug-drug interactions (DDIs).

Methods: A novel UHPLC-MS/MS assay was developed to quantify tizanidine and its 6 metabolites in human plasma using a QTRAP 6500+ mass spectrometer with an electrospray ionization source, coupled with a UHPLC system. MS optimization for compound- and instrument-dependent parameters for each analyte was performed. Data were acquired in positive mode, and quantification was made via MultiQuant software. Multiple reaction monitoring was used to measure Q1/Q3 transitions. To each 100 µL plasma sample, methanol was added to precipitate protein, with nevirapine as an internal standard. After centrifugation, aliquot of the supernatant was injected to UHPLC-MS/MS system. Chromatographic separation was achieved using a 150 × 4.6 mm, 5 µm C8 column with a mobile phase of aluminum acetate and methanol, delivered via gradient elution. The assay was fully validated, and its clinical utility was tested in a pharmacokinetic (PK) study of healthy subjects who received a single 4 mg oral dose of tizanidine.

Results: The developed method successfully quantified tizanidine, dehydro-tizanidine, guanidine-tizanidine, hydroxy-tizanidine, and additional metabolites (M3, M9, and M10). The assay demonstrated great linearity (R² > 0.99) for all analytes, with a lower limit of quantification of 0.01 ng/mL for tizanidine, dehydro-tizanidine, and guanidine-tizanidine, and 0.03 ng/mL for hydroxy-tizanidine. Except for hydroxy-tizanidine, intra- and inter-day variability of most analytes ranged from 2% to 16%, with accuracy between 85% and 130%. The method was successfully applied to determine plasma PK of tizanidine and its metabolites in 3 healthy volunteers who took tizanidine.

Conclusion: We have developed a novel UHPLC-MS/MS method to successfully quantify not only tizanidine but also for the first time 6 metabolites in human plasma. This reliable method can be used in in vitro and PK studies to understand mechanisms of intersubject variability and DDIs involving tizanidine.