Validation of an UHPLC–MS/MS Assay for the Quantification of Isoniazid and Four Major Metabolites in Human Plasma

Isoniazid (INH) is a key component of tuberculosis treatment regimens but is also associated with hepatotoxicity. This toxicity is mediated in part by its metabolites, but some degrade rapidly in plasma. A rapid freeze/thaw process with a methanol protein precipitation extraction was developed to slow the rapid degradation of acetylisoniazid (AcINH) into isonicotinic acid (INA) and acetylhydrazine (AcHZ) in human plasma. An assay consisting of two UHPLC-MS/MS methods was developed and validated to determine concentrations of INH and four of its metabolites in human plasma: the "polar" method covering AcINH and INA, and the "nonpolar" method covering p-tolualdehyde derivatized AcHZ, hydrazine (HZ), and INH. Polar and nonpolar method chromatographic separation was accomplished with biphenyl or C18 columns, respectively, mobile phases of 4 mM ammonium formate in water or acetonitrile, and method specific gradient conditions. The validated range for AcINH, INA, and INH was 10.0 to 5000 ng/mL and for AcHZ and HZ was 10.0 to 500 ng/mL from 0.1 mL of plasma, respectively. Though other groups have validated methods to quantitate some or all of these analytes, none have adequately characterized or addressed AcINH degradation. This validated assay will help facilitate a better understanding of INH metabolite disposition and relationships with toxicity.