Related Experiment Video
Updated: Aug 6, 2026
Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures
Published on: March 28, 2017
Validation of an UHPLC-MS/MS Assay for the Quantification of Isoniazid and Four Major Metabolites in Human Plasma
David Nerguizian1, Brandon Klein1, Farah Abdelmawla1
1Department of Pharmaceutical Sciences Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Abstract:
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.
Insights
Isoniazid (INH) treatment for tuberculosis can cause liver damage due to its metabolites. A new method stabilizes acetylisoniazid (AcINH) in plasma, enabling accurate measurement of INH and its key metabolites to understand toxicity.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Clinical Toxicology
Background:
- Isoniazid (INH) is a primary drug for tuberculosis treatment.
- INH is associated with hepatotoxicity, partly due to its metabolites.
- Rapid degradation of acetylisoniazid (AcINH) in plasma complicates metabolite analysis.
Purpose of the Study:
- To develop and validate a method to accurately quantify INH and its metabolites in human plasma.
- To address the challenge of rapid AcINH degradation during sample processing.
- To facilitate a better understanding of INH metabolite disposition and its link to toxicity.
Main Methods:
- A rapid freeze/thaw and methanol protein precipitation extraction was employed to stabilize AcINH.
- Two UHPLC-MS/MS methods were developed: one for polar metabolites (AcINH, isonicotinic acid) and one for nonpolar metabolites (acetylhydrazine, hydrazine, INH).
- Chromatographic separation utilized biphenyl and C18 columns with specific mobile phases and gradient conditions.
Main Results:
- The assay successfully quantified INH and four key metabolites: AcINH, isonicotinic acid (INA), acetylhydrazine (AcHZ), and hydrazine (HZ).
- Validated ranges were 10.0–5000 ng/mL for AcINH, INA, and INH, and 10.0–500 ng/mL for AcHZ and HZ.
- The developed method effectively mitigated AcINH degradation, unlike previous approaches.
Conclusions:
- A validated UHPLC-MS/MS assay allows for the accurate quantification of INH and its metabolites in human plasma.
- The method addresses the critical issue of AcINH degradation, improving metabolite analysis reliability.
- This assay will be instrumental in advancing research on INH-induced hepatotoxicity and personalized treatment strategies.
