Simultaneous determination of isoniazid and pyrazinamide in plasma by high performance liquid chromatography

Purpose: To develop and validate a new high performance liquid chromatographic (HPLC) method for the simultaneous determination of isoniazid (INH) and pyrazinamide (PZA) in plasma.
Methods: A 150 μL aliquot of plasma was mixed with 75 μL of 10 % trichloroacetic acid containing 100 mg/L of acetanilide as the internal standard (IS). After vortex mixing and centrifugation, 100 μL of the supernatant was reacted with 20 μL of 0.1 % trans-cinnamaldehyde for 10 min, and then 40 μL of 1M ammonium acetate was added. Finally, 20 μL was injected into the HPLC system. HPLC analysis was performed on reversed phase C18 column. The initial composition of the mobile phase was 4 % acetonitrile, and 96 % of 20 mM 1-hexane sulfonic acid (PH 2.7) delivered at a flow rate 1 mL/min.
Results: All calibration curves were linear (r² > 0.997). The method was accurate, and relative error (RE) was < 4.5 % for both drugs. Intra-day and inter-day precision was good for both drugs, with the highest relative standard deviation (RSD) being 8.51 %. The lower limit of quantification was 0.60 mg/L for isoniazid and 3.00 mg/L for pyrazinamide.
Conclusion: The method proposed here is precise, accurate, fast, simple and suitable for therapeutic drug monitoring of INH and PZA simultaneously.