Abstract
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 mu L aliquot of plasma was mixed with 75 mu L of 10 % trichloroacetic acid containing 100 mg/L of acetanilide as the internal standard (IS). After vortex mixing and centrifugation, 100 mu L of the supernatant was reacted with 20 mu L of 0.1 % trans-cinnamaldehyde for 10 min, and then 40 mu L of 1M ammonium acetate was added. Finally, 20 mu L 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 (r(2) > 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.