Analysis of Rifampicin in dried blood spots using high performance liquid chromatography

Background and Objective: Rifampicin (RIF) is one of the first-line anti-tuberculosis drugs combined with isoniazid in fixed dose combination (FDC) form. Tuberculosis patient using RIF has been correlated with treatment failure because of low blood drug concentrations. Therefore, the application of therapeutic drug monitoring (TDM) is recommended. Besides the conventional venipuncture bio-sampling method, dried blood spots (DBS) offer several potential benefits, which increase the patient comfort. The main objective of this research is to obtain an analysis method of RIF in DBS. Materials and Methods: A simpler analytical method was developed and validated to quantify RIF in DBS samples using a high performance liquid chromatography (HPLC). A linear regression was used as the statistical analysis method. Results: The optimum chromatographic condition was obtained using C-8 (Waters, Sunfire™ 5 μm; 250×4.6 mm) with the mobile phase consisted of 50 mM ammonium acetate buffer pH 4.5-acetonitrile-methanol (40:30:30), flow rate was 0.5 mL min^-1, column temperature was 40EC and was detected at 261 nm. The time of analysis was 16 min and cilostazole was used as an internal standard. The DBS extraction was done by protein precipitation technique using acetonitrile:methanol (1:4 v/v%) with 1000 μL as the volume. The method was linear at concentration range of 1.0-30.0 μg mL^-1 with r > 0.9984. Conclusion: The analysis method was fulfilled the acceptance criteria of the European Medicines Agency (EMA) Bio-analytical Method Validation Guideline, 2011 and it is applicable for TDM of RIF.