OBJECTIVES

In response to the need for a simple and fast way of ensuring that generic drugs especially those that contain rifampicin are bioequivalent with reference drugs, this study validated an ultra-high-performance liquid chromatography (UHPLC) method of quantifying rifampicin in human plasma. The study also validated the method's selectivity, linearity, sensitivity, accuracy, precision, and the absence of a carry-over effect adhering to the Philippine Food and Drug Administration guidelines.

METHODS

Plasma samples were prepared via protein precipitation using methanol containing ascorbic acid. Three microliters (3 uL) of the prepared samples were then analyzed in a Waters Acquity H-Class UPLC® system coupled to a tunable ultraviolet (TUV) detector with an attached UPLC® BEH C-18 column using a developed and optimized method. Briefly, the column temperature was set to 40°C and the sample temperature was set to 10°C. Elution was done using a linear gradient flow of a water-acetonitrile mixture that started with 45% acetonitrile increasing to 60% acetonitrile at 0.5 minutes and back to 45% acetonitrile at 3 minutes and having a constant flow rate of 0.5 mL/min. Detection was done at 340 nm. Method validation was performed following the ICH guidelines for Bioanalytical Method Validation, the same guidelines referenced by the ASEAN Guideline for Harmonisation of Standards and the Philippine Food and Drug Administration (FDA).

RESULTS

The method had an analysis time of 3 minutes wherein rifampicin eluted at 1.4 minutes while the internal standard, rifapentine (IS) eluted at 1.7 minutes. Since no co-eluting endogenous materials were observed for the rifampicin and the internal standard, the method was confirmed to be selective. Its linearity over the range of 2 ug/mL to 25 ug/mL has been validated where it has a limit of detection (LOD) and limit of quantification (LOQ) values of 0.64 ug/mL and 1.94 ug/mL, respectively. The interday and intraday precision, reported as % coefficient of variance (%CV), and interday and intraday accuracy, reported as %error all within the limits of ±20% for the LLOQ and ±15% for the rest indicating its reliability and reproducibility. Lastly, due to the nature of the injection of the sample into the system, wherein a blank immediately follows the highest concentration standard, the method has been cleared of a carry-over effect.

CONCLUSION

The study successfully validated a UHPLC method of quantifying rifampicin in human plasma. Due to the sample processing method used and the chromatographic conditions set, the method can prepare and analyze samples in a simple yet fast, sensitive, reliable, and reproducible manner. The method can be applied in bioavailability and bioequivalence studies of rifampicin.

Human ; Rifampin ; Rifampicin ; Bioequivalence ; Therapeutic Equivalency

Introduction: Mycobacterium tuberculosis resists rifampicin (RIF) because of mutations in the rpoB (the p subunit of RNA polymerase) gene, mostly in the 81 bp region. \r\n', u'Objectives: Identify the frequency and characteristics relative to drug - resistant rpoB gene mutation in RIF - resistant M. tuberculosis strains. \r\n', u'Subjects and method: 40 M. tuberculosis strains including 11 RIF - sensitive strains and 29 RIF - resistant strains. Some bio molecular techniques were used such as extracting mycobacterial DNA, PCR, cloning, sequencing and analyzing mutation related RIF - resistance on rpoB gene. \r\n', u'Results: No mutation was found on the 81 bp region of rpoB gene of the RIF - sensitive M. tuberculosis strains. The rate of mutation on rpoB gene of 29 RIF - resistant M. tuberculosis strains is 96.6%. We found 12 mutation codon positions on the 81 bp region of the rpoB gene, and the mutation codon positions with high frequency were 531 (51.7%) and 526 (31%). The mutation position found in only one strain is codon 519 (3.4%) but not found in other reports. There are 15 types of drug resistant mutations in which TCG531 TCG is the most common with 50%. Multi - drug resistance was seen in mutable and none mutable cases, with all codon positions and mutable forms. \r\n', u'Conclusion: No mutation was found on the 81 bp region of the rpoB gene of RtF - sensitive M. tuberculosis strains. The rate of mutation on the rpoB gene of RIF - resistant M. tuberculosis strains is 96.6%. The new mutation position found is codon 519. The mutation on the rpoB gene does not determine the multi - drug resistance of M. tuberculosis. \r\n', u'
Mutation ; rpoB gene ; Rifampicin - resistant M. tuberculosis

Background: Rifampicin, isoniazid, and pyrazinamide are oral essential anti-tuberculosis drugs on single or combined preparations. Worldwide research has shown that the plasma concentration of anti-tuberculosis drugs with daily therapeutic doses were seen significant lower than permitted in tuberculosis patients, especially for rifampicin and isoniazid. Objective: To investigate plasma concentration of rifampicin, isoniazid, and pyrazinamide in pulmonary tuberculosis and pleural tuberculosis patients. Methods: Determine plasma concentration of rifampicin, isoniazid, and pyrazinamide at 2 hours after administration in 168 tuberculosis patients by the HPLC method. Identify prevalence of low plasma concentrations of anti-tuberculosis drugs. Results: There was a wide range of plasma concentration of rifampicin, isoniazid, and pyrazinamide in the tuberculosis patients. The mean plasma concentration of rifampicin was 6.13 \xb1 4.66 microgram/ml, of isoniazid was 2.99 \xb1 1.94 microgram/ml, pyrazinamide was 38.98 \xb1 18.39 microgram/ml. There was no significant differences in the plasma concentration of rifampicin, isoniazid, and pyrazinamide in groups of pulmonary tuberculosis and pleural tuberculosis patients. Percentage of patients with plasma concentration below therapeutic concentration was 76.83% of rifampicin, 51.85% of isoniazid, 10.13% of pyrazinamide. There were 12.03% of patients who had pyrazinamide concentration higher than the therapeutic range. Conclusions: There was a wide range of plasma concentration in rifampicin, isoniazid, and pyrazinamide of tuberculosis patients. Low plasma concentration of rifampicin and isoniazid are common. It may be necessary to optimize the drug dose by therapeutic drug monitoring, especially in patients with an inadequate clinical response to chemotherapy.
tuberculosis ; rifampicin ; isoniazid ; pyrazinamide