RAPID SIMULTANEOUS LC/MS2 DETERMINATION OF RIFAMPICIN AND 25-DESACETYL RIFAMPICIN IN HUMAN PLASMA FOR THERAPEUTIC DRUG MONITORING

Abstract

A rapid and sensitive liquid chromatography coupled with tandem mass spectrometry (LC/MS²) method for the simultaneous quantification of rifampicin and its main active metabolite 25-desacetyl rifampicin in human plasma was developed and validated. The separation was performed on a Gemini NX C18 column under isocratic conditions using a mobile phase of 40:60 (V/V) methanol and 2mM ammonium formate in water, at 40^ºC, with a flow rate of 0.6 mL/min. The detection of rifampicin and its metabolite was performed in multiple reaction monitoring mode using an ion trap mass spectrometer with positive electrospray ionization. The human plasma samples (0.1 mL) were deproteinized with methanol and aliquots of 0.3 μL from supernatants obtained after centrifugation were directly injected into the chromatographic system. The method shows a good linearity (r > 0.993), precision (CV < 8.2%) and accuracy (bias < 6.3%) over the range of 411 - 19737 ng/mL for rifampicin and good linearity (r > 0.992), precision (CV < 10.1%) and accuracy (bias < 8.2%) over the range of 70 - 3379 ng/mL for 25-desacetyl rifampicin. The lower limit of quantification (LLOQ) was 411 ng/mL and recovery was between 90.3-108.2% for rifampicin, whereas for 25-desacetyl rifampicin the LLOQ was 70 ng/mL and recovery between 93.1-107.5%, respectively. The developed and validated method is simple, rapid and specific for the high throughput simultaneous determination of rifampicin and 25-desacetyl rifampicin in human plasma and was successfully applied in therapeutic drug monitoring of rifampicin in patients with tuberculosis.