Quantification of rifampicin in human plasma and cerebrospinal fluid by a highly sensitive and rapid liquid chromatographic-tandem mass spectrometric method

Abstract

A highly sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed to measure the levels of the antitubercular drug rifampicin (RIF) in human plasma and cerebrospinal fluid (CSF). The analyte and internal standard (IS) were isolated from plasma and CSF by a simple organic solvent based precipitation of proteins followed by centrifugation. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay was linear in the concentration range 25-6400 ng/mL with intra- and inter-day precision of <7% and <8%, respectively. The validated method was applied to the study of RIF pharmacokinetics in human CSF and plasma over 25 h period after a 10 mg/kg oral dose.

Fig. 1

Chemical structures of compounds.

Fig. 2

Representative MRM chromatograms of RIF (I) and RPT (II, IS) in: (A) a blank human plasma sample; (B) a blank human plasma sample spiked with RIF at the LLOQ of 25 ng/mL; (C) patient plasma sample 25 h after an oral dose of 10 mg/kg (RIF concentration; 31 ng/mL); (D) CSF sample from a patient receiving a 10 mg/kg daily oral dose of RIF (RIF concentration; 41.83 ng/mL). Sample preparation for 100 μl of (B)–(D) was performed using 300 μl of 50/50 ACN/methanol (including 250 ng/mL IS), whereas IS was not included in case of (A).

Fig. 3

MRM chromatograms obtained after injection of extracted (A) heat-treated and (B) untreated blank human plasma during post column infusion of RIF (I) and RPT (II). A mixture containing 250 ng/mL RIF and RPT in methanol was infused directly by syringe pump through a T-connector in to the MS at a constant flow rate of 10 μl/min, whilst the blank plasma extract was injected via the autosampler.