Sensitive quantification of rifaximin in human plasma by liquid chromatography–tandem mass spectrometry

A liquid chromatography–mass spectrometry method (LC–MS/MS) for the quantitative determination of rifaximin in human plasma was developed and validated. In the developed procedure, metoprolol was added to human plasma as an internal standard (IS) and acetonitrile was used to precipitate the plasma proteins before LC–MS/MS analysis. Chromatographic separation was obtained on a RESTEK Pinnacle C18 column (50 mm × 2.1 mm, 5 μm) with a mobile phase consisted of ammonium acetate solution (15 mM, pH 4.32) as buffer A and methanol as mobile phase B. Quantification was performed in positive mode using multiple reaction monitoring (MRM) of the transitions m/z 786.1 → 754.1 for rifaximin and m/z 268.3 → 116.1 for the IS. The assay has been validated over the concentration range of 0.5–10 ng/ml (r = 0.9992) based on the analysis of 0.2 ml of plasma. The assay accuracy was between 98.2% and 109%. The within-day and between-day precision was better than 3.9% and 8.9% at three concentration levels. The freeze–thaw stability was also investigated and it was found that both rifaximin and the IS were quite stable. This method provides a rapid, sensitive, specific and robust tool for the quantitative determination of rifaximin in human plasma, which is especially useful for the pharmacokinetic study of rifaximin.