Determination of Rhodamine 123 in rat plasma utilizing liquid chromatography–tandem mass spectrometry

Rhodamine 123 (R123), as a typical of P-gp substrate, was widely used to quantify P-glycoprotein (P-gp) functional efflux activity in vivo. A new, rapid and sensitive method was developed for quantifying R123 in rat plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS). R123 and Rhodamine 6G (R6G, the internal standard, IS) were extracted from aliquots of plasma with ethyl acetate and dichloromethane (4:1) as the solvent and chromatographic separation was performed using a Zorbax Eclipse Plus C18 column. The mobile phase was composed of A: ammonium formate–formic acid buffer containing 5 mM ammonium formate and 0.1% formic acid and B: methanol (A:B, 5:95, v/v). To quantify R123 and IS respectively, multiple reaction monitoring (MRM) transition of m/z 345.2 → 285.2 and m/z 443.3 → 415.2 was performed. The analysis time was 4 min in positive mode; the calibration curve was linear in the concentration range of 1–200 ng/ml. The lowest limit of quantification (LLOQ) reached 1 ng/ml. The intra and inter-day precision were less than 9.2% for the low quality control (QC) level, and 3.4% for other QC levels, respectively, while the intra and inter-day relative errors ranged between −7.4% and 9.1% for three QC concentration levels. The LC–MS/MS method proved to be simple, accurate, reliable and with a shorter running time and has been successfully applied to evaluate the functional activity of P-glycoprotein in an absorption experiment in the rat.

► A new LC–MS/MS method for the measurement of R123 in rat plasma has been established.
► The method proved to be specific, highly sensitive and accurate over a concentration range of 1–200 ng/ml.
► Liquid–liquid extraction was more suitable for disposing plasma samples and available for enhancing the sensitivity.
► The method was successfully applied to evaluate the functional activity of P-gp in an absorption experiment in the rat.