Validation of an electrospray ionisation LC–MS/MS method for quantitative analysis of telaprevir and its R-diastereomer

• Validated method for quantification of telaprevir and its R-diastereomer.
• The ex vivo stability of both diastereomers in plasma was evaluated.
• The assay was sensitive and highly reproducible, with good recovery (>85%).
• The method has been successfully applied to measure telaprevir and its R-diastereomer in patients receiving telaprevir 750 mg thrice daily.

A sensitive high-performance reverse phase liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of telaprevir and its inactive R-diastereomer (VRT-127394) in human plasma. The analytes and the internal standard (telaprevir-d11) were extracted from plasma by liquid–liquid extraction using tert-Butyl methyl ether (TBME). Chromatographic separation was achieved on a reversed-phase Accucore C18 column with a gradient programme consisting of water:ammonia (25%), 100:0.01 (v/v) (mobile phase A) and ACN:MeOH:ammonia (25%), 15:85:0.01 (v/v/v) (mobile phase B). The MS acquisition was performed with selective reaction monitoring mode using the respective [M+H]+ ions, m/z 680.59 → 322.42 for telaprevir and VRT-127394, and 691.15 → 110.13 for telaprevir-d11. The assay exhibited a linear dynamic range of 5–5000 ng/mL for telaprevir and VRT-127394. Acceptable precision (%RSD < 6.5%) and accuracy (94–108%) were obtained for concentrations over the range of the standard curve. A procedure was established to stabilise the plasma to prevent ex vivo interconversion of the isomers.