Simultaneous quantification of simvastatin and simvastatin hydroxy acid in blood serum at physiological pH by ultrahigh performance liquid chromatography–tandem mass spectrometry (UHPLC/MS/MS)

• A method was developed to quantify simvastatin and simvastatin hydroxy acid.
• The method employs a physiological pH to maintain the in vivo ratio of compounds.
• The method differs from those previously published, which use pH 4.5.
• The low pH causes conversion and non-physiologically relevant concentrations.
• The method was successfully applied to a mouse exposure study.

Simvastatin attenuates airway inflammation and hyperreactivity, hallmarks of asthma, in allergen-challenged mice. As such, it is under consideration as a novel therapeutic, thus it is important to quantify the levels of simvastatin and its pharmacologically active and interconvertible metabolite, simvastatin hydroxy acid, that can be attained in the body. Methods exist to measure the concentrations of these compounds in biological media; however they do not maintain a physiological pH, and as a result do not accurately measure the ratio of these two compounds that exists in vivo. We developed a new method to measure simvastatin and simvastatin hydroxy acid more accurately in serum from mice by ultra high performance liquid chromatography–tandem mass spectrometry. We minimized the time that the compounds were in aqueous solution, and buffered samples to a physiological pH value of 7.4. Limits of quantification (LOQ) were 0.16 ng mL−1 extract (1.3 ng mL−1 serum) for simvastatin, and 8.3 ng mL−1 extract (66 ng mL−1 serum) for simvastatin hydroxy acid, respectively. No interconversion was observed, based on spike-and-recovery experiments of solutions containing both compounds. The method was applied using biological samples from mice challenged with house dust mite extract and simultaneously treated with subcutaneous simvastatin injection. Simvastatin hydroxy acid concentrations became significantly increased after a 2 week pre-treatment regime, whereas simvastatin concentrations were below the LOQ for all serum samples.