Simultaneous determination of the combined and free concentrations of atorvastatin and its major metabolite in vitro and in vivo based on ultrafiltration coupled with UPLC-MS/MS method: an application in a protein binding rate and metabolism ability study in uremic hemodialysis patients.

INTRODUCTION: A rapid, accurate, and specific ultrafiltration with ultra-performance liquid chromatographic-tandem mass spectrometry method was validated for the simultaneous determination of the protein binding rate of atorvastatin in uremic patients. Methods: The plasma samples were centrifuged at 6,000 r/min for 15 min at 37°C and the ultrafiltrate was collected. An ACQUITY UPLC® BEH C18 Column with gradient elution of water (0.1% formic acid) and acetonitrile was used for separation at a flow rate of 0.4 ml/min. RESULTS: The calibration curves of two analytes in the serum showed excellent linearity over the concentration ranges of 0.05-20.00 ng/ml for atorvastatin, and 0.05-20.00 ng/ml for orthohydroxy atorvastatin, respectively. This method was validated according to standard US food and drug administration and European medicines agency guidelines in terms of selectivity, linearity, detection limits, matrix effects, accuracy, precision, recovery, and stability. This assay can be easily implemented in clinical practice to determine the free and combined concentrations of atorvastatin in the plasma of uremic patients. The final result showed that the average plasma protein binding rate in uremic patients was 86.58 ± 2.04%, relative standard deviation (RSD) (%) = 1.98, while the plasma protein binding rate in patients with normal renal function was 97.62 ± 1.96%, RSD (%) = 2.04. There was a significant difference in the protein binding rate in different types of plasma (P < 0.05), and the protein binding rate decreased with increasing creatinine until it stabilized at nearly 80%. The mean metabolite/prototype ratio of atorvastatin in patients with normal renal function and in patients with uremia was 1.085 and 0.974, respectively. DISCUSSION: The metabolic process of atorvastatin may be inhibited in uremic hemodialysis patients, but the total concentration of atorvastatin did not change significantly; due to the decrease of protein binding rate increase the drug distribution of atorvastatin in the liver or muscle tissue, which may increase the risk of certain adverse reactions. We recommend that clinicians use free drug concentration monitoring to adjust the dose of atorvastatin to ensure patient safety for uremic hemodialysis patients.