Bioanalytical Methods and Data Handling
A fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method determining yimitasvir concentration in phase 1 and phase 1b studies had been reported elsewhere [7, 8].
Plasma concentration of yimitasvir in phase 2 study was analyzed using a validated LC-MS/MS method equipped with a Shimadzu LC-30AD liquid chromatography-SCIEX API6500 mass spectrometer. Chromatographic retention and separation were achieved on a XBridge Peptide BEH C18, 50 × 2.1 mm, 3.5 μm column. Gradient elution was used with 2 mM ammonium acetate in water with 0.1% formic acid as mobile phase A and acetonitrile:methanol (30:70, v:v) as mobile phase B at a flow rate of 0.5 ml min-1. The column temperature was maintained at 45 °C. Quantitation was accomplished in positive mode with precursor-to-production pairs of m/z 428.5→315.3 for yimitasvir and m/z 432.5→319.5 for the internal standard d8-DAG (isotope-labelled yimitasvir), respectively.
The calibration curve was linear in the range of 5.00-5000 ng ml-1 for both methods, and the lower limit of quantitation (LLOQ) was 5.00 ng ml-1. Accuracy and precision were within the acceptable criteria of ±15% for quality control (QC) samples and of ±20% for LLOQs. Both methods were fully validated in accordance with National Medical Products Administration (NMPA) of China, U.S. Food and Drug Administration (U.S. FDA) and European Medicines Agency (EMA) guidelines. These two bioanalytical methods were not cross-validated.
Pharmacokinetic data with an absolute value of conditional weighted residual (CWRES) |CWRES| > 6 in the structural models were regarded as outliers. Outliers were omitted because these observations had a potential to negatively influence the convergence and/or poor estimation precision of parameters. If outliers were removed in the process of model development, the final model was re-run with or without the outliers to assess the potential influence on parameter estimates. If the frequency of LLOQ data was less than 10%, PK samples below LLOQ were excluded from model development. Otherwise, Beal’s M3 method was used for handling the LLOQ data [11]. For covariates with missing values in less than 10% of subjects, continuous covariates were imputed as the population median, while a new category of ‘missing’ is generated for categorical covariates. No formal covariate screen procedure would be conducted if the covariates missed in more than 10% of the subjects.