Method validation
The method validation was performed with K2EDTA human
plasma according to European Medicines Agency Guideline on Bioanalytical
Method Validation [8]. The method was validated for selectivity,
specificity, carry-over, linearity, precision and accuracy, recovery,
dilution integrity, influence of haemolysed and hyperlipidaemic plasma,
drug-drug interaction, matrix effect and stabilities.
The analytical curves were constructed from a blank sample (plasma
sample processed without IS), a zero sample (plasma processed with IS)
and eight concentrations of Favipiravir, including the LLOQ, ranging
from 80 to 30000 ng/mL. The concentrations were calculated using peak
area ratios and the linearity of the calibration curve was determined
using least squares regression analysis employing a weighted (1/x)
linear (y=mx+b) for Favipiravir. The acceptance criteria for each
calculated standard concentration was not more than 15% deviation from
the nominal value, except for the LLOQ which was set at 20%. The
within-batch precision and accuracy was evaluated by analyzing QC
samples at five different concentration levels (80 ng/mL (LLOQ), 240
ng/mL (QC Low), 1500 ng/mL (QC Medium), 12000 ng/mL(QC High) and 24000
ng/mL (ULLOQ)) with six replicates in a batch.The between-batch
precision and accuracy were determined by analyzing three different
batches. The within-batch and between-batch values did not exceed 15%
for QC samples, expected for LLOQ which did not exceed 20%.
The selectivity was studied by checking the chromatograms obtained from
eight different sources of human plasma including one haemolytic and one
lipemic plasma. By comparing the chromatograms of those plasma samples
spiked with Favipiravir and IS with the chromatograms of the blank
plasma samples, no peak was found at the retention time of Favipiravir
and IS in ten of the blank plasma samples.The recoveries were estimated
by comparing the peak areas of Favipiravir in three replicates of QC
samples with those of post-extraction blank matrix extracts at the
corresponding concentrations. The matrix effects of Favipiravir were
evaluated by comparing the peak areas of post-extraction blank plasma
that were spiked at certain concentrations of QC samples with the areas
obtained by the direct injection of the corresponding standard
solutions. The stability of Favipiravir in the plasma samples was
determined from three QC levels with six replicates each under the
following conditions: Long-term stability at -70°C for 60 days,
short-term stability at RT for 5 h, using processed samples in
autosampler vials for 24 h, and after four freeze/thaw cycles (-70 to
RT).
An in-house high performance liquid chromatography with tandem mass
spectrometry method (LC-MS/MS) was developed and validated to quantify
Favipiravir in plasma.
The plasma samples were maintained at -70°C during the assay. 0.1 mL of
thawed samples to room temperature were transferred in a polypropylene
tube and were prepared for analysis using protein precipitation
according to SOP of bioanalytical center.