2.10.4. Method validation

The method was validated in accordance with the industrial guidelines for bioanalytical method validation of the US Food and FDA [9]. Drug-free human plasma containing K2 EDTA anticoagulant was obtained from the blood bank and stored frozen at −80 ± 10 °C until analysis. The retention times of analytes in the interfering substances were absent from chromatograms obtained from six different sources of blank plasma spiked with target analytes at the lower limit of quantitation (LLOQ) levels. Representative chromatograms are shown in Fig. 1(A–C). The calibration curve regressions for withanoside IV, withanolide A and withaferin A exhibit strong linearity with coefficients of determination (r2) of 0.9990, 0.9969, and 0.9985 respectively. The LLOQ was calculated by the analyte response at the LLOQ ≥ five times the analyte response of the zero calibrator. The LLOQ of withanoside IV and withanolide A was 1.0 ng/ml and that of withaferin A 0.5 ng/ml in plasma. The LOD for withanoside IV, withaferinA and withanolide A is 0.5, 0.5 & 0.1 ng/ml. Intra/inter day precision and accuracy determined using QC samples at four concentrations with six replications spiked in the blank plasma were found to be within acceptable range (±20% & 80–120% for LLOQ, <15% & 85–115% for other QC’s of nominal concentration) (Table 2). The Intra batch precision (coefficient of variation) ranged from 6.95 to 17.98% for withanoside IV, 7.27–16.62% for withaferin A and 3.47–15.85% for withanolide A respectively. The accuracy (%) ranged from 89.18 to 96.63% for withanoside IV, 92.99–102.97% for withaferin A and 94.09–107.57% for withanolide A respectively. The Inter batch coefficients of variation ranged from 1.61 to 10.76% for withanoside IV, 0.06–6.77% for withaferin A and 1.46–5.30% for withanolide A. The accuracy ranged from 95.33 to 100.57% for withanoside IV, 93.05–100.21% for Withaferin A and 92.81–105.12% for withanolide A respectively. The percentage extraction recoveries ranged from 93.06 to 93.60% for withanoside IV, 91.72–93.54% for withaferin A and 91.23–91.61% for withanolide A respectively (Table 3). The percentage of matrix effects ranged from 91.00 to 94.00% for withanoside IV, 90.80–94.04% for withaferin A and 90.88–93.42% for Withanolide A respectively (Table 2, Table 3). Since the blood samples were collected in K2 EDTA tubes, the impact of anticoagulants on quantification was also analyzed and found no interfere with the assay performance. The stability of LQC, MQC & HQC with six replicate samples of withanoside IV, withaferin A and withanolide A were assessed under conditions such as long term stability (−80 °C for 60 days), three freeze (−20 °C) thaw cycles (at room temperature), short-term room temperature storage and autosampler stability (Table 4). In all stability tests, the samples exhibited a recovery rate of 90–105%. These results confirm the stability of withanoside IV, withaferin A, and withanolide A under the specified conditions.

Representative UPLC -ESI-MRM chromatograms of extracted blank human plasma (A) chromatogram of blank human plasma without internal standard (B) chromatogram of blank human plasma spiked with internal standard (C) chromatogram of blank human plasma spiked with withaferin A, withanolide A and withanoside IV at lower limit of quantitation (LLOQ) level.

Precision and accuracy data for withanoside IV, withanolide A and withaferin A.

LLOQ- Lower limit of quantification.

LQC- Lower quality control.

MQC-Middle quality control.

HQC- Higher quality control.

Extraction recovery and matrix effect of withanoside IV, withanolide A and withaferin A.

R1 represents analytes spiked before extraction.

R2 represents analytes spiked after extraction.

R3 represents analytes prepared in injection solvent.

Recovery (%) was calculated as R1/R2 × 100.

Matrix effect (%) was calculated as R2/R3 × 100.

Stability data of Withanoside IV, Withanolide A & Withaferin A

Data are expressed as means % ± SD (n = 6), Observed con/nominal con *100.

LQC- Lower quality control.

MQC-Middle quality control.

HQC- Higher quality control.