Analytical Method Validation For Quantitative Analysis of 23 Analytes in O. sanctum Sample and Their Herbal Formulations

This UHPLC-QqQLn-MS/MS method for quantitation of 23 analytes was validated according to ICH (2005) guidelines.

Linearity, LOD and LOQ

The IS method was employed to calculate the content of 23 bioactive markers in O. sanctum. The stock solution was diluted with acetonitrile to ten different concentrations for the construction of calibration curves. The linearity of calibration was determined using the analytes-to-IS peak area ratios versus the nominal concentration, and the calibration curves were constructed with a weight (1/a'2) factor by least squares linear regression. The applied calibration model for all curves was y = ax + b, where y = peak area ratio (analyte/IS), x = concentration of the analyte, a = slope of the curve and b = intercept. The LODs and LOQs were measured with S/N of 3 and 10, respectively, as criteria. All the calibration curves indicated good linearity with correlation coefficients (r2) from 0.9971 to 1.0000 within the test ranges. The LOD for 23 analytes varied from 0.041 to 0.357 ng/mL and LOQ from 0.124 to 1.082 ng/mL.

Precision, Stability and Recovery

The intra- and inter-day variations, which were chosen to determine the precision of the developed method, were investigated by determining twenty three analytes with IS in six replicates during a single day and by duplicating the experiments on three consecutive days. Variations of the peak area were taken as the measures of precision and expressed as % RSD. The overall intra- and inter-day precisions were not more than 1.98%. Stability of sample solutions stored at room temperature was investigated by replicate injections of the sample solution at 0, 2, 4, 8, 12 and 24h. The RSD values of stability of the 23 analytes are <2.91%.

A recovery test was applied to evaluate the accuracy of this method. Three different concentration levels (high, middle and low) of the analytical standards were added to the samples in triplicate, and average recoveries were determined. The developed analytical method had good accuracy with overall recovery in the range from 95.10% to 103.04% (RSD < 1.68%).

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