METHOD VALIDATION FOR HPLC ANALYSIS OF RELATED SUBSTANCES

Intrinsic Accuracy.Intrinsic accuracy indicates the bias caused by sample matrix

and sample preparation. In this approach, a stock solution is prepared by using

known quantities of related substance and drug substance. The stock solution

is further diluted to obtained solutions of lower concentrations. These solutions

are used to generate linearity results. In addition, these linearity solutions of dif-

ferent concentrations are spiked into placebo. The spiked solutions are prepared

according to the procedure for sample analysis. The resulting solutions, prepared

from the spiked solution, are then analyzed. If the same stock solution is used for

both linearity and accuracy and all of these solutions are analyzed on the same

HPLC run, the response of linearity (without spike into matrix) and accuracy

(with spike into matrix) can be compared directly. Any differences in response

indicate the bias caused by matrix interference or sample preparation. To deter-

mine the intrinsic accuracy at each concentration level, one can compare the peak

area of accuracy (with matrix) with that of linearity (without matrix) at the same

concentration (Figure 3.11). This is the simplest approach, and one would expect

close to 100% accuracy at all concentration levels.