Drug substances precise assay testing

For autosampler precision, 10 consecutive lO-pL injections of an eth-ylparaben solution (20 J,g/mL) are used (Figure 6). A Waters Symmetry column packed with 5- J,m particles is used. The manufacturer s specification for peak area precision at 0.5% RSD is adopted as the acceptance criterion. This stringent precision criterion is required for precise assay testing of drug substances typically specified at 98-102% purity. The linearity test is performed by single injections of 5, 10, 40, and 80 pL of the... 

Precision reflects a procedure s ability to reproduce the same, but not necessarily the correct or expected, result each time it is correctly performed. Precision is assessed by repetitively injecting a number of samples and statistically evaluating the resulting data. Important issues related to the precision determination include the number of replicates required and the type of sample to be tested. For the determination of repeatability, recommendations include 1) Five to ten replicates for release or stability assays 2) duplicate measurements made on 10 samples at each of three different analyte levels 3) five replicates at three levels (limit of quantitation, midrange, and upper calibration bound) 4) replicate samples at analyte levels of 80-120% of expected for dosage forms and drug substance tests. 

Precision, accuracy, and specificity have meaning only for the concentration tested. Therefore, it is imperative to test the range of concentrations of drug and potentially interfering substances that will be encountered during sample analysis. For example, a cross-reactivity of 1% with cortisone concentrations of 1-50 ng/mL is acceptable for a prednisone RIA because after a standard 20-mg dose of prednisone, the prednisone concentrations in plasma will exceed or equal the cortisone concentrations. In contrast, similar cross-reactivity with cholesterol at these concentrations would render the assay useless without prior separation of prednisone and cholesterol, because cholesterol is present in concentrations of 150-250 mg/mL in plasma (—1000 X prednisone). It is apparent that crossreactivity per se is not the problem. Cross-reactivity in combination with the anticipated concentrations of the cross-reacting substance determines the resultant assay interference. 

---