Chromatography Forum

I have a question about results of an USP assay. I did two injections of Octisalate(Sunscreen active). The first injection came out to be 96.2% and the second injection came out to be 94.6%. The average of the two are 95.4%. The requirment is between 95.0%-105.0%. My question is, does my run pass because the average passes or does it fail because the second injection is below 95.0%?

Thanks,
David

No you can not average fail Assay result with pass Assay result.

Thanks

Taken from the FDA guidance for industry:

C. Reporting Testing Results
Practices used in reporting and interpretation of test results include (1) averaging and (2) outlier tests.
1. Averaging
There are both appropriate and inappropriate uses of averaging test data during original
testing and during an OOS investigation:
a. Appropriate uses
Averaging data can be a valid approach, but its use depends upon the sample and its purpose. For example, in an optical rotation test, several discrete measurements are averaged to determine the optical rotation for a sample, and this average is reported as the test result. If the sample can be assumed to be homogeneous, (i.e., an individual sample preparation designed to be homogenous), using averages can provide a more accurate result. In the case of microbiological assays, the U.S. Pharmacopeia (USP) prefers the use of averages because of the innate variability of the biological test system.

Contains Nonbinding Recommendations
It should be noted that a test might consist of a specific number of replicates to arrive at a result. For instance, an HPLC assay result may be determined by averaging the peak responses from a number of consecutive, replicate injections from the same preparation (usually 2 or 3). The assay result would be calculated using the peak response average. This determination is considered one test and one result. This is a distinct difference from the analysis of different portions from a lot, intended to determine variability within the lot, and from multiple full analyses of the same homogenous sample. The use of replicates to arrive at a single reportable10 result, and the specific number of replicates used, should be specified in the written, approved test method. Acceptance limits for variability among the replicates should also be specified in the method. Unexpected variation in replicate determinations should trigger remedial action as required by § 211.160(b)(4). If acceptance limits for replicate variability are not met, the test results should not be used.

You can see that averaging consecutive replicate injections from the same preparation is allowed to be averaged to report as a result. You may not average the results from two different preparations. So if you made one sample, injected twice from the same HPLC vial on a system that had established and maintained suitability, you are ok. If you made two different sample preparations, then you have an Out-of-Specification event.

Thanks, that's exactly what I wanted. You rock!

The FDA guidance continues as follows:

In some cases, a series of complete tests (full run-throughs of the test procedure), such as assays, are part of the test method. It may be appropriate to specify in the test method that the average of these multiple assays is considered one test and represents one reportable result. In this case, limits on acceptable variability among the individual assay results should be based on the known variability of the method and should also be specified in the test methodology. A set of assay results not meeting these limits should not be used.

This seems to indicate that a test method written to require averages of multiple preps is acceptable. But this must be clearly laid out in the test method prior to sample analysis.

This seems to indicate that a test method written to require averages of multiple preps is acceptable. But this must be clearly laid out in the test method prior to sample analysis.

As said above-the specification and/or method needs to be very clear the results are to be averaged. If they do not you can't use the average. If the method specifies averages are to be used, be prepared to have justification (data) to support it. Assay (potency) is not typically an averaged result.

The requirment is between 95.0%-105.0%.

This specification is not based on the average.

USP

7. TEST RESULTS
7.10. Interpretation of Requirements
Analytical results observed in the laboratory (or calculated from experimental measurements) are compared with stated acceptance criteria to determine whether the article conforms to compendial requirements.
The reportable value, which often is a summary value for several individual determinations, is compared with the acceptance criteria. The reportable value is the end result of a completed measurement procedure, as documented.
Where acceptance criteria are expressed numerically herein through specification of an upper and/or lower limit, permitted values include the specified values themselves, but no values outside the limit(s). Acceptance criteria are considered significant to the last digit shown.