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I have a query regarding the regulatory acceptance of using internal
I am developing and validating a bioanalytical method in human plasma
using LC-MS-MS (API-4000 system).
I have tried a no. of internal stds with the drug in positive ion
mode, but in due course of time the internal std areas start varying
and therefore my PA batches and stability batches start failing.
Then I have selected an Internal standard which is structurally very
much similar to the NCE of interest. Eventhough it is structurally
similar I was not able to get M+1 peak (+ve ion mode), so I went for
-ve ion mode and able to get good intensity, peak shape and
When I have used this as internal standard in -ve mode and the NCE of
interest in +ve mode in the same LC-MS-MS method the results were
very good and very much reproducible.
Now my query is whether there is any regulatory concern for using an
internal std in negative mode and compound of interest in +ve mode
where all the QC performance is good.
I have run some PA batches and stability batches and all the
validation parameters are passing as per FDA guidelines.
please give your valuable suggestions and some references
Thanks and regards.
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The FDA guidance does not mention details about LC/MS/MS use of
internal standards. The guidance is mainly focused on the following
points when it comes to PA and QC samples when it comes to internal
1) Recovery of the analyte need not be 100%, but the extent of
recovery of an analyte and of the internal standard should be
consistent, precise, and reproducible.
2) A calibration curve should consist of a blank sample (matrix
sample processed without internal standard), a zero sample (matrix
sample processed with internal standard), and six to eight non-zero
samples covering the expected range, including LLOQ.
3) The stability of stock solutions of drug and the internal standard
should be evaluated at
room temperature for at least 6 hours. If the stock solutions are
refrigerated or frozen
for the relevant period, the stability should be documented. After
completion of the
desired storage time, the stability should be tested by comparing the
response with that of freshly prepared solutions.
4) The stability of the drug and the internal standard should be
over the anticipated run time for the batch size in validation
samples by determining
concentrations on the basis of original calibration standards.
I hope this helps.
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