Surprisingly, there are a number of drug development companies that do not obtain stable isotope-labeled internal standards (SIL-IS) for their bioanalytical methods. Instead they settle for using a surrogate internal standard (a compound that closely resembles the measured analyte) which can lead to unreliable data and be detrimental to a study. There are scientific, regulatory and financial reasons that justify a company’s investment in incorporating a SIL-IS for each measured analyte.

SCIENTIFIC

Matrix effect is the key reason for incorporating SIL-IS into bioanalytical methods. The variability of plasma, due to intra-subject and dietary variability, can dramatically affect the LC-MS/MS response of drugs and their metabolites by ion suppression or enhancement. The presence of a SIL-IS, that co-elutes with the analyte serves to normalize this effect. Further, the presence of co-medication and the possibility of sample hemolysis can have a similar effect when measured as the ratio. We have seen where the presence of drugs, such as ketamine, even at low levels have completely suppressed drug responses.

A surrogate internal standard may be able to track the drug or metabolite. However, there is always a risk the presence of co-medications will compromise the assay performance requiring an investigation and having to report the problems.

REGULATORY

The European Medicines Agency (EMA) is on record stating over 90% of submissions to their agency have incorporated SIL-IS in supportive assay validations. EMA has rejected several studies where they felt the surrogate internal standard was not a close analogue. Also, companies that might be submitting data to agencies outside of the US are advised to consider the position of the EMA, which is often adopted by other agencies.

The FDA is not on record for requiring SIL-IS in LC-MS/MS methods. However, the FDA has issued 483 citations to laboratories for not having procedures in place that adequately track internal standard (IS) responses within an analytical run. These 483 citations were most commonly noted when the mean IS response varies either between standards/QCs and subject samples or alternatively, between subjects. The FDA has also stated laboratories are expected to develop robust and reliable methods incorporating SIL-IS when available.

FINANCIAL

Assay bias can be observed using a surrogate internal standard during validation which can significantly increase both costs and timelines as investigations are conducted. If sponsor companies invest in a SIL-IS it often cuts method development time in half, offsetting the price of its initial synthesis. Money well spent.

Alone, any one of these reasons justifies the need for inclusion of SIL-IS into LC-MS methods. As critical reagents are essential in biologics assays, we believe a SIL-IS really is the “critical reagent” for LC-MS assays. For our clients who have not thought about this component of their drug development program and/or need help with the process, KCAS works diligently with synthesis groups to provide our clients with the appropriate SIL-IS for their drug compound. This ensures we are validating robust and reliable methods which save our clients time, regulatory hassle, and ultimately money.


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