The workflow for bioanalytical LC-MS/MS requires isolation of analytes of interest from biological samples prior to analysis. This usually involves an extraction procedure ahead of injection into the LC-MS/MS system where the target compounds are separated from other analytes & potential matrix co-extractants. Addition of an internal standard at a fixed level prior to extraction helps to compensate for any experimental sample to sample variation.

It’s important to recognize that different internal standards can be used depending on the development stage of a project. Discovery bioanalysis often needs rapid data delivery for analytes that may only be looked at in a limited number of studies. KCAS maintains a panel of compounds with a variety of functional groups for use as generic internal standards with unknowns. Our aim is to have an internal standard that elutes as closely as possible to the analytes of interest. It’s also not uncommon for us to use analogs if they are available from our customers.

As we move into regulated small molecule LC-MS/MS, we recommend using stable labeled internal standards whenever possible. It’s vital that sufficient labels are added to avoid interference from the unlabeled analyte although this can become challenging when the molecule contains multiple chlorines or other halogens.

Although considered the gold standard, not all stable labeled internal standards are equal in performance. Labeling with multiple deuteriums can lead to sufficient change in molecular behavior that we see chromatographic separation from the unlabeled compound so it’s not seeing exactly the same instrumental conditions as the analyte. In addition, there can be deuterium/hydrogen exchange so the fragmentation may not match the unlabeled compound. Labeling with heavier stable isotopes such as 13C or 15N shows no issues with chromatographic separation or stability.

The workflow for quantitation of large molecules by our Biopharma LC-MS/MS team usually involves immunoaffinity extraction followed by digestion to isolate a small surrogate of the larger entity. We have one assay that uses a labeled protein internal standard but they tend to be hard to source. For most cases, an internal standard is only introduced post extraction. The internal standard is usually a stable labeled mimic of the sequence around the target peptide with the addition of around 3 amino acids on each end. This means we can at least check for consistency of digestion and the downstream processes. The subsequent analysis follows the workflow of small molecule LC-MS/MS.

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