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Importance Of Evaluating Internal Standards (IS) For Effective LC-MS/MS Bioanalysis

The purpose of an internal standard (IS) is to enhance the accuracy and precision of quantitation in bioanalysis. IS adds to the robustness of bioanalytics by minimizing variance.

It is usual for any standard bio lab to apply an IS in assays that combine liquid chromatography with tandem mass spectrometry (LC-MS/MS).

How IS Makes LC-MS/MS More Effective?

The application of ISs minimizes variations in analytes during sample extraction and chromatography. ISs play a significant role in compensating for variable analyte ionization in the ion source of the MS detector.

A GLP (Good Laboratory Practice) oriented bio lab applies IS in its bioanalytics during the early stages of drug development. The aim is to neutralize the adverse impact of differences in recovery, ionization competency, and liquid handling.

A Good Internal Standard (IS)

A good IS can track the analyte under consideration in each of the stages of LC-MS bioanalysis:

●       Sample preparation

●       Separation through chromatography

●       Detection through mass spectrometry

These three stages, however, do not play an equally important role in the selection of an effective IS.

Types of Internal Standard

Any good bio lab with an effective quality control mechanism in place employs one of the following ISs:

●       Stable Isotope Labeled (SIL) ISs

●       Structural Analog (SA) ISs

Scientists prefer SIL ISs as they are more effective for GC-MS, LC-MS, and LC-MS/MS. Another term for SIL IS application fo the quantitation of any of these processes is Isotope Dilution Mass Spectrometry (IDMS). 

To be effective, a SIL IS should be equal to or greater than three mass units. Such an application neutralizes any isotope effect on the quantitation of bioanalysis.

However, SIL ISs are not always available. They can also be quite expensive. That makes it necessary to employ SA ISs. SA ISs consist of compounds with similar structures with marginal variations in the functional groups.

When we employ SA ISs, it is critical to check whether there is any significant discrepancy in the instrument response of the target analyte and its SA IS. Any major variation in this regard implies the need to look for a different SA IS.

IS For Larger Protein Molecules

The ideal IS for protein molecules also consists of a SIL form of the analyte protein. However, limited commercial availability of such a SIL or its cost implications may necessitate the use of the SIL form of a surrogate peptide as an IS.

 We can also use a cleavable SIL-peptide as an IS for protein-based bioanalytics.

Inconsistency In IR Response And Its Implications

Scientists, by design, add a constant amount of IS to all samples before the extraction process starts. That includes calibration standards (CALS) and quality controls (QCs). Even then, sometimes, there are variations in the IS response of samples analyzed in the same run.

The latest FDA guidance published in 2019 mentions that such variations are sometimes the result of human errors in handling. Such variations impact data accuracy in the following situations:

●       One or more subject samples reflect an IS response different from the majority of subject samples, and the CALS/QC.

●       There is no IS response in the QC, but a gradual increase or decrease in the IS response extending beyond the range of the subject samples.

It is vital to eliminate these variations in IS response before filing for an INDA / NDA.