Stable isotope-labeled internal standards are widely used in laboratory-developed clinical mass spectrometry tests. An equal amount of these internal standards is added to all samples in a batch, including calibrators, controls, and unknowns. The ratio of the analyte signal to the internal standard signal is then used to create the calibration curve for quantification. Proper use of internal standards can normalize variations in recovery throughout the entire analytical process, which includes sample preparation, injection, chromatography, and mass spectrometry ionization. Key criteria for selecting an internal standard for a small molecule analyte typically include: 1) an identical chemical structure except for the isotopes or at least a similar structure, 2) chemical stability, 3) a mass-to-charge ratio difference that can be easily distinguished by mass spectrometry, and 4) absence from the sample matrix. Despite following these general guidelines, pitfalls may still arise, and improper use of internal standards can lead to biased clinical results. In this presentation, two troubleshooting cases will be discussed as part of our laboratory's quality improvement projects for existing assays. The first case addresses a non-linear response issue in the urine 5-Hydroxyindoleacetic acid (5-HIAA) test, while the second addresses high imprecision issues in the Sirolimus test at low concentrations. Both cases were resolved by switching to more suitable internal standards, resulting in improved accuracy, linearity, and precision in the optimized assays.

Leaning Objectives:

• Understand the role of internal standards in isotope dilution LC-MS/MS tests
• Identify general criteria for selecting an internal standard in laboratory-developed LC-MS/MS tests
• Learn from two troubleshooting cases how improper use of internal standards can lead to biased results