Robust LC-MS/MS Method: Standard/Calibrator Curve, LMH Quality Controls, and Stable Isotope Labeled Internal Standard

Bioanalytical methods such as LC-MS and HPLC assays are widely used in the quantitative analysis of protein and drug molecules. LC-MS and HPLC testing services are rapidly employing their expertise in a broader range of applications. However, due to its inherent selectivity, shorter development time, high dynamic range, and multiplexing capabilities, LC-MS/MS analysis is highly used in a majority of bioanalytical analyses. Similar to HPLC method development, LC-MS method development and validation comprises optimization of several crucial parameters. Let us explore some of the critical parameters revolving around the precision and bias of an LC-MS/MS analysis.

Standard/Calibration curve

To use LC-MS/MS analysis for quantifying analyte concentrations, researchers should first prepare samples with known concentrations. Analyte quantification involves preparing blank samples with a range of concentrations, to generate calibration solutions that can be employed in the preparation of a standard/calibration curve. However, the matrix of blank samples must be similar to the sample matrix being analyzed.

LC MS method consists of analyzing liquids in the instrument, and hence calibration standards are a known amount of analyte solutions. Accurate standard preparation and its use in LC-MS analysis are the key factors governing reliable data generation. Some crucial considerations include selecting calibration standards, preparing and storing samples, using internal standards, the strategy used for calibration, and employing appropriate mathematical and statistical models.

Quality Controls (L-M-H)

Quality control (QC) samples are used at regular intervals to ensure the precision and bias of LC-MS/MS assays. Acceptance of assay results largely depends on the fact that QCs are successfully analyzed within predefined limits. It is increasingly becoming popular to reanalyze a set of test samples for demonstrating assay precision, as test samples are usually different from control samples.

It is common to employ three levels of quality controls; low, medium, and high. Both the low and high levels are demanding positions in a standard curve. Lower limits of QCs are necessary as at a lower range, carryover and interferences have the most detrimental effects. The odds of non-linearity are taken care of by the higher limits. The mid-level QC lies around the geometric mean of the calibration range, and it is either where the curvature skews to the positive or the negative end.

Stable isotope-labeled internal standard

An internal standard helps compensate for the variability encountered during sample processing and analysis. A stable isotope labeled-internal standard is an ideal choice for mimicking the analyte in LC-MS analysis. As both the stable isotope-labeled internal standard and analyte protein have similar physiochemical characteristics, the internal standard can efficiently monitor the analyte throughout the entire LC-MS procedure. For smaller analyte proteins, the stable isotope-labeled internal standard can be synthesized chemically. But for most analytes, it needs a cellular environment.


Ideal calibration standards, quality controls, and internal standards are prerequisites for the accuracy and precision of bioanalysis, especially during drug development and research. Whether it is LC-MS or HPLC testing, thoroughly developed assays should be the primary concern of all sponsors and relevant stakeholders.