Maximizing Sensitivity and Specificity in HPLC MS Quantitative Analysis: Addressing Limitations in Dynamic Range and Accuracy

HPLC MS Quantitative Analysis
HPLC MS Quantitative Analysis

LC-MS bioanalysis has emerged as a promising technique in several pharmaceutical assessments, including pharmacokinetics, toxicological, and IND studies. Mass spectrometry individually is very sensitive. However, isolating the analyte from the sample matrix is essential in biological sciences. Mass spectrometry alone cannot meet this requirement. Hence, liquid chromatography is combined with mass spectrometry to separate and quantify analytes in complex biological matrices.

Both LC and MS units have different working principles. The liquid chromatography unit differentiates compounds based on their physicochemical nature, while mass spectrometry differentiates compounds by their mass-to-charge ratio. This dual selectivity makes HPLC-MS analysis a robust and powerful analytical tool. The MS unit not only acts as a detector, ; in theory, through its unique mass spectrum, it can identify individual species corresponding to chromatographic peaks. The current article examines the limitations in the dynamic range and accuracy of HPLC-MS analysis. It began by discussing the LC-MS technique that HPLC labs employ for higher sensitivity and specificity before addressing limitations in dynamic range and accuracy.

Dynamic Range

SRM detection in LC-MS only scans individual transitions or integrates output at a specific m/z value. This robust non-scanning mode of SRM provides increased dynamic range or sensitivity compared to LC-MS bioanalysis in the “full scan” mode. However, this increase in sensitivity can still not cover the needed dynamic range for protein quantification in plasma. In theory, achieving high SRM sensitivity in biological matrices faces two primary challenges: a) the overall resolving power of HPLC-MS analysis and b) inadequate analyte signal.

Although new-generation MS detectors offer enhanced performance, traditional SRM techniques provide a dynamic range of 4 to 5 orders of magnitude. However, it only detects protein concentrations higher than 1 μg/ml in serum or plasma without fractionation or depletion. A simple sample preparation protocol, where whole serum is trypsin digested followed by enhancement of tryptic peptides using MCX resin on a solid phase extraction unit, QTRAP SRM3 can effectively extend the lower limit of quantitation and dynamic range of proteins to low ng/ml range.


MS-based proteomics has increasingly become crucial for biomarker discovery studies and systems biology applications. However, limitations in throughput and quantification accuracy for global discovery proteomics are well noted. Besides, these studies often generate large volumes of data with limited statistical confidence. One of the primary reasons for this constraint is the lack of instruments that can provide sufficient throughput and accuracy for biomarker analysis. As SRM detection of low-abundance analytes is limited by signal intensity, one may use ion funnels to improve quantification accuracy and detection sensitivity.

Also Read: How to Choose the Right Bioanalysis Services Provider That Can Help You Enable Early Clinical Decisions?

As discussed earlier, QTRAP SRM3 mode can extend the dynamic range of analyte quantification. However, one disadvantage of this mode while quantifying candidate biomarkers is its relatively long cycle time. This increase in time reduces its multiplexing capacities and decreases the number of data points for a given peptide. Hence, QTRAP SRM3 is not suitable for assessing large numbers of biomarkers. However, more than 1000 transitions can be multiplexed using traditional schedule SRM or intelligent SRM.