FGF‐21 biomarker detection at sub‐nanogram per mLlevel in human serum using normal‐flow liquid chromatography‐tandem mass spectrometry

RATIONALE: Quantitative detection of FGF-21 biomarker at sub-nanogram per mL level in human serum has generally been achieved using nanoflow LC/MS/MS due to its high sensitivity. However, a nano-LC/MS/MS-based assay can suffer from limited reproducibility and MS signal instability making it challenging to employ it as a robust analytical method for routine clinical applications. METHODS: To tackle these limitations, parallel reaction monitoring (PRM)-based targeted protein quantification using normal-flow liquid chromatography coupled with high resolution, accurate mass instrumentation was evaluated as a possible alternative. Different from the conventional selected reaction monitoring (SRM) using triple quadrupole MS, the proposed strategy used high resolution orbitrap MS coupled with conventional normal-flow liquid chromatography. The primary goal of this assay development effort is to significantly improve the robustness of LC/MS/MS-based assay while maintaining high sensitivity by the use of high-resolution MS and a large sample loading volume. RESULTS: The performance of the normal flow LC-MS/MS assay was evaluated by using it to quantify FGF-21 protein, a potential biomarker for non-alcoholic fatty liver disease, in serum samples. Multiple replicated PRM sample quantification results demonstrated the excellent reproducibility and operational robustness of the assay. A limit of quantification of less than 0.4 ng/mL for FGF-21 in a complex serum matrix could be achieved by using the heavy-isotope-labeled peptide technique, a result which is comparable with the nano-LC/SRM MS based assay sensitivity. CONCLUSIONS: The strategy offered an effective alternative to nano-LC/SRM MS for the quantification of protein biomarkers in complex biomatrix with much improved reproducibility and operational robustness.