A rapid sequential salting-out/isoelectric precipitation LC-MS/MS method for quantifying cytokeratin 18 in human ascitic fluid.

Cytokeratin 18 (CK18), a predominant hepatocyte intermediate filament protein released during hepatocyte death, has been investigated as a hepatic injury biomarker. Conventional immunoassays for CK18 suffer from non-specific cross-reactivity and insufficient standardization. Though liquid chromatography-tandem mass spectrometry (LC-MS/MS) could overcome such immunoanalytical drawbacks, accurate CK18 quantification in ascitic fluid remains analytically challenging because of the complex matrix, low target abundance, and variability introduced by protein enrichment and digestion. Herein, we developed a rapid and practical LC-MS/MS method for CK18 quantification in ascitic fluid. A two-step non-immunoaffinity enrichment strategy, integrating sequential ammonium sulfate salting-out and isoelectric point-driven acidic precipitation, was optimized to balance enrichment yield, pretreatment turnaround, and operational cost. Subsequent pretreatment workflow comprising urea denaturation, enzymatic hydrolysis, and solid phase extraction purification can be completed within 6 h, providing an economical, instrument-compatible procedure for LC-MS/MS analysis. Systematic analytical performance evaluation showed satisfactory linearity spanning 0.10-10.36 nmol/L, a limit of quantification of 0.10 nmol/L, a limit of detection of 0.02 nmol/L, total imprecision <10%, and recoveries ranging from 93.07% to 108.92%. The internal standard effectively compensated for LC-MS/MS instrumental variability and ionization-related matrix effects. Method comparison showed acceptable agreement with a published multiplexed cytokeratin MS method. Clinical analysis demonstrated significantly elevated ascitic-fluid CK18 concentration in cirrhotic patients versus non-cirrhotic controls (P = 0.001). This novel enrichment strategy preserves the simplicity and cost-efficiency of conventional precipitation methods while avoiding costly antibody capture reagents. Collectively, the validated LC-MS/MS method is appropriate for preliminary exploration of the clinical value of ascitic-fluid CK18 and can facilitate translational research in cirrhotic populations.