A sensitive and cost‐effective high‐performance liquid chromatography/tandem mass spectrometry (multiple reaction monitoring) method for the clinical measurement of serum hepcidin

Rationale Hepcidin is a peptide hormone that plays a central role in regulating iron metabolism. It is a potential biomarker for the diagnosis, monitoring and treatment of iron metabolism disorders. Serum hepcidin level can differ by 3 orders of magnitude depending on the patient's condition. Existing liquid chromatography/mass spectrometry (LC/MS) assays lack clinical sensitivity or require costly sample preparation steps. A simple, sensitive, robust and cost‐effective assay for serum hepcidin quantitation in routine clinical laboratories is needed. Methods A high‐performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable‐isotope‐labeled hepcidin as the internal standard. The method was validated according to CLSI‐C62A guidelines. Calibrators were prepared with hepcidin‐free serum. Clinical samples were separately processed and compared using solid‐phase extraction (SPE) and acetonitrile (ACN) protein precipitation. Results The calibration curve was validated over the range of 0.1–100 nmol/L with R 2 >0.99. Both the SPE and the ACN precipitation methods had excellent and comparable reproducibility. The intra‐day and inter‐day coefficients of variation (CVs) were <3% and <6%. There was 89% and 88% hepcidin recovery by SPE and ACN preparation. Measurement of secondary reference material using non‐traceable calibrators yielded up to 30% positive bias, comparable with values obtained by an external comparator. Hepcidin was stable in serum at ambient temperature and at 4°C. The relative errors (REs) were ≤1.2% and ≤4.4%, respectively. The freeze–thaw (−70°C) stability after 3 cycles showed a relative error (RE) of ≤1.8%. The impact on hepcidin recovery due to hemolysis (4+), lipemia (4+) and Icterus (4+) was <3%. Conclusions We have developed and validated a simple, sensitive, robust and cost‐effective HPLC/MS/MS method for the quantitation of serum hepcidin. The method uses ACN protein precipitation for sample preparation and reversed‐phase normal‐flow HPLC. Sample preparation is inexpensive; it can be automated with a liquid handling system to allow high‐throughput application.