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

Abstract

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 R2 >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.