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To the Editor:Hepcidin-25, a liver-produced peptide hormone, was initially isolated from human urine and blood ultrafiltrate. Hepcidin-25 is thought to be the central regulator of iron metabolism (1 ). Iron deficiency is associated with low hepcidin-25 concentrations and anemia of chronic disease with high concentrations, but the true diagnostic value of hepcidin-25 is still under investigation. A recently published review (2 ) stated that only semiquantitative methods for comparative studies based on mass spectrometry (MS) have been used for the determination of hepcidin in serum and urine.Isotope-dilution MS is generally accepted as yielding high analytical specificity and accuracy. We report an isotope-dilution-micro-HPLC-tandem MS (MS/MS) method that allows the quantification of hepcidin-25 present at less than nanomole-per-liter concentrations.During sample preparation, hepcidin-25 undergoes strong nonspecific binding to surfaces. A stable isotope-labeled internal standard helps to compensate for any matrix effects. Micro-HPLC-MS/MS with monolithic capillary columns ensures highest resolution and limits of quantification (3 ).Native hepcidin-25 (M r 2789) was purchased from Bachem AG. Blood samples were collected and anonymized in-house according to the Roche Diagnostics policy, and informed consent was obtained from all sample donors; results were not used for regulatory purposes.Calibrators with concentrations of 0.1-100.0 nmol/L were prepared from hepcidin-25. For sample preparation we added 5 L concentrated formic acid and 50 L internal standard solution (1450 nmol/L in water, 0.04% acetic acid) to 45 L human serum or calibrator. Samples were ultrafiltered with a Microcon Ultracel YM-10 filter (Millipore). The flow-through was transferred into HPLC vials. A Thermo Electron Quantum-Ultra triple-quadrupole mass spectrometer, Dionex Ultimate 3000 micro-HPLC, and PS-DVB Monolithic 200-m internal diameter ϫ 5 cm column (P/N 161409) were used for online micro-HPLC-MS/MS. A 2-L sample was injected. The mobile phases were (eleunt A) 1% formic acid/0.025% trifluoroacetic acid in water and (eleunt B) 1% formic acid/0.025% trifluoroacetic acid in acetonitrile, flow rate 3 L/min, with a linear gradient from 0% to 80% eleunt B during 7 min and then held at 80% eleunt B until minute 11.We used microelectrospray ionization in the positive mode; recorded selected reaction-monitoring transitions were m/z 930.83 1145.5 and m/z 935.531152.6, collision energy 33 V, and argon collision gas 2.0 mTorr. Carryover was excluded by blank injections. Samples were kept in long-term storage at Ϫ80°C and were stable for at least 2 days at 6°C. Processed samples were stable for 48 h at 6°C. We observed no ion suppression attributable to changing elution conditions and monitoring-signal intensities; postcolumn infusion ex-

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Objectives To develop an isotope dilution-liquid chromatography-tandem mass spectrometry-(ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for quantification of methotrexate in human serum and plasma. Methods Quantitative nuclear magnetic resonance (qNMR) was used to determine absolute methotrexate content in the standard. Separation was achieved on a biphenyl reversed-phase analytical column with mobile phases based on water and acetonitrile, both containing 0.1% formic acid. Sample preparation included protein precipitation in combination with high sample dilution, and method validation according to current guidelines. The following were assessed: selectivity (using analyte-spiked samples, and relevant structural-related compounds and interferences); specificity and matrix effects (via post-column infusion and comparison of human matrix vs. neat samples); precision and accuracy (in a five-day validation analysis). RMP results were compared between two independent laboratories. Measurement uncertainty was evaluated according to current guidelines. Results The RMP separated methotrexate from potentially interfering compounds and enabled measurement over a calibration range of 7.200–5,700 ng/mL (0.01584–12.54 μmol/L), with no evidence of matrix effects. All pre-defined acceptance criteria were met; intermediate precision was ≤4.3% and repeatability 1.5–2.1% for all analyte concentrations. Bias was −3.0 to 2.1% for samples within the measuring range and 0.8–4.5% for diluted samples, independent of the sample matrix. RMP results equivalence was demonstrated between two independent laboratories (Pearson correlation coefficient 0.997). Expanded measurement uncertainty of target value-assigned samples was ≤3.4%. Conclusions This ID-LC-MS/MS-based approach provides a candidate RMP for methotrexate quantification. Traceability of methotrexate standard and the LC-MS/MS platform were assured by qNMR assessment and extensive method validation.