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A reference method that specifically measures hemoglobin (Hb) A1c is an essential part of the reference system for the international standardization of Hb A1c/glycohemoglobin. We have developed a new method for quantification, based on the specific N-terminal residue of the hemoglobin β-chains. Enzymatic cleavage of the intact hemoglobin molecule with endoproteinase Glu-C has been optimized to obtain the β-N-terminal hexapeptides of Hb A1c and Hb A0. These peptides have been separated by reversed-phase HPLC and quantitated by electrospray ionization-mass spectrometry (method A) or by capillary electrophoresis (method B). With these peptides and hyphenated separation techniques, it has been possible to overcome the insufficient resolution of currently used protein separation systems for Hb A1c.

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-

Background: The aim of our work was to develop and validate a reliable LC-MS/MS-based measurement procedure for the quantification of vancomycin in serum, to be applied in the context of efforts to standardize and harmonize therapeutic drug monitoring of this compound using routine assays. Methods: Sample preparation was based on protein precipitation followed by ultrafiltration. In order to minimize differential modulation of ionization by matrix constituents extended chromatographic separation was applied leading to a retention time of 9.8 min for the analyte. Measurement was done by HPLC-ESI-MS/MS. For internal standardization the derivative vancomycin-glycin (ISTD) prepared by chemical synthesis was used, HPLC conditions ensured coelution of ISTD with the analyte. Results: In a bi-center validation total CVs of < 4% were observed for quality control material ranging from 5.3 mg/L to 79.4 mg/L; accuracy was ± 4%. No relevant ion suppression was observed. Comparative measurement of aliquots from 70 samples at the two validation sites demonstrated close agreement. Conclusions: Employing a closely related homologue molecule for internal standardization and the use of MS/ MS following highly efficient sample pre-fractionation by HPLC, the method described here can be considered to offer the highest level of analytical reliability realized so far for the quantification of vancomycin in human serum. Thus, the method is suitable to be used in a comprehensive reference measurement system for vancomycin.

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