Liquid Chromatography–Isotope Dilution–Mass Spectrometry as a New Basis for the Reference Measurement Procedure for Hemoglobin A1c Determination

Abstract Background: Standardization of hemoglobin (Hb)A1c measurements is a process of considerable interest for quality assurance in diabetes management. To contribute to continuous quality improvement and fulfillment of the requirements for reference measurement procedures according to the standards of the International Organization for Standardization, we developed a calibration system of highest metrological order using isotope dilution–mass spectrometry with a reference material. Method: Samples were prepared by enzymatic cleavage based on the IFCC reference measurement procedure for LC-MS analysis. After digestion the samples were spiked with [D7]-labeled glycated and nonglycated hexapeptides as internal standards for quantification. LC-MS analysis was performed by using a C12 reversed-phase column and a gradient of acetonitrile/H2O containing 0.1% formic acid. Results: Calibration systems for HbA1c determination based on liquid chromatography–isotope dilution–mass spectrometry (LC-ID-MS) and on the IFCC reference measurement procedure were compared. A linear regression analysis demonstrated a correlation of r2 = 1.00 between the 2 different calibration systems. Mean deviation was 5.5% for the calibration and 3.3% for hemolysate samples, with a mean expanded uncertainty of 4.9%. Conclusions: This LC-ID-MS procedure allows the current IFCC reference measurement procedure for HbA1c to be raised to a higher order of accuracy.

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Proposed Serum Cholesterol Reference Measurement Procedure by Gas Chromatography–Isotope Dilution Mass Spectrometry

Clinical Chemistry ◽

10.1373/clinchem.2010.158766 ◽

2011 ◽

Vol 57 (4) ◽

pp. 614-622 ◽

Cited By ~ 30

Author(s):

Selvin H Edwards ◽

Mary M Kimberly ◽

Susan D Pyatt ◽

Shelton L Stribling ◽

Kara D Dobbin ◽

...

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Serum Cholesterol ◽

Isotope Dilution ◽

Isotope Dilution Mass Spectrometry ◽

Measurement Procedure ◽

Standard Reference Materials ◽

Reference Measurement ◽

Reference Measurement Procedure ◽

The Mean

BACKGROUND Our purpose was to establish a mass spectrometry reference measurement procedure (RMP) for cholesterol to use in the CDC's standardization programs. We explored a gas chromatography–isotope dilution mass spectrometry (GC-IDMS) procedure using a multilevel standard calibration curve to quantify samples with varying cholesterol concentrations. METHODS We calibrated the mass spectrometry instrument by isotope dilution with a pure primary standard reference material and an isotopically enriched cholesterol analog as the internal standard (IS). We diluted the serum samples with Tris-HCl buffer (pH 7.4, 0.05 mol/L, 0.25% Triton X-100) before analysis. We used 17 serum pools, 10 native samples, and 2 standard reference materials (SRMs). We compared the GC-IDMS measurements with the CDC's modified Abell–Levy–Brodie–Kendall (AK) RMP measurements and assessed method accuracy by analyzing 2 SRMs. We evaluated the procedure for lack of interference by analyzing serum spiked with a mixture of 7 sterols. RESULTS The mean percent bias between the AK and the GC-IDMS RMP was 1.6% for all samples examined. The mean percent bias from NIST's RMP was 0.5% for the SRMs. The total %CVs for SRM 1951b levels I and II were 0.61 and 0.73%, respectively. We found that none of the sterols investigated interfered with the cholesterol measurement. CONCLUSIONS The low imprecision, linear response, lack of interferences, and acceptable bias vs the NIST primary RMP qualifies this procedure as an RMP for determining serum cholesterol. The CDC will adopt and implement this GC-IDMS procedure for cholesterol standardization.

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Feasibility of Standardization of Serum C-Peptide Immunoassays with Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry

Clinical Chemistry ◽

10.1373/clinchem.2005.062505 ◽

2006 ◽

Vol 52 (6) ◽

pp. 1193-1196 ◽

Cited By ~ 29

Author(s):

Diego Rodríguez Cabaleiro ◽

Dietmar Stöckl ◽

Jean M Kaufman ◽

Tom Fiers ◽

Linda M Thienpont

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Isotope Dilution ◽

Method Comparison ◽

Measurement Procedure ◽

Tandem Ms ◽

C Peptide ◽

Reference Measurement ◽

Reference Measurement Procedure ◽

Lc Tandem Ms

Abstract Background: Serum C-peptide concentrations reflect pancreatic function in different clinical and diagnostic settings; however, the utility of C-peptide testing is limited by the lack of standardized commercial immunoassays. Standardization can best be done by split-sample comparison with a hierarchically higher reference measurement procedure with a set of native sera. For serum peptides, isotope-dilution liquid chromatography–mass spectrometry (ID-LC/MS) is recommended as a reference measurement procedure. Methods: We evaluated the analytical performance characteristics of an ID-LC/tandem MS procedure for measurement of serum C-peptide after a 2-step solid-phase extraction. To investigate the feasibility of this procedure for use in standardization, we also performed a method comparison with 3 representative commercial assays. Results: The ID-LC/tandem MS procedure showed maximum within-run, between-run, and total CVs on dedicated sera (C-peptide concentrations, 1.6 and 4.0 μg/L) of 2.1%, 2.5%, and 2.9%, respectively; an accuracy of 94.6%–104.1%; a minimum trueness of 98.1% (95% confidence interval, 96.2%–100.0%), and limits of quantification and detection of 0.15 and 0.03 μg/L, respectively. Deming linear regression analysis of the method-comparison data showed that the immunoassays correlated well with ID-MS and were specific, but lacked intercomparability and trueness. We propose that the deficiencies can be resolved by recalibration on the basis of the method comparison. Conclusions: The ID-LC/tandem MS procedure is suitable for specific and accurate measurement of basal and stimulated serum concentrations of proinsulin C-peptide fragment 33–63 and is suitable for use in standardization of C-peptide immunoassays.

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