Quantitation of glycated albumin by isotope dilution mass spectrometry

Glycated albumin is considered an alternative glycemic indicator in certain situations where HbA1c does not accurately reflect glycemic status. These patient cases are usually associated with decreased erythrocyte lifespan, gestational diabetes, or end-stage renal disease. The aim of our study was to develop an assay for absolute quantitation of glycated albumin based on isotope dilution liquid chromatography-mass spectrometry.

The plasma samples were reduced/alkylated, spiked with isotope-labeled standards RQIKKQTALV(D₈)E and RQIKK(fructosyl)QTALV(D₈)E and enzymatically digested by Glu-C. The samples were analyzed on an LC-MS system. Two MRM transitions (M³⁺ → (b₉-3H₂O)²⁺ and M³⁺ → (b₁₀-3H₂O)²⁺ or M³⁺ → b₉²⁺ and M³⁺ → b₁₀²⁺) were used for each peptide, then the percentage of glycation (MS GA%) was calculated.

The comparison study demonstrated a good linear correlation between our LC-MS/MS and Lucica method with r² = 0.95. The intra-day CV for the low HbA1c sample was 2.2%, while CV for the high HbA1c sample was 0.64%. Inter-day CV for low HbA1c sample was 5.6%, while the CV for the high HbA1c sample was 5.7%. We found the LLOQ to be 0.12 nmol/ml for the non-glycated and glycated peptide. No interference from hemoglobin was observed up to 500 mg/dL concentration.

This is the first implementation of isotope dilution LC-MS assay for glycated albumin with simultaneously quantitation of glycated and non-glycated peptides. The method includes a simple sample preparation and has demonstrated a good analytical performance.