Quantification of urinary albumin by using protein cleavage and LC-MS/MS

BACKGROUND: Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). METHODS: We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, ¹⁵N-labeled recombinant HSA (¹⁵NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides ⁴²LVNEVTEFAK⁵¹ and ⁵²⁶QTALVELVK⁵³⁴ from albumin and 15NrHSA were referenced to the calibration curve. RESULTS: The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13-200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%-11.0% for the peptide ⁴²LVNEVTEFAK⁵¹, and 1.9%-12.3% for the ⁵²⁶QTALVELVK⁵³⁴ peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%-14.8% for the ⁴²LVNEVTEFAK⁵¹ peptide, and 6.4%-14.1% for the ⁵²⁶QTALVELVK⁵³⁴ peptide. For the ⁴²LVNEVTEFAK⁵¹ peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R² value of 0.97 and a regression line of γ = 0.99x + 23.16. CONCLUSIONS: Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a ¹⁵NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.