Urine albumin is a fundamental diagnostic used for classification, risk assessment, and monitoring treatment of patients with chronic kidney disease. Clinical practice guidelines for urine albumin categories are the following: A1 <30 mg/24 hrs or <30 mg/g creatinine as normal to mildly increased; A2 30-300 mg/24 hrs or 30-300 mg/g creatinine as moderately increased; A3 >300 mg/24 hrs or >300 mg/g creatinine as severely increased.1 Unfortunately, urine albumin results are not standardized and may exceed >40% difference between immunometric methods.2, 3 This lack of standardization confounds guideline application throughout health systems globally. Alternative assays devoid of antibody reagents, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), have been suggested as a potential reference measurement procedure4 along with the stringent standardization criteria needed.5 The National Institute of Standards and Technology (NIST) has developed a Standard Reference Material (SRM) 2925 as a primary reference calibrator for urine albumin to value assign external calibrators.
This effort focuses on the development of a candidate reference measurement procedure for urine albumin using LC-MS/MS.
Calibrators spanning 3 to 200 mg/L were derived from recombinant human serum albumin (HSA). Specimens were prepared by adding 40 µL calibrator, control or urine specimen to a 1.8 mL conical tube followed by the addition 135 µL of 0.1M ammonium bicarbonate, and by adding 20 µL of internal standard (15N recombinant HSA, 200 mg/L). Specimens were then reduced (TCEP at 60°C for 1 hour), alkylated (iodoacetamide 0.5 hour in the dark) and digested (trypsin at 37oC for 1 hour). This process was repeated in quadruplicate with results averaged. Analysis was performed using a XR HPLC system (Shimadzu) interfaced to a QTRAP 6500 mass spectrometer (SCIEX). Linear-gradient chromatography was employed using water and acetonitrile with 0.1% formic acid mobile phases. The following peptides and their fragment ions were analyzed in addition to their corresponding 15N analogs: DLGEENFK y6/b2, LVNEVTEFAK y8/y5, YLYEIAR y5/a2, AEFAEVSK y6/b2, LVTDLTK b2/y5, and QTALVELVK y5/y1.
Calibration curves derived of HSA from purified human serum displayed unacceptable linearity for several peptides, LVTDLTK and YLYEIAR, along with accuracy exceeding >10% of the expected values. Linearity and accuracy improved greatly after calibration using recombinant HSA calibration material. The LVNEVTEFAK y8 transition inter-assay imprecision for the quadruplicate analysis was 4.79 %CV and 2.84 %CV at the 2.99 mg/L level and 24.1 mg/L versus the single analysis procedure, which found imprecision to be 6.39 %CV and 4.29 %CV at the 2.99 mg/L level and 24.1 mg/L respectively.
Selection of calibration material is extremely important in urine albumin analysis. Use of recombinant HSA calibrators improved accuracy and imprecision and allowed for quantification of all six HSA peptides. The four analytical replicates for all calibrators, controls and specimens improved imprecision and suggests this method is suitable as a candidate reference measurement procedure for urine albumin.
1.Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney Int 2013; Suppl: 1-150.
2.Bachmann LM, et al. State of the art for measurement of urine albumin: comparison of routine measurement procedures to isotope dilution tandem mass spectrometry. Clin Chem 2014; 60: 471-480.
3.Seegmiller JC, et al. Moving Toward Standardization of Urine Albumin Measurements. EJIFCC 2017; 28: 258-267.
4.Seegmiller JC, et al. Quantification of urinary albumin by using protein cleavage and LC-MS/MS. Clin Chem 2009; 55: 1100-1107.
5.Miller WG, et al. Standardization of Urine Albumin Measurements: Status and Performance Goals. JALM 2017; 2: 423-429.