Table 1. Overview of the Multiplex LC-MRM-MS Test Results.
| parameter | strategy | desirable criteria | performance results |
|---|---|---|---|
| selectivity and interferences | proteotypic peptides per protein | min. 2 | two to three proteotypic peptides per protein |
| multiple MRM transitions per peptide | min. 3 | one quantifier and two qualifier product ions | |
| ion ratio monitoring in biological matrix urine for agreement with SSS | H0: μQual ratioSSS = μQual ratiourine | no difference (p > 0.05), except for cubilin, TGF-β1, and calbindin | |
| CV < 20% | CV < 20%, except for cubilin, TGF-β1, and calbindin | ||
| carryover | blank after SSS | <1% | ≤0.9% for all peptides |
| specimen-to-specimen carryover (EP10) | carryover <3*low-low SD | carryover < than error limit (pass)a | |
| matrix effects (MEs) | postextraction spiking of IS to asses matrix effect ion suppression in ESI | absolute ME < 15% | ±15% signal intensity reduction compared with signal in LC eluent A |
| area CV < 15% | CV < 12% for all quantifier peptides | ||
| admixing samples with high total protein and hematuria samples | r > 0.975 | r ≥ 0.958, quantitation was not affected by proteinuria or hematuria | |
| linearity | admix sample low and sample high, N = 9 in triplicate | r > 0.975 (range specified per measurand) | r ≥ 0.975 for NGAL,TIMP2, KIM-1, NGAL, SLC22A2, and CXCL9; r = 0.824 for nephrin |
| by dilution of CAL1 (N = 5) in duplicate | r > 0.975 | r ≥ 0.995 for uromodulin and IGFBP7; no linear correlation obtained for cubilin and TGF-β1 | |
| peptide agreement | r > 0.975 | r ≥ 0.991 for NGAL,TIMP2, IGFBP7, KIM-1, nephrin, SLC22A2, CXCL9, and UMOD; no linear correlation obtained for cubilin and TGF-β1 | |
| imprecision | within-run imprecision by measuring five QC samples in five replicates | CVwr < 15% | AV CV = 13.3% for NGAL, 4.7% for IGFBP7, 11.4% for KIM-1, 9.7% for TIMP2, 8.2% for nephrin, 8.3% for CXCL9, 34.2% for SLC22A2, 15.1% for UMOD, 19.6% for TGF-β1, and 20.3% for cubilin |
| total imprecision by measuring five QC samples in five replicates over 5 days | CVtot < 15% | average CVtot = ±26% depending on the measurand and concentration level |
a
No data for peptides from TGF-β1 or cubilin due to poor response (S/N < 10) in urine of healthy individuals. AV, average over five QC samples; CVwr, within-run imprecision; CVtot, total imprecision; CV, coefficient of variance; r, Pearson’s r in linear regression.