Ultra-sensitive Quantification of Mid-regional Proadrenomedullin in Human Plasma Using Micro-liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry

Abstract

Mid-regional proadrenomedullin (MR-proADM) has been suggested to be a powerful biomarker for the onset, progression, and mortality of various diseases. To achieve sensitive quantification of MR-proADM without cross-reactivity, we established micro-liquid chromatography coupled to the quadrupole time-of-flight mass spectrometry (μLC-QTOF/MS) method using IonKey technology. 100 μL of plasma sample was pretreated by protein precipitation followed by solid-phase extraction. Using the established μLC-QTOF/MS method, plasma MR-proADM concentrations were measured in 198 individuals of the Japanese general population, 13 patients with chronic kidney disease (CKD) stage G3b-G4 (nondialysis), and 12 patients with CKD stage G5 (dialysis). The novel assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.05 ng/mL. Recovery rates from human plasma and matrix effects were 89.6-114.2% and 95.3-111.7%, respectively. Median plasma MR-proADM concentrations were 0.48, 1.16, and 2.75 ng/mL in general population, CKD stage G3b-G4, and CKD stage G5, respectively (p < 0.0001). All measured concentrations were within the calibration range (0.05-100 ng/mL). The robust Passing-Bablok regression plot between MR-proADM concentrations measured by μLC-QTOF/MS and by immunoluminometric assay showed systemic and proportional bias between methods. The values measured by the μLC-QTOF/MS method tended to be lower than those by immunoluminometric assay. An ultrasensitive and selective method using μLC-QTOF/MS for measuring plasma MR-proADM was established. The novel method can be used in the general population and patients and may have better specificity for MR-proADM than the immunoluminometric assay; thus, it may improve the performance of MR-proADM as a biomarker for predicting the prognosis of patients with various diseases.

1

Mass spectra of product ions of mid-regional proadrenomedullin (MR-proADM) (a) and internal standard (¹³C₂₅, ¹⁵N₅-MR-proADM) (b). P* denotes ¹³C and ¹⁵N-labeled proline.

2

Selected chromatograms of mid-regional proadrenomedullin (MR-proADM; left) and internal standard (¹³C₂₅, ¹⁵N₅-MR-proADM; right) obtained from micro-liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (μLC-QTOF/MS) analysis of pretreated plasma samples: (a) blank plasma sample, (b) plasma sample at lower limit of quantitation (LLOQ: 0.05 ng/mL), (c) high-quality control plasma sample (HQC: 75 ng/mL), (d) plasma sample from an individual of general population (calculated MR-proADM concentration: 0.45 ng/mL), and (e) plasma sample from a patient with chronic kidney disease (CKD) (calculated MR-proADM concentration: 0.97 ng/mL).

3

Plasma mid-regional proadrenomedullin (MR-proADM) concentrations in Japanese general population (n = 198), patients with chronic kidney disease (CKD) stage G3b-G4 (n = 13), and patients with CKD stage G5 (n = 12).

4

Ratios of quantifier/qualifier transitions for in the Japanese general population (n = 198), patients with chronic kidney disease (CKD) stage G3b-G4 (n = 13), and patients with CKD stage G5 (n = 12).

5

Passing–Bablok plot between plasma mid-regional proadrenomedullin (MR-proADM) concentrations measured by micro-liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (μLC-QTOF/MS) and by immunoluminometric assay (a) and Bland–Altman plot for plasma MR-proADM concentrations measured by μLC-QTOF/MS and immunoluminometric assay (b).