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Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis
Published on: April 27, 2021
An ID-HPLC-MS/MS based candidate reference measurement procedure for the quantification of ceramides in human serum
Huoyan Ji1, Xiaozhen Zhang2, Xiaofan Yin3
1Department of Clinical Laboratory, Affiliated Hospital of Nantong University, Jiangsu 226001, PR China.
Background:
Ceramides have emerged as promising biomarkers for predicting residual cardiovascular risk in addition to traditional lipid parameters. We developed and validated a candidate reference measurement procedure (RMP) based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for the accurate quantification of four ceramide subspecies (Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1)) relevant to cardiovascular risk in human serum.
Methods:
Primary reference materials (PRMs) were characterized by quantitative nuclear magnetic resonance (qNMR) to establish SI traceability. A ChromCore AQ C18 column with gradient elution was used. Method validation followed Clinical and Laboratory Standards Institute (CLSI) guidelines, assessing specificity, matrix effects, linearity, limits of quantification (LOQ) and detection (LOD), trueness, precision, and stability.
Results:
Excellent linearity was observed across the following ranges: 19.90-4081.48 nmol/L for Cer(d18:1/16:0), 9.92-2022.39 nmol/L for Cer(d18:1/18:0), 49.99-9877.01 nmol/L for Cer(d18:1/24:0), and 49.94-9873.91 nmol/L for Cer(d18:1/24:1), with R2 = 1.00 for all analytes. LOQs were 15.22 nmol/L, 7.63 nmol/L, 37.09 nmol/L, and 38.44 nmol/L, respectively. Recovery rates ranged from 95.04% to 101.15%, with intra-day and inter-day precision (CV) below 3.0% and 4.0%, respectively. Analysis of NIST SRM 1950 yielded Z-scores of 0.9-1.7. Expanded uncertainties ranged from 4.72% to 8.52% (k = 2). Reference intervals (P2.5-P97.5) were 187.10-442.50 nmol/L, 35.30-140.05 nmol/L, 1374.63-4522.55 nmol/L, and 585.30-1906.66 nmol/L, respectively.
Conclusion:
The candidate RMP was successfully developed and validated for the accurate quantification of four ceramide subspecies relevant to cardiovascular risk in human serum, with SI traceability.
