Fully validated LC–MS/MS method for quantification of homocysteine concentrations in samples of human serum : a new approach

Ghassabian, Sussan, Rethwan, Nur Syazwani Ahmad, Griffiths, Lyn, & Smith, Maree T. (2014) Fully validated LC–MS/MS method for quantification of homocysteine concentrations in samples of human serum : a new approach. Journal of Chromatography B, 972, pp. 14-21.

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Abstract

Reported homocysteine (HCY) concentrations in human serum show poor concordance amongst laboratories due to endogenous HCY in the matrices used for assay calibrators and QCs. Hence, we have developed a fully validated LC–MS/MS method for measurement of HCY concentrations in human serum samples that addresses this issue by minimising matrix effects. We used small volumes (20 μL) of 2% Bovine Serum Albumin (BSA) as surrogate matrix for making calibrators and QCs with concentrations adjusted for the endogenous HCY concentration in the surrogate matrix using the method of standard additions. To aliquots (20 μL) of human serum samples, calibrators or QCs, were added HCY-d4 (internal standard) and tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) as reducing agent. After protein precipitation, diluted supernatants were injected into the LC–MS/MS. Calibration curves were linear; QCs were accurate (5.6% deviation from nominal), precise (CV% ≤ 9.6%), stable for four freeze–thaw cycles, and when stored at room temperature for 5 h or at −80 °C (27 days). Recoveries from QCs in surrogate matrix or pooled human serum were 91.9 and 95.9%, respectively. There was no matrix effect using 6 different individual serum samples including one that was haemolysed. Our LC–MS/MS method has satisfied all of the validation criteria of the 2012 EMA guideline.

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ID Code: 80852
Item Type: Journal Article
Refereed: Yes
DOI: 10.1016/j.jchromb.2014.09.032
ISSN: 1570-0232
Divisions: Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
Copyright Owner: Copyright 2014 Elsevier
Deposited On: 22 Jan 2015 02:15
Last Modified: 27 Jan 2015 02:00

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