A novel fully validated LC–MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood
Ghassabian, Sussan, Griffiths, Lyn, & Smith, Maree T (2015) A novel fully validated LC–MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood. Journal of Chromatography B, 1000, pp. 77-83.
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Quantification of pyridoxal-5´-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20 µL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was > 92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80 °C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.
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|Item Type:||Journal Article|
|Keywords:||Pyridoxal-5′-phosphate, LC-MS/MS, Method development, Method validation, Endogenous compounds, Bioanalysis|
|Divisions:||Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
|Copyright Owner:||Copyright 2015 Elsevier B.V.|
|Copyright Statement:||This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/|
|Deposited On:||04 Apr 2016 22:44|
|Last Modified:||05 Apr 2016 21:08|
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