A novel fully validated LC-MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood

被引:12
作者
Ghassabian, Sussan [1 ]
Griffiths, Lyn [2 ]
Smith, Maree T. [1 ,3 ]
机构
[1] Univ Queensland, Ctr Integrated Preclin Drug Dev, Brisbane, Qld 4029, Australia
[2] Queensland Univ Technol, Inst Hlth & Biomed Innovat, Brisbane, Qld 4001, Australia
[3] Univ Queensland, Sch Pharm, Brisbane, Qld 4029, Australia
来源
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2015年 / 1000卷
关键词
Pyridoxal-5 '-phosphate; LC-MS/MS; Method development; Method validation; Endogenous compounds; Bioanalysis; PERFORMANCE LIQUID-CHROMATOGRAPHY; PYRIDOXAL 5'-PHOSPHATE; VITAMIN-B6; VITAMERS; 4-PYRIDOXIC ACID; HUMAN PLASMA; HUMAN SERUM; RED-CELLS; ASSAY; PYRIDOXAL-5-PHOSPHATE;
D O I
10.1016/j.jchromb.2015.07.019
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 mu L) 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 17 h or at -80 degrees 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. (C) 2015 Elsevier B.V. All rights reserved.
引用
收藏
页码:77 / 83
页数:7
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