title: A novel fully validated LC-MS/MS method for quantification of pyridoxal-5'-phosphate concentrations in samples of human whole blood.
abstract: 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 (20μL) 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.
authors.affiliation: Centre for Integrated Preclinical Drug Development, University of Queensland, Brisbane, Queensland, Australia. Electronic address: s.ghassabian@uq.edu.au.
authors.forename: Sussan
authors.initials: S
authors.lastname: Ghassabian
authors.affiliation: Institute for Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.
authors.forename: Lyn
authors.initials: L
authors.lastname: Griffiths
authors.affiliation: Centre for Integrated Preclinical Drug Development, University of Queensland, Brisbane, Queensland, Australia; School of Pharmacy, University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Maree T
authors.initials: MT
authors.lastname: Smith
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D016014:Linear Models; D011732:Pyridoxal Phosphate / Q000097:blood* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D012710:Serum Albumin, Bovine; D011732:Pyridoxal Phosphate
keywords: Bioanalysis; Endogenous compounds; LC–MS/MS; Method development; Method validation; Pyridoxal-5′-phosphate