A stable isotope dilution LC–ESI-MS/MS method for the quantification of pyridoxal-5′-phosphate in whole blood

Vitamin B6 is a cofactor in numerous biologic processes that include gluconeogenesis, neurotransmitter synthesis and amino acid metabolism. The aim of this study was to develop a method to measure the concentration of the biologically active form of vitamin B6 (pyridoxal-5′-phosphate, PLP) in whole blood with stable isotope dilution LC–ESI-MS/MS and compare this new procedure with an established HPLC method based on derivatization of pyridoxal-5′-phosphate. 50 μl of stable isotope (PLP-d3) was added to 250 μl of sample, followed by deproteinization with 10% trichloroacetic acid. After centrifugation, 20 μl of the supernatant was injected into the LC–ESI-MS/MS. Reversed phase chromatography was performed on a UPLC system, using a Waters™ Symmetry C18 column, with a gradient of 0.1% formic acid in methanol. PLP was measured on a tandem MS with a mass transition of 247.8 > 149.8 in the positive ion mode with a collision energy of 14 eV. The chromatographic run lasted 4 min. The method was linear from 4 to 8000 nmol/l. The intra-day and inter-day precision ranged between 1.7–2.8% and 3.0–4.1%, respectively. The mean absolute matrix-effect was 99.3% [97–102%]. The relative matrix-effect was 98.8%. The mean recovery was 98% [89–103%]. The lower limit of quantification was 4 nmol/l. The comparison of the LC–ESI-MS/MS method with our current HPLC method yielded the following equation: LC–ESI-MS/MS = 1.11 [confidence interval, CI: 1.03–1.20] × HPLC + 4.6 [CI: −1.3 to 11.0] (r2 = 0.94). This LC–ESI-MS/MS based method is characterized by simple sample processing and a short run time. The comparison with the current HPLC method is excellent although a significant proportional bias was detected. To conclude, the LC–ESI-MS/MS method is an appropriate method to determine PLP in whole blood.

► Development of an LC–MS/MS-method for the determination of pyridoxal-5′-phosphate in whole blood. ► The method is characterized by short run times and simple sample preparation. ► Validated showed precision CV < 5%, mean recovery of 98%, no significant matrix effects and an LLOQ of 4 nmol/l. ► The method showed an excellent correlation with an existing HPLC method.