Method(s): LC-MS/MS (1).
What is measured: Pyridoxal 5'-phosphate (PLP), pyridoxal (PL), 4-pyridoxic acid (PA), and pyridoxine (PN). The prevailing vitamin B6 form in serum/plasma is PLP.
Performance of the assay
Lower limit of detection (LOD) (PLP/PL/PA/PN): 0.2/0.2/0.5/1 nmol/L.
Within-day CV: 3/3-5/3-4/3-5 %; between-day CV: 6-7/5-6/7-11/7 %.
Assessment of vitamin B6 status. Different combinations of circulating vitamin B6 species have been suggested as indicators of vitamin B6 status: PLP, PLP in combination with PL, and PLP in combination with PA. PLP is the most commonly used (2).
Specimen, collection and processing
Patient/subject: Effect of prandial status on PLP not reported.
Matrix: B6 vitamers are most stable in EDTA plasma.
Volume: Minimum volume is 60 µL, but 200 µL is optimal and allows reanalysis.
Preparation and stability: The plasma fraction must be separated from the blood cells, preferentially within 30 minutes. Samples should be put on ice, and protected from light. PLP is light sensitive. In serum, citrate plasma or heparin plasma, PLP is converted to PL. Measurement of the sum of PLP plus PL is an option in samples that have not been adequately treated (3).
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Frozen, on dry ice.
Reported values, interpretation
Reported values (PLP/PL/PA): 17-102/6-28/11-88 nmol/L.
Intraclass correlation coefficient (ICC) (PLP/PL/PA): 0.70 /0.62/0.58.
1. Midttun, O., Hustad, S., and Ueland, P.M. (2009). Quantitative profiling of biomarkers related to B-vitamin status, tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Sp 23, 1371-79.
2. Ueland, P.M., Ulvik, A., Rios-Avila, L., Midttun, Ø., and Gregory, J.F. (2015). Direct and Functional Biomarkers of Vitamin B6 Status. Annu Rev Nutr 35, 33-70.
3. Hustad, S., Eussen, S., Midttun, O., Ulvik, A., van de Kant, P.M., Morkrid, L., Gislefoss, R., and Ueland, P.M. (2012). Kinetic modeling of storage effects on biomarkers related to B vitamin status and one-carbon metabolism. Clin Chem 58, 402-410.