Development and Clinical Evaluation of a High-Throughput LC-MS/MS Assay for Vitamin B6 in Human Plasma and Serum

Pyridoxal 5'-phosphate (PLP) is the primary circulatory form of vitamin B6, an essential cofactor for numerous biochemical enzymatic reactions. Conventional PLP analysis using high-performance liquid chromatography (HPLC) with fluorescence requires derivatization and long injection-to-injection time. Development of high-throughput LC-MS/MS assays is desirable.

Stable isotope labeled internal standard was added to aliquots of samples, proteins were precipitated using trichloroacetic acid, and supernatants were analyzed by multiple reaction monitoring using LC-MS/MS in positive ion mode. Analysis time for PLP was 3.0 min using single column HPLC separation and 2.4 min using alternating column regeneration (ACR). Clinical evaluation of the method included review of results (n = 102 386) from routine performance of the assay.

The assay was linear to 500 nmol/L; limit of quantification was 5 nmol/L. Imprecision (CV) of the assay was <5%. Equivalent performance was observed for single HPLC column and ACR. In 62% of routinely analyzed patient samples, PLP concentrations were within the reference interval; higher PLP concentrations were observed in samples from males than from females. Vitamin B6 deficiency was lowest in children and highest in elderly adults. Lower PLP concentrations were observed in samples collected during winter/spring than during summer/fall. We observed lower concentrations in plasma collected in lithium heparin tubes, suggesting PLP degradation caused by the anticoagulant.

This LC-MS/MS method allows PLP determination using simple sample preparation and short analysis time. We observed association of PLP concentrations with age, sex, and season of sample collection. Our data indicate that lithium heparin anticoagulant tubes reduce measured PLP concentration.