Assays - Pyridoxal

High-Performance Liquid Chromatography Determination of Total Pyridoxal in Human Plasma

Author: H. J. Mascher
Publisher: Methods Enzymol. Vol. 280 (Eds. D.B. McCormick, Academic Press 1997)

Vitamin B6 occurs in three interconvertible forms: pyridoxine (PN), pyridoxamine (PM), and pyridoxal (PL). There is also a 5´-phosphate for each of these forms (PNP, PMP, PLP). From the published method information on the endogenous levels of the vitamin analogs and their phosphated, it was apparent that PLP and PL might well constitute the majority of the vitamin analogs in plasma following administration of pyridoxine. We decided to determine PLP and PL as total PL following enzymatic cleavage with acid phosphatase. Preliminary trials had shown that sensitive analysis of PL was possible only with either extensive sample preparation or lang analysis times, because of the strong polarity of PL. For this reason we attempted to improve the selectivity and sensitivity of detection. Published papers prompted us to attempt postcolumn derivatization with semicarbazide.

Determination of total Pyridoxal in human plasma following oral administration of vitamin B6 by high-performance liquid chromatography with post-column derivatization

Author: H.J. Mascher
Publisher: J. Pharm. Sci., 82 (1993), 972-974

An HPLC method for determining total pyridoxal from plasma was developed for a relative bioavailability comparison of two oral vitamin B6 (pyridoxine HCl) preparations. After cleavage of the pyridoxal-5-phosphate with the acid phosphatase enzym, the total pyridoxal was determined by HPLC. Pyridoxal was separated on a reversed-phase column, post-column derivatized to pyridoxal-semicarbazide, and then detected by fluorescence and quantified. The limit of detection was 2 ng/ml and interday variation (3 days) over the whole concentration range (13 - 215 ng/ml spiked) was < 4.1 %. In the relative bioavailability study, 16 human subjects were put on a low vitamin B6 diet for a period of 3 days. On the 2nd and 3rd days, 14 blood samples were taken per subject at the same times each day. The drug was administered on the 3rd day. Total endogenous pyridoxal detected on the 2nd day varied in plasma between 13 and 17 ng/ml. Pharmacokinetic parameters corrected with the Hoffmann-La roche preparation are, respectively: AUC0-24, 369.2 and 352.6 ng x h / ml; AUC24-48, 1638.2 and 1662.3 ng x h / ml; net Cmax, 193.0 and 197.1 ng/ml; tmax, 1.25 and 1.44 h; and relative bioavailability, 97.9 % (Westlake, 88 - 112 %)