Improving the Detection of Pyridoxal Phosphate in Biological Fluids
Challenge: Diteba was contracted to develop and validate a specific quantitative analytical method capable of simultaneously detecting and quantifying all three analytes (vitamers) in different biological fluids and support a clinical study as well as for toxicological studies on small animal models. It should be noted that previously made attempts by a reputable US-based contract research organization to develop a suitable method for quantification of PLP, PL and 4-PA by Liquid Chromatographic Tandem Mass Spectrometry (LC/MS/MS) technique had failed due to the complexity of the project. All three analytes appear at high endogenous levels in biological fluids which in turn complicates the analytical procedure.
Solution: PLP is the primary biologically active form of vitamin B6, serving as a coenzyme in numerous bio-processes and also acts as a coenzyme in the catabolism of homocysteine to cystathionine (β-synthase) and ultimately cysteine. As a result, vitamin B6 plays a role in lowering homocysteine levels and its associated risk with atherosclerosis and coronary heart disease. Low PLP status may be associated with an increased risk of stroke and transient ischemic attack.
Historically, several analytical methodologies such as fluorometric, enzymatic and microbiological have been used for determining PLP and other B6 vitamers, but the use of High Performance Liquid Chromatography (HPLC) technology has been the most successful in determining vitamin B6 in its multiple forms in plasma and serum. Fluorescence detection has been used almost exclusively in HPLC determinations of vitamin B6 due to its selectivity and high sensitivity. Although most forms of vitamin B6 and its metabolites fluoresce adequately for HPLC/Ultra Performance Liquid Chromatography (UPLC) analysis, the natural fluorescence of PLP is low. This has led to the development of several chemical derivatization methods aimed at improving PLP fluorescence detection. Examples include semicarbazone formation, bisulfite adduct formation and catalyzed oxidation by using cyanide. The targeted method sensitivity was 0.5ng/ml but natural fluorescence detection can only achieve 20ng/mL. Diteba was able to achieve a 100 times improvement in sensitivity and obtained 0.2 ng/mL sensitivity.
A post column chlorite oxidation mechanism is the notable exception among these derivatization methods. This approach not only avoids the toxicity potential of the oxidation procedure but also allows the separation of the B6 vitamers on the basis of their primary chemical structures. A successful selection of a suitable internal standard and sample extraction method that minimizes calculation error for the analytes was implemented. Another challenging obstacle of this project was the high endogenous level of analytes presented in all patients' and animals' bio samples.
In the past, most HPLC methodologies developed for determining vitamin B6 in biological matrices were reversed-phase systems that used octadecylisilyl C18 stationary phases and buffered phosphate mobile phases. A similar approach was used to develop the current method. Chlorite post-column derivatization increases the fluorescence of PLP by converting the molecule from an aldehyde to a carboxylic acid by way of oxidation. Conditions for achieving this were adapted by Diteba from those general procedures described in publications for the aldehyde class of compounds. The use of carboxylic acid provided stronger fluorescence than an aldehyde.
A notable exception was the introduction by Diteba of a new post column derivatization method by using chlorite. The oxidation of PLP to a more fluorescent 4-pyridoxic acid 5-phosphate was prepared by using 10% solution of sodium chlorite instead of the more commonly used cyanide anions. This approach not only avoided the toxicity potential of the cyanide-catalyzed oxidation procedure but also allows for the separation of the B6 vitamers (PL, PE, PLP, 4-PA) on the basis of their primary chemical structures.
The relatively high fluorescence intensity of the derivatized analytes resulted in low limits of detection, ~100 times than that typically found in existing methods. This high sensitivity, combined with excellent within-run repeatability, good run-to-run reproducibility, and high sample throughput, ensures the developed method is ideal for routine clinical and toxicological determination of PLP, PL and 4-PA in large studies.
Result: The method was validated for human and cross-validated for various animals' species in plasma/serum matrices.
This is the first-time an endogenous background subtraction method has been successfully employed during the generation of standard calibration curve and the calculation of quality control samples. The validated method developed by Diteba was successfully implemented for processing a variety of clinical and toxicology study samples.