A history of the isolation and identification of vitamin B(6).

In the 1930s, Rudolf Peters showed that young rats kept on a semi-synthetic diet with added thiamin and riboflavin but no other supplement developed 'rat acrodynia', a condition characterized by severe cutaneous lesions. In 1934, Paul György showed that the factor which cured 'rat acrodynia' was vitamin B(6). Other studies soon showed that vitamin B(6) deficiency produced convulsions in rats, pigs, and dogs, and a microcytic anemia in certain animals. Samuel Lepkovsky isolated and crystallized vitamin B(6) in 1938. The following year, Leslie Harris and Karl Folkers, and Richard Kuhn and his associates independently showed that vitamin B(6) was a pyridine derivative, 3-hydroxy-4,5-dihydroxy-methyl-2-methyl-pyridine. György proposed the term pyridoxine for this derivative. Esmond Snell developed a microbiological growth assay in 1942 that led to the characterization of pyridoxamine, the animated product of pyridoxine, and pyridoxal, the formyl derivative of pyridoxine. Further studies showed that pyridoxal, pyridoxamine, and pyridoxine have largely equal activity in animals and owe their vitamin activity to the ability of the organism to convert them into the enzymatically active form pyridoxal-5-phosphate. Pyridoxal-5-phosphate plays a role in a wide variety of enzyme systems, especially in the metabolic utilization and transformation of amino acids.

Esmond E. Snell--the pathfinder of B vitamins and cofactors.

Esmond E. Snell (1914-2003) was a giant of B-vitamin and enzyme research. His early research in bacterial nutrition had lead to the discovery of vitamins such as lipoic acid and folic acid, and an anti-vitamin avidin. He developed microbiological assay methods for riboflavin and other vitamins and amino acids, which are still used today. He also investigated the metabolism of vitamins, discovered pyridoxal and pyridoxamine as the active forms of vitamin B(6) and revealed the mechanism of transamination and other reactions catalysed by vitamin B(6) enzymes. His research in later years on pyruvoyl-dependent histidine decarboxylase unveiled the biogenesis mechanism of this first built-in cofactor. Throughout his career, he was a great mentor of many people, all of whom are inspired by his philosophy of science.

Determination of B2 and B6 vitamers in serum by capillary electrophoresis-molecular fluorescence-charge coupled detector.

A method for the separation by capillary zone electrophoresis and determination of vitamers of two important water-soluble vitamins, i.e., B2 and B6, is proposed here. The working conditions for optimal separation were obtained by a multivariate methodology in order to succeed in the best resolution in the shortest analysis time. The optimization of the buffer composition together with other variables, such as analysis and injection voltage, temperature of the capillary cassette, and injection time, resulted in a solution of 30 mM KH2PO4 adjusted to pH 8.5 with formic acid. Concerning the detection step, the target analytes were quantified by molecular fluorescence, for which two different detectors, a photomultiplier tube (PMT) and a charge-coupled detector (CCD), were compared in terms of resolution, sensitivity, and precision. A Xe-Hg lamp was used as an irradiation source in the two cases. The best option was the CCD, which provides three-dimensional electropherograms and enables to solve the overlapped peaks. Besides, the sensitivity of the CCD was similar to that of the PMT, due to the treatment data, obtaining limits of detection and quantification from 1.16 to 27.1 ng/g and from 3.83 to 89.4 ng/g, respectively. The method was applied to the serum samples for which a prior liquid-liquid extraction using ethanol in an acid medium was mandatory for eliminating the interferences and concentrating the analytes by a factor of 5. The rapidity of the analysis (13 min for the electrophoretic separation) and the excellent precision (repeatability and within-laboratory reproducibility between 2.86 and 4.11% and 7.03 and 8.45%, respectively, both expressed as relative standard deviation) demonstrated the capability of the proposed method for a clinical routine analysis.

Separation of B6 vitamers with micellar liquid chromatography using UV and electrochemical detection.

Separation of six vitamers of vitamin B6 was performed by RP-HPLC using micellar mobile phase, UV and electrochemical detection. Effect of temperature, type and amount of organic modifier in mobile phase on efficiency and asymmetry factor showed that, the appropriate conditions were temperature of 35 degrees C and 3.0-5.0% (v/v) 1-butanol in mobile phase. Variations of selectivity factor versus 1-butanol concentration, pH of mobile phase, and SDS concentration was investigated and the following optimized conditions were selected for the separation: 3.0% (v/v) 1-butanol, pH=5.5 and 65 mM SDS in mobile phase. Electrochemical behavior of vitamers in optimized mobile phase was investigated using cyclic voltammetry, and potential of +1.2 V versus Ag/AgCl(Sat.) was chose as working potential. Finally, separation of B6 vitamers using UV detection at 254 nm and electrochemical detection at +1.2 V was compared.

[Preliminary study on separation of three vitamins by capillary zone electrophoresis and micellar electrokinetic chromatography with amperometric electrochemical detection].

In this paper, separation of water soluble vitamins C (VC), B1 (VB1) and B6 (VB6) has been studied preliminarily by capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) with a home-made amperometric electrochemical detector at the working electrode [carbon fiber (5 microm o.d.)] potential of 510-540 mV (vs. SCE). It has been shown that each of the three vitamins has more than one peak in NH3-NH4Cl (pH 9.0) which were mainly caused by the decomposition products in weak base medium and by heat except those from impurities. The electrochemical detector has better performance for the three vitamins from CZE and MECC. They can be separated either by CZE or by MECC. The separation by CZE is better than that by MECC with sodium dodecyl sulfate (SDS). SDS and buffer acidity can influence the retention time, peak sequence and height. The separation efficiency of CZE in 0.01 mol/L NH3-NH4Cl was 468 800 theoretical plates for VC.