Rapid determination of the various native forms of vitamin B6 and B2 in cow's milk using ultra-high performance liquid chromatography.

As the formation of pyridoxal phosphate, the active cofactor of vitamin B6, is dependent on riboflavin 5-phosphate, we propose a fast and simple ultra-high performance liquid chromatography method for the simultaneous determination of the native B6 vitamers pyridoxal, pyridoxine, pyridoxamine, their mono phosphorus esters and 4-pyridoxic acid as well as vitamin B2 as riboflavin and its phosphorus ester riboflavin 5-phosphate in milk. Separation was achieved under 6.0min by reversed-phase and pH gradient elution. Sample preparation was optimized regarding various acids and pH levels. Changes in those parameters led to significant deviations of sample matrix breakdown efficiency. The optimized method was then validated regarding specificity, accuracy, precision, linearity, range, detection and quantification limits. As the method performed satisfactory, is was used to study commercial liquid cow's milk (n=31), regarding effects of the employed preservation technique (pasteurization, extended shelf-life, ultra-high temperature) on the composition and content of B6 and B2 vitamers. In cow's milk, vitamin B6 mostly consists of pyridoxal and its phosphate ester, with pyridoxal phosphate being the bulk component. The catabolite of the vitamin B6 metabolism, 4-pyridoxic acid was present in significant amounts in all studied samples, with up to 2.69µmolL-1. Vitamin B2 was present as riboflavin and its phosphate ester up to 12.86µmolL-1.

Reversed-phase high-performance liquid chromatography method with coulometric electrochemical and ultraviolet detection for the quantification of vitamins B(1) (thiamine), B(6) (pyridoxamine, pyridoxal and pyridoxine) and B(12) in animal and plant foods.

A rapid and sensitive high-performance liquid chromatography (HPLC) method with coulometric electrochemical and ultraviolet detections for analysis of vitamin B (B(1), B(6) and B(12)) in animal and plant foods has been developed. A combination of acid digestion and enzymatic extraction to release protein bound and phosphorylated vitamins followed by HPLC analysis was applied. The analyses were carried out on a LC18 column 5 microm (25 cm x 4.6 mm), using the mobile phase consisting of methanol-phosphate buffer (10:90) and 0.018 M trimethylamine, pH 3.55, following at 1.0 ml min(-1). The method offers excellent linearity with regression coefficient r>0.998, good repeatability and reproducibility and relatively short analysis time (17 min). The relative standard deviation (RSD) of the intermediate precision was satisfactory for all the vitamins studied and amounted to <7.3%. The repeatability of the method was <4.7%. The limits of quantification (LOQ) for pyridoxamine, pyridoxal, pyridoxine, vitamins B(1) and B(12) in seafood products were as follows: 2.1, 2.01, 0.99, 62.0 and 0.11 ng ml(-1), respectively. The mean recovery values from a food products spiked with all five vitamins ranged from 92.3 to 101.3%, with a relative standard deviation less than 3.4%. The proposed separation and detection procedures were successfully applied for the simultaneous determination of vitamins B(1), B(6) and B(12) in fruit juices and vitamins B(6) and B(12) in seafood.

Simultaneous fluorometric resolution of pyridoxal, pyridoxamine and 4-pyridoxic acid using chemometric methods.

An alternatively minimizing covariant matrix error (AMCME) algorithm, newly proposed by the present authors, was applied to the simultaneous fluorometric determination of pyridoxal, pyridoxamine and 4-pyridoxic acid without loss of sensitivity. The experimental results illustrate that the profiles of spectra and concentration can be accurately resolved using the AMCME algorithm with a high sensitivity and stable repeatability. That is to say, the closely overlapping problem of the spectra could be resolved owing to the characteristic features of the AMCME algorithm.

Micellar liquid chromatography in clinical chemistry: application to the monitorization of B6 vitamins.

BACKGROUND: A micellar reversed-phase liquid chromatographic procedure was developed for the determination of B6 group vitamins, i.e. pyridoxine, pyridoxal and pyridoxamine, in human serum. METHODS: Chromatographic conditions used were a C18 column, isocratic mode, flow-rate of 1 ml/min and UV-detection at 290 nm. Optimization of the composition of the mobile phase was performed using an interpretative strategy. RESULTS: After modeling, the composition of the selected mobile phase was 150 mM sodium dodecyl sulphate (SDS)--2% (v/v) pentanol-dihydrogenphosphate buffer 10 mM at pH 3. In this mobile phase, serum samples were injected without pretreatment and analysis time was below 14 min. Calibrations for the three vitamins were linear, with coefficient regression better than 0.999, and intra- and inter-day precision, achieved according to ICH, offering values below 4.3% and 3.2%, respectively. The method was applied to the determination of the B6 vitamins in spiked serum samples, with recoveries around 100%, and in the pharmacokinetic determination of pyridoxine half-life in serum, which was found to be 47.5 +/- 3.2 min (n = 5). The procedure was also applied for the analysis of pyridoxine in human serum spiked with several pharmaceutical preparations that contain other drugs which do not produce any kind of interference. Finally, solutions of B6 vitamins kept at -201 degrees C are stable for up to 3 months. CONCLUSIONS: Using the method proposed here, with an SDS-pentanol mobile phase, it is possible to carry out the fast sensitive determination of B6 vitamins in serum following direct injection, without sample pretreatment.

Determination of vitamin B(6) in cooked sausages.

A reverse-phase high-performance liquid chromatography (HPLC) method has been described for the determination of various active forms of vitamin B(6) in meat products. Different extracting agents were tested to solubilize fully the analyte for quantification. The best data were obtained by extracting the samples with 5% (w/v) metaphosphoric acid. Separation by HPLC was performed with fluorescence detection (excitation, 290 nm; emission, 395 nm), on a 10 cm x 0.46 cm i.d. Hypersil BDS C(18) 5 microm column using a mixture of 50 mM phosphate buffer (pH 3.2) and acetonitrile (99:1, v/v) as mobile phase. Precision of the method was 0.5% (within a day) and 4.3% (between days). The detection limits were 0.020 mg/100 g for pyridoxal and pyridoxamine, 0.017 mg/100 g for pyridoxamine phosphate, 0.500 mg/100 g for pyridoxal phosphate, and 0.033 mg/100 g for pyridoxol, with a signal-to-noise ratio of 3. The recovery ranged from 92.0 to 100.0%.

Determination of vitamin B6 (pyridoxamine, pyridoxal and pyridoxine) in pork meat and pork meat products by liquid chromatography.

A liquid chromatographic method for determining vitamin B6 compounds in pork meat and pork meat products is examined. It uses the same extraction procedure as that applied for thiamin and riboflavin determination, followed by a liquid chromatographic separation on a reversed-phase C18 column with 0.01 M H2SO4 as mobile phase at 30 degrees C. 4-Deoxypyridoxine is used as internal standard. The analytical parameters linearity, precision of the method (R.S.D. = 7.3 and 6.9% for pyridoxamine and pyridoxal, respectively) and accuracy obtained by recovery assays (99 and 85.1% for pyridoxamine and pyridoxal, respectively) show that the studied method is useful to measure these compounds in pork meat.

[The effect of different vitamin B6 supplies on the vitamin B6 status (pyridoxine, pyridoxal and pyridoxamine) of the liver and the body of lactating rats]. / Untersuchungen zum Einfluss einer unterschiedlichen Vitamin-B6-Versorgung auf den Vitamin-B6-Status (Pyridoxin, Pyridoxal und Pyridoxamin) der Leber und des Körpers laktierender Ratten.

Eighty female Sprague-Dawley rats were fed a semisynthetic diet during gravidity which was supplemented with 5 mg vitamin B6 per kg diet. The daily food intake was 14 g. During the following lactation the rats were assigned to one of 10 vitamin B6 treatment groups (0, 3, 6, 9, 12, 15, 18, 36, 360 and 3,600 mg per kg diet). The feed was given ad libitum. At day 14 of lactation the rats were decapitated. Parameters for determination of the vitamin B6 status were concentration of pyridoxine, pyridoxal and pyridoxamine in liver and body analyzed by using HPLC. Body was defined without the gastroenteral tract that was divided into carcass (extrahepatic compartments without liver) and total body (extrahepatic compartments plus liver). The mean weight of liver was 13 g with a dry mass of 33%; there was no difference between the treatment groups. The vitamin B6 concentration was lowest in rats fed 0 mg vitamin B6/kg diet (5 micrograms/g fresh matter, FM) and highest in the rats fed 3600 mg vitamin B6/kg diet (10.9 micrograms/g FM). The total vitamin B6 consisted on the average of 38% pyridoxal and 62% pyridoxamine. This was only changed significantly at the highest supplementation level, where 20% pyridoxine were detected instead of pyridoxamine. The mean weight of carcass averaged 212 g at a dry matter content of 31%. The vitamin B6 concentration ranged in the treatment groups from 0 mg to 360 mg vitamin B6/kg diet between 2.1 micrograms/g FM and 2.8 micrograms/g FM. It was highest in the 3600 mg vitamin B6 treatment group at 7.5 micrograms/g FM. The total vitamin B6 consisted of 63% pyridoxal and 37% pyridoxamine. It was only significantly affected in the 3600 mg vitamin B6 treatment group, where also pyridoxine could be found in the amount of 56%. The results indicate that alimentary vitamin B6 supply had more influence on liver vitamin B6 concentration than on carcass concentration. Total body concentration is very similar carcass concentration, as 95% of vitamin B6 is located there. The suitability of the parameters by the evaluation of the vitamin B6 requirement was confirmed the comparison of two statistical methods. It is concluded that a vitamin B6 supply of 5 to 6 mg/kg diet is necessary to meet the requirements during lactation.

The regulation of alanine and aspartate aminotransferase by different aminothiols and by vitamin B-6 derivatives.

We examined the effects on alanine aminotransferase and aspartate aminotransferase of different aminothiols (L-cysteine, D-cysteine, cysteamine, L-cysteine ethyl ester, L-cysteine methyl ester) and several vitamin B-6 derivatives (pyridoxal, pyridoxamine, pyridoxol, pyridoxol 5'-phosphate), before and after treatment with KOCN, which transforms these molecules into the corresponding carbamoyl derivatives. Only GPT, and not GOT, was specifically inhibited by L-cysteine and, to a lesser extent, by D-cysteine. The association reaction: PLP + apo GPT<-->holo GPT was inhibited by the vitamin B-6 derivatives, and this inhibition was prevented by pretreatment of the vitamin B-6 derivatives with KOCN. All the observed effects occurred at pH 7, 37 degrees C, at mM and even lower concentrations of reagents. Hence, they all potentially play a physiological role, in the regulation of the PLP dependent enzymes and of the vitamin B-6 levels in the cell.

[Determination of water-soluble vitamins B1, B2, B6 and B12 in milk using HPLC]. / Bestimmung der wasserlöslichen Vitamine B1, B2, B6 und B12 in Milch durch HPLC.

Simple methods of determining the water-soluble vitamins B1, B2, B6 and B12 in milk by HPLC are described. Compared to existing procedures, the following improvements can be realized. The oxidation of vitamin B1 to thiochrome is stopped by the addition of sodium sulphite. This step significantly increases repeatability. Thiochrome is then extracted with butan-1-ol, which results in fewer co-extracts and greater selectivity. After the hydrolysis of the 5-phosphates of the vitamin B6 (pyridoxine, pyridoxal and pyridoxamine), these three vitamers are determined by isocratic HPLC as DDS-ion-pairs and with fluorimetric detection. As only microbiological methods have so far been used for the determination of minute quantities of vitamin B12 in milk, a new HPLC procedure is proposed with a detection limit of 0.2 micrograms vitamin B12/L milk.

Determination of vitamin B6 vitamers and pyridoxic acid in biological samples.

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.

Relationship between blood, liver and brain pyridoxal phosphate and pyridoxamine phosphate concentrations in mice.

Plasma pyridoxal 5'-phosphate (PLP) concentrations are considered to be the most reliable single indicator of vitamin B-6 nutritional status and are thought to reflect tissue PLP and pyridoxamine 5'-phosphate (PMP) levels. We investigated the relationship between dietary level of pyridoxine hydrochloride (PN-HCl) and concentrations of PLP in blood and PLP and PMP in liver and brain of mice. Female heterogeneous stock mice, 60 to 90 d old, were fed purified diets containing 0.5, 1.0, 2.0, 3.0, 5.0, or 7.0 mg PN-HCl/kg diet for 5 wk. PLP and PMP concentrations were determined by a spectrophotometric apotryptophanase assay. PLP content of plasma, erythrocytes, whole blood, liver and brain and PMP levels in liver and brain were highly correlated with dietary level of PN-HCl (r values ranged from 0.81 to 0.94, n per correlation = 32 to 43). By using the entire range of dietary levels of PN-HCl, both plasma and erythrocyte PLP were found to be significantly correlated with tissue PLP and PMP concentrations. For any one dietary level, however, correlations between plasma or erythrocyte PLP and tissue PLP and PMP concentrations were low and nonsignificant. These results suggest that plasma PLP levels may be suitable to determine vitamin B-6 status of populations, but not to reliably predict tissue concentrations of PLP or PMP in individuals.

Reaction of pyridoxamine-5-P with pyrroloquinoline quinone (coenzyme PQQ).

PQQ catalyzes the oxidation of pyridoxamine (PM) and pyridoxamine-5-P (PMP) to pyridoxal and pyridoxal-5-P (PLP) at 37 degrees C in the absence of micelles and proteins. The time course of conversion of PMP into PLP was monitored by absorption spectroscopy; a rate of 10 nmol PLP/min was determined. The product of the reaction was identified by TLC, HPLC and its ability to restore the catalytic activity of apoaspartate aminotransferase. The conversion of PMP into PLP by free PQQ is more efficient than reactions catalyzed by the enzymes plasma amine oxidase and pyridoxamine-5-P oxidase at optimal pH values.

Complexes of vitamin B6XX: equilibrium and mechanistic studies of the reaction of pyridoxal-5'-phosphate with pyridoxamine-5'-phosphate in the presence of copper(II).

The interaction of Cu(II) with pyridoxamine-5'-phosphate (PMP) and pyridoxal-5'-phosphate (PLP) was studied potentiometrically. The titration data were assessed by MINIQUAD program. Several protonated and nonprotonated complexes have been found to exist in solution. The reaction of PLP with Cu(II)-PMP has been studied kinetically, using the stopped-flow technique. Two rate steps have been observed. The first step has been attributed to the formation of a Schiff's base metal complex. The second step may be due to the formation of a ternary complex formation. A mechanism was suggested.

A simple preparation method for apoaspartate aminotransferase from Escherichia coli B, and its application for the assay of pyridoxal and pyridoxamine 5'-phosphate.

A simple and rapid preparation method for apoaspartate aminotransferase from Escherichia coli B was developed. A crude extract of the bacterial cells was treated batchwise with DEAE-cellulose. The enzyme fraction obtained was then applied to a pyridoxamine-Sepharose column. Apoaspartate aminotransferase was eluted with 50 mM potassium phosphate buffer (pH 7.0), and found to be electrophoretically homogeneous. The apoenzyme preparation thus obtained showed very low holoenzyme activity (only 0.4% of the activity seen in the fully saturated condition with pyridoxal 5'-phosphate) and was successfully used for assaying pyridoxal and pyridoxamine 5'-phosphate.

Analysis of B-6 vitamers in human milk by reverse-phase liquid chromatography.

A simple, accurate reverse-phase high performance liquid chromatography (RPLC) method was introduced for the analysis of B-6 vitamers in human milk. The assay consisted of a phosphate buffer (pH 2.9) delivered isocratically through 5 micron ODS column packing, followed by post-column bisulfite derivatization to enhance the detection of PLP (and to a minor extent PL). The vitamers were detected using a fluorescence spectromonitor. Sample run time was less than 30 min. The sensitivity of the method was such that PL, PN, and PLP were detectable to 30 pmol/ml milk and PM and PMP to 5 pmol/ml milk. Total vitamin B-6 content in milk analyzed by RPLC correlated well with the microbiological assay. B-6 vitamer distribution in human milk was similar to values obtained from two different ion-exchange HPLC systems. The RPLC procedure is simpler and faster than the HPLC systems and is suggested for future use in analysis of B-6 vitamer concentrations in human milk.

Two homogeneous immunoassays for pyridoxamine.

Protein conjugates of pyridoxal have been used to elicit anti-vitamin B6 antibodies in rabbits. These antibodies have been incorporated into 2 homogeneous assays systems, a spin immunoassay, using a paramagnetic derivative of the vitamin as ligand, and a fluorescence enzyme immunoassay, using beta-galactosidase conjugated to vitamin B6 as the indicator molecule. These assay systems do not require fractionation steps, and could be the basis of analytical methodology for nutritional research or clinical diagnosis.

Quantities of B6 vitamers in human milk by high-performance liquid chromatography. Influence of maternal vitamin B6 status.

A rapid, sensitive procedure is described for the analysis of the B6 vitamers pyridoxal, pyridoxamine, and pyridoxine in human milk from women taking and not taking supplements containing the vitamin using high-performance liquid chromatography with fluorometric detection. Vitamer values represent the sum of their phosphorylated and unphosphorylated forms. Minimum detectable quantities were 1-3 ng. Excellent recoveries of these vitamers in milk were obtained. Similar B6 vitamer concentrations of milk were obtained using the developed high-performance liquid chromatographic and the accepted microbiological techniques. Pyridoxal, actually consisting of pyridoxal plus pyridoxal phosphate, was the predominant B6 vitamer in human milk. The concentration of B6 vitamers in milk was reflective of the maternal vitamin B6 status.