Analysis of B6 vitamers in plasma by reversed-phase column liquid chromatography

The seven vitameric forms of B6 can be measured in biologic fluids by high-performance liquid chromatography. Use of a reversed-phase column, with optimized solvent and buffer conditions, combined with fluorometric detection, allowed linear detection of vitamers from 0.4 to 20 ng. All vitamers from plasma, urine or tissue extracts can be analyzed within 50 min. Reproducibility and recovery studies indicate a selective and sensitive procedure, which greatly enhances studies on vitamin B6 metabolism.     

Simple isocratic high-performance liquid chromatographic method for the separation of the six vitamers of vitamin B6

A simple, sensitive and fast isocratic high-performance liquid chromatographic method has been developed for the separation of all six biologically active forms (vitamers) of vitamin B6. The separation is accomplished using a strong cation-exchange column and a mobile phase of 0.1 M ammonium dihydrogenphosphate adjusted to pH 4.0. All six vitamers are separated within 20 min at a flowrate of 1 ml/min. The concentration of the vitamers is determined with a fluorescence detector (excitation 290 nm; emission 389 nm). The within-run precision of the method expressed as the coefficient of variation is below 5% at the 25 pmol level. Pyridoxal 5'-phosphate can be determined using either pre- or post-column derivatization with sodium bisulfite. Application of the method to cell-free yeast culture media is presented.     

Determination of vitamin B5 in human urine by high-performance liquid chromatography coupled with mass spectrometry

In the present work, we have developed a simple and rapid liquid chromatography/mass spectrometry (LC/MS) method for the identification and quantification of vitamin B5 in human urine. Urine was spiked with vitamin B5 internal standard, hopantenic acid (HOPA), and then diluted with the LC mobile phase prior to its analysis by LC/MS. The quantification was performed in single ion monitoring mode. The calibration curve was linear (r2 = 0.999) between 0.25 to 10 microg/mL. With a limit of detection of 0.1 microg/mL the method was sensitive enough to determine low levels of vitamin B5 in urine. The overall quantitative efficiency of the method was evaluated by spiking urine samples with four different concentrations of vitamin B5; the intra-assay coefficient of variation was below 5% and the recoveries were between 96 to 108%. The results of the present study show that the proposed method is selective and sensitive enough for the quantification of vitamin B5 in urine.     

Determination of folate vitamers in food and in Italian reference diet by high-performance liquid chromatography.

A trienzyme treatment (conjugase, alpha-amylase, protease) followed by affinity chromatography and reversed-phase HPLC with UV and fluorescence detection was performed for the quantification of folate vitamers in legumes (chickpea and beans), processed meats (salami Milano and Parma ham) and in an Italian reference diet. This method allowed a good separation of six folate vitamers: 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, folic acid, 10-formylfolic acid, 10-formyldihydrofolate and tetrahydrofolate within 30 min. Recovery, reproducibility and limits of detection of the method are reported. HPLC results were 24-52% lower than the microbiological assay findings.     

Determination of vitamin B12 in multivitamin tablets by multimode high-performance liquid chromatography

Quantitative determination of vitamin B₁₂ in B-complex tablets was performed by using multimode high-performance liquid chromatography. The multivitamin tablets (B₁, B₆ and B₁₂) were sonicated for 30 min in methanol–water (50:50, v/v) and diluted to appropriated volume with the same solvent. The resulting solution was filtered and the filtrate was analysed on a phenylpropanolamine bonded silica column (15 cm×4.6 mm I.D., 5 μm). The optimized mobile phase was 30 mM phosphate buffer (pH 3.00) containing 6% (v/v) acetonitrile at a flow-rate of 1 ml min⁻¹ and the detection was measured at 361 nm. The calibration graph prepared using standards was linear from 0.05 to 0.25 μg. The determination limit was 25 ng, the relative standard deviation was 0.47% and recovery from tablet solution was 100%. An analysis was completed in 5 min. The new method is simple, rapid and precise.     

Separation and quantification of the B6 vitamers in plasma and 4-pyridoxic acid in urine of adolescent girls by reversed-phase high-performance liquid chromatography

The vitamin B6 status of seemingly healthy adolescent girls was determined using several accepted and proposed parameters in an effort to establish guidelines for status evaluation. High-performance liquid chromatography-derived plasma B6 vitamers (pyridoxal phosphate, PLP; pyridoxine phosphate. PNP; pyridoxamine phosphate, PMP; pyridoxal, PL; pyridoxine, PN; and pyridoxamine, PM) and 4-pyridoxic acid (4-PA) concentrations and urinary 4-PA levels of 28 white adolescent females, 12-15 years, having radiomonitored plasma PLP concentrations and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate status were determined. Mean vitamin B6 and protein intakes were 1.48 mg and 78.3 g. Ranges for plasma B6 vitamer and 4-PA concentrations (nmol/l) were: PLP, 40.9-122.2; PNP, non-detectable (ND)-16.1; PMP, ND-8.1; PL, ND-15; PN, ND-21.9; PM, ND-17.8; and 4-PA, ND-55.7. PLP was the only vitamer found in plasma of all subjects. Urinary 4-PA concentrations ranged from 0.11 to 2.50 mumol/mmol of creatinine. B6 vitamer values of these girls should be of use in the establishment of normal ranges for vitamin B6 status parameters.     

Determination of coenzyme Q10 in human breast milk by high-performance liquid chromatography

An isocratic HPLC method was developed for the determination of coenzyme Q(10) (CoQ(10)) in human breast milk. After a single-step liquid-liquid extraction, the milk extract was injected directly into the HPLC system. The analytical method is based on pre-column inline treatment of CoQ(10). Chromatographic separation of CoQ(10) and coenzyme Q(9) (CoQ(9)) internal standard was achieved using a reversed-phase Microsorb-MV C(18) analytical column. CoQ(10) and CoQ(9) were monitored by an electrochemical detector (ECD). An excellent linearity (r = 0.999) was observed for CoQ(10) in the concentration range 0.06-2.5 micromol L(-1) in breast milk. The limit of quantitation (LOQ) was 60 nmol L(-1). Coefficients of variations (CVs) for intra-day and inter-day assay precisions were less than 5%. A total of 194 breast milk samples were analyzed for the CoQ(10) concentration; the mean value was 0.32 +/- 0.21 micromol L(-1).     

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.     

Determination of 6-methyladenine in DNA by high-performance liquid chromatography.

A method for the determination of 6-methyladenine (6MA) by high-performance liquid chromatography (HPLC) has been developed. DNA bases were separated by using the strong cation-exchange resin Zipax SCX. Purine bases were obtained by hydrolysis and dialysis of DNA and analysed by HPLC. 6MA in DNA from Escherichia coli was determined by the proposed method. It is suggested that the method could be applicable to analyses of 6MA from other biological sources.     

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.     

Phenotyping of bovine milk proteins by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the separation of the most common and some less common genetic variants of the bovine caseins is described. When the method is used for analysing clarified skim milk, simultaneous identification of casein variants and major they protein variants can be effected in a single run. The potential of the method for quantitative application is discussed.     

Determination of cephalosporins in raw bovine milk by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method based on solid-phase extraction was developed for the determination of cefazolin, cefoperazone, cefquinome and ceftiofur in raw bovine milk. The milk fat was removed by centrifugation and the cephalosporins were extracted in acetonitrile. The extract was cleaned up by solid-phase extraction on an octadecyl sorbent. The compounds were separated by ion-paired gradient HPLC on a phenyl column with ultraviolet detection at 270 nm. The limits of detection estimated by a conservative model were 11 microg/kg for cefazolin and cefoperazone and 7 microg/kg for cequinome and ceftiofur. The mean recoveries were 86-88% for cefazolin, 91-93% for cefoperazone, 69-72% for cefquinome and 84-88% for ceftiofur in the concentration range 20-200 microg/kg.