Determination of B-group vitamins in Turkish honey using ultra-performance liquid chromatography with electrospray ionization coupled to tandem mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry method with electrospray ionization (UPLC–ESI–MS/MS) for analysis of B-group vitamins in honey has been presented. Aim of this study is the characterization of different types of Turkish honeys according to B-group vitamins. Vitamins were determined in 17 different types of Turkish honey samples by UPLC–ESI–MS/MS. Heather honey samples were distinguished among the studied honeys with the richest vitamin content with 286.10?mg/kg, and it is followed by sunflower honey and thyme honey with the total vitamin contents of 206.01 and 163.27?mg/kg, respectively. The presence of vitamin B₁ (thiamine), vitamin B₂ (riboflavin), vitamin B₃ (nicotinamide, B₃N and nicotinic acid, B₃H), vitamin B₆ (pyridoxine), and vitamin B₉ (folic acid) was detected in all the honey samples analyzed. Moreover, vitamin B₅ (pantothenic acid) was observed in most of them. Vitamin B₁₂ (cyanocobalamin) and vitamin B₇ (biotine) were not detected in the studied honey samples. Turkish honey samples showed efficacious vitamin content for the consumers.     

RP-HPLC determination of water-soluble vitamins in honey

The assessment and validation of reliable analytical methods for the determination of vitamins in sugar-based matrices (e.g. honey) are still scarcely explored fields of research. This study proposes and fully validates a simple and fast RP-HPLC method for the simultaneous determination of five water-soluble vitamins (vitamin B₂, riboflavin; vitamin B₃, nicotinic acid; vitamin B₅, pantothenic acid; vitamin B₉, folic acid; and vitamin C, ascorbic acid) in honey. The method provides low detection and quantification limits, very good linearity in a large concentration interval, very good precision, and the absence of any bias. It has been successfully applied to 28 honey samples (mainly from Sardinia, Italy) of 12 different botanical origins. While the overall amount of the analytes in the samples is quite low (always below 40 mg kg⁻¹), we have observed a marked dependence of some of their concentrations (i.e. vitamin B₃ and vitamin B₅) and the botanical origin of the honey. This insight might lead to important characterization features for this food item.     

Study on the inclusion interactions of β-cyclodextrin and its derivative with dyes by spectrofluorimetry and its analytical application

The inclusion interactions of β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) with dyes were developed by spectrofluorimetry, and the inclusion constants of inclusion complexes were determined by direct fluorescence technique. The main factors (the host molecule, the guest molecule, and the pH) for the inclusion interaction were discussed in detail. At the same time, the inclusion interaction of HP-β-CD and vitamin B₆ (VB₆) was investigated with the competitive fluorescence inclusion method and the inclusion constant of HP-β-CD and vitamin B₆ (VB₆) was obtained by indirect fluorescence technique. On the basis of the linear relationship between the change of fluorescence intensity (ΔF) and the concentration of VB₆, a competitive fluorescence inclusion method was used to the determination of VB₆. The method has been successfully applied to the analysis of VB₆ in synthetic samples, tablets and injections with satisfactory results.     

Liquid chromatographic analysis of vitamin B6 in soy-based infant formula

A liquid chromatographic (LC) method is described for determination of total vitamin B6 in soy-based infant formula. Total vitamin B6 is quantitated by using ion-pair LC after precolumn transformation of phosphorylated and free vitamers into pyridoxol. The limit of detection is 0.3 ng and the limit of quantitation is 1.0 ng on-column (injection volume = 100 microL). Linear response ranged from 39 to 616 ng/mL (r2 = 0.99986). Analysis of a soy-based infant formula control fortified at 6 different concentration levels gave recoveries that averaged 104%. Assay of SRM 1846 gave results within the certified range (8.6 +/- 0.086 mg/kg versus the certified value of 8.4 +/- 1.0 mg/kg). The method provides a rapid and specific assay for the analysis of total vitamin B6 in fortified soy-based infant formula.     

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.     

LC Simultaneous Determination of the Free Forms of B Group Vitamins and Vitamin C in Various Fortified Food Products

An LC–UV screening method for simultaneous determination of ascorbic acid (C), and the free forms of thiamine (B₁) riboflavin (B₂), niacin (B₃), pyridoxine (B₆) in enriched food products was developed and validated. The chromatographic separation was accomplished within 18 min using a gradient of water with 0.1% formic acid (pH 2.5) and methanol with 0.1% formic acid on a C₁₈ reverse phase column (5 μm, 150 × 3.2 mm) while detection was performed at two wavelengths (266 and 290 nm). Sample preparation was based on an extraction method originally developed for vitamin C. This procedure besides extracting vitamin C was extended to the extraction of the free forms of vitamins B₁, B_2, B_3, B₆ and B₉. The developed analytical method was successfully applied for the simultaneous determination of the vitamin C content along with the free vitamin B forms of three different enriched food products.

     

LC Simultaneous Determination of the Free Forms of B Group Vitamins and Vitamin C in Various Fortified Food Products

An LC–UV screening method for simultaneous determination of ascorbic acid (C), and the free forms of thiamine (B₁) riboflavin (B₂), niacin (B₃), pyridoxine (B₆) in enriched food products was developed and validated. The chromatographic separation was accomplished within 18 min using a gradient of water with 0.1% formic acid (pH 2.5) and methanol with 0.1% formic acid on a C₁₈ reverse phase column (5 μm, 150 × 3.2 mm) while detection was performed at two wavelengths (266 and 290 nm). Sample preparation was based on an extraction method originally developed for vitamin C. This procedure besides extracting vitamin C was extended to the extraction of the free forms of vitamins B₁, B_2, B_3, B₆ and B₉. The developed analytical method was successfully applied for the simultaneous determination of the vitamin C content along with the free vitamin B forms of three different enriched food products.     

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.     

Determination of Vitamin B12 in Multivitamin Tablets by High Performance Liquid Chromatography

Abstract

Two procedures for separation and determination of vitamin B₁₂ in multivitamin tablets by reversed phase high performance liquid chromatography are proposed. Sample preparation is very simple: tablets are dissolved in distilled water, centrifuged and filtered. The sample solution is directly applied in the sample loop injector and chromatograms are obtained with gradient elution using water-methanol and water-acetonitrile as solvents. The peak of vitamin B₁₂ from samples of B-complex tablets is well separated with the two procedures. For multivitamin tablets, however, only the procedure with water and methanol as solvents was good for separation and quantification of vitamin B₁₂. Both procedures were verified by the standard addition method and also compared to a previously developed method using electrothermal atomic absorption spectrometry for vitamin B₁₂ determination.     

HPLC-ESI-MS analysis of Vitamin B12 in food products and in multivitamins-multimineral tablets

Methods for the determination of Vitamin B₁₂ remain limited due to their low sensitivity and poor selectivity. In the present work, a simple and sensitive HPLC-ESI-MS method for determining Vitamin B₁₂ in food products and in multivitamin-multimineral tablets was developed. Vitamin B₁₂ was extracted from food products with 50 mM sodium acetate buffer (pH 4.0) in the presence of sodium cyanide. Total Vitamin B₁₂ content in food product can be obtained by efficient enzymatic hydrolysis to release the bound Vitamin B₁₂. Vitamin B₁₂ was quantified with ginsenoside Re as internal standard (I.S.) after their separations on a C₁₈ column with a gradient of mobile phase made of water and acetonitrile. MS with SIR mode at m/z 930.8 was used for Vitamin B₁₂ quantification. The calibration graphs plotted with five concentrations of Vitamin B₁₂ was linear with a regression coefficient r² = 0.9994. The intra-assay R.S.D. and the inter-assay R.S.D. were 2.6% (n = 5) and 3.5% (n = 6), respectively. The recoveries evaluated at spiking three different concentrations on fortified products were above 93%. The method has been applied successfully in the determination of Vitamin B₁₂ in fortified food products and multivitamin-multimineral tablets.     

Water-soluble vitamins

Simultaneous Determination of Vitamins.--Klejdus et al. described a simultaneous determination of 10 water- and 10 fat-soluble vitamins in pharmaceutical preparations by liquid chromatography-diode-array detection (LC-DAD). A combined isocratic and linear gradient allowed separation of vitamins in 3 distinct groups: polar, low-polar, and nonpolar. The method was applied to pharmaceutical preparations, fortified powdered drinks, and food samples, for which results were in good agreement with values claimed. Heudi et al. described a separation of 9 water-soluble vitamins by LC-UV. The method was applied for the quantification of vitamins in polyvitaminated premixes used for the fortification of infant nutrition products. The repeatability of the method was evaluated at different concentration levels and coefficients of variation were <6.5%. The concentrations of vitamins found in premixes with the method were comparable to the values declared. A disadvantage of the methods mentioned above is that sample composition has to be known in advance. According to European legislation, for example, foods might be fortified with riboflavin phosphate or thiamin phosphate, vitamers which are not included in the simultaneous separations described. Vitamin B2.--Vi?as et al. elaborated an LC analysis of riboflavin vitamers in foods. Vitamin B2 can be found in nature as the free riboflavin, but in most biological materials it occurs predominantly in the form of 2 coenzymes, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD). Several methods usually involve the conversion of these coenzymes into free riboflavin before quantification of total riboflavin. According to the authors, there is growing interest to know flavin composition of foods. The described method separates the individual vitamers isocratically. Accuracy of the method is tested with 2 certified reference materials (CRMs). Vitamin B5.-Methods for the determination of vitamin B5 in foods are limited because of their low sensitivity and poor selectivity. Pakin et al. proposed a post-column derivatization of pantothenic acid as a fluorescent compound and used this principle in a specific and sensitive method for the determination of free and bound pantothenic acid in a large variety of foods. A French laboratory invited European laboratories to participate in a series of collaborative studies for this method, which will be carried out in 2005/2006. A more sophisticated method was described by Mittermayer et al. They developed an LC-mass spectrometry (LC/MS) method for the determination of vitamin B5 in a wide range of fortified food products. Application of the method to various samples showed consistent results with those obtained by microbiology. Vitamin B6.-Method 2004.07, an LC method for the analysis of vitamin B6 in reconstituted infant formula, was published by Mann et al. In contrast with this method, which quantifies vitamin B6 after converting the phosphorylated and free vitamers into pyridoxine, Vi?as et al. published an LC method which determines 6 vitamin B6 related compounds, the 3 B6 vitamers, their corresponding phosphorylated esters, and a metabolite. Accuracy was determined using 2 CRMs. Results were within the certified ranges. Vitamin C.-Franke et al. described an extensive study to vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. Vitamin C was determined by measuring ascorbic acid in its reduced state by LC and coulometric detection along with UV absorbance detection at 245 nm. No attempts were made to assess levels of dehydroascorbic acid. Most recent research revealed that cell uptake of dehydroascorbic acid is unlikely to play a major role, which may explain the very low vitamin C activity of orally administered L-dehydroascorbic acid in rats. The food levels found by Franke et al. are variably lower, higher, or equal in comparison to other studies. Iwase described a method for the determination of ascorbic acid in foods using L-methionine for the pre-analysis sample stabilization. Electrochemical detection was used for the quantification. Traditionally, metaphosphoric acid was proven to be a useful dissolving agent for the determination of ascorbic acid. However, it dissolves in water very slowly, it is hygroscopic, and accurate weighing is not easy. Adjustment at pH 2-3 takes a long time. It appeared to be possible to replace metaphosphoric acid by 0.2% phosphoric acid. Methionine played an important role on the stability of ascorbic acid. The method seemed to be applicable to the routine analysis of ascorbic acid in foods. Folic Acid.-Microbiological analysis of total folate in foods is often considered as the golden standard compared to other methods based on, for example, LC. Koontz et al. showed results of total folate concentrations measured by microbiological assay in a variety of foods. Samples were submitted in a routine manner to experienced laboratories that regularly perform folate analysis fee-for-service basis in the United States. Each laboratory reported the use of a microbiological method similar to the AOAC Official Method for the determination of folic acid. Striking was, the use of 3 different pH extraction conditions by 4 laboratories. Only one laboratory reported using a tri-enzyme extraction. Results were evaluated. Results for folic acid fortified foods had considerably lower between-laboratory variation, 9-11%, versus >45% for other foods. Mean total folate ranged from 14 to 279 microg/100 g for a mixed vegetable reference material, from 5 to 70 microg/100 g for strawberries, and from 28 to 81 microg/100 g for wholemeal flour. One should realize a large variation in results, which might be caused by slight modifications in the microbiological analysis of total folate in foods or the analysis in various (unfortified) food matrixes. Furthermore, optimal combination of enzymes and reaction conditions may vary depending on the composition of the food. Padrangi and Laborde showed recently that treatment with alpha-amylase had no significant effect on measured folate in spinach, although addition of protease significantly increased the release of folate. LC/MS applications gain increasing attention because of their specificity. Rychlik used stable isotope dilution assays for the determination of the folate content of broccoli and bread. Compared to data in the literature and food data bases, amounts were significantly lower. Pawlosky et al., however, found comparable values for 5-methyltetrahydrofolic acid and folic acid by HPLC analysis with fluorescent detection and HPLC/MS. Among samples analyzed were CRMs and broccoli. Besides folic acid, other water-soluble vitamins were also determined by LC/MS/MS by Leporati et al. The method was applied to the quantitative analysis of the natural content of vitamins in typical Italian pasta samples, as well as in fortified pasta samples produced for the U.S. market. Biotin.-A paper from Staggs et al. included the assertion that existing biotin data in food composition tables are inaccurate because the majority are based on bioassays with all relevant disadvantages. Data in most cases are overestimated with consequences for recommendations for dietary biotin intake. An HPLC/avidin-binding assay was used to analyze 87 foods to support the hypothesis mentioned.     

An immediate luminescence enhancement method for determination of vitamin B1 using long-wavelength emitting water-soluble CdTe nanorods

A method for determination of vitamin B₁ has been developed that is based on the enhancement effect of vitamin B₁ on the luminescence of water-soluble CdTe nanorods modified with thioglycolic acid and cysteine. The effect of variables including the size of the nanorods on the enhancement of luminescence have been investigated. A preliminary mechanistic study showed that the passivating action of vitamin B₁ on the surface of the CdTe nanorods is likely to be responsible for the enhancement. Interferences by shortwave fluorescence are effectively eliminated because measurements are performed in the near-infrared. Due to the near-infrared measurement character, the fluorescence interference of vitamin B₂ can be effectively eliminated. Under the optimum conditions, the extent of luminescence enhancement is proportional to the concentration of vitamin B₁ in the range from 0.1 to 3.0 μmol L^?1 and the detection limit is 0.03 μmol L^?1. The relative standard deviation for 2.0 μmol L^?1 vitamin B₁ is 1.3% (n?=?6). The method is highly sensitive and selective, avoids the sample treatment needed in other procedures, and can be applied to the determination of vitamin B₁ in real samples with satisfactory results.     

Dipstick based immunochemiluminescence biosensor for the analysis of vitamin B12 in energy drinks: A novel approach

In this article, we describe a dipstick based immunochemiluminescence (immuno-CL) biosensor for the detection of vitamin B₁₂ in energy drinks. The method is a direct competitive type format involving the immobilization of vitamin B₁₂ antibody on nitrocellulose membrane (NC) followed by treatment with vitamin B₁₂ and vitamin B₁₂–alkaline phosphatase conjugate to facilitate the competitive binding. The dipstick was further treated with substrate disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate (CDP-Star) to generate chemiluminescence (CL). The number of photons generated was inversely proportional to the vitamin B₁₂ concentration. After systematic optimization, the limit of detection was 1 ng mL⁻¹. The coefficient of variation was below 0.2% for both intra- and inter-assay precision. Vitamin B₁₂ was extracted from energy drinks with recovery ranged from 90 to 99.4%. Two different energy drinks samples were analyzed, and a good correlation was observed when the data were compared with a reference enzyme linked immuno sorbent assay (ELISA) method. The developed method is suitable for an accurate, sensitive, and high-throughput screening of vitamin B₁₂ in energy drinks samples. The dipstick technique based on immuno-CL is suitable for the detection of several analyte in food and environmental samples.     

Determination of vitamin B<Subscript>1</Subscript> in seawater and microalgal fermentation media by high-performance liquid chromatography with fluorescence detection

A highly sensitive high-performance liquid chromatographic method with fluorescence detection has been developed for determination of vitamin B₁. Vitamin B₁ was converted into a fluorescent compound by treatment with hydrogen peroxide–horseradish peroxidase and the derivative was subsequently analyzed by HPLC on a Waters Spherisorb ODS2 column (250 mm×4.6 mm ID, 5 μm) with 40:60 methanol–pH 8.5 acetate buffer solution as mobile phase and fluorescence detection at 440 nm (with excitation at 375 nm). The calibration graph was linear from 5.00×10⁻¹⁰ mol L⁻¹ to 5.00×10⁻⁷ mol L⁻¹ for vitamin B₁ with a correlation coefficient of 0.9991 (n=9). The detection limit was 1.0×10⁻¹⁰ mol L⁻¹. The method was successfully used for determination of vitamin B₁ at pg mL⁻¹ levels in microalgal fermentation media and seawater after solid-phase extraction. Recovery was from 89 to 110% and the relative standard deviation was in the range 1.1 to 4.3%.     

Indirect Determination of Organic Compounds by Atomic Absorption Spectrophotometry: The Study of Vitamin B12 in Pharmaceutical Dosage Forms

Abstract

A method for the determination of vitamin B₁₂ in pharmaceutical dosage forms is presented. The method involves, dissolution of the vitamin B₁₂ and subsequent determination of the complexed cobalt by atomic absorption spectrophotometry. The method is both rapid and sensitive giving applications to quality, control of this type of formulation.     

Investigation on the Inclusion Behavior of ApoCopC with Vitamin B6

In neutral phosphate buffer solution of pH 7.0, the interaction between apoCopC and Vitamin B₆ has been investigated in detail by means of fluorescence spectroscopy. According to the change of Vitamin B₆ fluorescence spectra and fluorescence polarization, we can conclude that a novel supramolecular system is generated. ApoCopC can form a 1:5 host-guest inclusion supramolecular complex with Vitamin B₆, and the formation constant has been calculated to be (2.24 ± 0.08) × 10⁴ M^? 1. It suggests the strong inclusion ability of apoCopC to the guest molecules. In addition, the mechanism of the apoCopC protein fluorescence quenching by Vitamin B₆ was also discussed. And based on the Stern-Volmer equation, the apparent quenching constant was estimated to be (2.75 ± 0.05) × 10⁴ M^? 1.     

Liquid chromatography of vitamin B6 with electrochemical detection using a carbon fibre electrode

A flow-through electrochemical detector with a carbon fibre electrode was fabricated. A chromatographic method for the determination of vitamin B₆ using this detector is described, and the results are compared with those obtained by detection with a thin-layer glassy carbon electrode or spectrophotometry. The detection limit of vitamin B₆ is 2.5 ng for pyridoxine, 1 ng for pyridoxal and 1 ng for pyridoxamine. The technique was applied to the determination of vitamin B₆ in tablets.     

Effects of γ-irradiation and cooking on vitamins B6 and B12 in grass prawns (Penaeus monodon)

The effects of irradiation doses, irradiation temperature and a combined treatment of irradiation and cooking on the vitamin B₆ and B₁₂ contents of grass prawns have been studied. Grass prawns were irradiated at refrigerated (4°C) or frozen (-20°C) temperatures with different doses. A domestic cooking procedure was followed after irradiation. The changes in vitamins B₆ and B₁₂ of both raw and cooked grass prawns were evaluated. Results showed no significant changes of vitamin B₆ and B₁₂ in grass prawns with a radiation dose up to 7 kGy at either 4°C or -20°C. Irradiation at 4°C caused more destruction of vitamin B₁₂ but not vitamin B₆ than did irradiation at -20°C in grass prawns. There was significant destruction of both vitamins B₆ and B₁₂ in unirradiated samples during cooking. The introduction of the irradiation process before cooking had no effect on either vitamin. These results indicate that the loss of vitamins B₆ and B₁₂ in the combined treatments was caused mainly by thermal destruction.     

Validation of analytical methods for determining mycotoxins in foodstuffs

The European Union (EU) has established demanding regulatory limits for controlling aflatoxins B₁, B₂, G₁ and G₂, in cereals, nuts, nut products and dried fruit, aflatoxin M₁ in milk, and ochratoxin A in cereals. These limits are likely to be extended in the future to additional commodities and other mycotoxins. For enforcement purposes and in particular for resolving any disputes between parties, it is essential that validated methods are available, with performance characteristics that meet certain minimum criteria. As such methods were not available and had not previously been validated either for matrices of interest in Europe or at the low European limits compared to the USA, the EU funded a method-validation project to fulfil this requirement. Immunoaffinity column clean-up methods with HPLC determination were established for aflatoxins B₁, B₂, G₁ and G₂ in peanut butter, pistachios, fig paste and paprika, aflatoxin B₁ in baby food, aflatoxin M₁ in liquid milk, and ochratoxin A in roasted coffee and baby food. For patulin in apple juice and apple puree, solvent extraction and solid-phase clean-up HPLC methods were developed. To undertake collaborative studies, particular care was taken in preparation of naturally-contaminated test materials containing the toxins at levels close to regulatory limits and in demonstrating the homogeneity of batches of material. To ensure that participants in the validation exercise could follow the procedures to be tested, videos of the methods were prepared showing, in particular, any critical steps. Prior to undertaking the method validation, participants were invited to collaborative study workshops to ensure that they fully understood the methods and their role in the study. This care in planning and executing the collaborative studies led to impressive performance characteristics and adoption of six procedures by AOAC International as First Action Methods and seven methods by CEN as European standards. The valuable lessons learned in undertaking these validation exercises are now being put to further use in studies aimed at validating methods for mycotoxins in foodstuffs, which are appropriate for developing countries based on TLC as the end determination but use more modern sample clean-up techniques.     

Voltammetric determination of B<Subscript>1</Subscript> and B<Subscript>6</Subscript> vitamins using a pencil graphite electrode

Due to the importance of B₁ and B₆ vitamins for human health it is useful to develop new cheap and rapid methods for their determination. Voltammetric behavior of these vitamins on a pencil graphite electrode was investigated using cyclic voltammetry in different media. Direct quantitative determination of the two vitamins, one in the presence of the other, was done by differential pulse voltammetry. Vitamin B₁ was electroactive only in a NaOH solution generating two irreversible oxidation peaks; the first peak obtained at 250 mV is well-defined and was used in quantitative determinations. In case of vitamin B₆, a well-defined oxidation peak was observed in all investigated supporting electrolytes except for HCl. The linear concentration ranges were 10^?5–10^?3 M for vitamin B₁ in a NaOH solution and 5 × 10^?6–10^?3 M for vitamin B₆ in an acetate buffer solution. The obtained detection limits were 5.34 × 10^?6 M and 2.81 × 10^?6 M for vitamin B₁ and vitamin B₆, respectively. The developed method is simple and rapid and it was successfully applied in the determination of the two vitamins in pharmaceuticals.