Plasma B-6 vitamer and plasma and urinary 4-pyridoxic acid concentrations in young women as determined using high performance liquid chromatography.

Plasma B-6 vitamer and plasma and urinary 4-pyridoxic acid concentrations of 21 young white women, 21-27 years, having radiomonitored pyridoxal 5'-phosphate and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate vitamin B-6 status were determined in an effort to establish normal ranges for plasma B-6 vitamers. B-6 vitamers and 4-pyridoxic acid were quantitated using reversed phase high performance liquid chromatography with fluorometric and ultraviolet detection. Pyridoxal phosphate values obtained by radioenzymatic and chromatographic, fluorometric and ultraviolet, assays were highly correlated as were pyridoxine phosphate values determined using both detectors. The B-6 vitamer and 4-pyridoxic acid values of these subjects should be of use in the establishment of normal ranges of these congeners in women.     

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 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.     

Plasma B6 vitamer and plasma and urinary 4-pyridoxic acid concentrations of middle-aged obese black women.

Plasma B6 vitamer and plasma and urinary 4-pyridoxic acid (4-PA) concentrations of fifteen middle-aged obese black women were determined by high-performance liquid chromatography (HPLC). Estimated protein and vitamin B6 intakes of the subjects, aged 27-52 years, were 64.5 +/- 15.6 g and 1.21 +/- 0.68 mg (mean +/- S.D.), respectively. Mean HPLC-derived plasma B6 vitamer and 4-PA concentrations for these subjects were 68.9, 3.1, 1.2, 4.1, 3.4, 7.2 and 2.0 nmol/l for pyridoxal 5'-phosphate (PLP), pyridoxine 5'-phosphate, pyridoxamine 5'-phosphate, pyridoxal, pyridoxine, pyridoxamine and 4-PA, respectively. The mean urinary 4-PA/creatinine ratio of the women was 0.88 mumol/mmol. All subjects had plasma PLP levels indicative of adequate vitamin B6 status. Vitamin B6 status parameters of the middle-aged obese black women were similar to those previously reported for white nonobese women having adequate vitamin B6 status.     

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 pyridoxal 5'-phosphate in human serum by reversed phase high performance liquid chromatography combined with spectrofluorimetric detection of 4-pyridoxic acid 5'-phosphate as a derivative

A very sensitive spectrofluorimetric method for the determination of pyridoxal 5'-phosphate (PLP) in human serum is described. The specificity is based on the selective oxidation of PLP to 4-pyridoxic acid 5'-phosphate with potassium cyanide. Separation of the highly fluorescent 4-pyridoxic acid 5'-phosphate is achieved by reversed-phase high-performance liquid chromatography. Specificity is improved by a careful choice of fluorescence filters, maximised at the excitation (325 nm) and emission (418 nm) wavelengths of 4-pyridoxic acid 5'-phosphate. The detection limit for the reaction is 0.22 ng ml(-1). For quantification, the serum is spiked with PLP before protein precipitation with 3.3% m/V trichloroacetic acid. The method can be used for the determination of PLP in serum, even in vitamin B6 deficient patients. The mean value for human serum PLP from 30 healthy adults was found to be 14.6 +/- 4.8 ng ml(-1) (mean +/- standard deviation).     

‘Mass spectrometry of vitamin B6: Different forms of the vitamin,its metabolites,antimetabolites, and analogs’

The mass spectra of the different forms of vitamin B₆, some metabolites and antimetabolites, and some analogs are reported. The free bases are suitable for study, but the 5-phosphates are not sufficiently volatile. The hydrochloride salts dissociate thermally in the inlet system into the free base and HCl and give the spectrum of the free base with the spectrum of HCL superimposed on it. The compounds studied are: pyridoxol, pyridoxamine and pyridoxal, which are different forms of vitamin B₆; 4-pyridoxic acid and its lactone, 5-pyridoxic acid lactone, isopyridoxal, 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic acid, and 3-hydroxy-2-methylpyridine-4,5-dicarboxylic acid, which are vitamin B₆ metabolites; α⁴-and α⁵-deoxypyridoxol, 3-deoxypyridoxol, and 4- and 5- norpyridoxol, which are antimetabolites and analogs of vitamin B₆.     

Photophysics and Photochemical Studies of the Vitamin B6 Group and Related Derivatives

The photophysics and photochemical properties of vitamin B6 constituents and analogs were studied as function of pH and solvent. The p K of the phenolic oxygen and the pyridine ring nitrogen depends on the electron donor-acceptor ability of the 4-substituent, and agrees with the calculated proton affinity. For all studied compounds, the fluorescence properties showed that the phenolic oxygen is 8 units more acidic in the lowest singlet excited state than in the ground state. The pyridine N-atom is slightly more basic in the excited state. At pH of biological significance, pH 6–8, pyridoxamine and 4-pyridoxic acid are the more efficient chromophores with higher fluorescence yield and longer lifetime. Spectroscopic studies showed that the tautomeric equilibrium depends on the nature of the 4-substituent. The quenching of the singlet excited state of pyridoxamine and 4-pyridoxic acid by amino acids, free or in a peptide, and DNA bases at pH 7 was studied by time-resolved fluorescence techniques. The quenching rate constants are well correlated with the redox properties of the pyridoxinic compound and amino acids, and are related to the free energy change in the electron transfer process. Guanosine and pyrimidine bases also are efficient quenchers, involving an electron transfer reaction.     

Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in Human Hair Using LC-MS/MS

Water-soluble B vitamins participate in numerous crucial metabolic reactions and are critical for maintaining our health. Vitamin B deficiencies cause many different types of diseases, such as dementia, anaemia, cardiovascular disease, neural tube defects, Crohn’s disease, celiac disease, and HIV. Vitamin B3 deficiency is linked to pellagra and cancer, while niacin (or nicotinic acid) lowers low-density lipoprotein (LDL) and triglycerides in the blood and increases high-density lipoprotein (HDL). A highly sensitive and robust liquid chromatography–tandem mass spectroscopy (LC/MS-MS) method was developed to detect and quantify a vitamin B3 vitamer (nicotinamide) and vitamin B6 vitamers (pyridoxial 5′-phosphate (PLP), pyridoxal hydrochloride (PL), pyridoxamine dihydrochloride (PM), pridoxamine-5′-phosphate (PMP), and pyridoxine hydrochloride (PN)) in human hair samples of the UAE population. Forty students’ volunteers took part in the study and donated their hair samples. The analytes were extracted and then separated using a reversed-phase Poroshell EC-C18 column, eluted using two mobile phases, and quantified using LC/MS-MS system. The method was validated in human hair using parameters such as linearity, intra- and inter-day accuracy, and precision and recovery. The method was then used to detect vitamin B3 and B6 vitamers in the human hair samples. Of all the vitamin B3 and B6 vitamers tested, only nicotinamide was detected and quantified in human hair. Of the 40 samples analysed, 12 were in the range 100–200 pg/mg, 15 in the range 200–500 pg/mg, 9 in the range of 500–4000 pg/mg. The LC/MS-MS method is effective, sensitive, and robust for the detection of vitamin B3 and its vitamer nicotinamide in human hair samples. This developed hair test can be used in clinical examination to complement blood and urine tests for the long-term deficiency, detection, and quantification of nicotinamide.     

Stereoselective high-performance liquid chromatographic assay with fluorometric detection for the isomers of mivacurium in human plasma.

A high-performance liquid chromatographic assay with fluorometric detection was developed for the analysis of the stereoisomers of mivacurium, a new short-acting neuromuscular blocker, in plasma. The isomers were isolated from plasma by solid-phase extraction with C18 and anion-exchange cartridges. The extracts were chromatographed on a LiChrosphere 60 RP Select B column (125 mm x 4.6 mm I.D.) using a mobile phase of acetonitrile-water (4:6, v/v) containing 0.005 M octanesulfonic acid. The fluorescence excitation and emission wavelengths were 202 and 320 nm, respectively. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from true values and the percentage coefficient of variation, respectively, were less than or equal to 10% at all concentrations except for the percentage coefficient of variation at the lower limit of quantitation (5 ng/ml). The assay has been successfully used for the analysis of plasma samples from a pharmacokinetic study in human volunteers.     

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.     

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.     

Quantitative profiling of biomarkers related to B‐vitamin status,tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry

Vitamins B₂ and B₆ serve as cofactors in enzymatic reactions involved in tryptophan and homocysteine metabolism. Plasma concentrations of these vitamins and amino acids are related to smoking and inflammation, and correlate with other markers of immune activation. Large‐scale studies of these relations have been hampered by lack of suitable analytical methods. The assay described includes riboflavin, five vitamin B₆ forms (pyridoxal 5′‐phosphate, pyridoxal, 4‐pyridoxic acid, pyridoxine and pyridoxamine), tryptophan and six tryptophan metabolites (kynurenine, kynurenic acid, anthranilic acid, 3‐hydroxykynurenine, xanthurenic acid and 3‐hydroxyanthranilic acid), cystathionine, neopterin and cotinine. Trichloroacetic acid containing 13 isotope‐labelled internal standards was added to 60 µL of plasma, the mixture was centrifuged, and the resulting supernatant used for analysis. The analytes were separated within 5 min on a stable‐bond C8 column by a gradient‐type mobile phase containing acetonitrile, heptafluorobutyric acid and high concentration (650 mmol/L) of acetic acid, and detected using electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The mobile phase ensured sufficient separation and high ionization efficiency of all analytes. Recoveries were 75–123% and within‐day and between‐day coefficients of variance (CVs) were 2.5–9.5% and 5.4–16.9%, respectively. Limits of detection ranged from 0.05 to 7 nmol/L. The method enables quantification of endogenous plasma concentrations of 16 analytes related to B‐vitamin status and inflammation, and may prove useful in large‐scale epidemiological studies. Copyright © 2009 John Wiley & Sons, Ltd.     

A new selective reagent for the spectrofluorometric determination of beryllium

The fluorescence properties of the beryllium and aluminum complexes with 2, 4-dioxo-4-(4-hydroxy-6-methyl-2-pyrone-3-yl) butyric acid ethyl ester (Ligand) were studied and optimal conditions for their fluorometric determination were established. Beryllium can be determined in the linearity range of 0.5–2.0 μg/ml and aluminum 0.5–1.5 μg/ml. The effect of diverse ions on the determination of beryllium is discussed.A simple procedure for the fluorometric determination of beryllium in human blood plasma in the concentration range of 5–50 μg Be/ml is described.     

维生素B1、B6与金属离子关系的光化学研究

分别研究了必需微量元素铁、锌、铜、锰、硒、常量元素钙、镁、非必需元素铝、锂，有毒元素尔，铅，镉对维生素B1、B6的紫外吸收光谱的影响，并对其机理进行了初步探讨。所得结果对于金属离子与维生素B1、B6的关系及其在人体中的生化、生理功能和生物有效性提供了一定的理论依据。     

Determination of vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection

A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.     

Synthesis of vitamin b6 derivatives. II. 3-Hydroxy-4-hydroxymethyl-2-methyl-5-pyridine acetic acid and related substances

The syntheses of 3-hydroxy-4-hydroxymethyl-2-methyl-5-pyridineacetic and -5-pyridine-propionic acids and several related compounds are described. Although acid hydrolysis of α⁴,3-O-isopropylidene-5-pyridoxic acid gives 5-pyridoxic acid lactone (α-pyracin), its higher homolog α⁴,3-O-isopropylidene-pyridoxylformic acid gave a corresponding free alcohol whose carboxylic acid proton was shown to be exchanged rapidly with the 3-phenolic and -4-alcoholic - protons in nuclear magnetic resonance studies. Inter-molecular hydrogen bonding between the side chain carboxylic acid and pyridine nitrogen atoms is suggested in the solid state.     

A solid-phase extraction method for high-performance liquid chromatographic determination of salvianolic acid B in rabbit plasma: application to pharmacokinetic study

A sensitive solid-phase extraction/high-performance liquid chromatographic method with ultraviolet detection was established for the analysis of salvianolic acid B in rabbit plasma. The analyte was separated on a reversed-phase column with trifluoroacetic acid-methanol-acetonitrile (70:10:20, v/v/v) as mobile phase at a flow rate of 1 mL/min, and ultraviolet detection at 315 nm. The calibration curve for salvianolic acid B was linear over the range 35-1400 microg/L with coefficients of correlation >0.999. The inter-day and intra-day precisions of analysis were <15%, and assay accuracy ranged from 95.3 to 109.1%. This method is suitable for determining salvianolic acid B in plasma and thus investigating the pharmacokinetics of salvianolic acid B.     

反相高效液相色谱法同时测定人血浆中的西诺沙星和萘啶酸

提出了同时测定人血浆中的西诺沙星和萘啶酸的高效液相色谱方法。采用反相C18柱、254nm紫外检测,乙腈－甲醇－0．01mol/L草酸（30：5：65,V／V;pH4．55）体系,实现了二者的良好分离。分别考察了磷酸缓冲溶液和草酸缓冲溶液作为流动相弱溶剂,结果草酸缓冲溶液能有效改善峰形、降低检测限。在不同pH值（pH2．80,4．08,4．55）进行了实验,pH4．55获得最佳分离,最低检测量分别为0．14ng（西诺沙星）和0．24ng（萘啶酸）,比文献报道的同类药物最低检测量低10倍。     

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.