Headspace conditions and ingredients can affect artefactual benzene formation in beverages.

A headspace sampling-gas chromatography/mass spectrometry (HS-GC/MS) method using mild HS conditions (40 °C, 30 min) was established, validated in terms of specificity, linearity (1.75-87.65 ng mL-1), precision (0.3-9.1% RSD), and accuracy (81.1-117.7%); and applied for the monitoring of 900 commercial beverage samples of six different types. These mild (low-temperature) conditions were compared with 1) optimized (high-temperature) conditions and 2) a liquid-phase microextraction method involving no heat treatment. This method was desirable because a high equilibrium temperature induced artefactual benzene formation from benzoate and ascorbic acid. In a 2IV8-3 fractional factorial design, eight variables-ascorbic acid, benzoate, benzaldehyde, Cu2+, Fe2+, riboflavin, pyridoxine, and heat treatment-were tested as potential factors affecting benzene formation. All variables except Fe2+ and pyridoxine significantly affected benzene formation, both individually and interactively. The present study suggests an accurate and reliable method for benzene analysis and provides strategies to prevent unintentional benzene formation in beverages.

The effect of UV-protected ethylene vinyl acetate (EVA) bags on the physicochemical stability of pediatric parenteral nutrition admixtures.

BACKGROUND: The safe administration of parenteral admixtures should be considered under the headings of physical and chemical stability. Vitamins are considered to be most susceptible to chemical degradation. OBJECTIVES: To evaluate the protective effect of UV-protected monolayer ethylene vinyl acetate (EVA) bags in comparison with that of EVA bags without UV protection, on the physicochemical characteristics and stability of the light sensitive vitamins in pediatric parenteral admixtures stored under various temperature and light conditions. METHODS: Four different parenteral pediatric admixtures (with trace elements and vitamins) in two types of ethylenovinylacetate (EVA) monolayer containers (with - yellow one and without - transparent one UV protection) were assessed. The physicochemical analyses such as visual inspection, pH and potential zeta measurements, lipid globules size distribution and vitamins concentration were performed at 0 h, 24 h, 8 days and 8 days+24 h after the preparation of the TPN admixtures. In order to quantify ascorbic acid, thiamine and pyridoxine levels, HPLC was used. RESULTS: No differences (p < 0.05) in physicochemical stability of TPN admixtures were noted between two types of EVA bags, with the compositions assessed; stored 8 days (4 °C ± 2) without light plus 24 h at room temperature and light exposure. However significant differences were noticed in ascorbic acid, thiamine and pyridoxine content after 8 days+24 h in comparison with t = 0. This was noted for both for UV-protected bags and bags without UV-protection, Nevertheless, amounts were still within the pharmacopeial range. CONCLUSIONS: Both EVA bags under test (with and without UV-protection) ensure physicochemical stability up 8 days at 4 °C ± 2 °C without light exposure and then 24 h at room temperature with light exposure for the total pediatric parenteral admixtures, intended for home parenteral nutrition. Graphical abstract Scheme of physicochemical analysis of parenteral admixtures.

Thermal decomposition of pyridoxine: an evolved gas analysis-ion attachment mass spectrometry study.

RATIONALE: Pyridoxine is an important vitamer in food and pharmaceutical products. Heat treatments applied during preparation or storage of the products cause the decomposition of pyridoxine. Identification and understanding of the degradation products of pyridoxine and studying its decomposition kinetics are essential in the preparation and preservation of pyridoxine-containing foods and pharmaceuticals. METHODS: Real-time, non-isothermal decomposition of pyridoxine was studied using evolved gas analysis-Li(+) ion attachment mass spectrometry (EGA-Li(+) IAMS). Arrhenius parameters for the thermal decomposition of pyridoxine were obtained via the total ion monitoring (TIM) curve. RESULTS: Most of the pyridoxine evaporated in molecular form, but the formation of pyridoxal and o-quinone methide, both biologically important species, was also observed from the solid-phase degradation of pyridoxine. The observation of o-quinone methide, a species possessing anticancer activity, was particularly noteworthy due to its chemical instability. The activation energy (E(a) ) for pyridoxine decomposition determined by EGA-IAMS was found to be 20.0 kcal mol(-1) , and the pre-exponential factor (A) was 5.7 × 10(9) min(-1) . CONCLUSIONS: The calculated kinetic parameters are important for predicting the thermal stability of pyridoxine vitamer. The estimated lifetime (t(90%,25°C) ) of 1.7 × 10(-2) years in nitrogen was also obtained from the EGA-IAMS experiment.

Chemical analysis and quality control of Ginkgo biloba leaves, extracts, and phytopharmaceuticals.

The chemical analysis and quality control of Ginkgo leaves, extracts, phytopharmaceuticals and some herbal supplements is comprehensively reviewed. The review is an update of a similar, earlier review in this journal [T.A. van Beek, J. Chromatogr. A 967 (2002) 21-55]. Since 2001 over 3000 papers on Ginkgo biloba have appeared, and about 400 of them pertain to chemical analysis in a broad sense and are cited herein. The more important ones are discussed and, where relevant, compared with the best methods published prior to 2002. In the same period over 2500 patents were filed on Ginkgo and the very few related to analysis are mentioned as well. Important constituents include terpene trilactones, i.e. ginkgolide A, B, C, J and bilobalide, flavonol glycosides, biflavones, proanthocyanidins, alkylphenols, simple phenolic acids, 6-hydroxykynurenic acid, 4-O-methylpyridoxine and polyprenols. In the most common so-called "standardised" Ginkgo extracts and phytopharmaceuticals several of these classes are no longer present. About 130 new papers deal with the analysis of the terpene trilactones. They are mostly extracted with methanol or water or mixtures thereof. Supercritical fluid extraction and pressurised water extraction are also possible. Sample clean-up is mostly by liquid-liquid extraction with ethyl acetate although no sample clean-up at all in combination with LC/MS/MS is gaining in importance. Separation and detection can be routinely carried out by RP-HPLC with ELSD, RI or MS, or by GC/FID or GC/MS after silylation. Hydrolysis followed by LC/MS allows the simultaneous analysis of terpene trilactones and flavonol aglycones. No quantitative procedure for all major flavonol glycosides has yet been published because they are not commercially available. The quantitation of a few available glycosides has been carried out but does not serve a real purpose. After acidic hydrolysis to the aglycones quercetin, kaempferol and isorhamnetin and separation by HPLC, quantitation is straightforward and yields by recalculation an estimation of the original total flavonol glycoside content. A profile of the genuine flavonol glycosides can detect poor storage or adulteration. Although the toxicity of Ginkgo alkylphenols upon oral administration has never been undoubtedly proven, most suppliers limit their content in extracts to 5 ppm and dozens of papers on their analysis were published. One procedure in which a methanolic extract is directly injected on a C8 HPLC column appears superior in terms of sensitivity (<5 ppm), separation, simplicity and validation and will be incorporated in the European Pharmacopoeia. Alternatively GC/MS and ELISA methods can be used. A sharp contrast to the plethora of papers on terpene trilactones, flavonol glycosides, and ginkgolic acids forms the low number of papers on biflavones, proanthocyanidins, simple phenolics, simple acids, and other constituents that make up the remaining 70% of Ginkgo standardised extracts. More research in this direction is clearly needed. For the analysis of Ginkgo proanthocyanidins (7%) for instance, no reliable assays are yet existing. Finally the growing literature on pharmacokinetic and fingerprinting studies of Ginkgo is briefly summarised.

Plasma folate as marker of folate status in epidemiological studies: the European Investigation into Cancer and Nutrition (EPIC)-Potsdam study.

Folate deficiency is often discussed as a potential risk factor for CVD and some cancers. Reliable assessment of folate status in large-scale epidemiological studies is therefore of major importance. The present study assessed the value of plasma folate (PF) compared with erythrocyte folate (EF) as a marker of folate status in 363 participants in the European Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort. EF and PF, total homocysteine (tHcy), pyridoxine, cobalamin, creatinine, total protein and packed cell volume were determined; glutamate carboxypeptidase (GCP) C1561T, reduced folate carrier (RFC) G80A and methylenetetrahydrofolate (MTHFR) C677T polymorphisms were analysed. Anthropometric measurements were taken and dietary intake was assessed with the EPIC-Potsdam food-frequency questionnaire. Comparison of EF and PF with factors that may modulate their concentrations was performed. Cross-classification of blood folates in quintile categories resulted in correct classification into the same or adjacent category of 75.5 % of all subjects. Age, BMI, pyridoxine and cobalamin, fruit and vegetable intake, and vitamin supplementation 24 h before blood draw were positively associated with EF and with PF. For tHcy an inverse association was found. Participants with the MTHFR 677TT genotype showed significantly elevated EF concentrations compared with those with 677CT genotype; EF and PF were more strongly correlated (r 0.78, P<0.0001) for participants with MTHFR 677TT genotype than for those with the 677CC or 677CT genotype. In summary, our present results indicate that plasma folate seems to be a suitable marker for assessment of folate status for use in large-scale epidemiological studies.

Uptake, hydrolysis, and metabolism of pyridoxine-5'-beta-D-glucoside in Caco-2 cells.

An important dietary source of vitamin B-6, pyridoxine-5'-beta-D-glucoside (PNG), exhibits only partial bioavailability, which is limited by the extent of enzymatic cleavage of the beta-glucosidic bond to release metabolically available pyridoxine (PN). This laboratory showed that the intestinal hydrolysis of PNG is catalyzed by cytosolic PNG hydrolase (PNGH) and brush border lactase-phlorizin hydrolase (LPH). LPH-catalyzed PNG hydrolysis in vitro is competitively inhibited by lactose. In the present study, the uptake and hydrolysis of PNG were examined in Caco-2 human colon carcinoma cells, which express a functional LPH but exhibit no PNGH activity. PNG uptake at 37 degrees C was linear over 5-500 micromol/L PNG. Uptake was not significantly reduced when Na(+) was substituted with K(+), Li(+), or Tris in the medium. Increasing PNG concentration in the medium did not change intracellular concentrations of PN, pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP), or pyridoxal 5'-phosphate (PLP); however, intracellular pyridoxal (PL) concentration increased. Intracellular PNG concentration was not significantly reduced in the presence of lactose, but the concentration of PL declined in proportion to extracellular lactose (P = 0.01). These results indicate that PNG can be absorbed intact in a Na(+)-independent process and is taken up by passive diffusion. The presence of lactose in this in vitro model of intestinal uptake reduced the enzymatic hydrolysis of PNG by lactase.

Simultaneous determination of melatonin and pyridoxine in tablets by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method for the qualitative and quantitative determination of melatonin plus pyridoxine commercial tablets is described. Melatonin and pyridoxine were simultaneously determined by GC-MS after extraction from ground tablets with methanol and derivatization with N-methyl-N-N-trimethlylsilyltrifluoroacetamide (MSTFA). The mass chromatograms were generated using 232 m/z ion for melatonin and 280 m/z ion for pyridoxine, respectively. Splitless injection offers good reproducibility with a standard deviation of 2%. The developed method was applied to analyze the melatonin and pyridoxine content from two different tablet formulations. Also, recovery, detection and quantification limits are reported.

Validación del método analítico para la determinación de 3 vitaminas hidrosolubles en un suplemento vitamínico / Validation of the analytical method for the determination of 3 vitamins hydrosoluble in a vitamin supplement

Se presentan los resultados obtenidos en la validación de un método analítico por cromatografía líquida de alta resolución, para la determinación de tiamina mononitrato, piridoxina clorhidrato y nicotinamida en el suplemento nutricional neovitamin II, el cual se diseñó para separar las vitaminas entre sí, con la utilización de una columna RP-18 de 25 cm y un detector UV-Visible. Dicho método se empleó para el control de la calidad y la estabilidad de este producto. El método fue validado siguiendo una metodología de trabajo elaborada previamente en un Protocolo de Validación, donde se analizaron diferentes parámetros como son: linealidad, exactitud, precisión, selectividad, límites de detección y cuantificación, adecuación del sistema y estabilidad de las soluciones. Se obtuvieron resultados satisfactorios y se comprobó de esta forma la validez del método analítico

[The criteria for the vitamin B2 and B6 allowances of children suffering from celiac disease]. / Kriterii obespechennosti vitaminami B2 i B6 detei, stradaiushchikh tseliakiei.

The relationship between the blood concentrations of vitamins B2 and B6 and excretion of their metabolites with the urine were studied in 34 children of both sexes aged 7 to 10 suffering from celiac disease. The study revealed that the criteria of adequate supply of these vitamins in the patients and healthy age-matched children differ. Riboflavin concentration of at least 90 ng/ml in red cells, at least 15 ng/ml in the blood plasma, and urinary excretion of at least 7 micrograms/h should be accepted as the criteria of this vitamin sufficiency in the organism. For vitamin B6 these values are 13 ng/ml pyridoxal phosphate in the blood plasma and urinary excretion of at least 47 micrograms/h of 4-pyridoxic acid.

Adenosine-N6-diethylthioether-N1-pyridoximine 5'-phosphate. A novel marker for human cancer detection.

The Schiff base conjugate of vitamin B6 with adenosine-N6-diethylthioether was originally reported as unknown compound B6X and considered to function as a storage form for vitamin B6 utilization by tumor cells. This novel compound is present in tumor cells in culture, the blood of normal and tumor-bearing animals, and the circulation of healthy individuals and patients with various ailments including malignancies. However, its level in the blood of cancer patients is significantly much greater -a desirable feature for cancer detection. Using HPLC pair-ion, reverse phase chromatography blood samples from patients with various malignancies and ailments were screened on a blind basis for the novel compound following its extraction at pH 4.2. The results show that the level of the vitamin B6 metabolite in the blood of cancer patients is up to 4x or higher than levels seen in the blood of normal volunteers, patients in remission or patients with other diseases. In addition, patients receiving treatment had lower levels than before treatment. Normal volunteers, cancer patients prior to treatment, cancer patients on therapy, cancer patients at remission and patients with other ailments had levels of 162.2; 601.7; 497.9; 216.5 and 179.3 (SEM range +/- 18.76-46.60), respectively. A comparison of the control, remission and other ailments groups with the cancer patient groups shows them to be significantly different (P < 0.00001) and strongly supports the use of the novel vitamin B6 conjugate metabolite for detection of human cancers.

Neural tube defects and elevated homocysteine levels in amniotic fluid.

OBJECTIVE: Our purpose was to study maternal blood and amniotic fluid concentrations of homocysteine and relevant vitamins in relation to neural tube defects. STUDY DESIGN: Concentrations of total homocysteine, folate, and vitamins B12 and B6 were measured in maternal blood and amniotic fluid of 27 women carrying a fetus with a neural tube defect and 31 control women carrying a healthy fetus. RESULTS: The mean total homocysteine concentration in amniotic fluid of the study group was significantly higher than that of the control group. The mean concentrations of total homocysteine in blood and the vitamins folate, B12, and B6 in, respectively, blood and amniotic fluid were not significantly different between the groups. The mean concentrations of homocysteine and vitamin B6 were significantly lower in amniotic fluid than in blood in both groups, whereas vitamin B12 in amniotic fluid was higher than in blood. CONCLUSION: These results support the hypothesis that at least the cause of a subset of neural tube defects could reside in a primary or secondary maternal or fetal derangement of homocysteine metabolism.

Abnormal vitamin B6 status in rheumatoid cachexia. Association with spontaneous tumor necrosis factor alpha production and markers of inflammation.

OBJECTIVE: To compare vitamin B6 levels in rheumatoid arthritis (RA) patients and healthy control subjects. METHODS: We measured levels of vitamin B6 in 23 adults with well-controlled RA, and in 23 healthy control subjects matched for age, sex, race, and weight. RESULTS: Although plasma folate and vitamin B12 concentrations and erythrocyte B6 activity coefficients were similar in the patients and controls, plasma levels of pyridoxal-5'-phosphate (PLP) were lower in the RA patient group (mean +/- SD 46.1 +/- 48.1 versus 69.3 +/- 58.4 nmoles/liter; P < 0.004). In multivariate analyses, PLP was inversely associated with tumor necrosis factor alpha (TNF alpha) production by peripheral blood mononuclear cells (PBMC) (P < 0.001), after adjustment for age, pain score, erythrocyte sedimentation rate, and use of nonsteroidal antiinflammatory drugs. CONCLUSION: PLP levels are reduced in patients with RA. This reduction is associated with TNF alpha production by PBMC.

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.

Human vitamin B-6 pools estimated through muscle biopsies.

Previous estimates of total vitamin B-6 pools in humans based on extrapolations from tracer studies yielded values of 107-190 mumol when the tracer was administered orally and 345-725 mumol when the tracer was administered intravenously. To obtain a more direct estimate of vitamin B-6 pools, muscle biopsies from five female and seven male adults were analyzed by cation-exchange chromatography. Total muscle mass was estimated from creatinine excretion and the assumption that muscle is 40% of the body weight. The total muscle vitamin B-6 pool was estimated to be 917 +/- 319 mumol in the females and 850 +/- 216 mumol in the males. Because muscle accounts for approximately 80% of the vitamin B-6 in the body, the total body pool of vitamin B-6 in adult humans is probably approximately 1000 mumol.

Separaçäo de vitaminas pela eletroforese em papel / Separation of vitamins by paper electrophoresis

As vitaminas A (retino), B1 (tiamina), B6 (piridoxina), B12 (cianocobalamina), C (ácidoo ascórbico) e E (alfa-tocoferol), foram analisadas por eletroforese inidimensional em papel (qualitativo Klabin), usando como eletrólito soluçöes aquosas de carbonato de amônio 0,050, 0,075 e 0,100 M, sob um gradiente de potencial de 12,6V/cm. As melhores separaçöes ocorreram com o eletrólito nas concentraçöes 0,050 M (30 e 45 minutos) e 0,100 (45 e 60 minutos), quando 05 (cinco) vitaminas foram perfeitamente separadas: A (ou B6)-B1-B12-C e E

Water-soluble vitamins in cancer patients on parenteral nutrition: a prospective study.

Forty-three patients with mild weight loss were studied prospectively to determine whether the parenteral water-soluble vitamin doses in a commercially available preparation (MVI concentrate; USV Laboratories, Tarrytown, NY) maintained serum, red blood cell (RBC), and urinary concentrations of water-soluble vitamins in stressed cancer patients receiving total parenteral nutrition (TPN). Patients were divided into three groups: (1) oral diet, no intravenous vitamins given; (2) TPN plus 5 ml MVI; and (3) TPN plus 10 ml MVI. Vitamins C, B1, B2, B3, B6, and niacin were measured initially and weekly during a 6-week study period. Caloric and nitrogen balances were quantified. Most of the patients in all three groups had normal blood or urine levels of all water-soluble vitamins. No clinical evidence of vitamin deficiency or MVI toxicity was detected. The recommended parenteral dosages of vitamin C (100 mg/day) and B3 (15 mg/day) provided measurably adequate levels in all patients. Levels of vitamins B1, B2, B6, and niacin that were less than the normal range were noted in 4-40% of patients receiving the recommended daily dosages of 3 mg, 3.6 mg, 4 mg, and 40 mg, respectively. These deficiencies appeared to improve in group III patients who received twice the recommended parenteral vitamin dosages, although they did not completely disappear. Niacin deficiency appeared to be the most prevalent, occurring in 40% of patients studied. Since intravenous doses of B1, B2, B6, and niacin are safe and well tolerated, it appears that increased daily amounts of these vitamins should be given to cancer patients on parenteral nutrition.

Investigations on the nutritional status of advanced breast cancer patients. The influence of long-term treatment with megestrol acetate or tamoxifen.

The nutritional status of three groups of postmenopausal women (age 41-80 yrs) with advanced breast cancer was investigated with special reference to vitamin B6. The interference of hormonal treatment was studied with respect to the progestin megestrol acetate (Group MA, n = 14) and the antiestrogen tamoxifen (Group TAM, n = 15) compared with untreated patients (Group U, n = 11). Healthy postmenopausal women served as controls (Group C, n = 16). Nutritional status was judged from body mass index (BMI), vitamin and trace element status, hematology, and clinico-chemical parameters. Intake of nutrients was calculated from a food record. Hormonal status was studied by analysis of LH, FSH, and prolactin in plasma and of steroids and catecholamines and their metabolites in 24-hour urine. Compared with values for Group C, nutrient intake, hematology, clinico-chemical parameters, and 24-hour urinary excretion of catecholamines and their metabolites of patient groups (U, TAM, and MA) were not significantly different. The BMI of patients was significantly higher (by about 10%; 60% showed an overweight) than that of controls. With respect to fat-soluble vitamin status, significantly lower plasma levels of vitamin A (at least 40% lower, with deficient levels in more than 50% of the patients), D (40% lower), and E (20% lower) were found for Group U. However, water-soluble vitamin status of the four groups was fairly similar. A significantly higher excretion of xanthurenic acid in 24-hour urine, after an oral tryptophan load, was observed for Groups TAM and MA. This is most probably the result of hormonal treatment without affecting vitamin B6 status. Small, but significant, differences between groups were found for trace element status, especially with respect to lower plasma selenium of Group U (25% lower). LH, FSH, and prolactin in plasma and excretion of steroids in 24-hour urine showed levels that could be expected for controls and for untreated and hormonally treated patients. We concluded that the nutritional status of all patients is reasonably adequate. Hormonal treatment did not influence vitamin B6 status, although levels of vitamins A, D, and E and of selenium seem to be elevated.