Determination Of Thiamine Research Articles

A simplified approach to the determination of thiamine and riboflavin in meats using reverse phase HPLC

A simplified procedure for the determination of thiamine and riboflavin contents in meat products is proposed. The vitamins were extracted from the meats by digestion with 0.1 M hydrochloric acid followed by an enzymatic hydrolysis step in which phosphorylated forms of both vitamins were hydrolysed using an acid phosphatase enzyme of defined activity (0.5 U/mg) derived from wheat germ. Riboflavin in the filtered hydrolysates was determined directly by high performance liquid chromatography (HPLC) on a reverse phase (C 18) column using a methanol:water (40:60) mobile phase and a fluorescence detector (excitation wavelength, 450 nm; emission wavelength, 510 nm). After conversion to thiochrome and partitioning into isobutanol, thiamine was determined on the same column by fluorescence (excitation wavelength, 366 nm; emission wavelength, 434 nm) using 80:20 methanol:water as the eluent. Measured levels of both vitamins were found to be very close to certified values provided with a lyophilized reference liver sample, while the contents determined for a range of meats were also in good agreement with published values.

Simultaneous Determination of Water‐Soluble Vitamins in Human Urine by Fluorescence in a Flow‐Injection Analysis

Fluorescence in flow‐injection analysis is described for the simultaneous determination of thiamine, riboflavin, and folic acid. Its detection limit linearity and reproducibility were examined. The kinetic method is based on the enhancing effect of thiamine and riboflavin on the fluorescence generated by oxidizing to thiochrome with alkaline ferricyanide. Different parameters affecting this reaction were thoroughly studied. The procedure was applied to the determination of thiamine and riboflavin and folic acid in human urine samples.

Simultaneous Spectrophotometric Determination of Group B Vitamins Using Parallel Factor Analysis: PARAFAC

In this work, a simple and rapid analytical procedure was applied for simultaneous determination of folic acid (vitamin B 0 ), thiamin (vitamin B 1 ), riboflavin (vitamin B 2 ) and pyridoxal (vitamin B 6 ) based on the absorbance data in the pH range 2.0-12.0 at 25 °C using parallel factor analysis (PARAFAC). The effect of the pH as the most important factor on the sensitivity of the determination was studied. The spectral data were recorded in 400-650 nm intervals and a 2-12 pH range for all four vitamins. The calibration set was constructed in the concentration ranges of 4-22, 1-20, 6-26, and 4-20 pg mL -1 for B 6 , B 2 , B 1 and B 0 , respectively. The root mean squares errors of prediction for the prediction set, (RMSEP), are 0.65, 0.63, 1.13 and 0.34 for B 0 , B 1 , B 2 and B 6 , respectively. The recovery percent for the validation set are in the range of 90.6 to 107.0%. The effect of the experimental conditions and diverse species were discussed. The optimum values of these factors were searched according to the relative standard deviation of the prediction set of mixtures solutions.

Small Bowel Bacterial Overgrowth Is a Potential Mechanism for Thiamine Deficiency Identified after Roux-En-Y Gastric Bypass Surgery

Purpose: Present evidence supports a saturable, carrier-mediated exchange mechanism for thiamine transport across jejunum. It is proposed that thiamine deficiency after gastric bypass surgery results from prolonged nausea and emesis. We observed that individuals after gastric bypass surgery developed thiamine deficiency and elevation of serum folate levels, a proposed marker for small bowel bacterial overgrowth. We hypothesized that small bowel bacterial overgrowth interferes with absorption of thiamine from the small intestine. Methods: We performed a retrospective review of consecutive patients who underwent Roux-en-Y gastric bypass surgery from 2002–2004 in our institution. To eliminate immediate, post-operative changes, we included patients who received evaluation at 3 months or longer after surgery. There were 75 patients with determination of serum thiamine and folate levels. This included 48 females and 27 males with an average age of 46 years (range 21 to 68). Five patients with low serum thiamine and elevated serum folate levels had undergone glucose-hydrogen breath testing. Results: There were 37 patients with serum folate levels greater than the upper limit of normal; among these patients, 23 (62%) had serum thiamine levels below the lower limit of normal. There were 38 patients with serum folate levels within normal range; 11 (29%) had serum thiamine levels below the lower limit of normal (Chi-squared test comparing thiamine deficiency in the normal folate group to the elevated folate group: p< .01). All 5 patients who underwent glucose-hydrogen breath testing had abnormal findings supporting a diagnosis of small bowel bacterial overgrowth: 4 patients had a significant rise (>10 ppm) in breath hydrogen within 45 minutes after glucose ingestion, while 1 had an elevated fasting breath hydrogen level of 57 ppm. Conclusions: Thiamine deficiency is common after Roux-en-Y gastric bypass surgery. Small bowel bacterial overgrowth appears to be common after gastric bypass surgery, as determined by elevation of serum folate levels or the presence of abnormal glucose-hydrogen breath testing. The results support the hypothesis that small bowel bacterial overgrowth following gastric bypass surgery can interfere with thiamine absorption.

Research Report: Determination of Riboflavin Content in Goat and Cow Milk by High Performance Liquid Chromatography (HPLC)

A high performance liquid chromatography method has been developed to determine riboflavin content in goat and cow whole milk. The present method involves acidification of the milk to precipitate bulk of the proteins and centrifugation affording a supernatant, which was diluted with 2% acetic acid to a constant volume. This solution was subjected to HPLC analysis using an ODS column. The method is simple, rapid and sensitive for riboflavin determination. The results obtained by the method compared well with the results of analysis by the \"Official Methods of Analysis\" of the Association of Official Analytical Chemists (AOAC). Several analytical methods for the determination of riboflavin have been reported (Kamman, 1980). Most of the procedures involve a pretreatment to liberate the protein bound vitamin from flavoproteins. Fluorometric methods of detection are considered the most desirable because of their sensitivity, rapidity, and specificity. However, they require extra treatments to separate riboflavin from interfering fluorescent compounds. In the official methods of analysis of the Association of Official Analytical Chemists (AOAC, 1996, method 970.65), samples are treated with potassium permanganate (KMnO4) and hydrogen peroxide (H2O2) to remove background fluorescence. Rashid and Potts (1980) treated milk samples with acidified lead acetate solution to separate extraneous protein and minimize background fluorescence. Fellman et al. (1982) have reported a C18 reversed phase separation for the simultaneous determination of thiamin and riboflavin in selected foods including milk. This method required the removal of interfering substances using a 0.01M phosphate buffer. Journal of Agriculture, Science and Technology Vol.3(2) 2001: 96-101

Implementation of flow-through solid phase spectroscopic transduction with photochemically induced fluorescence: determination of thiamine

For the first time, flow-through solid phase spectroscopic transduction has been implemented with photochemically induced fluorescence. The system developed allows the sensitive, selective and rapid determination of thiamine in the presence of the rest of B-complex vitamins. By UV-irradiation in a single-line flow injection device, thiamine is converted on-line into a strongly fluorescent photoproduct, which is retained on C 18 silica gel filling the flow cell placed in the detector. The fluorescence of the photoproduct retained on C 18 is measured at excitation and emission wavelengths of 367 and 443 nm, respectively. The sorption of the photoproduct on the solid support improves noticeably sensitivity and selectivity with regard to previously reported photochemical–fluorimetric methods. Two calibration lines are constructed for 300 and 600 μl of injection volume. Concentrations of the vitamin of 0.18–80 ng ml −1 (300 μl) and 0.09–50 ng ml −1 (600 μl) are determined; the detection limits are 55 and 28 ng l −1 and the R.S.D.s are 1.4 and 2.4%, respectively. The method allows the analysis of 52 and 36 samples per hour, respectively. The applicability of the method for the determination of thiamine in pharmaceuticals and human serum and urine was demonstrated by investigating the effect of potential interferences and by the analysis of real samples. The accuracy of the proposed method was checked by applying a reference method and a recovery test. Recovery levels were between 95 and 105%.

Separation of thiamin and its phosphate esters by capillary zone electrophoresis and its application to the analysis of water-soluble vitamins

A capillary zone electrophoresis method for the separation and determination of thiamin and its phosphate esters (free thiamin, thiamin monophosphate, and thiamin pyrophosphate) was developed and optimized. The efficiency achieved with boric acid–sodium tetraborate decahydrate buffer (pH 8.24; 65–8 mM) at an applied potential of 30 kV gave the detection limit (S/N = 3) and the limit of quantitative measurement (S/N = 10) of thiamin and its phosphate esters ranging from 10 −4 to 6 × 10 −4 mM and from 6 × 10 −4 to 1.2 × 10 −3 mM, respectively. The effects of pH on separation and migration times of thiamin and its phosphate esters are described. The method was validated and applied to the quantitative determination of thiamin in commercial tablets containing both a massive and a normal dose of thiamin.

Simultaneous Spectrophotometric Multicomponent Determination of Folic Acid, Thiamine, Riboflavin, and Pyridoxal by Using Double Divisor–Ratio Spectra Derivative–Zero Crossing Method

A graphical method based on the double divisor–ratio spectra derivative–zero crossing was proposed for the spectrophotometric multicomponent analysis of synthetic mixtures containing folic acid (vitamin B0), thiamine (vitamin B1), riboflavin (vitamin B2), and pyridoxal (vitamin B6) in the presence of strongly overlapping signals without any chemical separation. The graphical method is based on the use of derivative signals of the ratio spectra by using double divisor. The selected data of ultraviolet‐visible absorption spectra consisted of points corresponding to the spectral region 225–475 nm. The linear determination ranges were 1–26 µg mL−1 of B0, 4–50 of µg mL−1 of B1, 1–28 µg mL−1 of B2, 6–42 µg mL−1 of B6 in a buffer solution at (pH 5.8). The proposed method applied to the simultaneous determination of four vitamins by using synthetic quaternary mixtures. The obtained results were statistically compared with each other.

Determination of thiamine and its phosphate esters in rat tissues analyzed as thiochromes on a RP-amide C16 column

A new reversed-phase chromatographic method is described for the separation and quantification of thiamine (T), thiamine monophosphate (TMP) and diphosphate (TDP) in rat tissues. Sample extraction with perchloric acid (HClO 4) was found more suitable than extraction with trichloroacetic acid (TCA), as regards convenience and background fluorescence. Derivatization of thiamine vitamers to thiochromes was optimized and complete separation of TDP and TMP thiochromes was obtained on a RP-amide C16 column in isocratic elution, with T thiochrome eluting in less than 10 min. The precision and the accuracy of the HPLC procedure were assessed: ranging from 0.5 to 7.7% for intra-day and from 2.0 to 9.4% for inter-day precision, a recovery average of 101% was determined (range 90–111%). Mean values of recovery for TDP, TMP or T were 91, 96 and 90% for liver extracts, respectively. Analysis of vitamers in tissues of rat submitted to 8 days thiamin deficiency, followed by a 14 days repletion, showed a significant reduction of TPP after 8 days of depletion in liver (−67%), brains (−50%), kidneys (−60%), followed by a complete recovery upon repletion.

Determination of thiamine and its phosphorylated forms in human plasma, erythrocytes and urine by HPLC and fluorescence detection: a preliminary study on cancer patients

In man, neurotoxicity associated to ifosfamide treatment can be reversed by intravenous thiamine administration. Trying to explain this clinical finding, we decided to study possible changes in thiamine availability and activation in patients exposed to ifosfamide. Free thiamine and its phosphate esters levels were measured in plasma, erythrocytes and urine by an ion-pair HPLC method with pre-column derivatization, which allowed separation of the fluorescent compounds in less than 10 min. The method was validated by linearity, sensitivity and reproducibility studies, whose values met the demands for bioanalytical assays. This method was applied to assess thiamine status in cancer patients exposed to ifosfamide therapy for advanced disease.

Liquid chromatographic determination of thiamin in dairy products

Concentrations of thiamin were determined in pasteurized and UHT-sterilized milks, yoghurt and kefir samples by high-performance liquid chromatography using a reversed-phase C-18 column connected to fluorescence detector. The linearity, recovery and reproducibility of the method were satisfactory. The thiamin content ranged from 0.305±0.02 to 0.252±0.03 mg/l in market milks. Yoghurt samples had higher concentration of thiamin (0.355±0.04 and 0.404±0.02 mg/l) than values found in market milks. The least thiamin content was found in kefir (0.175±0.03 mg/l) among the samples analyzed.

Simultaneous determination of thiamin and riboflavin in the sea urchin, Paracentrotus lividus, by high-performance liquid chromatography

Thiamin and riboflavin were determined in the sea urchin, Paracentrotus lividus, by reversed-phase liquid chromatography with fluorescence detection and a mobile phase consisting of 72% 0.005 M NH4OAc and 28% MeOH at a flow rate of 1.450 ml/min. The total analysis time never exceeded 20 min. After acid hydrolysis and enzymatic digestion, thiamin was oxidized to thiochrome and the oxidized fraction was purified and concentrated by solid-phase extraction. Limits of detection were 9 pg/ml and 80 pg/ml for thiamin and riboflavin, respectively. Good results were obtained with respect to repeatability (RSD%<2.4) and recovery (>91.3%).

Determination of thiamine in blood serum and urine by high-performance liquid chromatography with direct injection and post-column derivatization

Thiamine (vitamin B 1) was determined in human serum and urine by HPLC with fluorimetric detection of its oxidation product, thiochrome. The samples were injected directly into the chromatographic system without previous treatment or dilution. A column filled with an ultra-high molecular weight surface-modified polyethylene (PE) was able to separate matrix components from analyte and also to allow a good chromatographic resolution of thiamine. The interaction of thiamine, thiocrome and both matrices (serum and urine) with PE was studied off- and on-line to determine the optimal procedure for vitamin B 1 determination. When carried off-line, matrix adsorption yield was 49 mg serum proteins/g polymer and components of 1000 μl urine/g polymer. In an on-line arrangement, the yield dropped to 10 mg/g and 150 μl/g, respectively. The matrix/analyte separation was carried out in an on-line procedure on a 50×4.6-mm, 25-μm PE column, using a water–sodium phosphate–methanol gradient elution. Part of the matrix was eluted within the first 2 min and thiamine after 3.8 min. The rest of the matrix retained on the column was eluted after thiamine at the last step of the gradient elution. Analysis time was 12 min. The within-day and day-to-day precision gave C.V. varying from 3.6% to 14.5% and recoveries from spiked samples were in the range of 84.8–98.8%.

Obese individuals as thiamin storers.

To investigate thiamin and its phosphoester content in plasma and erythrocytes for a complete picture of thiamin status in obese individuals. Comparative study of the thiamin status of obese vs normal individuals. In all, 10 healthy, overweight, fertile age women (age: 33.1+/-5.1 y; BMI: 47.0+/-0.2 kg/m(2)) and 10 normal women (age: 30.1+/-3.5 y; BMI: 22.8+/-0.2 kg/m(2)). a high-pressure liquid chromatography (HPLC) method for the determination of thiamin and its phosphoesters in the plasma and erythrocytes of the subjects. The major findings were: (1) significant decrease of plasma thiamin, its monophosphate and total thiamin contents in obese vs normal women; (2) significant decrease of thiamin pyrophosphate ester and total thiamin content in obese vs normal women; (3) significant increase in plasma thiamin/thiamin monophosphate ratio (in practice, it was inverted) and corresponding decrease of the plasma thiamin monophosphate/erythrocytes thiamin pyrophosphate ratio in obese vs normal women, where plasma thiamin monophosphate and erythrocytes thiamin pyrophosphate contents are an index of thiamin status. This study advances the hypothesis that obese women maintain higher levels of thiamin compared to normal weight subjects by storing greater amounts of thiamin in cells through preferential intracellular thiamin recycling to compensate for relatively lower levels of thiamin.

Simultaneous determination of thiamine and riboflavin in edible marine seaweeds by high-performance liquid chromatography.

This study presents a high-performance liquid chromatography (HPLC) method for simultaneous determination of thiamine and riboflavin and the results of its application to a number of edible seaweeds that are sampled in dried form (Himanthalia elongata, Laminaria ochroleuca, Undaria pinnatifida, Palmaria sp., and Porphyra sp.) or as canned food (H. elongata and Saccorhiza polyschides). Samples are prepared by acid and enzymatic hydrolysis. Optimized conditions for reversed-phase HPLC with fluorescence detection are as follow: column, Kromasil 100 C18; column temperature, 35 degrees C; mobile phase, a 72:28 (v/v) mixture of 0.005 M ammonium acetate (pH 6.7)-methanol; and flow rate, 1.35 mL/min. With these conditions, recovery is 95.52% for thiamine and 90.08% for riboflavin, and the method precision (relative standard deviation) is 2.66% for thiamine and 2.21% for riboflavin. On a dry weight basis, thiamine contents range from 0.14 microg/g in dried H. elongata to 2.02 microg/g in dried Porphyra and riboflavin contents from 0.31 microg/g in canned H. elongata to 6.15 microg/g in dried Porphyra.

Flow‐Injection Chemiluminescent Determination of Thiamine in Pharmaceutical Samples by On‐line Photodegradation

A simple, sensitive, and precise method for the determination of thiamine hydrochloride in a flow‐injection system is described. The method is based on the UV irradiation of thiamine in acid medium. Then, the photo‐fragments are oxidized by permanganate in acid medium, and the resultant chemiluminescent intensity is measured. The optimum conditions for the photoreaction and for the chemiluminescence emission were investigated. The method allows the determination of thiamine, over the range 0.05(LOD)–84 mg · l−1, with a throughput of 30 h−1, and a RSD (n, 20) at 20 and 0.5 mg · l−1 of the thiamine level of 2.5 and 1.3%, respectively. The method was applied to pharmaceutical preparations.

Successive determination of thiamine and ascorbic acid in pharmaceuticals by flow injection analysis

A simple and rapid fluorimetric method for the determination of mixtures of thiamine and ascorbic acid is proposed. The procedure is based on the oxidation with mercury(II) of the B 1 and C vitamins to form thiochrome (TC) and quinoxaline derivate, respectively. Both reaction products exhibit fluorescence at the same wavelengths ( λ ex=356 and λ em=440 nm). The procedure is optimised in a flow injection (FI) system and applied with excellent results in the determination of B 1 and C vitamins in commercial pharmaceutical preparations. The calibration graphs were linear over the range 2–100 μg ml −1 for thiamine and 5–100 μg ml −1 for ascorbic acid. The throughput was 25 samples per hour.

Determination of thiamine and its esters in beers and raw materials used for their manufacture by liquid chromatography with postcolumn derivatization.

Thiamine and its mono- and pyrophosphate esters were determined in beer and the raw materials used for its manufacture (brewer's yeast, malt, raw grain, and hops) after separation using reversed-phase liquid chromatography. The method used fluorescence detection and a new amide-based stationary phase, which avoids the need to form ion pairs, leading to narrower peaks and a simpler mobile phase. Analyses were performed by isocratic elution with a phosphate buffer mobile phase and using a postcolumn derivatization system based on the oxidation of thiamine to fluorescent thiochrome with potassium ferricyanide in alkaline solution. Only thiamine was found in the beers and raw products, especially in brewer's yeast and malt. A stability study pointed to a faster decrease in the thiamine content of samples stored at room temperature and in sunlight.

Optimization of Oxidation Steps Used in Fluorometric Determination of Thiamin in Soft Wheat Flour

Optimization of Oxidation Steps Used in Fluorometric Determination of Thiamin in Soft Wheat Flour

Determination of thiamine in pharmaceutical preparations by sequential injection renewable surface solid-phase spectrofluorometry.

Fluorometric determination of thiamine requires the conversion of the analyte to fluorescent thiochrome by hexacyanoferrate(III) oxidation in alkaline solution and the isolation of the produced thiochrome from the reaction medium by solvent extraction. It was observed that thiochrome could be concentrated and separated from the reaction medium by solid-phase extraction. The thiochrome sorpted on the surface of octadecyl-alklylated poly[styrene/divinylbenzene] (C18-PS/DP) microbeads emitted strong fluorescence upon excitation, the maximum excitation and emission wavelengths being 385 nm and 433 nm, respectively. Based on this observation, a sequential injection renewable surface solid-phase spectrofluorometry was developed for the determination of thiamine. A sequential injection system on-line coupled to a chip-based flow-through cell was employed to handle the chemical reaction, bead injection and discharging, and adsorption of thiochrome. Solid-phase fluorometric detection was realized by coupling the chip-based flow-through cell to a spectrofluorometer with a multistrand bifurcated optical fiber. Under the optimized condition, a detection limit of 0.03 microg ml(-1) was achieved at the sample throughput of 30 h(-1) and consumption of 1 mg C18-PS/DP microbeads for each run. Eleven runs of a 2 microg ml(-1) thiamine standard solution gave a relative standard deviation of 1.0%. The developed approach was successfully applied for the determination of thiamine contents in pharmaceutical preparations.