Determination Of Thiamine Research Articles

Green synthesis of self-oriented flower-like Ag@Ag2O nanostructures functionalized with L-Tryptophan for colorimetric simultaneous determination of ultra-trace level of thiamin and riboflavin

The study focuses on the green synthesis of Ag@Ag2O nanostructures using Padina algae extract and functionalizing them with L-tryptophan to enhance their properties as a colorimetric sensor for simultaneous detection of ultra-trace levels of thiamin and riboflavin. The nanostructures are characterized using techniques like XRD, FESEM, FTIR, TEM, AFM, and DLS to understand their morphology, structure, and interactions with target molecules. FESEM analysis revealed the hierarchical flower-like Ag@Ag2O nanostructures. The TEM image shows the formation of core-shell nanostructures. Also, DLS analysis and surface zeta potential spectra illustrated the aggregated nature of fabricated nanocomposites in the presence of vitamins. The study is the first to report simultaneous determination of thiamin and riboflavin using a colorimetric sensor based on Ag@Ag2O-L-Try nanocomposites using partial leas square (PLS). The dynamic range of thiamin and riboflavin was achieved in 0.1 mol L−1 acetate buffer pH 4 and the ratio Ag@Ag2O: L-try 1:1. The Ag@Ag2O-L-Try sensor exhibited two linear ranges of 0.1- 1.0 and 3-350 µMol L− 1 for riboflavin and a linear range 3.0–60 µMol L− 1 for thiamin. Also, low detection limit of 1.92 µMol L− 1 and 0.048 µMol L− 1 was obtained for riboflavin and thiamin, respectively. The results indicated that the success of the method depends on the selective and sensitive colorimetric assay of the sensor along with the simultaneous determination by the PLS algorithm. Hence, the proposed technique can be used for the accurate and precise determination of vitamins in different pharmaceutical syrup and tablet samples.

Developing a highly efficient fluorescence strategy for thiamine detection in real samples

This research presents the first report of a simple fluorescent approach for determination of thiamine (TH, Vitamin B1) with great sensitivity and selectivity by manganese dioxide (MnO2) nanosheets. These nanoparticles have several advantages such as activity of oxidase like and easy, low-cost preparation. They can successfully oxidize nonfluorescent TH to intense fluorescence thiochrome at physiological pH and room temperature. Under optimized conditions and in the presence of EDTA and HCl, a linear relationship was gained between fluorescence intensities and thiamine concentrations from 10 to 5000 nM. This fluorometric method demonstrated high sensitivity with a low detection limit (5 nM). In order to confirm the selectivity of the proposed system, related interfering agents were investigated with the MnO2 nanosheets and it was verified that the fluorescence response is greatly selective to TH. This method exhibited acceptable outcomes to evaluate TH in pharmaceutical, food, urine and serum specimens. Therefore, we suggest that the current study could be appropriate for TH measurement in biological, food and drug samples.

Liquid Chromatography Mass Spectrometric Method and a Fluorometric 96 Well Plate Assay for Determination of Thiamine in Salmonid Eggs.

Thiamine deficiency is a large contributor to reduced reproduction success among salmonids throughout the northern hemisphere. In Scandinavia, this reproduction disorder is known as M74; while in North America, it is known as early mortality syndrome (EMS). The disorder fluctuates in magnitude from year to year. During years with high prevalence of the disorder, salmonid hatcheries that stock various aquatic systems to maintain the population size experience difficulties filling their quotas without thiamine treatment of alevins. The disorder is monitored both by observing the survival rate and by measuring the thiamine content of prefertilized eggs in the hatcheries. Here, a simple extraction procedure is presented, which allows for quantitative determination of the various phosphorylated forms of thiamine using liquid chromatography mass spectrometry but also allows for extraction in 96 deep-well plates and measurement of the total thiamine content using fluorescence monitoring with a plate reader, following oxidation of thiamine to thiochrome. The latter procedure could also be integrated into a highly portal system where the thiochrome is determined using the DeNovix QFX analyzer. The newly developed extraction procedure and cleanup method for fluorescence measurement represent the most versatile and simple methods to date for monitoring of thiamine in salmonid eggs. The methods produced accurate and precise data with quantification limits below the limit where the deficiency causes 100% lethality.

A robust method for simultaneous determination of eight B vitamins in human serum by liquid chromatography tandem mass spectrometry.

The level of vitamin B group in human serum is an important index of human health. Among B vitamins, cyanocobalamin in serum is unstable and its content is extremely low. Rapid and simultaneous detection of multiple B vitamins including cyanocobalamin is a challenge. Herein, we have developed a rapid and stable method that can realize the determination of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin simultaneously in 6 min. The method was established based on protein precipitation with methanol and then chromatographic separation was achieved using Waters acquity ultra-high-performance liquid chromatography high strength silica T3 column, which was stable and sensitive especially for cyanocobalamin. Limit of quantification, precision, trueness, and matrix effect were validated according to the European Medicines Agency and United States Food and Drug guidelines and Clinical and Laboratory Standards Institute guidelines on bioanalytical method. The limit of quantification for thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin was 0.4, 0.4, 0.8, 2.0, 0.4, 0.1, 0.4, and 0.04ng/mL separately, respectively. Intra- and interday precisions were 1.1%-12.4% and 2.0%-13.5%, respectively. The relative errors were between 0.3% and 13.3%, and the matrix effects were between 2.6% and 10.4%.

A fast, accurate and comprehensive LC-MS/MS method validation for the sensitive quantification of water-soluble vitamins in walnut, almond, hazelnut and pistachio fruits

Evaluating nutritional values of food products such as nuts has been of great importance nowadays. In the current study, development and validation of a robust and fast LC-MS/MS method was carried out for simultaneous determination of pantothenic acid (B5), riboflavin (B2), biotin (B7), thiamine (B1), cyanocobalamine (B12), nicotinamide (B3), nicotinic acid (B3), pyridoxine (B6) and folic acid (B9) in walnut, almond, hazelnut and pistachio samples from Turkey. Isotope labeled vitamin standards were used to compensate the sensitivity losses and increase the degree of precision. The validation parameters of the developed analytical method were accuracy (recovery), linearity, limit of quantification (LOQ), intra-day and inter-day precision (repeatability), relative standard uncertainty (U% at 95% confidence level (k = 2)), and limit of detection (LOD). Chromatographic parameters were optimized to have optimum separation of nine water soluble vitamins (WSVs) and MS detection was performed using a tandem mass spectrometer. Linearity of the established LC–MS/MS method was in the range of 25–1000 ng/mL for the studied vitamins. The coefficients of determination for the vitamins were between 0.9973 and 0.9999. LOD/LOQ values of the analytes were 10.48/12.54, 13.08/19.59, 5.77/7.70, 12.48/17.71, 12.38/14.90, 6.55/10.08, 8.08/9.81, 13.03/16.38, 13.08/19.59 for B1, B2, nicotinamide (B3), nicotinic acid (B3), B5, B6, B7, B9 and B12, respectively.

Gold Nanoparticles/Nanographene-Based 3D Sensors Integrated in Mini-Platforms for Thiamine Detection.

Vitamins are essential for sustaining daily activities and perform crucial roles in metabolism, such as preventing vascular events and delaying the development of diabetic nephropathy. The ultrasensitive assessment of thiamine in foods is required for food quality evaluation. A mini-platform utilizing two 3D sensors based on nanographene and gold nanoparticles paste modified with protoporphyrin IX and protoporphyrin IX cobalt chloride is proposed for the detection of thiamine in blueberry syrup, multivitamin tablets, water, and a biological sample (urine). Differential pulse voltammetry was utilized for the characterization and validation of the suggested sensors. The sensor modified with protoporphyrin IX has a detection limit of 3.0 × 10-13 mol L-1 and a quantification limit of 1.0 × 10-12 mol L-1, whereas the sensor modified with protoporphyrin IX cobalt chloride has detection and quantification limits of 3.0 × 10-12 and 1.0 × 10-11 mol L-1, respectively. High recoveries (values greater than 95.00%) and low RSD (%) values (less than 5.00%) are recorded for both 3D sensors when used for the determination of thiamine in blueberry syrup, multivitamin tablets, water, and urine, demonstrating the 3D sensors' and suggested method's high reliability.

A simple, sensitive, and specific method for the extraction and determination of thiamine and thiamine phosphate esters in fresh yeast biomass

Thiamine is an essential vitamin for most living organisms, of which yeasts are a rich nutritional source. In this study we developed a thiamine extraction and determination method to detect thiamine in fresh yeast biomass. The thiamine determination method combines the derivatization of thiamine to a highly fluorescent product, with chromatographic separation (HPLC) and fluorescence detection. The method specifically detects free thiamine (T), thiamine phosphate (TP), and thiamine pyrophosphate (TPP). It has a high sensitivity of 2 ng/ml for TPP and TP, and 1 ng/ml for T, excellent instrumental repeatability, and low day-to-day variation in retention time of the different phosphate forms. We demonstrated the robustness of the method by proving that the fluorescence signals of the derivatised samples are stable for at least 82 h after derivatization, and by showing that the final pH of the samples does not influence the fluorescent response. In addition, we developed and validated a thiamine extraction method consisting of beads beating the fresh yeast biomass in 0.1 M HCl using a lysing matrix composed of 0.1 mm silica spheres. The performance of this method was compared to extraction via heat treatment at 95 °C for 30 min, and a combination of beads beating and heat treatment carried out in different order. We demonstrated that thiamine extraction via beads beating is the only method that prevents the biologically active form thiamine pyrophosphate to be degraded to thiamine phosphate, therefore, the extraction method developed and described in this study is preferred when the different thiamine vitamers need to be detected in their actual proportions. The combination of the extraction via beads beating, the conversion of all vitamers to the thiochrome derivatives, and the separation of these compounds on the reversed phase HPLC with a fluorescence detector, yielded a sensitive, specific, repeatable, and robust method for extraction and determination of vitamin B1 in fresh yeast biomass.

Determination of thiamine and pyridoxine in food supplements by a green ultrasensitive two-dimensional capillary electrophoresis hyphenated with mass spectrometry

Determination of thiamine and pyridoxine in food supplements by a green ultrasensitive two-dimensional capillary electrophoresis hyphenated with mass spectrometry

W178 Optimization of chromatographic and sample pretreatment conditions for thiamine determination in human plasma using an internally developed reversed high performance liquid chromatography coupled with fluorescence detector method

W178 Optimization of chromatographic and sample pretreatment conditions for thiamine determination in human plasma using an internally developed reversed high performance liquid chromatography coupled with fluorescence detector method

Voltammetric and sequential injection amperometric determination of B group vitamins on electrodes modified with binary systems based on oxides of manganese, bismuth and gold

It was found that oxides of manganese (MnOx), bismuth (ВiOx), gold (AuOx), and ВiOx–MnOx and MnOx–AuOx binary systems electrodeposited on the surface of planar carbon electrodes (SPE) exhibited catalytic activity during electrooxidation of thiamine, riboflavin, pyridoxine and cyanocobalamin in acidic media. Immobilization of the binary systems on the surface of the SPE working electrode allowed to achieve a joint selective voltammetric determination of two-component systems of the vitamins considered: riboflavin and pyridoxine on SPE with BiOx–MnOx (ВiOx–MnOx–SPE) and MnOx–AuOx (MnOx–AuOx–SPE); and riboflavin and thiamine or riboflavin and cyanocobalamin on MnOx–AuOx–SPE. The calibration dependences obtained for the considered vitamins using voltammetry on BiOx–MnOx–SPE and MnOx–AuOx–SPE electrodes were observed in the range from 1×10–6 to 1×10–3 M. Detection limit of thiamine, pyridoxine and cyanocobalamin was decreased by an order of magnitude compare to static conditions in a sequential injection system (SI-system). To implement two- and three-component analysis of biologically active additives (BAA) and drugs in the SI-system, the surface of the working electrode modified with MnOx–AuOx binary system was covered with poly-2-vinylpyridine (PVP) or Nafion (Nf) polymer films. SPE with two working electrodes (DSPE) modifies with MnOx–AuOx were used in the two-component analysis of BAA samples. PVP film was deposited on the electrode for thiamine determination and Nf film was deposited on the electrode for pyridoxine determination. Combination of SPE and DSPE with polymer composites containing MnOx–AuOx binary system and PVP or Nf in a two-detector SI-system made it possible to implement a three-component analysis of drugs containing thiamine, pyridoxine and cyanocobalamin. Excipients present in them did not interfere with the results of the determination. Application of the proposed SI-system scheme increased selectivity, speed and productivity of the analysis to 540 determinations per hour.

UV-VIS spectrophotometric-assisted chemometric calibration models for simultaneous determination of thiamine and pyridoxine vitamins in powdered infant formula

Thiamine and pyridoxine, which are hydrophilic B vitamins, are compounds that play an important role in many biochemical events for the health of infants and adults. Recently, because of the complexity of food and pharmaceutical composition, chemometric models are preferred to obtain relevant information from multivariate measured data. The aim of this study was to determine the simultaneous quantitative analysis of thiamine and pyridoxine in powdered infant formula and pharmaceutical formulations by the UV-visible spectrophotometric method coupled with chemometric calibration models (partial least squares (PLS) and principal component regression (PCR)). These vitamins were extracted by addition of trichloroacetic acid (precipitation of proteins) after samples of powdered infant formula was dissolved in Milli-Q water. Factorial design was used to prepare the calibration (25 samples) and validation (8 samples) sets containing mixture of thiamine and pyridoxine within their linear range (2.5-25 μg/mL). Spectra of obtained mixtures and samples were recorded between wavelengths of 200-360 nm with the intervals Δλ=1 nm. In regression models created by using PLS and PCR algorithm, the results had acceptable recovery 98.14% for both compounds. These chemometric models predict the quantification of both, thiamine and pyridoxine in powdered infant formula samples via interpreting the UV-Vis spectrum. The average thiamine and pyridoxine contents of powdered infant formula samples were found to be 5.02±0.065 μg/g and 4.32±0.086 μg/g for PLS model and 4.98±0.078 μg/g and 4.03±0.091 μg/g for PCR model, respectively.

Metabolic engineering of rice endosperm towards higher vitamin B1 accumulation.

SummaryRice is a major food crop to approximately half of the human population. Unfortunately, the starchy endosperm, which is the remaining portion of the seed after polishing, contains limited amounts of micronutrients. Here, it is shown that this is particularly the case for thiamin (vitamin B1). Therefore, a tissue‐specific metabolic engineering approach was conducted, aimed at enhancing the level of thiamin specifically in the endosperm. To achieve this, three major thiamin biosynthesis genes, THIC, THI1 and TH1, controlled by strong endosperm‐specific promoters, were employed to obtain engineered rice lines. The metabolic engineering approaches included ectopic expression of THIC alone, in combination with THI1 (bigenic) or combined with both THI1 and TH1 (trigenic). Determination of thiamin and thiamin biosynthesis intermediates reveals the impact of the engineering approaches on endosperm thiamin biosynthesis. The results show an increase of thiamin in polished rice up to threefold compared to WT, and stable upon cooking. These findings confirm the potential of metabolic engineering to enhance de novo thiamin biosynthesis in rice endosperm tissue and aid in steering future biofortification endeavours.

Fluorometric Detection of Thiamine Based on Hemoglobin-Cu3(PO4)2 Nanoflowers (NFs) with Peroxidase Mimetic Activity.

Component analysis plays an important role in food production, pharmaceutics and agriculture. Nanozymes have attracted wide attention in analytical applications for their enzyme-like properties. In this work, a fluorometric method is described for the determination of thiamine (TH) (vitamin B1) based on hemoglobin–Cu3(PO4)2 nanoflowers (Hb–Cu3(PO4)2 NFs) with peroxidase-like properties. The Hb–Cu3(PO4)2 NFs catalyzed the decomposition of H2O2 into ·OH radicals in an alkaline solution that could efficiently react with nonfluorescent thiamine to fluoresce thiochrome. The fluorescence of thiochrome was further enhanced with a nonionic surfactant, Tween 80. Under optimal reaction conditions, the linear range for thiamine was from 5 × 10−8 to 5 × 10−5 mol/L. The correlation coefficient for the calibration curve and the limit of detection (LOD) were 0.9972 and 4.8 × 10−8 mol/L, respectively. The other vitamins did not bring about any obvious changes in fluorescence. The developed method based on hybrid nanoflowers is specific, pragmatically simple and sensitive, and has potential for application in thiamine detection.

Comparative analysis of riboflavin and thiamine in raw and commercial honey

The paper describes the determination of riboflavin and thiamine in the best-selling types of 13 raw and 8 commercial monofloral and multifloral honeys originating from Serbia. It was found that there is a difference in average riboflavin and thiamine content between raw and commercial honey, as well as between different honey floral varieties. The results showed that forest, meadow and oregano honey had a significantly higher content of both B vitamins compared to acacia, linden and lavender honey. On the other hand, a very low content of riboflavin and thiamine was found in honeydew honey. Among the commercial products, royal jelly was the richest in thiamine and riboflavin, followed by forest honey. In general, the quality of all of the commercial honey samples regarding these vitamins was lower than that of the raw domestic honeys.

Spectrophotometric Determination of Thiamine hydrochloride Via Oxidative Coupling Reaction Using 4-Aminoantipyrine

A sensitive spectrophotometric method has been developed for the determination of thiamine hydrochloride by oxidative coupling reaction of thiamine with the reagent 4-aminoantipyrine (4-AAP) in the presence of copper sulphate as oxidizing agent in alkaine medium forming a reddish brown colour. The product show maximum absorption at 335 nm. The molar absorptivitiy is 26410.59 l/mol.cm for concentrations obeyed Beer’s law in the range 0.2-18 μg.ml-1. The recovery was 100.93 % with relative standard deviation < 2.0 % for thiamine hydrochloride.Thiamine and reagent 4-AAP product was formed in the ratio of 1:1. The stability constant of the product was 3.39×106 l.mol-1 for thiamine hydrochloride indicating the good stability of this product. The optimum conditions for full colour development are described and the proposed method was applied successfully for determination of thiamine in the pharmaceutical preparation (Neurorubine). The common excipients used as additives in pharmaceutical do not interfere in the proposed method.

Rapid Method for the Determination of Thiamine and Pantothenic Acid in Infant Formula and Milk-Based Nutritional Products by Liquid Chromatography‒Tandem Mass Spectrometry.

Thiamine and pantothenic acid play a critical role in numerous metabolic reactions and are typically supplemented in infant and adult nutritional formulas as thiamine chloride hydrochloride and calcium pantothenate salts. A rapid compliance method for the analysis of thiamine and pantothenic acid applicable to infant formula and milk-based nutritional products is described. Proteins are removed by centrifugal ultrafiltration, followed by analysis by reversed-phase liquid chromatography‒tandem mass spectrometry (LC-MS/MS), with quantitation accomplished by internal standard technique. The method was shown to be accurate, with acceptable recovery (thiamine, 99.3-101.1%; pantothenic acid, 99.2-108.6%). A certified reference material (NIST 1849a), showed no statistical bias (α = 0.05) for thiamine (P = 0.64); although a statistically significant bias (P < 0.01) for pantothenic acid was found, the nominal bias was only 4.7% (mean = 7.1 mg/hg; certified value = 6.8 mg/hg). A comparison of results by LC-MS/MS and current methods showed negligible bias (mean bias: thiamine, 0.01 mg/hg; pantothenic acid, 0.17 mg/hg) and no statistical significance (α = 0.05; thiamine, P = 0.399; pantothenic acid, P = 0.058). Acceptable precision was demonstrated with a repeatability of 7.2% repeatability relative standard deviation (RSDr) (HorRat: 0.6) and an intermediate precision of 7.0% RSD for thiamine, and a repeatability of 5.7% RSDr (HorRat: 0.5) and an intermediate precision of 6.1% RSD for pantothenic acid. This rapid method is intended for use in high-throughput laboratories as part of routine product compliance release testing of thiamine and pantothenic acid in manufactured infant and milk-based nutritional products.

Riboflavin and thiamine content in extracts of wild-grown plants for medicinal and cosmetic use

Products derived from wild-grown plants represent a growing segment of the modern pharmaceutical industry. In recent years, particular attention has been paid to their application in the cosmetic industry. The content of B group vitamins in these products is important because they affect the quality of the skin, hair and nails. The determination of thiamine and riboflavin in natural products is not so simple, as it requires complex sample preparation procedures and very sensitive analytical methods. In this paper, the HPLC method with fluorescence detection was applied to the determination of riboflavin and thiamine in the aqueous, ethanol and methanol extracts of elderberry, rosehip, rowanberry, wild garlic and blackthorn. Also, the influence of the extraction solvents on the vitamin’s concentration in obtained extracts was examined. It was found that the content of these vitamins in aqueous extracts is higher than that in alcoholic extracts. Riboflavin concentrations were in the range of 0.84–20.4 μg/g of the dry extract, while thiamine levels were from 0.06 to 0.67 μg/g of the dry extract. The highest riboflavin concentration was found in wild garlic extract followed by rosehip and elderberry extracts. On the other hand, thiamine content was higher in elderberry extract, while in rosehip it was not detected. In this work, the content of riboflavin and thiamine was determined for the first time in extracts of wild garlic, elderberry and rowanberry.

Development of an Isotope Dilution Mass Spectrometry Method for Accurate Determination of Thiamine in Diverse Food Matrices

Many foods contain thiamine as a natural or fortified nutrient. In order to realize accurate measurement of thiamine in various food matrices, analytical methods should be developed on the basis of careful examination of the matrix and the analytes in the matrix. An isotope dilution liquid chromatography–mass spectrometry (ID-LC-MS/MS) method was developed for the analysis of thiamine in various samples such as multivitamin tablets, infant formula, rice, and wheat flour. After using spiked isotope-labeled thiamine HCl (13C4-thiamine HCl) as an internal standard, samples were analyzed by LC/MS in a selected reaction monitoring mode to detect ions at m/z 265 → 122 and m/z 269 → 122 for thiamine and 13C4-thiamine, respectively. To improve the accuracy and precision of this method, the preparation procedure was optimized depending on the chemical form of thiamine present in each sample. Thiamine in multivitamin tablets, infant formula, rice, and wheat flour was analyzed after buffer extraction, acid hydrolysis, and continuous acid and enzyme hydrolyses, respectively. The instrumental analyses were also optimized for individual samples, facilitating excellent repeatability (0.16–0.81%) and reproducibility (0.1–4%). The accuracy of the developed method was verified by comparing the experimental data with the certified values of certified reference materials. Thus, optimization of the preparation procedures and ID-LC-MS/MS method allowed the accurate determination of thiamine in diverse food matrices.

La vitamine B1 : la première vitamine identifiée

B1 vitamin, or thiamin, is a water-soluble vitamin of dietary origin. It acts as a co-factor of metabolic reactions and is involved in the neurotransmission phenomena. The body has a limited stockpile of thiamine, about 25-30 mg, which is found mainly in the liver, but also in the heart, kidneys and muscles. Thiamine is found in many foods (wheat, raw ham, nuts…), and the deficiency is exceptional in industrialized countries where the diet is varied. Absorption defect due to gastric surgery, excessive elimination, chronic alcoholism, pregnancy, elderly subject, genetic disease, rare lack of intake (anorexia). Clinical manifestations of vitamin B1 deficiency or “beriberi” are varied in adults. Gayet-Wernicke’s encephalopathy is an acute neuropsychiatric complication whose progression to Korsakoff syndrome (disorientation, anterograde amnesia and polyneuritis) is irreversible in the absence of treatment. No B1 vitamin overload has been evidenced. B1 vitamin status can be objectively assessed by the determination of transcetolasic activity and/or the determination of thiamine and its intra-erythrocytic phosphoric esters using enzyme and chromatographic techniques. The daily intake required was established in France in 2001 on the basis of sex and age and is approximately 1.1 mg. If a thiamine deficiency is highlighted, a B1 vitamin supplementation will be put in place.

Development of an IPRP-LC-MS/MS method to determine the fate of intracellular thiamine in cancer cells

Understanding the mechanisms underlying cancer cell survival is critical toward advancing drug discovery efforts in this field. Supplemental vitamins have been proposed to play a role in cancer cell metabolism because the increased supply of nutrients is thought to provide cofactors supporting the higher metabolic rate of cancer cells. Particularly, the role of thiamine (vitamin B1) in many biochemical pathways that supports cancer cell metabolism has been investigated. Consequently, the analysis of thiamine and its derivatives in a manner that reflects its dynamic response to genetic modification and pathophysiological stimuli is essential. In this work, we developed a mass spectrometry based-analytical method to track metabolites derived from stable isotope tracers for a better understanding of the metabolic fate of thiamine in cancer cells. This method used ion-pair reversed phase liquid chromatography to simultaneously quantify underivatized thiamine, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) in cells. Hexylamine was used as an ion-pairing agent. The method was successfully validated for accuracy, precision and selectivity in accordance with U.S. FDA guidance. Furthermore, the method was then applied for the determination of thiamine and its derivatives with stable isotope labeling to explore the metabolic fate of intracellular thiamine in cancer cells. The finding shows that thiamine is rapidly converted to TPP however, the TPP does not return to thiamine. It appears that TPP may be utilized for other purposes rather than simply being an enzyme cofactor, suggesting unexplored roles for thiamine in cancer.