Determination of Thiamine Hydrochloride Content in Sample Vitamin B1 Using High Performance Liquid Chromatography

Complied by Group 57: Mizan Muhammad Toyyibun 09.55.06503

Abstract Determination of Thiamine Hydrochloride Content in Sample Vitamin B1 Using High Performance Liquid Chromatography (HPLC). Thiamine dissolve in buffer phosphate ph 4,5. Separation modes of thiamin done by injecting sample solution into HPLC system using stationary phase C18 column (HP Zorbax ), and mobile phase is run as mixed solvent buffer phosphate:methanol (55:45) running at a constant ratio with a flow rate 0.5 mL/minute (isocratic) with setting of UV-detector at wavelength 254 nm. Comparing the sample area with the standard area, the content of thiamin can be determined. The standard thiamine solution 1000 ppm and then diluted to 30 ppm using buffer phosphate, and filtered by Millipore filter paper. The sample preparation is weighed 2 grams of sample vitamin B1and then dissolved in 100 ml volumetric flask using buffer phosphate and filtered with filter paper 41. And then pipette 5 ml filtrate and diluted into 50 ml volumetric flask. After that filtered the diluted solution with Millipore filter paper and injected into the HPLC. The result of analysis is 23,05 mg/tablet and compared by the label of vitamin B1 is 25 mg/tablet. Keyword: Thiamine, High Performance Liquid Chromatography, buffer phosphate:methanol (55:45), isocratic, Millipore filter paper. INTRODUCTION Thiamine (vitamin B1, aneurin) deficiency results in the disease called beriberi, which has been classically considered to exist in dry (paralytic) and wet (oede-matous) forms. Beriberi occurs in human-milk-fed infants whose nursing mothers are deficient. It also occurs in adults with high carbohydrate intakes (mainly from milled rice) and with intakes of anti-thiamine factors, such as the bacterial thiaminases that are in certain ingested raw fish. Beriberi is still endemic in Asia. In relatively industrialized nations, the neurologic manifestations of WernickeKorsakoff syndrome are frequently associated with chronic alcoholism in conjunction with limited food consumption. Some cases of thiamine deficiency have been observed with patients who are hypermetabolic, are on parenteral nutrition, are undergoing chronic renal dialysis, or have undergone a gastrostomy. Thiamine deficiency has also been observed in Nigerians who ate silk worms, Russian school children (Moscow), Thai rural elderly, Cubans, Japanese elderly, Brazilian Xavante Indians, French Guyanese, southeast Asian school children who were infected with hookworm, Malaysian detention inmates, and people with chronic alcoholism. LITERATURE REVIEW High Performance Liquid Chromatography History of HPLC Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. During 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time, pressure liquid chromatography began to be used to decrease flow through time, thus reducing purification times of compounds being

isolated by column chromatography. However, flow rates were inconsistent, and the question of whether it was better to have constant flow rate or constant pressure was debated. High pressure liquid chromatography was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In the late 1970's, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds. By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above the previous techniques. Computers and automation added to the convenience of HPLC. Improvements in type of columns and thus reproducibility were made as such terms as micro-column, affinity columns, and Fast HPLC began to immerge. Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as for the pharmaceutical industry, these fields currently comprise only about 50% of HPLC users. Currently HPLC is used by a variety of fields including cosmetics, energy, food, and environmental industries. Stationary Phase The stationary phase in HPLC refers to the solid support contained within the column over which the mobile phase continuously flows. The sample solution is injected into the mobile phase of the assay through the injector port. As the sample solution flows with the mobile phase through the stationary phase, the components of that solution will migrate according to the non-covalent interactions of the compounds with the stationary phase. The chemical interactions of the stationary phase and the sample with the mobile phase, determines the degree of migration and separation of the components contained in the sample. For

example, those samples, which have stronger interactions with the stationary phase than with the mobile phase, will elute from the column less quickly, and thus have a longer retention time, while the reverse is also true. Normal Phase columns operate on the basis of hydrophilicity by using a polar stationary phase and a less polar mobile phase. Thus hydrophobic compounds elute more quickly than do hydrophilic compounds. Reverse Phase operates on the basis of hydrophobicity by using a stationary phase that consists of silica-based packings with n-alkyl chains covalently bound to them. For example, C-8 signifies an octyl chain and C-18 an octadecyl ligand in the matrix. The more hydrophobic the matrix on each ligand, the greater is the tendancy of the column to retain hydrophobic compounds. Thus hydrophilic compounds elute more quickly than do hydrophobic compounds. Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied to the column, or stationary phase. The mobile phase acts as a carrier for the sample solution. A sample solution is injected into the mobile phase of an assay through the injector port. As a sample solution flows through a column with the mobile phase, the components of that solution migrate according to the noncovalent interactions of the compound with the column. The chemical interactions of the mobile phase and sample, with the column, determine the degree of migration and separation of components contained in the sample. For example, those samples, which have stronger interactions with the mobile phase than with the stationary phase, will elute from the column faster, and thus have a shorter retention time, while the reverse is also true. The mobile phase can be altered in order to manipulate the interactions of the sample and the stationary phase. Mobile phase can be run as a single or mixed solvent running at a constant ratio and flow rate (isocratic) or two separate mobile phases can be

pumped simultaneously at varying concentrations to facilitate washing compounds from the stationary phase (gradient). Separation modes in HPLC Normal Phase Stationary and Mobile Phases: In normalphase HPLC the stationary phase is a polar adsorbent such as bare silica or silica to which polar nonionic functional groupsalcoholic hydroxyl, nitro, cyano (nitrile), or amino-have been chemically attached (e.g., R = -CH2CH2CH2NH2, aminopropyl). Mobile phase: - a nonpolar solvent, such as hexane, to which is added a more polar modifier, such as methylene chloride, to control solvent strength and selectivity. Applications of Normal-Phase HPLC: Normal-phase HPLC is best applied to the separation of compounds that are highly soluble in organic solvents, such as fatsoluble vitamins, or suffer from low stability in aqueous mobile phases, such as phospholipids. Reversed Phase (70 percent of all HPLC separations). Stationary and Mobile Phases: Uses a nonpolar stationary phase (chemically bonded phases) and a polar mobile phase. Many silica-based reversed-phase columns are commercially available, and differences in their chromatographic behavior result from variation in the following: 1. Type of organic group bonded to the silica matrix, such as C18 versus phenyl; 2. Chain length of organic moiety, such as C8 versus C18; 3. Amount of organic moiety per unit volume of packing; 4. Support particle size and shape; 5. Matrix surface area and porosity; 6. Bonded-phase surface topology, such as monomeric versus polymeric; and 7. Concentration of free silanols.

Mobile Phase: Reversed-phase HPLC utilizes polar mobile phases, usually water mixed with methanol, acetonitrile, or tetrahydrofuran. Solutes are retained due to hydrophobic interactions with the nonpolar stationary phase and are eluted in order of increasing hydrophobicity (decreasing polarity). Applications of Reversed-Phase HPLC: Many - for example - plant proteins, waterand fat-soluble vitamins, carbohydrates, soft drinks (caffeine, aspartame, etc.), lipids (including triglycerides and cholesterol, chlorophylls, carotenoids, and anthocyanins). Instrumentation of HPLC

Solvents must be degassed to eliminate formation of bubbles. The pumps provide a steady high pressure with no pulsating, and can be programmed to vary the composition of the solvent during the course of the separation. The liquid sample is introduced into a sample loop of an injector with a syringe. When the loop is filled, the injector can be inject the sample into the stream by placing the sample loop in line with the mobile phase tubing. The different types of HPLC columns are described in a separate document. The presence of analyte(s) in the column effluent is recorded by detecting a change in refractive index, UV-VIS absorption at a set wavelength, fluorescence after excitation with a suitable wavelength, or electrochemical response. Mass spectrometers can also be interfaced with liquid chromatography to provide structural information and help identify the separated analytes.

Thiamine

History of Thiamine In the 19th century discovered the disease beriberi is edemis in Japan, China, and Southeast Asia. Takaki (1906) suggests that this Japanese sailor disease can be reduced by replacing some of the white rice that has been eaten, the bread was made from wheat. Eykman (1897) in Batavia / Jakarta, Indonesia observed that chickens eat white rice remnants of the prison experience severe weakness. Funk (1911) succeeded in isolating factors dedek antiberi-beri of rice and eat vitamins. Jansen and donuts (1926) in the laboratory succeeded in isolating crystalline form Eykman Thiamine and performed experiments on birds. The chemical structure and synthesis of thiamine for the first time successfully carried out by Williams and Cline in 1936. Deficiency Thiamine (vitamin B1, aneurin) deficiency results in the disease called beriberi,which has been classically considered to exist in dry (paralytic) and wet (oede-matous) forms. Beriberi occurs in human-milk-fed infants whosenursing mothers are deficient. It also occurs in adults with high carbohydrateintakes (mainly from milled rice) and with intakes of anti-thiamine factors,such as the bacterial thiaminases that are in certain ingested raw fish.Beriberi is still endemic in Asia. In relatively industrialized nations, the neurologicmanifestations of WernickeKorsakoff syndrome are frequently associatedwith chronic alcoholism in conjunction with limited food consumption. Some cases of thiamine deficiency have

been observed with patients whoare hypermetabolic, are on parenteral nutrition, are undergoing chronic renaldialysis, or have undergone a gastrectomy. Thiamine deficiency has alsobeen observed in Nigerians who ate silk worms, Russian schoolchildren(Moscow), Thai rural elderly, Cubans, Japanese elderly, Brazilian XavanteIndians, French Guyanese, southeast Asian schoolchildren who wereinfected with hookworm, Malaysian detention inmates, and people withchronic alcoholism. Toxicity Thiamine toxicity is not a problem because renal clearance of the vitamin is rapid. Role in human metabolic processes Thiamine functions as the coenzyme thiamine pyrophosphate (TPP) in the metabolism of carbohydrates and branched-chain amino acids. Specifically the Mg2+-coordinated TPP participates in the formation of a-ketols (e.g. among hexose and pentose phosphates) as catalysed by transketolase and in the oxidation of a-keto acids (e.g. pyruvate, aketoglutarate, and branchedchain a-keto acids) by dehydrogenase complexes. Hence, when there is insufficient thiamine, the overall decrease in carbohydrate metabolism and its interconnection with amino acid metabolism (via a-keto acids) has severe consequences, such as a decrease in the formation of acetylcholine for neural function. Biochemical indicators Indicators used to estimate thiamine requirements are urinary excretion, erythrocyte transketolase activity coefficient, erythrocyte thiamine, blood pyruvate and lactate, and neurologic changes. The excretion rate of the vitamin and its metabolites reflects intake, and the validity of the assessment of thiamine nutriture is improved with load test. Erythrocyte transketolase activity coefficient reflects TPP levels and can indicate rare genetic defects. Erythrocyte thiamine is mainly a

direct measure of TPP but when combined with high performance liquid chromatography (HPLC) separation can also provide a measure of thiamine and thiamine monophosphate. Thiamine status has been assessed by measuring urinary thiamine excretion under basal conditions or after thiamine loading; transketolase activity; and free and phosphorylated forms in blood or serum. Although overlap with baseline values for urinary thiamine was found with oral doses below 1mg, a correlation of 0.86 between oral and excreted amounts was found by Bayliss et al. The erythrocyte transketolase assay, in which an activity coefficient based on a TPP stimulation of the basal level is given, continues to be a main functional indicator, but some problems have been encountered. Gans and Harper found a wide range of TPP effects when thiamine intakes were adequate (i.e. above 1.5 mg/day over a 3-day period). In some cases, the activity coefficient may appear normal after prolonged deficiency. This measure seemed poorly correlated with dietary intakes estimated for a group of English adolescents. Certainly, there are both interindividual and genetic factors affecting the transketolase. Baines and Davies suggested that it is useful to determine erythrocyte TPP directly because the coenzyme is less susceptible to factors that influence enzyme activity; there are also methods for determining thiamine and its phosphate esters in whole blood. Factors affecting requirements Because thiamine facilitates energy utilization, its requirements have traditionally been expressed on the basis of energy intake, which can vary depending on activity levels. However, Fogelholm et al. found no difference in activation coefficients for erythrocyte transketolase between a small group of skiers and a less physically active group of control subjects. Also, a study with thiamine-restricted Dutch males whose intake averaged 0.43 mg/day for 11 weeks did not reveal an association between short bouts of intense exercise

and decreases in indicators of thiamine status. Alcohol consumption may interfere with thiamine absorption as well. Evidence used to derive recommended intakes Recommendations for infants are based on adequate food intake. Mean thiamine content of human milk is 0.21 mg/l (0.62 mmol/l), which corresponds to 0.16 mg (0.49 mmol) thiamine per 0.75 l of secreted milk per day. The blood concentration for total thiamine averages 21053 nmol/l for infants up to 6 months but decreases over the first 1218 months of life. A study of 1314 year old children related dietary intake of thiamine to several indicators of thiamine status. Sauberlich et al. concluded from a carefully controlled depletionrepletion study of seven healthy young men that 0.3 mg thiamine per 4184 kJ met their requirements. Intakes below this amount lead to irritability and other symptoms and signs of deficiency. Anderson et al. reported thiamine intakes of 1.0 and 1.2 mg/day as minimal for women and men, respectively. Hoorn et al. reported that 23% of 153 patients aged 6593 years were deemed deficient based on a transketolase activation coefficient greater than 1.27, which was normalized after thiamine administration. Nichols and Basu found that only 57% of 60 adults aged 65 74 years had TPP effects of less than 14% and suggested that ageing may increase thiamine requirements. An average total energy cost of 230 MJ has been estimated for pregnancy. With an intake of 0.4 mg thiamine/4184 kJ, this amounts to a total of 22 mg thiamine needed during pregnancy, or 0.12 mg/day when the additional thiamine need for the second and third trimesters (180 days) is included. Taking into account the increased need for thiamine because of an increased growth in maternal and fetal compartments and a small increase in energy utilization, an overall additional requirement of 0.3mg/day is considered adequate. It is estimated that lactating women transfer 0.2 mg thiamine to their infants through their milk each day. Therefore, an

additional 0.1 mg is estimated as the need for the increased energy cost of about 2092 kJ/day associated with lactation. EXPERIMENTAL Equipment Analysis was carried out used in determination of thiamin in vitamin B1. HPLC Agilent 1100, 100 ml and 50 ml of volumetric flask, 400 ml of beaker glass, watch glass, stirrer, wash bottle, Millipore filter paper Chemical Reagent All solvent were HPLC grade, used for determination of thiamin in vitamin B1. Thiamin-HCl pure analysis, phosphate buffer, and distillated water. Sample preparation Percentage of Thiamin in vitamin B1 can be detected by dissolving sample in buffer phosphate and then filtered with Millipore filter paper and injected into the HPLC and carried by phosphate buffer and methanol in the ratio 55:45 as mobile phase and thiamin-HCL used as standard. Experimental method

has been ready to be injected as a standard for HPLC. 3. Preparation of sample Weighed about 2 grams of vitamin B1 as sample and then dissolved in 100 ml volumetric flask and then diluted and homogenous the solution after that marked up to the line with phosphate buffer. Then pipette 5 ml of sample and put into 50 ml volumetric flask and marked up to the line with phosphate buffer and filtered with Millipore filter paper and the sample has been ready to be injected as sample into the HPLC. OBSERVATION DATA Dilution factor Sample Absorbance Standard Absorbance SD Retention RSD Retention SD Area RSD Area : 100/50 = 20x : 584,80356 : 770,88042 : 7,07x10 : 1,2x10
-4

-4

: 3,7656 : 0,6439

1. Preparation of standard solution 1000 ppm

thiamine CALCULATION The Content of Thiamine

Thiamin-HCL of pure analysis is weighed about 0.1 grams and then dissolved in 100 ml of volumetric flask and diluted with phosphate buffer and then homogenous the solution after that marked up to the line and standard thiamine solution 1000 ppm has been prepared. 2. Preparation of thiamin 30 ppm Pipette 3 ml of standard thiamin solution 1000 ppm into 100 ml volumetric flask and then diluted with phosphate buffer then marked up to the line then filtered with Millipore filter paper and standard

The Content of Thiamine (mg/tablet)

3. RESULT AND DISCUSSION Thiamine can be determined by UV absorption spectrophotometry at a wavelength below 380 nm. The percentage result is 23,05 mg/tablet. While there is negative error that we should considered on several step; Set the HPLC instrument properly. At this setting, the mobile phase used was a mixture of buffer phosphate:methanol (55:45) with stationary phase C18 column and measured at a wavelength of 254 nm. Syringes were once made of metal or glass, and required cleaning and sterilization before they could be used again. Syringe has to be cleaned and rinsed by the sample or standard solution. The amount of injected solution should be more than the amount of sample entered, so do not need mark up to the scale (less excess), because the injected excess solution will be automatically discarded. When the syringe to be injected, press quickly so that no liquid left in the mouth of injector which can cause a negative error. CONCLUTION Analysis result percentage of thiamin in sample Vitamin B1 by using High Performance Liquid Chromatography we get 23,05 mg/tablet. By comparing with the label of the Vitamin B1 25 mg/tablet, there are human errors on the procedure. REFERENCES 1. Anderson SH, Charles TJ, Nicol AD. Thiamine deficiency at a district general hospital: report of five cases. Quarterly Journal of Medicine, 1985, 55:1532. 2. Bailey AL et al. Thiamin intake, erythrocyte transketolase (EC

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