Chemistry Seminaring Paper

By : Dania Puri Oktaviani

Title : Quantitative Simultaneous Estimation of Water Soluble Vitamins, Riboflavin, Pyridoxine, Cyanocobalamin and Folic Acid in Neutraceutical Products by HPLC



ABSTRACT

Water soluble vitamins e.g. riboflavin (B2), pyridoxine (B6), cyanocobalamin (B12) and folic acid in neutraceutical product have been determined simultaneously by using a rapid, precise and time saving new high performance liquid chromatographic method and its validation. The method involves gradient elution of mobile phase through C18 discovery column (Supelco, Sigma-Aldrich) in a reverse phase chromatography with UV detection at 254 nm at ambient temperature. The ranges for quantification for B2, B6, B12 and folic acid were 0.13 mg g-1 (0.57-131 μg g-1), 0.235 mg g-1 (3-235 μg g-1), 7.94 x 10-2 mg g-1 (8-80 μg g-1) and 9.66 x10-2 mg g-1 (10-97μg g-1), respectively. For the validation of the method, linearity, precision, accuracy and robustness have been performed. The repeatability was measured in terms of RSD value. The RSD for all vitamins was below 1%. Recovery of vitamins ranges from 98.6 to 100.5%.

Key words : HPLC, riboflavin, pyridoxine, cyanocobalamin, folic acid

A. INTRODUCTION
Nutraceutical is a food or food product that provides health and medical benefits, including the prevention and treatment of disease. Such products may range from isolated nutrients, dietary supplements and specific diets. This products were considered alternative medicine for many years. Dietary suplements which is exist in neutaceutical products can be contains vitamins. Vitamins in general, play a very important role in our health, even though they only make up a very small part of the food we eat each day Diets which do not contain adequate amounts of these vitamins often result in deficiency diseases .
Nutraceuticals have become a more mainstream supplement to the diet, now that research has begun to show evidence that these chemicals found in food. And in this paper describes simultaneous estimation of water soluble vitamins, riboflavin, pyridoxine, cyaconobalamin and folic acid.
Vitamin B2 or pyridoxine is identified from eggs in a pure crystalline state and named ovovlavin in 1933 by Kuln and co-workers. Vitamin B2 is an easily absorbed micronutrient with a key role in maintaining health in humans and animals. It is the central component of the cofactors FAD and FMN, and is therefore required by all flavoproteins. As such, vitamin B2 is required for a wide variety of cellular processes. It plays a key role in energy metabolism, and for the metabolism of fats, ketone bodies, carbohydrates, and proteins. Milk, cheese, leafy green vegetables, liver, kidneys, legumes, tomatoes, yeast, mushrooms, and almonds are good sources of vitamin B2. Riboflavin can be assayed by chemical, microbiological, and biological methods. Both fluorometric and microbiological assays are official methods of the Association of Official Analytical Chemicals (AOAC). High performance liquid chromatography (HPLC) has been applied to the determination of riboflavin in a variety of foods by reverse-phase HPLC method. Its use as a column additive is approved by the Food and Drug Administration 21 CFR (73:450).
Pyridoxine is one of the compounds that can be called vitamin B6, along with pyridoxal and pyridoxamine The analysis Pyridoxine is not normally found in plants and plants are not the principal source of this vitamin. This vitamin is made by certain bacteria. Some vegetarians may get adequate pyridoxine simply from eating plants that have traces of soil (like potato skins). Most people get their supply of this vitamin from either milk or meat products. A very good source of pyridoxine is dragon fruit from South East Asia. Lack of pyridoxine may cause anemia, nerve damage, seizures, skin problems, and sores in the mouth. . The analysis of vitamin B6 in food is complicated by the fact that six forms (vitamers) are found in nature, therefore microbiological, colorimetric and HPLC methods are currently used.
Cyanocobalamin is an especially common vitamin of the vitamin B12 family. It is the most famous vitamer of the family, because it is, in chemical terms, the most air-stable. It is the easiest to crystallize and, therefore, easiest to purify after it is produced by bacterial fermentation. Cyanocobalamin is usually prescribed for the following reasons: after surgical removal of part or all of the stomach or intestine to ensure there are adequate levels of vitamin B12 in the bloodstream; to treat pernicious anemia; vitamin B12 deficiency due to low intake from food; thyrotoxicosis; hemorrhage; malignancy; liver or kidney disease. Cyanocobalamin injections are often prescribed to gastric bypass patients having had part of their small intestine bypassed, making it difficult for B12 to be absorbed via food or vitamins. Vitamin B12 can be determined by microbiological, radioisotope dilution spectrophotometric, chemical or biological methods.
Vitamin B9 (folic acid and folate inclusive) is essential to numerous bodily functions ranging from nucleotide biosynthesis to the remethylation of homocysteine. The human body needs folate to synthesize DNA, repair DNA, and methylate DNA as well as to act as a cofactor in biological reactions involving folate. Leafy vegetables are a principal source, although, in Western diets, fortified cereals and bread may be a larger dietary source. A lack of dietary folic acid leads to folate deficiency (FD). This can result in many health problems, the most notable one being neural tube defects in developing embryos. Methods for determining folic acid in food include biological, microbiological, chemical, chromatographic and radiometric assays
Analysis of water soluble vitamins B2, B12, folic acid, biotin and pantothenic acid based on biosensor-based vitamin analysis technology, this method is sensitive but did not analyze the vitamin B6 simultaneously. In another method simultaneous determination of seven water soluble vitamins nicotinamide, thiamin, riboflavin, pyridoxine, pyridoxal, pyridoxamine, cyanocobalamine and folic acid were carried out by using ion-pair chromatography. Nevertheless, the detection time is very high and the UV detector has to set at different wavelengths. Literature showed simultaneous determination of four B-group vitamins, B1, B3, B6 and B12 but B12 has been detected separately at 550 nm. In this paper, will be discussed a new method which simultaneously analyses four water soluble vitamins B6, folic acid, B12, and B2 in a complex mixture (neutraceutical) by HPLC.

B. Quantitative Simultaneous Estimation of Water Soluble Vitamins, Riboflavin, Pyridoxine, Cyanocobalamin and Folic Acid in Neutraceutical Products by HPLC
1. Literature Study
a. High Performance Liquid Chromatographic
High Performance Liquid Chromatography (HPLC) is one mode of chromatography, the most widely used analytical technique. HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture is resolved into its components. The interaction on the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.


The type of HPLC that is used for experiment is liquid-liquid partition of chromatograhy. In liquid-liquid partition chromatography an inert support is coated with a polymeric layer or with a liquid that is insoluble in the mobile phase. This seperation is based on the relative solubility’s of the solutes in the mobile and stationary phase.
In HPLC, there are two operational modes isocratic mode and gradient elution mode. In isocratic mode, mobile phase composition always same when the measurement of progress. On the contrary, in the gradient mode the mobile phase composition was varied during the measurement of progress. In various experiments, using a gradient mode, the result that the separation took place more perfect than using isocratic mode.
There are two types of partition chromatography namely normal phase and reverse phase. Normal phase’s chromatography make us of highlypolar stationary phases such as hexane for the mobile phase. Here the least polar component is eluted where an increase in the polarity of mobile phase will decrease the retention times. Reverse-phase chromatography is used to seperate highly polar analytes, which give problems of long retention times and peak tailingwith conventional absorption chromatography. In this case a non polar stationary phase such as a hydrocarbonis conjunction with a relatively polar mobile phase.
b. Validation of The Method
Linearity is the method's ability to obtain results which are either directly, or after mathematical transformation proportional to the concentration of the analyte within a given range. Linearity is determined by calculating the regression line using a mathematical treatment of the results (ie least mean squares) vs analyte concentration.
The precision of an analytical method is the degree of agreement among individual test results obtained when the method is applied to multiple sampling of a homogenous sample . Precision is a measure of the reproducibility of the whole analytical method (including sampling, sample preparation and analysis) under normal operating circumstances. Precision is determined by using the method to assay a sample for a sufficient number of times to obtain statistically valid results (ie between 6 - 1 0). The precision is then expressed as the relative standard deviation.

Accuracy is a measure of the closeness of test results obtained by a method to the true value. Accuracy indicates the deviation between the mean value found and the true value. lt is determined by applying the method to samples to which known amounts of analyte have been added. These should be analysed against standard and blank solutions to ensure that no interference exists. The accuracy is then calculated from the test results as a percentage of the analyte recovered by the assay.
Robustness measures the capacity of an analytical method to remain unaffected by small but deliberate variations in method parameters. It also provides some indication of the reliability of an analytical method during normal usage. Parameters that should be investigated are percent organic content in the mobile phase or gradient ramp; pH of the mobile phase; buffer concentration; temperature; and injection volume. These parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment.
c. Neutraceutical Products
Nutraceutical is a food or food product that provides health and medical benefits, including the prevention and treatment of disease. Such products may range from isolated nutrients, dietary supplements and specific diets to genetically engineered foods, herbal products, and processed foods such as cereals, soups, and beverages. Dietary suplements are defined as any product (other than tobacco) that is intended to supplement the diet and contains one or more of the following: a vitamin, mineral, herb or other botanical; an amino acid or metabolite; an extract; or any combination of the previously mentioned items.
d. Riboflavin, Pyridoxine, Cyanocobalamin and Folic Acid
Chemical structure of vitamin B2 is as shown in (a). in addition to coenzyme FAD, riboflavin is also a shaper mononucleotide flavin (FMN), which has the formula shown below (b)


Vitamin B2
Vitamin B2 deficiency symptonology includes keilosis. Rich sources of vitamin B2 are milk, meat, egg, and fish.
Vitamin B6 is actually a mixture of pyridoxine, pyridoxal, and pyridoxamine; name “pyridoxine” is generally accepted to this mix because of all these compounds are to each other change. This vitamin acts as a coenzyme for the decarboxylation of amino acids, plays a role in reactions that occur in the nervous system, and also act as coenzymes for the reaction of certain transminase.

Vitamin B12 also called cobalamin, is a water soluble vitamin with a key role in the normal functioning of the brain and nervous system, and for the formation of blood. It is one of the eight B vitamins. It is normally involved in the metabolism of every cell of the human body, especially affecting DNA synthesis and regulation, but also fatty acid synthesis and energy production. As the largest and most structurally complicated vitamin, it can be produced industrially only through bacterial fermentation-synthesis.

Vitamin B12
Folic acid (also known as vitamin B9 or folacin) and folate (the naturally occurring form), as well as pteroyl-L-glutamic acid and pteroyl-L-glutamate, are forms of the water-soluble vitamin B9. Folic acid is itself not biologically active, but its biological importance is due to tetrahydrofolate and other derivatives after its conversion to dihydrofolic acid in the liver.

Vitamin B9
2. Experiment
a. Materials and methods
Potassium dihydrogen phosphate (BDH, Anala R), formic acid (BDH, Anala R) and methanol (BDH, Anala R) were used. Standard solutions of riboflavin (B2), pyridoxine (B6), cyanocobalamin (B12) and folic acid were freshly prepared. Acetic acid (BDH, Anal R), HCl (reagent grade and water (deionized) was used.
b. HPLC Method
The high performance liquid chromatographic system used was equipped with a solvent delivery 200 HPLC pump with online degasser, UV/VIS detector chromatography interface, and a data processing unit compaq. HPLC column discovery C18, 25cm x 4.6mm, 5μm was used for the separation of vitamins. A gradient of methanol and buffer (30:70, in eight minutes) of 50 mM (0.05M) potassium dihydrogen phosphate having pH 4.2 + 0.1, adjusted with formic acid was used as mobile phase. The flow rate was maintained at 1 ml min-1. Wave length of detection was 254 nm. An injection volume of 20μL was chromatographed, and the whole chromatography was performed at ambient temperature.
c. Preparation of Standard Solution
The standard samples B2, B6, B12 and folic acid 100 mg (each) were accurately weighed and transferred into three 100 mL volumetric flask separately and 100 mg of B2 was transferred into 250 mL volumetric flask. Initially 7 mL of acetic acid and 50 mL of methanol were added to each flask; the contents were dissolved by sonication for 10 min and allowed to cool to ambient temperature. The contents were diluted to volume with water and thoroughly mixed. These solutions were used as reference working standard solution (Fig. 1). Prior to injecting into the liquid chromatograph, the solution was filtered through 0.45 μm membrane filter. The
samples were quite stable at room temperature. The stock solutions of standards were kept in a refrigerator for further use and remain unchanged for a period of a month
d. Preparation of Sample Solution
15.07 g of neutraceutical enriched with vitamins was accurately weighed and transferred into a 250 mL round bottom flask. Initially about 10 mL of 0.1 N HCl and 80 mL water was added and then reflux on boiling water bath for 15 min. After completion of refluxing period the flask was cooled and volume made up to 100 mL in a volumetric flask. The content was centrifuged (1400 rpm) to remove suspended material. The supernatant solution was first filtered through a Whatman No. 1 filter paper and the resulting filtrate was again filtered through 0.45 μm membrane filter before injection into LC system (Fig. 2). The stock solutions of sample were kept in a refrigerator for further use and remain unchanged for a period of a month.
C. Results and Discussions
Standard solutions containing B2, B6, B12 and folic acid equal to 20, 50 for linearity, 80, 100, 120, 150 and 180% were prepared and examined by the assay procedure. The peak area responses measured for B2, B6, B12 and folic acid were plotted versus concentration and a linear response was obtained over the range of concentrated studied for all four ingredients. The slope of calibration curve and proximity of all points to the calibration curve demonstrates that the method has adequate sensitivity to the concentrated of vitamins B2, B6, B12 and folic acid.
The accuracy of the assay procedure was determined by carrying out recovery experiments by spiking the standard. Amounts of B2, B6, B12 and folic acid equivalent to 20, 50, 80, 100, 120 and 150% of the theoretical assay concentration were added to the formula amount of neutraceutical preparation and the mixtures were subjected to the assay procedure. The results so obtained are summarized in Table 1. The recovery experiment shows that the method is sufficiently accurate and there is no significant interaction between the active components and excepients.
The precision of assay method was determined under repeatability condition by an experiment in which six preparations were made from the same batch of formulation and were analyzed by one operator on a single occasion. The results are presented in Table 2. The intermediate precision was assessed by another experiment in which two analysts on two different instruments with six independent determinations assayed the same batch of formulation. The results are statistically valid as shown in Table 3.
The robustness of the assay method was assessed with respect to alternations in flow rate, column (same column but of different batch) and change in wave length of UV of the standard and sample, as stability of working standard and test solutions stored in amber glass at ambient temperature. The results show that the new HPLC method is robust to small changes in flow rate, change in column, and the solutions exhibited a good degree of stability. The LOD for B2, B6, B12 and folic acid were found to 0.57, 3, 8 and 10 μg g-1,
respectively.
Neutraceutical preparation omitting B2, B6, B12 and folic acid was examined by the assay procedure. No peak due to excipients in the formulation was observed at the typical retention times for B2, B6, B12 and folic acid. Therefore, it is concluded that the assay method is specific for both active ingredient in the presence of excipients of formulated product. The suitability of the system was defined by determining the value of column efficiency, tailing factor and resolution factor using the method in VSP. Column efficiency was greater than 1000 per column, tailing factor was not more than 2 and resolution factor is greater than 3 for B2, B6, B12 and folic acid (Table 4).



The dependence of retention time on flow rate has been observed for both standard and sample and it was found that a increase in flow rate decrease the retention time of B2, B6, B12 and folic acid in a regular manner. The HPLC method has been found to be time saving with a high degree of precision and accuracy.



D. Conclusions
Based on experiment we can concluded that HPLC is a simple, rapid, economic and accurate quantitative simultaneous estimation of water soluble vitamins, riboflavin, pyridoxine, cyanocobalamin and folic acid in neutraceutical products. A validation of this method was carried out and showed that specificity, robustness and precision are guaranteed.












BIBLIOGRAPHY

Anonim. 2010. Cyaconobalamin. [Online]. Avalilable : http://en.wikipedia.org/wiki/ Cyaconobalamin. [October 5th 2010].
Anonim. 2010. Folic Acid. [Online]. Avalilable : http://en.wikipedia.org/wiki/Folic acid. [October 5th 2010].
Anonim. 2010. Neutraceutical. [Online]. Available: http://en.wikipedia.org/wiki/ Nutraceutical. [October 5th 2010].
Anonim. 2010. Pyridoxine. [Online]. Avalilable : http://en.wikipedia.org/wiki/ Pyridoxine. [October 5th 2010].
Anonim. 2010. Riboflavin. [Online]. Avalilable : http://en.wikipedia.org/wiki/ Riboflavin. [October 5th 2010].
HAM, Mulyono. 2005. Kamus Kimia. Jakarta : Bumi Aksara.
Hendayana, Sumar. 2006. Kimia Pemisahan. Bandung : PT Remaja Rosda Karya.

Mulry, Mary. 2000. Functional Foods and Neutraceutical. [Online]. Available: http://www.chiro.org/nutrition/FULL/Functional_Foods.shtml [October 10th 2010].
P. Elmer, Hearn. 2010. A guide validation in HPLC. [Online]. Available : http://152.106.6.200:8080/dspace/bitstream/10210/379/11/11Ch3HPLC.pdf
Perveen, Shahnaz. Et al. 2009. Quantitative Simultaneous Estimation of Water Soluble Vitamins, Riboflavin, Pyridoxine, Cyanocobalamin and Folic Acid in Neutraceutical Products by HPLC. The Open Analytical Chemistry Journal, 2009, 3, 1-5 [Online]. Available : www.bentham-open.org. [September 21st 2010].
Poedjiadi, Anna. 2005. Dasar-Dasar Biokimia. Jakarta : Penerbit Universitas Indonesia (UI-Press).
Shabir, Ghulam A. 2004. HPLC Method Development and Validation for Pharmaceutical Analysis. [Online]. Available : http://pharmtech.findpharma.com/pharmtech/article/articleDetail.jsp?id=89002.