Analysis of vitamin tablets using voltammetry

Vitamins are essential to life.

They are compounds that cannot be manufactured by the body and must therefore be obtained, ideally through the human diet or alternatively via supplementation with vitamin tablets.

Vitamins are characterised by the material in which a vitamin will dissolve, there are two categories; water soluble and fat soluble vitamins.

Water soluble vitamins include the B-complex group and vitamin C and travel through the bloodstream.

The water soluble vitamins that are not used by the body are eliminated during urination which means that a continuous supply is required in the human diet.

Vitamin B2 (also known as riboflavin) is required by the body for growth and red cell production.

It works with other vitamins in the B-complex to process calories from carbohydrates, protein and fat and an adequate intake promotes healthy skin and good vision. Voltammetry offers an alternative analytical technique to methods that are currently used for the quantification of vitamin B2; these include fluorimetric techniques, affinity chromatography, and microbiological methods.

The theory behind the voltammetric determination of vitamin B2 is that riboflavin is reduced to 1,5-dihydroriboflavin following two successive one-electron steps at the mercury electrode.

Manufacturers of formulations that contain vitamin B2 need to ascertain that quality control tolerance values are strictly adhered to and voltammetry can provide this information within ten minutes on the prepared sample.

The role of vitamin B2.

Vitamin B2 was discovered in 1920 and first isolated in egg albumen in 1933.

The chemical structure was determined in 1935 and has two distinct parts; a ribose sugar unit and a three ring flavin structure known as lumichrome. B-complex vitamins have multi faceted functions and vitamin B2 works hand in hand with other B-complex vitamins in the breakdown of carbohydrate, protein and fat for energy.

It is thought that vitamin B2 is helpful in the absorption of iron in treating anaemia and it is needed in the conversion of the amino acid tryptophan to niacin for normal cell function.

Thus an absence of vitamin B2 will impair the other nutrient's ability to function.

Vitamin B2 makes its possible for oxygen to be used by the human body and is unique because it aids the formation of antibodies and red blood cells helping to maintain cell respiration which is good for the preservation of eyesight, skin, nails and hair.

Vitamin B2 eases watery eye fatigue and is helpful in the prevention and treatment of cataracts.

It is required for the health of mucus membranes in the digestive tract and aids activation of vitamin B6.

Vitamin B2 is the substance in vitamins that gives urine its yellow-green fluorescent glow indicating that the body is absorbing and using the vitamin. Vitamin B2 deficiency.

Vitamin B2 deficiency can occur in alcoholics and may be present also in people with sickle cell anaemia.

In developing countries insufficient vitamin B2 has been found to be a risk factor for the development of pre-eclampsia in pregnant woman.

A deficit of vitamin B2 can occur as a result of trauma and has been observed in those patients with chronic debilitating disorders, diabetes and cirrhosis of the liver. Other groups at risk include the elderly, women taking oral contraceptives, children from poor backgrounds, children with chronic heart disease and those individuals that exclude milk products from their diet.

If an individual is subjected to physical and mental stress then this can deplete the body's limited store of vitamin B2.

Reported symptoms associated with an inadequate intake of vitamin B2 include nerve tissue damage, drying of the skin, burning or itching sensation within the eyes, inflammation of the mucous membranes in the mouth and a swollen or reddened tongue.

Cataracts were shown to be associated with a deficiency of vitamin B2 in animals as long ago as the 1930s; studies conducted since then have shown this to be the case in humans too.

A recent evaluation concluded that the chance of developing cataracts were cut in half in people consuming the highest quantities of vitamin B2 compared to those consuming lower amounts.

It is thought that vitamin B2 levels are low in as many as 80% of cataract patients and an estimated 30% of the elderly population is said to consume insufficient quantities of the vitamin.

Sources of vitamin B2.

Vitamin B2 widely occurs in nature and is present in all animal and plant cells but few sources are rich in the vitamin.

The highest concentrations are in yeast and liver but other good sources include enriched breads and cereals, green leafy vegetables, milk and dairy products. Vitamin B2 is one of the vitamins added to flour and bakery products to compensate for losses due to processing; it is also used to enrich milk, breakfast cereals and dietetic products. Vitamin B2 is heat stable and is not easily destroyed during ordinary cooking processes unless the food is exposed to light, in which case up to 50% may be lost.

Because of its light sensitivity, vitamin B2 will disappear rapidly from milk kept in glass bottles exposed to bright sunlight at a rate of up to 85% within two hours.

Sterilisation of foods by irradiation may also cause destruction of vitamin B2.

For individuals requiring supplementation then vitamin B2 is available as oral preparations, alone and in multivitamin combinations with or without other minerals.

At supplementary and dietary levels, vitamin B2 has been found to be non-toxic.

Vitamin tablets or supplements should be stored at room temperature in a dry environment.

It is thought that women should consume about 1.1 milligrams of vitamin B2 daily and men around 1.7 milligrams. Production of vitamin B2.

Vitamin B2 has been produced commercially by chemical synthesis, by fermentation and by a mixed fermentation/chemical synthesis route.

Fermentation is the most recent and cost effective method due to the fact that chemical syntheses involve the use of toxic reagents necessitating stringent environmental control of the derived waste products thereby increasing the cost of production.

The chemical producers use different basic raw materials, a source of carbon is the significant building block for producing vitamin B2 and this can be derived from vegetable oil, glucose, or molasses. The fermentation plant is relatively straight forward as it typically uses simple mixing/stirring vessels and conventional purification technologies.

After separation of the biomass then evaporation followed by drying of the concentrate, an enriched product with a vitamin B2 content of up to 80% can be obtained.

A fermentation plant requires an effective production strain of micro-organism to convert the raw material into vitamin B2 and numerous strains of bacterium, fungus or yeast are used by the different manufacturers.

In contrast to the fermentation/chemical synthesis process, the method of fermentation makes little use of toxic solvents.

The main waste products are edible residues of the production micro-organism and its growth medium which can be used as animal feed or dealt with by conventional sewage treatment plants.

A brief overview of voltammetry.

Jaroslav Heyrovsky first introduced the concept of polarography circa 1922.

The term voltammetry is applied to designate the current-voltage measurement obtained at a given electrode. Polarography is a special case of voltammetry referring to the current-voltage measurement acquired using a dropping mercury electrode with a constant flow of mercury drops.

With the Metrohm Multi Mode Electrode (MME) the mercury is hermetically sealed in the reservoir and suffices for around 200,000 drops ensuring low laboratory running costs.

The Dropping Mercury Electrode (DME) is an electrode mode of the MME.

It is the classical mercury electrode where the mercury flows freely from the glass capillary until the mercury drop is knocked off by a tapping mechanism after each voltage step time set in the measurement mode.

Method for analysis of vitamin B2 in vitamin tablets.

A single vitamin tablet was dissolved in 100ml of 0.03M potassium hydroxide.

19ml of a potassium chloride/potassium carbonate electrolyte and 1ml of the vitamin tablet solution was added to the reaction vessel in the Metrohm 757 VA Computrace.

The role of the electrolyte and additional solutions in voltammetry is crucial as many determinations are pH dependent and the electrolyte can increase the conductivity and selectivity of the solution.

The solution was then degassed with nitrogen for a period of five minutes to remove the electrochemically active oxygen, before the vitamin B2 content was determined with two standard additions using the DME. Conclusion.

The voltammetric method for the determination of vitamin B2 in vitamin tablets is straightforward.

For vitamin tablets, voltammetry requires little sample preparation and the result determined by standard addition obtained in less than ten minutes.

The advantage of using standard addition as a means of calibration and quantification is that matrix effects present in the sample are taken into account.

It would potentially be possible to determine vitamin B2 in a variety of other sample matrices as long as the sample was in a homogenous, aqueous form after sample treatment had been performed.

Over recent years, voltammetry has undergone a tremendous surge in popularity and today represents a refined, clean simple technique that offers outstanding limits of detection and is now the fastest growing analytical technique for trace analysis and is eminently suited to vitamin B2 analysis of vitamin tablets, whereby quality control of the finished product is required to ensure compliance within strictly defined tolerance values.

Voltammetry requires no specialist laboratory infrastructure like expensive fume extraction all that is required is a sturdy bench top on which to mount the instrument and a regulated flow of an inert gas.

The running and maintenance costs of voltammetry are minimal ensuring a cost effective analytical solution to surpass the demands required by those organisations interested in quantifying the levels of vitamin B2 present in vitamin tablets.