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Human medicines are defined by Medicines and Healthcare products Regulatory Agency as Any substance or combination of substances which may be used in or administered to human beings either with a view to restoring, correcting or modifying physiological functions by exerting a pharmacological, immunological or metabolic action, or to making a medical diagnosis. However, there are products that distinguished from the category of medicinal product as they do not possess the properties as defined by MHRA and any pharmacological active ingredient. These products are known as "borderline preparation", for instance, dietary supplements. Dietary supplements (multivitamin preparation or mineral supplement) are regarded as "food" which is readily ingested by human without having the ability to treat, prevent, diagnose or cure any physiological disorder. Hence, they are defined as food substances formulated in dose form which are supplementary to normal diet and contain intense source of desired nutrients with a nutritional value or physiological effect. Moreover, they do not require approval from marketing authorization or product license issued by MHRA and their effectiveness will not be evaluated prior marketing in United Kingdom. Conversely, they are subjected to Trade Description Act 1968, Food Safety Act 1990, and Food Labeling Regulation 1996. These Acts and Regulation has implemented the requirement of safety, goods described or labeled as to its nature and quality, prohibition of medicinal claims, as well as make it an offence when the requirements are not satisfied. For these reason, together with its definition, the borderline preparation has become the target of companies to make it as their commercial product.
Among the preparations, multivitamin and mineral pills are most commonly found on the shelves of the markets. The reason for this is the perception on primary role vitamins and minerals have been extensively known as essential dietary sources for the normal growth, metabolism, self-maintenance and physiological functioning of human body. In the case of vitamins, they can be further classified as fat-soluble and water-soluble vitamins. The water-soluble vitamins consist of thiamine (B1), riboflavin (B2), nicotinamide (B3), pantothetic acid (B5), pyridoxine (B6), Biotin (B8), Folic acid (B9), Cyanocobalamin (B12), Ascorbic acid (C); fat-soluble vitamins include Retinol (A), Alpha-tocopherol (E), Cholecalciferol (D3), and naphthoquinones (K). They are utilized as cofactors which involved in various metabolic pathways and possess relatively specific function in human health and growth. However, almost all of the vitamins are not synthesized by human cells, except vitamin D and vitamin K whereas all minerals. This fact, together with the essential need for adequate vitamins and minerals, has lead to the raise of perception of involving the dietary supplements into human daily diet for propose of improving the quality of life. On contrary, excessive intake or deficient of vitamins and minerals generate various symptoms and diseases, which may eventually jeopardize physiological functions of human body. The relative functions and diseases are summarised in Table 1.
Vitamins or minerals
Antioxidant, essential in visual process
Promotion of bone formation, prevention of osteoporosis
Hypercalcaemia and calcification of soft tissues
Rickets (children), osteomalacia(adult)
Increase risk of bleeding in vitamin K-deficient people.
No clearly defined syndrome.
Cofactor in metabolism and production of energy; aids nerve conduction.
No toxic effect with high oral doses.
Cofactor in metabolism of carbohydrates, lipids, proteins
Not toxicity effect with high oral doses.
Cofactor in metabolism of carbohydrates, lipids, proteins
Toxic effect on peripheral nervous system
Nervous disorder, dermatitis and anaemia
Folic acid (B9)
Essential in DNA synthesis.
Safe at high doses
Impaired cell division, megaloblastic anaemia
Essential in blood formation, metabolism of folic acid and production of myelin around nerve.
Safe at high doses
Megaloblastic anaemia, demyelination
Ascorbic acid (C)
Antioxidant; essential in synthesis of collagen, noradrenaline; enhance iron absorption from diet.
Gastro-intestinal disturbance and increase stone formation in kidney and bladder in some cases.
Fatigue, weakness, loss of appetite. Severe deficiency cause scurvy
prevent and treat iron-deficient anaemia
catalytic co-factor for cellular enzymes in redox reactions
Excess copper ion will cause cell damage
Metabolism of lipids, carbohydrates, proteins
Low blood pressure, weak muscle and appetite, short of breath
loss of appetite, nausea, vomiting, fatigue, and weakness
Essential for protein synthesis cell membrane integrity
Rough skin;Impaired taste & smell acuity; poor appetite;
Delayed wound healing
Essential in bone formation
cardiac arrhythmias & arrest; renal failure
Ricket ( children) ,Osteoporosis ( adult)
Table 1: summaries of functions, disorder caused by excessive doses and symptoms of deficiency of various vitamins and minerals with Recommended Daily Allowance (RDA)
In United Kingdom, dietary supplements are widely consumed by adult, most commonly in the 55+ years age group (Ransley et al., 2001). It was reported in Full Regulatory Impact Assessemnt that there is nearly half of the commercial food supplements have been made up by commercial multivitamin products, vitamin C products and mineral supplement and they have respectively take up approximately 25%, 13.6%, 7.7% of dietary supplement market. The rise of awareness in good consumption habits and the increasing interest in the vital role of vitamins and minerals in growth and health have also played a major role to the broad sale of commercial dietary supplements. Besides, increased sale and intake of preserved food due to high demand from the growth of human population and the wrong perception of achieving balance diet by compensating with multivitamin pills are also factors that trigger the employment of pharmaceutical multivitamin and minerals product to the market. Moreover, reduction or destruction of vitamin and mineral through chemical reaction during storage and food processing by the manufacturer has give rise to the importance of introducing more available preparation to compensate the possible insufficient of vitamins and minerals in the daily diet. The high availability and demand of dietary supplements, along with the food labeling regulations, has contributed to the development of analytical separation and detection methods for the quality control of the complex constituents. However, the complexity, instability and varies concentration of matrices in a single product has remained as challenges to the research work and simultaneous quantification, therefore, it is crucial to have both accurate and efficient analytical methods for the determination and quality control of the vitamins and minerals supplements.
There are various of analytical method that have been described for determining the vitamins, including the polarographic, fluorimetric, enzymatic, microbiological and spectrophotometric procedures. These methods are, sometimes lack of specificity and time-consuming as they may involve complex chemical, physical, or biological reaction in the preparation of sample as to remove interference occurs during the analytical procedure. In contrast, it has been reported by several paper that there are some easier, uncomplicated, and time-efficient methodologies have been published recently in determining and quantifying vitamins. These methods are liquid chromatography and capillary electrophoresis ( L. Fosting et al., 1997). The high-pressure liquid chromatography (HPLC) has found to be most commonly used in simultaneous determination and individually analysis of vitamins (P P.Moreno & V.Salvado, 2000 ; Edwina M.P. Sau et al., 1997; D.Ivanovic et al.,1999 ). This methodology is reported to be more sensitive, rapid and precise in measuring the content of vitamins and its ability to determine small amount of analyte together with easy coupling with other technique, has made it been extensively used in pharmacopoeia assays. Chromatographic analysis of vitamin with different settings have been describe in several journals and reference books, this include the ion-exchange ( R.C. Williams et al.,1973), normal phase (A.P. De Leenheer, 1992), ion-pairing (D.Ivanovic et al.,1999), and among the them, the reverse phase chromatography is most widely be used (F. Zonta et al., 1982; R.M. Kothari et al., 1982; M. Amin et al., 1987) . Besides that, most paper with chromatographic analysis involves the utilization of complex buffered mobile phase (Zaman et al.1993), a gradient elution program (Blanco et al., 1994; Papadoyannis et al., 1997; (P.Moreno & V.Salvado, 2000), electrochemical (Delgado et al., 1992) or fluorimetric detector (MacCrehan and Schonberger, 1995) , two or three different detectors (Craft et al., 1988; Chase et al., 1995), or changing the detection wavelength during the run ( Lee et al., 1992). The diode arrays detectors were also used in the determination of water and fat soluble vitamin ( Blanco et al., 1994; BoÅ™ivoj et al., 2004). With all these complex settings, the sensitivity and selectivity of HPLC can be intensively improved for analytic process.
High-pressure liquid chromatography (HPLC) is an application most commonly used in analytical method to provide accurate, precise and robust outcome. The typical HPLC system is set up by using a liquid phase which is pumped into a stainless steel column containing compounds of stationary phase. The analyte is loaded into loop injector and transferred onto the head of the column. Subsequently, the analyte is pumped through the column. From settings and mechanisms, they imply that the principle of HPLC is based on the extent of interaction between the analyte and stationary phase which results in relative time spent by analyte in the stationary phase, which term as retention time. Together with UV/visible detector, the detection and separation of sample contents can be obtained by alternating the wavelength of detector. Both mechanisms provide qualitative determination of sample and quantitative analysis of different constituents contain in sample products. In addition, the stability of the pure analyte can be acknowledged with quantitative detection of any degradation product in the sample undergoing elution. HPLC is easily controlled and precise injection of the content ensures quantitative precision. Besides that, it is intensively used in other analytical research works recently which lead to improved columns, detectors and software control. The varieties of stationary phase, mobile phase and different types of detector have contributed to the high selectivity of HPLC with readily adjustable method. In comparison to gas chromatography (GC), HPLC has less risk of sample degradation as it does not involve the heating process in order to vaporize the sample in GC column. Other advantage of HPLC is that it is readily automated which reduce the random error occurs in the process. However, there are still some limitations remain in HPLC system. High amount of organic solvent will be disposed when the retention time of certain analyte is long and leads to the increase cost of analytical research. Additionally, sample with fat-soluble and water-soluble compounds will require separation through extraction and distinct settings in HPLC stationary and mobile phases.
There are several approaches in sample preparation and analysis have been developed in determining the multielement content from different sources. The methods includes the flame atomic absorption spectrometry (S. Soriano. 2007), inductively coupled plasma optical emission spectroscopy (ICP-OES) method (Davidowski, 2005), neutron activation analysis ( I.O. Abugassa et al., 2008). However, only a few papers have been published in related to the determination of compound in multimineral supplements. In spite of different methodologies, the techniques in sample preparation are still remain as the key-step and time-consuming process. For these reason, various ways of extraction procedure have been use as the comparative parameter. Extraction through dry ashing was found to be slower than wet-acid digestion, although the latter one takes 2-3 hours for sample preparation (E. Olveira, 2003). Lost of volatile substances is still as a weak point in dry ashing method which leads to in accurate results obtained. Whereas in wet-acid digestion, it has been reported that large amount of solvents were used and this method required constant supervision (E. Olveira, 2003). Total digestion was also used as it claimed to be not involving acid and heat during sampling process as it only uses a closed-vessel microwave oven device. George et al. (2007) prepared the samples as slurries in aqueous acidic media and his work concluded that slurry suspension sample introduction technique was more reliable in routine qualitative determination of multielement in multimineral tablets. In addition, 20 different elements were simultaneously determined through inductively coupled plasma atomic emission spectrometry (ICP-AES). Incorporation of magnetic or ultrasound stirring in sample preparation was done in S. Soriano et al. (2007) work, but no significant variation was observed in comparison with normal mode (without stirring). Many methodologies have been published in determination of macro, micro, or trace elements, but the downside of them are still remained.
Atomic absorption spectrophotometry (AAS) is used limit tests in determining metallic ingredient contain in the analyte. The principle of AAS is that the atoms of metal undergo volatilization and absorb the emitted energy from the radiation produce by hollow cathode lamp. This mechanism gives rise to the excitation of metallic atoms from ground state so that they produce atomic spectra in order to return to resting state. The absorbed or emitted atomic spectra are extremely narrow and, upon filtration, is then analysed by the detector. It is found to be more sensitive than atomic emission spectrophotometry (AES). The reason is that the numbers of volatilised atom in ground state is higher and more readily to absorb energy from the radiation in AAS, whereas in AES, there is lesser number of atoms which volatilised to excited stage and emit light upon return to ground state. However, the analytical process includes the use of flame, providing a temperature of ca 2500Â°C, in order to volatilise the metal atom. This has limited the use of AAS only applicable to analysis of metallic elements. Other limitation of this method also include the specificity of the hollow cathode lamp, which only coated with the element being analysed, is required to be change every time when different metallic compound is being analysed and simultaneous determination cannot be done in this system. These disadvantages have led to the increase of cost of research and time required as more than one mineral can be found in most dietary supplements.
Up to now, there is no paper has been published which includes the analysis of borderline preparation on both vitamins and minerals. In present work, the evaluation on the "active" ingredient contained in a "borderline" formulation will be carried out with the utilization of different methodologies. A commercial multivitamin/mineral tablets is used as the target sample in the assay. In particular, reverse phase-HPLC system will be used in simultaneously determination of various vitamins, whereas the AAS is used for metal ions analysis. The primary goal of this research work comprises the development and validation of assays for determination of the active ingredients in borderline preparation. This experiment also includes the quantitative and qualitative analysis of the sample ingredients with respective methods. Additionally, the presence of impurities and degraded compounds should also be identified as they affect the quality of the product. Other objectives include the determination the extent of compliance of measured active compound concentration found in the products to pharmacopoeia monographs and the printed content on container's label.