Riboflavin is said to be unique among the water-soluble vitamins. what makes riboflavin unique?

12.3.2 Water-Soluble Vitamins

Water-soluble vitamins are formed by a wider group of compounds with different functions in humans. In fact, the number of water-soluble vitamins is higher than the fat-soluble vitamins. The analysis of these compounds by LC usually implies different extraction/sample treatment steps due to the possibility of finding these vitamins in foods bound to other components. Besides, the chemical nature of most of water-soluble vitamins impairs also their analysis because of the similarity with other components that may be also co-extracted and analyzed.

Vitamin C, l-ascorbic acid, is one of the most important water-soluble vitamins. The main function of this compound is to prevent and treat scurvy and to act as an antioxidant, although it is also related to other functions. As a result of this activity, this compound is regarded as a potential protective agent against cancer and atherosclerosis, among other diseases. In foods, l-ascorbic acid is widely distributed, above all, in fruits and vegetables. This compound is very prone to degradation of oxidation. This is one of the major problems when analyzing vitamin C by LC. It is usual that typical sample preparation steps are able to degrade this compound. For this reason, acids are frequently employed both in the sample preparation phase as well as in the LC mobile phases to avoid the degradation of this vitamin. RP-LC is the method of choice. For its detection, either UV-based detectors or MS detectors are used. The MS detector provides with better specificity, considering that the UV-Vis absorption of l-ascorbic acid is quite similar to other natural compounds frequently found in food with this component, thus, making difficult its identification.

Vitamin B1 (thiamin) is related to beriberi, a disease associated to the deficiency of this vitamin. In fact, thiamin is a coenzyme in different biochemical reactions. Pork, legumes, as well as liver and kidney products are regarded as excellent sources of this vitamin. Thiamin, as well as other water-soluble vitamins, is frequently found bound to proteins or carbohydrates or even phosphorylated. Thus, before their analysis, the application of a sample treatment to release all the free forms of the vitamin is common. A typical extraction protocol for water-soluble vitamins includes the autoclaving of the sample with hydrochloric acid for the acid hydrolysis of the vitamins followed by an adjustment in the pH to values around 4.0–4.5, adequate for an enzymatic treatment. This vitamin can be, subsequently, separated by ion-pair RP chromatography and detected with a fluorescence detector after a postcolumn oxidation to thiochrome. MS detection through electrospray ionization may also be employed although the separation pH should be wisely adjusted to maximize the ionization of the vitamin.

Riboflavin (vitamin B2) acts also as a cofactor and it is a precursor for the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These coenzymes are widely distributed in the metabolism and catalyze numerous oxidation-reduction reactions. Among the good dietary sources for riboflavin, most animal-derived products, milk and dairy products, are pointed out. Foods are usually pretreated before analysis of riboflavin following similar procedures to those described for vitamin B1. Similarly, fluorescence detection is mostly employed (370 nm ex., 520 nm em.) after RP separation.

Pantothenic acid, also known as vitamin B5, is widely distributed in food since it is a component in the coenzyme A structure. Therefore, it is essential to all organisms being its deficiency not very usual. Besides, being part of this coenzyme, for the total vitamin B5 determination, an enzyme hydrolysis is necessary before analysis. Foods richest on pantothenic acid are organ meats, egg yolk and whole grains. RP separations are employed to analyze pantothenic acid, which does not possess any specific UV-Vis absorption, thus, being its UV detection difficult. To overcome this problem, either fluorescence detection or MS detection may be employed.

In contrast to pantothenic acid, folic acid (vitamin B9) deficiency is more common. Normally, under the term vitamin B9 a series of compounds formed by the same folic acid structure differing on the number of glutamate residues attached to their structure are included. These molecules are used as cofactors and serve as acceptors and donors of one-carbon unit in a variety of reactions involved in amino acid and nucleotide metabolism. Although folates are present in most foods, legumes, leafy green vegetables, citrus and other fruits, and liver are regarded as good sources. The determination of folates is often performed by RP-LC, although UPLC can significantly increase the sensitivity. If total folate is to be determined, enzymatic hydrolysis is needed to deconjugate all the forms to the corresponding monoglutamates. Besides, given the complexity of most of food samples, an SPE clean-up protocol is also frequent. UV-Vis detection (at 290 nm) may be enough if the sample is concentrated. If not, fluorescence detection has to be used to enhance sensitivity.

Niacin refers to a group of compounds also known as vitamin B3, presenting similar biological activity including nicotinamide, nicotinic acid, as well as other pyridine nucleotide structures. In the body, these compounds act as cofactors in oxidation-reduction reactions. To determine the total vitamin B3 content, either an acid or an alkaline hydrolysis is necessary. The separation is normally performed under RP conditions with fluorescence (322 nm ex., 380 nm em.) or UV detection (254 nm).

Although pyridoxine, pyridoxamine, and pyridoxal are three different forms of vitamin B6, pyridoxal-5′-phosphate is the metabolically active form, acting as a cofactor in different reactions involving amino acids, mainly transamination. Good food sources of this vitamin include meats, poultry, fruits, potatoes, and some vegetables. From the different forms in which vitamin B6 is found in food, phosphorylated pyridoxal is generally the most frequent. Thus, acid hydrolysis followed by enzymatic hydrolysis is the most frequent sample preparation protocol before LC analysis. The conditions employed comprise the use of RP methods including buffers in the mobile phases to keep a low pH. The detection may be performed by UV or fluorescence.

Vitamin B12 comprises a family of compounds generically called cobalamin. This coenzyme is particularly rich in animal products, such as meat, seafood, eggs, and milk. Therefore, as cobalamin is normally found with proteins, different steps for its purification have to be carried out before chromatographic analysis. Among them, enzymatic hydrolysis is frequently used, together with a purification protocol using SPE. The LC analysis is usually carried out under RP conditions, being water, methanol, or acetonitrile typical solvents. The UV detection of these components is also possible.

There is an obvious interest on the development of analytical methods able to determine multiple vitamins simultaneously. Nevertheless, as described in the previous section, each water-soluble vitamin has different optimum conditions for their extraction and analysis. Therefore, the most employed strategy is the development of protocols for different groups of vitamins that can be simultaneously and properly analyzed under the same analytical conditions, grouping vitamins that can be treated and extracted under the same conditions. For instance, the combination of an RP separation coupled with MS allowed the simultaneous determination of seven water-soluble vitamers, including thiamin, pyridoxine, pyridoxal, nicotinamide, nicotinic acid, panthotenic acid, and riboflavin in different wheat-based flours and food products [20].

4.09.3.3 Extraction of Water-Soluble Vitamins from Food

Water-soluble vitamins are widely distributed in a variety of foods (as observed in Table 5), although these compounds are often not present at high concentrations. There exist in the literature very well-established methods directed to individually determine the content of one specific vitamin; no significant new applications have been developed using newer advanced techniques.

Water-soluble vitamins in foods are often bound to proteins or carbohydrates, or even phosphorylated. For this reason, the extraction procedures commonly applied before their determination are aimed at releasing all the free forms of the vitamins. A typical extraction protocol for water-soluble vitamins includes the autoclaving of the sample with HCl for the acid hydrolysis of the vitamins. The particular conditions have to be optimized for each matrix, although temperatures of approximately 120 °C for 20 min are commonly used. The next step includes adjusting the pH to values around 4.0–4.5 to subsequently apply an enzymatic treatment. Sodium acetate and takadiastase, respectively, are frequently used for these two steps. The incubation time for this reaction is long, with the purpose of obtaining completely dephosphorylated vitamins. Incubation times for up to 18 h are used at temperatures near 40 °C.

To a great extent, this basic methodology is used for the extraction of thiamin (vitamin B1), riboflavin (vitamin B2), and vitamin B6 from food. In this latter case other enzymes instead of takadiastase can be also used, such as acid phosphatase. In fact, depending on the nature of the sample, some enzymes could provide better results than others; it has been observed that for foods of animal origin, acid phosphatase is the preferred enzyme after acid hydrolysis for the extraction of vitamin B6, whereas for products of vegetal origin, a combination of acid phosphatase and β-glucosidase allows the determination of free pyridoxine and β-glycosilic forms of pyridoxine. Although HCl is the acid most frequently used to carry out the hydrolysis, other strong acids can also be used, such as perchloric acid, metaphosphoric acid, or sulfuric acid. However, in some foods such as milk, this procedure can be omitted and replaced by a simple precipitation of the protein by acidification at room temperature for the determination of thiamin and riboflavin. In this case only nutritionally active thiamin would be detected, since no enzymatic treatment is performed, thus leaving phosphated forms intact. On the other hand, it should be assumed that free riboflavin will be the greatly predominant flavin form. For vitamin B6 determination, it has been observed that homogenization using a 5% metaphosphoric acid solution for 2 min followed by centrifugation of the samples could be enough to extract this vitamin from meat products before analysis.60

Vitamin C can be also extracted from foods by homogenizing with an aqueous solution containing a small proportion of metaphosphoric acid. As already mentioned, under these conditions proteins are denatured and precipitate, thus inactivating enzymes. In addition, this acidic pH favors the stability of both ascorbic and dehydroascorbic acid.

In general, the analysis of vitamin B5 in foods requires the liberation of pantothenic acid bound as coenzyme A. Free pantothenate can be extracted by autoclaving an aqueous suspension of the food product at temperatures around 100 °C for 20 min. However, acid hydrolysis may produce significant loss of pantothenic acid. If the total pantothenate is to be analyzed, further enzymatic treatments are necessary in order to release all of the pantothenic acid bound to its different conjugates. This is mainly performed using a combination of different enzymes, such as pepsin, pantotheinase, or alkaline phosphatase. On one hand, the phosphatase splits the coenzyme A molecule into a phosphate-containing moiety and pantothiene. On the other hand, the action of pantotheinase hydrolyzes this latter molecule into pantothenic acid and β-mercaptoethylamine.

A combination of several enzyme activities is also the preferred method for the extraction and determination of total folates from food. In this case, the trienzyme procedure is based on the use of protease, α-amylase, and folate conjugase enzymes. The particular procedure has to be carefully optimized so that carbohydrate and protein-bound folates are perfectly released from the food matrix while glutamyl peptides of folates are hydrolyzed to mono- or diglutamyl derivatives. In this sense, response surface methodology can be a useful tool to determine the best conditions for each enzyme treatment and incubation time;61 the use of all the enzymes or only some of them will depend on the particular type of food. For instance, for analyzing fortified instant noodles in which the added form of folate exists in the monoglutamic form of folic acid, the use of conjugase is not actually necessary. In this particular case, considering that instant noodles are basically formed by starch, folic acid can be liberated by using only α-amylase.62

Niacin can be determined as readily available niacin or total niacin, although it must be kept in mind that some bound forms of niacin naturally present in food may not be nutritionally available. In this regard, it seems preferable to carry out a treatment with NADase to hydrolyze NAD and NADP to nicotinamide, and to be able to analyze this compound together with nicotinic acid. On the other hand, acid hydrolysis could provide higher levels of determined niacin, although this difference would be caused by the release of bound vitamin forms that are not nutritionally available.63 In addition, acid hydrolysis could produce partial degradation of nicotinamide in some food products. A similar situation can be found when analyzing cobalamin (vitamin B12). This group of vitamins is one of the most complex among the water-soluble vitamins. In food, vitamin B12 exists mainly as coenzyme forms, which are normally bound to proteins that have to be denatured prior to extraction. Therefore, typically α-amylase and pepsin are used to liberate vitamin B12 from the bound proteins. Later, vitamins are extracted using an acetate buffer (pH 4.0) and a small amount of sodium cyanide for a given time to promote the conversion of labile, naturally occurring forms to the more stable form cyanocobalamin. Later still, the mixture is heated to 100 °C for 35 min and diluted as needed for its subsequent analysis.

Sometimes the sample treatment and the vitamins extracted will depend on the expected amounts that could be found and/or on the type of sample. For example, for biotin extraction from fortified samples, which naturally do not have this vitamin, an alkaline extraction using diluted ammonium hydroxide can be enough. However, to fully extract the biotin contained in foods rich on these compounds the extraction conditions have to be more drastic, since the vitamin has to be released from its numerous physical and chemical bonds. In this latter case, a combination of acid hydrolysis and enzymatic digestion is widely preferred. For the acidic hydrolysis, a strong acid (mainly sulfuric acid) at high temperatures is used, as described above, although in this particular case less concentrated acid and shorter hydrolysis times can be applied for the biotin extraction of foods of vegetal origin. Animal tissues generally require more severe conditions. Subsequently enzymatic digestion takes place using, for biotin, papain, which has been shown to be an adequate enzyme for digestion under 37 °C for 16 h.

When extraction and simultaneous determination of several water-soluble vitamins is required, extraction conditions have to be optimized, as a compromise to maximize the extraction of each individual vitamin. For instance, the basic procedure for extracting vitamin C was slightly modified and applied to the extraction of vitamins B1, B2, B3, and B6 besides ascorbic acid from fortified cereals and commercial fruit juices.64 This procedure was based on the homogenization of the sample in a 2% metaphosphoric acid solution and sonication for 10 min, followed by centrifugation. The use of a precipitation solution theoretically able to precipitate proteins and fats in samples such as vitamin-enriched milks has demonstrated its usefulness. The use of a precipitation solution containing zinc and wolframium salts in an acidic medium, plus a centrifugation step, allows the correct extraction of water-soluble vitamins (B1, B2, niacin, pantothenic acid, B6, folic acid, B12, and vitamin C) from enriched milks.65 In other cases, acid hydrolysis and takadiastase digestion steps are carefully optimized to simultaneously extract several water-soluble vitamins, minimizing the losses due to degradation during the extraction.66,67

The aforementioned vitamin extraction mechanisms are sometimes slightly modified, including SPE steps to eliminate interferences from the matrix before the analysis. The possibility of using C18 cartridges in combination with strong cation exchange (SCX) to isolate and concentrate niacin from meat and fish samples after extraction has been explored.68 The two columns are conditioned with methanol and water. Once the sample is loaded into the cartridges, water is passed. Then the C18 cartridge is discarded and the nicotinic acid is removed from the SCX column with 2% ammonium hydroxide in methanol.68

The influence of the nature of the SPE cartridge has been further studied for the extraction of folates. Different SPE cartridges, phenyl-bonded silica, and cyclohexyl-bonded silica have been tested after folate extraction in a wide variety of food matrices.69 It seems that the best results are attained through the combination of a strong anion exchange and a phenyl end-capped cartridge. However, the load capacity of the cartridges can be increased in order to extend this application to other food samples by using a styrene–divinylbenzene-based SPE column after trienzyme extraction of folates.70

C18 SPE cartridges can also be used for the analysis of trace amounts of cyanocobalamin from fatty foods.71 This vitamin is eluted from the C18 cartridges using acetonitrile/water.

Considering the polar nature of water-soluble vitamins, SFE is obviously not the most adequate technique to extract them; however, SFE can be used to extract unwanted compounds from the samples, allowing an easier vitamin analysis from the nonextracted sample. SFE can also be used to remove fats and other lipid-soluble substances from the sample using CO2 as supercritical solvent. The residue left after extraction can be solubilized in water and the content of the vitamins directly analyzed. This cleanup strategy has been applied to water-soluble vitamin extraction from orange juice72 as well as for the determination of riboflavin from other food samples, such as chicken liver or powdered milk.73

Methods of Extraction and Cleanup

Water-soluble vitamins have been determined using a wide range of analytical methods: biological (including microbiological), chemical, physicochemical, chromatographic, and immunological. The need for purification and the method of cleanup for analytical samples greatly depends on the vitamin and/or vitamers to be measured, the analytical method that is to be used, and on the sample material. The physicochemical properties of vitamins may place limitations on the methodology. Furthermore, the sample matrix must be taken into consideration. The vitamin/vitamer concentration in a sample may also vary greatly: pharmaceutical vitamin preparations typically have the highest concentrations, and in biological samples their concentration may be very low. In the latter case, concentration and efficient separation from other interfering substances is a paramount requirement for successful analysis.

Manipulation in the analysis of biological samples (plasma, urine, tissue samples) is designed to decrease the amount of protein and other interfering material, and it may be necessary also to control the oxidation/reduction state of the vitamin. The chemical characteristics of the vitamins (Table 1) are of major importance when choosing the method. Reduction of the protein content may be accomplished by precipitation using acid, heating, or organic solvent. The release of some vitamins from the corresponding esters or conjugates (e.g., folate polyglutamates, thiamin phosphate esters) may demand enzymatic or acid hydrolysis.

The use of solid-phase extraction cartridges is now well established in the analysis of clinical specimens. However, although this method provides efficient purification of the sample, it may lead to a loss of protein-bound vitamins. Direct injection of plasma samples into liquid chromatography (LC) columns is possible in some applications. Dilute filtered or centrifuged urine can be injected in certain LC applications, as is the case in urinary riboflavin assay.

The analysis of foods follows a similar procedure. Liquids (e.g., milk, juice, and infant formula) often require the same process as is applied for plasma or urine, although here lipid removal may also be needed. Solid food is mechanically disrupted or homogenized and hydrolyzed when necessary, followed by extraction or dissolution in a suitable buffer or solvent plus hydrolytic enzyme treatment to release the vitamins from their bound forms, especially those in vitamin-containing enzymes. The analytical method may be directly applied to a clarified supernatant or may require further concentration and purification of the sample, e.g., on a solid-phase extraction cartridge prior to analysis. Table 2 shows an outline of the sample preparation scheme for the determination for thiamin and riboflavin in different foods. This sample preparation procedure efficiently reduces sample matrix interferences and excess oxidizing reagent is flushed off. The extraction cartridge concentrates the vitamins when very low levels are to be analyzed. The subsequent LC analysis is shown in Figure 1 for selected foods.

Table 2. Outline of the sample preparation for determination of thiamin and riboflavin in foods

Adapted from Millipore Corp., Milford, MA.

Pharmaceutical multivitamin preparations can often be analyzed after minimal manipulation: in chromatographic analysis, the finely powdered material is dissolved, or liquid extract is diluted in the mobile phase containing the internal standard. After clarification of the supernatant, appropriate aliquots are injected directly into the LC column. Moreover, the number of vitamers, i.e., different chemical forms used in pharmaceuticals, is generally, rather, limited.

Water-Soluble Vitamins

Water-soluble vitamins are in general more stable than fat-soluble vitamins, although vitamin B2 and to a lesser extent vitamin B12 (folic acid) are light sensitive. All manipulations should therefore be performed in subdued light. No special treatment of samples in pharmaceutical preparations is required before chromatography. In biological fluids or foodstuffs prepurification and/or derivatization of the compounds are necessary before LC separation. The methods include acid extraction followed by enzymatic hydrolysis with takadiastase, papain or acid phosphatase, sometimes with pre-column or post-column chromatography. Trichloroacetic, perchloric and metaphosphoric acids are usually preferred for acid extraction. Depending on the aim of the investigation, vitamins can be determined in their free forms or in both free and phosphorylated forms. In the latter case the enzymatic hydrolysis step is omitted.

In blood, plasma and food, vitamin B1 in protein-free extract is oxidized by agents such as [Fe(CN)6]3−, cyanogen bromide or mercuric chloride to thiochrome in a pre- or post-column chromatography reactor. For pre-column chromatography two different procedures are used. In the first, the thiochrome extract is neutralized by concentrated phosphoric acid to ensure a pH level compatible with the C18 column used for the separation and to eliminate possible pH-dependent alkaline degradation of thiochrome to its disulfide. It is then centrifuged and the supernatant injected into the HPLC. In the second procedure, isobutyl alcohol is used to extract thiochrome after alkaline oxidation. Aliquots of the extracts are then chromatographed.

After acid extraction vitamin B2 is readily detected owing to its intense fluorescence.

Since vitamin B6 is present in six chemical forms, there are methods for the simultaneous separation of the three free forms and the three phosphorylated forms as well as methods for determining the sum of all the forms. Pyridoxamine is transformed into pyridoxal by reaction with glyoxylic acid in the presence of Fe2+ as catalyst. The pyridoxal is then reduced to vitamin B6 pyridoxol by the action of sodium borohydride in an alkaline medium before LC separation. Semicarbazide is also used for post-column derivatization of vitamin B6.

In multivitamin–multimineral preparations, vitamin B12 or cyanocobalamin is extracted with a mixture of dimethyl sulfoxide (DMSO) and water, or ammonium pyrrolidine dithiocarbamate and citric acid in DMSO and water. The extract is centrifuged and the supernatant is diluted with water before concentration and clean-up by solid-phase extraction using a quaternary amine and a phenyl column in series before LC separation. There are few LC methods for the determination of vitamin B12 in human plasma and food.

In biological fluids and foodstuffs a treatment for removing protein is a major requirement for vitamin C assay. Protein precipitation may be done by organic reagents (methanol or acetonitrile) or mineral acids (perchloric, metaphosphoric acid, etc.). Aqueous solutions of vitamin C are rapidly oxidized on exposure to air. Stabilizers such as hydrogen sulfide and 1,4-dithio-dl-threitol have also been employed. The deproteinization may be followed by an enzymatic oxidation of ascorbic acid to dehydroascorbic acid, which is transformed with 1,2-phenylenediamine to its quinoxaline derivative for final separation.

Biotin, or vitamin H, is very stable. However, the limitation of HPLC lies in the lack of a suitable detection system. There are applications of LC to pharmaceutical products containing at least 300 μg biotin per tablet. In pharmaceutical products and feed premix, biotin is extracted from the matrix with buffer, followed by purification and concentration by solid-phase extraction and separation by LC. However, there are few LC methods for the estimation of biotin in biological samples.

Folic acid (pteroylglutamic acid; also called vitamin M) and its derivatives are stable substances. Folic acid may be determined simultaneously with other water-soluble vitamins in pharmaceutical preparations. In food products folates are extracted from the matrix with buffer and enzymes (e.g. hog kidney and chicken pancreas, or rat plasma conjugase, α-amylase and protease together), followed by purification and concentration by solid-phase extraction or with affinity chromatography before final separation.

In pharmaceutical preparations panthenol, panthotenic and its salt (vitamin B5) are extracted with a phosphate solution. The extract is centrifuged, filtered and separated by LC. There are few methods for the determination of pantothenic acid and its salt in food products and biological fluids.

Niacin (or nicotinic acid) and nicotinamide are the two different forms of vitamin PP (so called for its pellagra-preventive factor). Nicotinamide is the form of the vitamin generally found in human plasma. Plasma is deproteinated with acetone/chloroform, the organic layer evaporated to dryness, and the methanolic extract of the residue separated by a reversed-phase HPLC. Isonicotinic acid is used as an internal standard. Urine is purified by extraction with chloroform, the aqueous phase evaporated, and taken for separation by LC. In foods vitamin PP is present mostly in its phosphorylated forms. Hydrolysis is necessary to break the ester bonds, releasing the total vitamin PP content of the food for assay. In food products niacin is extracted with buffer and enzyme. The sample extracts are purified through an ion exchange column (e.g. Dowex 1-X8 resin) before HPLC.

In multivitamin preparations 0.1 mol L−1 hydrochloric acid is used to extract the vitamins and DMSO containing anhydrous citric acid is used to disperse the multivitamin–multimineral preparation, since vitamin B6 is not completely extracted by either 0.1 mol L−1 hydrochloric acid or DMSO owing to adsorption of the vitamin to the minerals. The extraction of nicotinamide is not impaired by the addition of citric acid to DMSO.

Choline in plant material is extracted with isopropanol containing internal standards and p-nitrobenzylhydroxylamine hydrochloride for the formation of p-nitrobenzyl oximes. The extract is purified by solid-phase extraction (C18 and ion exchange), after which the choline fraction is benzoylated to yield UV-absorbing derivatives. In biological samples choline is extracted with formic acid in acetone containing an internal standard. After purification the sample is separated by LC.

For the analysis of inositol mono- and diphosphate isomers in foods the method involves extraction of samples with hydrochloric acid and separation of inositol phosphates by anion exchange chromatography.

V. Summary

Human milk contents of the water-soluble vitamins of well-nourished women and respective intakes of their exclusively breast-fed infants provide the primary knowledge base for estimates of infant water-soluble vitamin requirements and recommended levels of intakes and for the formulation of human milk substitutes. Our knowledge of contents and forms of the water-soluble vitamins secreted and factors capable of having an impact is far from complete. Many of the techniques used to assay the water-soluble vitamins in human milk are insensitive, nondiscriminatory, and inaccurate. Newer methods are available that can furnish accurate determination of the vitamins and their active metabolite and their application to human milk analyses are warranted. There is an amazing lack of data on milk levels secreted in advanced lactation (> 3 months) even though human milk feeding is recommended for the entire first year of life. Investigations on mechanisms of water-soluble vitamin secretion are virtually nonexistent. These are areas where further research is not only warranted, it is necessary.

Riboflavin

Riboflavin is a water-soluble vitamin of group B (B2), which is a precursor of flavoprotein, a key component of mitochondrial complexes I and II. Riboflavin also acts as a cofactor in other key enzymatic reactions, including fatty acid oxidation and the Krebs cycle. The use of riboflavin in an average daily dose of 100 mg contributed to the improvement of the clinical condition of patients with a deficiency of complex I activity caused by acyl-Coa dehydrogenase 9 mutation (ACAD9 is a FAD-containing enzyme) [87]. Also, the positive effect of riboflavin on changes in the activity of complex I was confirmed in a culture of neuronal cells with a mutation in the ACAD9 gene. Riboflavin or riboflavin containing drugs can be added as a means of complex therapy of ischemic or degenerative cerebral pathology [88].

Alternative strategies for the treatment of mitochondrial diseases include biochemical “shunting” of specific respiratory chain complexes, for example, using succinate (an intermediate of the Krebs cycle, which is an electron donor directly to FAD, thus partially “bypassing” complex I) [89] or combinations of vitamins C and K (shunting of complex III) [90]; a decrease in the cytotoxic effect of ROS (N-acetylcysteine, vitamins C and E, lipoic acid, and dimethylglycine) [91], as well as the strategy of “energy buffering” (using creatine and its analogues to increase ATP accumulation through the creatine phosphokinase system) [92].

Thiamine and dichloroacetate, in addition to succinate and vitamins C and K, will belong to drugs that have a “shunting” effect of biochemical processes that occur in mitochondria.

Streptavidin/Avidin–Biotin

Biotin is a small, water-soluble vitamin with a molecular mass of 244. It has an exceptionally high affinity for the egg white protein, avidin (affinity constant 1015 L/mol) in free solution. One of the biological functions of biotin is to prevent absorption of avidin from raw eggs in the gut, which has harmful properties if taken into the circulation. The strength and speed of the binding between avidin and biotin may be used to link molecules together to create signal generation systems. In general, a bacterial source of the protein, streptavidin, is used in preference to avidin. Unlike avidin, streptavidin has a neutral isoelectric point and does not contain carbohydrates. These properties make streptavidin more inert in assay systems, resulting in lower nonspecific binding and hence greater sensitivity. Streptavidin has a molecular mass of 60 kDa.

Each molecule of streptavidin has four binding sites for biotin. Biotin is normally used as the label, in place of a radioisotope or enzyme. Biotinylation of protein is a gentle reaction that usually does not reduce the biological activity. At the end of the assay, a conjugate of streptavidin linked to a signal-generating substance is added. Examples of suitable conjugates are streptavidin-alkaline phosphatase, streptavidin-HRP, streptavidin-125I, streptavidin-fluorescein, and streptavidin-rhodamine. The conjugate and biotin quickly bind together, labeling the bound fraction with the signal source (see Fig. 10). In an immunometric assay, each molecule of the labeled antibody may have many of the small biotin molecules attached to it. This provides a means of attaching extra molecules of enzyme to amplify the final level of signal. Extra amplification may be achieved by using streptavidin conjugates that are labeled with two or three molecules of enzyme. One advantage of using a streptavidin-enzyme conjugate is that it can act as a generic signal generation reagent that can be applied to a range of analyte tests, simply by biotinylation of the relevant analyte-specific antibodies (immunometric format) or analyte (competitive format).

Streptavidin has been incorporated into macromolecular complexes containing large numbers of chelation sites for europium ions, providing several 1000-fold signal amplification of each biotin label (streptavidin-based macromolecular complexes).

Perhaps the greatest benefit of streptavidin-biotin systems has been for the speed and simplicity of assay development. Use of generic signal generation reagents labeled with streptavidin avoids the need to develop individual conjugation methods for each assay. Streptavidin and biotin have also been used by some companies to develop generic capture systems. The solid phase (e.g., microtitration plate) is coated with streptavidin, and the capture antibody is biotinylated. This minimizes the need for new coating methods and facilitates the use of antibodies with high affinities for analyte but poor coating properties (see Separation Systems). For a review of the use of biotin-streptavidin systems in immunoassays, see Diamandis and Christopoulos (1991).

16.3.9 Simultaneous determinations of water-soluble vitamins

The simultaneous determination of several water-soluble vitamins has been a difficult task. There have been various publications on such determinations using HPLC [8]. Although this technique requires gradient elution and suffers from poor efficiency and peak tailing, the use of reversed-phase chromatography for the analysis of formulations containing vitamins has gained wide acceptance from Quality Assurance laboratories of pharmaceutical industries [313,314]. Recently, improvements in the HPLC separation have been achieved using either narrow-bore columns [315,316] and/or by hyphenating HPLC with MS [315,317-319].

Thiamine, riboflavin, pyridoxine, nicotinic acid, nicotinamide and folic acid can be determined simultaneously within 18 min using a Spherisorb ODS-2 (100 × 2.1 mm i. d., 3 μm) microcolumn at 25 °C, and 5 mM sodium hexanesulfonate, 20% methanol, 0.1 % triethylamine and 0.01 M KН2РO4/Н3 PO4, pH 2.8 as mobile phase in a flow gradient from 0.2 ml min− 1 at 5 min to 0.3 ml min− 1 at 5.5 min (Fig. 16.22) [316]. In the monitoring of nicotinic acid, nicotinamide, thiamine and riboflavin at 254 nm, and of pyridoxine and folic acid at 280 nm, the LODs are lower than those obtained with normal-bore columns. Some examples for narrow-bore columns are nicotinic acid, 0.125 ng; nicotinamide, 0.185 ng; pyridoxine 0.26 ng; thiamine, 0.43 ng; folic acid and riboflavin, 0.465 ng, compared with nicotinic acid, 0.38 ng; nicotinamide, 0.56 ng; pyridoxine 0.62 ng; thiamine, 1.26 ng; folic acid, 1.7 ng, and riboflavin, 1.8 ng with the normal-bore columns.

Iida and Murata [318,319] have studied a new buffer system containing formic acid and ammonium formate as a strongly acidic mobile phase for thermospray-LC–MS analysis of thiamine, nicotinic acid, pyridoxine, nicotinamide and pantothenic acid using a Shim-pack CLC-ODS(M) column (150 × 4.6 mm i.d., 5 μm) at 50 °C. This new buffer system reduces peak tailing and retains acids better than the most common volatile electrolytes employed in thermospray-LC–MS, i.e., ammonium acetate solutions, with LODs of 10 pg for thiamine and nicotinic acid, 25 pg for pyridoxine, and 100 pg for nicotinamide and pantothenate. This method has been applied successfully to a commercial health drink and a fermented soybean paste miso analysis, by injecting 5 μl of sample with no pretreatment [319] (Fig. 16.23).

Thiamine, riboflavin, ascorbic acid, dehydroascorbic acid, biotin, nicotinic acid, nicotinamide, pantothenic acid, pyridoxal, pyridoxamine and pyridoxine can be detected simultaneously by HPLC–particle beam-MS [315]. The reversed-phase chromatographic method makes use of a mobile phase consisting of a gradient of methanol and 0.02 M ammonium formate, pH 3.75, at a flow of 0.15 ml min− 1, and of a narrow-bore Ultracarb ODS(20) column (250 × 2.0 mm, 5 μm). With such a system, all analytes except dehydroascorbic acid, pyridoxamine, and ascorbic acid are well resolved; riboflavin is not detected, even at microgram levels, thus indicating that loss of this highly polar compound occurs during transmission of the analyte molecules through the particle-beam interface (Fig. 16.24). Operating in positive-ion chemical-ionization–SIM mode, the LODs are: nicotinamide, 5 ng; nicotinic acid, 4 ng; ascorbic acid, 15 ng; biotin, 250 ng; dehydroascorbic acid, 85 ng; pantothenic acid, 100 ng; pyridoxamine, 400 ng; pyridoxine, 225 ng and thiamine, 90 ng, while in negative-ion chemical ionization–SIM a LOD of 6 ng is obtained for pyridoxal. Comparison of the HPLC–atmospheric pressure chemical ionization spectra with the Ion Spray spectra of the above-mentioned eleven water-soluble vitamins shows that the atmospheric pressure chemical ionization has advantages over ion spray, especially for dehydroascorbic acid [317].

By using electromigration methods (Table 16.9), the simultaneous determination of water-soluble vitamins is greatly improved, especially in clinical and pharmaceutical analysis, because automation of the assays is now available at costs which are considerably lower than other methods. The main features of CE, and its application to the analysis of water-soluble vitamins in clinical [320], pharmaceutical [321,322] and food [323] samples have been reviewed.

Table 16.9. Simultaneous Determinations of Water-Soluble Vitamins: Electromigration Methods

Thiamine, nicotinamide, pyridoxine, pantothenate, ascorbic acid, folic acid, orotic acid, and nicotinic acid are separated by CZE in phosphate buffer at pH 7.0, without assistance from micelles [324]. The buffer pH has a minor impact on thiamine, nicotinamide and nicotinic acid standard separation, whilst the ionic strength and the buffer type strongly affect the electro-osmotic flow, the electrophoretic mobility of the solutes, and the peak shape. The quantitative analysis of these eight vitamins is carried out by using spectral suppression: spectral analysis and peak purity tests for peak identification are very useful when analysing samples where there is an interfering matrix which influences the migration times and peak shapes (Fig. 16.25).

Thiamine, riboflavin, pyridoxal, pyridoxine and pyridoxamine can be determined simultaneously by CZE in phosphate buffer, pH 9.0, by injecting a 5 μl sample via split injection [325]. This method shows a relative standard deviation for peak areas from 2.1 to 6.3 % owing to the split injection, so it can be applied to pharmaceutical products, where the concentrations of analytes are high: it can not be applied to the determination of trace amounts of vitamins in foodstuffs. CZE in phosphate buffer, pH 8.0, is also useful in determining thiamine, nicotinamide, d − (+)-biotin, ascorbic acid, and nicotinic acid in pharmaceutical preparations, native fruit juices, and fruit beverages [326]. The addition of l-cysteine to the samples as an antioxidant is necessary in order to obtain reliable results for ascorbic acid, and the peak areas have to be corrected for the retention times to compensate for the matrix effect. Under these conditions, the LODs are: thiamine, 2.7 μg ml− 1; nicotinamide, 1.6 μg ml− 1, biotin, 3.4 μg ml− 1; ascorbic acid, 1.2 μg ml− 1, and nicotinic acid, 0.9 μg ml− 1. Boonkerd et al. [327] compared two CE modes, CZE and MECC, with the HPLC method given in the US Pharmacopoeia [328], to quantify thiamine, riboflavin, pyridoxine, and nicotinamide in various pharmaceutical formulations (Fig. 16.26). Under the conditions used in the CZE mode, the four vitamins are completely resolved within 6 min, with the following migration sequence: thiamine, nicotinamide, riboflavin, and pyridoxine: paracetamol (internal standard) elutes between nicotinamide and riboflavin. In MECC the separation is completed within 13 min and the order of elution is: nicotinamide, paracetamol, pyridoxine, riboflavin, and thiamine. When either the migration time or the injection volume varies, the precision of the peak area is greatly improved by using the internal standard technique, which gives an RSD of 1%: without an internal standard the RSD is 7-10 %.

Water-soluble vitamins in multivitamin integrators can be separated by MECC [329], using a 70 cm × 100 μm i.d. capillary at 25 °C, operated with 50 mM sodium borate–22.5 mM SDS, 10% (v/v) methanol, at pH 8.0 and 16 kV. By extracting the active ingredients by solid-phase extraction, there are recoveries ranging from 92 to 103 %, with a relative coefficient of variation below 5%.

Boso et al. [330] studied microemulsions consisting of SDS or trimethyl-tetradecyl-ammonium bromide (TTAB), using as test mixture both water-soluble (nicotinamide, pyridoxol, nicotinic acid, thiamine) and fat-soluble-vitamins (vitamin E and vitamin A). The critical pair, pyridoxol – nicotinamide, is not resolved with 20 mM SDS only; on changing the micellar phase to an n-hexane, n-heptane, or cyclohexane-containing emulsion there is a resolution of 1.0 or higher (Fig. 16.27). With TTAB, the best separations are obtained either in the micellar mode only, or in the presence of diethyl ether as the microemulsion component; other types of microemulsions do not improve the resolution of pyridoxol – nicotinamide (Fig. 16.28).

5.1.5.1 Enhancement of Oral Absorption

Various therapeutic agents, such as water-soluble vitamins, structural analogu of natural purine and pyrimidine nucleoside, dopamine, antibiotics, such as ampicillin and carbenicillin, phenytoin, and cardiac glycoside, such as gitoxin, suffer from poor GI absorption. The prime cause of the poor absorption of these agents is their highly polar nature, poor lipophilicity, and/or metabolism during the absorption process. In contrast, gitoxin, a cardiac glycoside, has very poor oral bioavailability due to limited aqueous solubility. This problem could be manipulated successfully by using the prodrug approach. The absorption of water-soluble vitamin was enhanced by derivatization of thiolate ions to form lipid-soluble prodrugs. Dopamine was made useful by making its precursor l-dopa. Although l-dopa is highly polar, it is actively transported through a specific l-amino acid active transport mechanism and regenerates dopamine by decarboxylation.

This approach has been adopted with various penicillin antibiotics. Ampicillin is zwitterionic in the pH range of the GI tract and is only about 20–60% absorbed following oral dosing. Esterification of the carboxyl group of ampicillin to form the prodrugs pivampicillin, bacampicillin, or talampicillin alters the polarity of the molecule and successfully improves oral bioavailability.

7.7.6 Ascorbic Acid

Ascorbic acid (vitamin C) is a water soluble vitamin. It is naturally occurring in considerable amounts in several root crops. In general, yams contain 6–10 mg of vitamin C and may vary up to 21 mg/100 g (FAO, 1997). As reported by the Nutritional Food Survey Committee, potatoes serve as a principal source of vitamin C in British diets, providing 19.4% of the total requirement (FAO, 1997). In addition, the vitamin C content of potatoes varies similar to those of sweet potatoes and cassava. The level of ascorbic acid in TRCs could be reduced during the process of cooking, and their content should be maintained in the diet. However, the concentration of vitamin C varies with the species of TRCs, location, crop year, maturity at harvest, soil, and nitrogen and phosphate fertilizers.