Riboflavin extraction

Solid-phase extraction is routinely used to clean up extracts prior to quantitation (19,42,70, 80-82). Alternatively, endogenous fluorescent artifacts in food samples can be eliminated by oxidation with potassium permanganate/hydrogen peroxide/sodium metabisulphite. Benzyl alcohol has been used to extract riboflavin selectively without the coenzymes, permitting the determination of free riboflavin. 

Riboflavin (E101). This is vitamin B2. Riboflavin can be extracted from yeast but is normally encountered as a nature identical substance. Unfortunately, riboflavin has an intensely bitter taste. The colour produced is an orange yellow. It is stable to acid but is unstable in water. Riboflavin is sometimes used for panned goods. 

The SP procedure of water-soluble vitamins from multivitamin tablets is particularly challenging due to the diverse analytes of varied hydrophobicities and pfC. Water-soluble vitamins (WSVs) include ascorbic acid (vitamin C), niacin, niacinamide, pyridoxine (vitamin B ), thiamine (vitamin Bj), folic acid, riboflavin (vitamin B2) and others. While most WSVs are highly water soluble, riboflavin is quite hydrophobic and insoluble in water. Folic acid is acidic while pyridoxine and thiamine are basic. In addition, ascorbic acid is light sensitive and easily oxidized. The extraction strategy employed was a two-step approach using mixed solvents of different polarity and acidity as follows ... 

More recently [635], a unique extraction step in supplemented foods, by using hot water and a precipitation solution, following by HPLC-ELD/UV analysis has been performed for the simultaneous determination of pyridoxine, thiamine, riboflavin, niacin, pantothenic acid, folic acid, cyanoco-balamin, and ascorbic acid. The mobile phase consisting of phosphate buffer and methanol has been modified in order to perform ion-liquid chromatography by adding l-octanesulfonic acid sodium salt. Furthermore, triethylamine has been also added to improve peak symmetry. 

Juices extracts (liquorize), spirits (orange, lemon), syrups (black currant), tinctures (ginger), and aromatic waters Mineral pigments (iron oxides), natural colorants, anthocyanins, carotenoids, chlorophylls riboflavine, red beetroot extract, and caramel synthetic organic dyes azo compounds... 

Riboflavin [83-88-5] M 376.4, m 295-300°(dec), [aln -9.8 (H2O), -125 (c 5, 0.05N NaOH). Crystd from 2M acetic acid, then extracted with CHCI3 to remove lumichrome impurity. [Smith and Metzler JACS 85 3285 7965]. Has also been crystd from water. 

Babior, who has studied this enzyme at several stages of its purification, found in lysates of PMNs which were activated with zymosan that of eight potential biological reductants only reduced pyridine nucleotides supported the formation of O ". The K , for NADPH was less than the K , for NADH and the activity was decreased in preparations from three patients with chronic granulomatous disease. In accord with predictions based on reaction 7, 0.55 molecule of O7 was measured per molecule of NADPH oxidized under conditions of saturating concentrations of cytochrome c The enzyme which was extracted with Triton X-100 from a granule-rich fraction from activated PMNs, required an external source of FAD for the formation of O from NADPH . Riboflavin and FMN would not substitute. Flavin adenine dinucleotide was proposed as a necessary cofactor, which was probably lost when the enzyme was treated with the detergent. 

More than one century ago a yellow, fluorescent pigment was isolated from whey by Blyth In the subsequent years yellow pigments were extracted from various biological materials. Depending either on the source of isolation or the physical appearance, these natural products were named e.g. lactochrome , lycochrome , ovoflavin , lactoflavin , hepatoflavin , or verdoflavin . Later, it became evident that all these compounds are riboflavin (vitamin B2). 

Yeast extract, liver extract, or cornmeal can be added to whey, thus achieving normal fermentation and avoiding addition of iron. The presence of these added solids (1%) in whey ensures normal fermentation and high yields of riboflavin. 

Exempt colors do not have to undergo formal FDA certification requirements, hut are monitored for purity. The colorants exempt from FD C certification are annatto extract, /i-carolene, beet powder, 0-apo—8 -carntenol. canlhaxanthin. caramel, carmine, carrot oil. cochineal extract, cottonseed Hour, ferrous gluconate, fruit juices, grape skin extract, paprika, paprika oleoresin. riboflavin, saffron, titanium dioxide, turmeric, turmeric oleoresin. ultramarine blue, and vegetable juices. See also Colorants (Foods). 

A recent interlaboratory comparison of HPLC and microbiological methods for total riboflavin revealed significant variability between the 13 participating laboratories (42). The extraction and hydrolysis of the riboflavin coenzymes were cited as the most likely sources of this variability. A later intercomparison (70) of riboflavin methods showed lower variability between laboratories, although coefficients of variability (CV) of 12-40% were still reported. 

LF Russell, JT Vanderslice. Non-degradative extraction and simultaneous quantitation of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in foods by HPLC. Food Chem 43 151-162, 1992. 

AK Hewavitharana. Method for the extraction of riboflavin for high-performance liquid chromatography and application to casein. Analyst 121 1671-1676, 1996. 

Oliveira HM et al (2010) Exploiting automatic on-line renewable molecularly imprinted solid-phase extraction in lab-on-valve format as front end to liquid chromatography application to the determination of riboflavin in foodstuffs. Anal Bioanal Chem 397(1 ) 77-86... 

Lumichrome (Figure 10.1), a water-soluble degradation product of riboflavin,157 appears to be the first characterized gregarious (produced by conspecifics) cue for induction of metamorphosis of a marine invertebrate. Lumichrome is exuded into aqueous media from cultures of larvae of the ascidian Halocynthia roretzi, and was also found in the eggs, gonads, and tunic of the adults following extraction in methanol.157 Metamorphosis of larvae was induced by purified lumichrome... 

Many ascidians live gregariously, and for some of these species conspecific chemical cues may play an important role in gregarious settlement of the larvae. The extracts of conspecific adults, larvae, or their conditioned seawater have been shown to contain metamorphosis inducers which have never been characterized.74-75 Recently, Fusetani and co-workers elucidated the structure of the metamorphosis inducer for the solitary ascidian Halocynthia roretzi, which was isolated from seawater conditioned by ascidian larvae.10 The compound isolated from the medium was identical to lumichrome, a compound known to be a degradation product of riboflavin (Figure 13.1). The origin of lumichrome in //. roretzi is not known at present. 

To illustrate potential problems that may occur with real samples, the effect of flow rate was investigated. To demonstrate this effect, riboflavin was trace-enriched from 10 mL of an aqueous solution. A fresh solid-phase extraction device was properly conditioned and used for each experiment. The surface of the solid phase must be solvated so that the cartridge can be properly conditioned. Directions for solvating the surface can be found in the operating instructions that accompany the cartridge. 

TABLE 6-8. Recovery of Riboflavin from a Solid-Phase Extraction Cartridge"... 

Photolysis of riboflavin leads to the formation of lumiflavin in alkaline solution and lumichrome in acidic or neutral solution (see Figure 7.2). Because lumiflavin is chloroform extractable, photolysis in alkaline solution, followed by chloroform extraction and fluorimetric determination, is the basis of commonly used chemical methods of assaying riboflavin. The photolysis proceeds by way of intermediate formation of cytotoxic riboflavin radicals, and the addition of riboflavin and exposure to light has been suggested as a means of inactivating vimses and bacteria in blood products (Goodrich, 2000). 

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