Vitamin fluorescence detection

An automatic method for the separation and determination of RF vitamin in food samples (chicken liver, tablet, and powder milk) is proposed by Zougagh and Rios [2], The method is based on the online coupling of supercritical fluid extraction (SFE) with a continuous flow-CE system with guided optical fiber fluorometric detection (CE-CE-ED). The whole SFE-CF-CE-FD arrangement allowed the automatic treatment of food samples (cleanup of the sample followed by the extraction of the analytes), and the direct introduction of a small volume of the extracted material to the CE-ED system for the determination of RF vitamins. Fluorescence detection introduced an acceptable sensitivity and contributed to avoidance of interferences by nonfluorescent polar compounds coming from the matrix samples in the extracted material. Electrophoretic responses were linear within the 0.05-1 pg/g range, whereas the detection limits of RE vitamins were in the 0.036-0.042 pg/g range. 

Fluorescence is much more widely used for analysis than phosphorescence. Yet, the use of fluorescent detectors is limited to the restricted set of additives with fluorescent properties. Fluorescence detection is highly recommended for food analysis (e.g. vitamins), bioscience applications, and environmental analysis. As to poly-mer/additive analysis fluorescence and phosphorescence analysis of UV absorbers, optical brighteners, phenolic and aromatic amine antioxidants are most recurrent [25] with an extensive listing for 29 UVAs and AOs in an organic solvent medium at r.t. and 77 K by Kirkbright et al. [149]. 

Fig. 2.37. Gradient LC separation of the retinoid solution components and retinoic acid isomers by (A) UV-DAD detection (350 nm) and (B) fluorescence detection with on-line photoreactor switched (a) off and (b) on with irradiation at 366 nm. Peak identification 1 = 13-civ retinoic acid 2 = 9-civ retinoic acid 3 = all-fraws retinoic acid 4 = vitamin A palmitate 5 = /1-carotene. Reprinted with permission from R. Gatti el al. [85].

When fluorescent detection is used, the mobile phase contains zinc chloride as the reduction agent for vitamin K derivatization. The most used mobile phases are methanol and dichloromethane or water. 

Werner ER, Wachter H, Werner Felmayer G (1997) Determination of tetrahydrobiopterin biosynthetic activities by high-performance liquid chromatography with fluorescence detection. In McCormick DB, Suttie JW, Wagner C (eds) Methods in Enzymology Vitamins and Coenzymes. Academic Press, San Diego, pp 53-61... 

The fluorescent intensities of the E vitamers are highly dependent on the solvent. Polar solvents such as diethyl ether and alcohols provide greater intensities compared with hexane. The fluorescence is negligible when the compounds are dissolved in chlorinated hydrocarbons (137). The inclusion of an ether or an alcohol in the hexane mobile phase increases the sensitivity of vitamin E detection measurably in normal-phase HPLC. 

For the determination of supplemental vitamin E in infant formulas, Woollard and Blott (222) employed a radially compressed Radial-PAK cartridge. This enabled lipid material to be rapidly cleared by stepping up the mobile-phase flow rate from 2 ml/min to 10 ml/min after elution of the a-tocopheryl acetate. Fluorescence detection, using a filter-type fluorometer, allowed the indigenous a-tocopherol to be conveniently estimated, while UV absorbance detection was used to quantify the a-tocopheryl acetate. Supplemental retinyl acetate could be assayed simultaneously with either added or indigenous vitamin E using the appropriate detection mode. With the aid of a dual-monochromator spectrofluorometer, a-tocopheryl acetate and a-tocopherol could be determined simultaneously with wavelengths of 280 nm (excitation) and 335 nm (emission), but the increased selectivity eliminated detection of the vitamin A esters (233). 

Reversed-phase HPLC with fluorescence detection is the preferred system for the routine determination of total a-tocopherol in vitamin E-supplemented foods after saponification. The use of NARP chromatography with a predominantly hexane mobile phase allows aliquots of hexane extracts of the unsaponifiable matter to be injected directly onto the column, thus avoiding the evaporation step necessary when a semiaqueous mobile phase is used (234). 

Fig. 13 HPLC of vitamin E. (A) Standards of vitamin E vitamers. Column, 5-p.m Supelcosil LC-Si (250 X 4.6-mm ID) mobile phase, isooctane/ethyl acetate (97.5 2.5), 1.6 ml/min fluorescence detection, excitation 290 nm, emission 330 nm. Peaks (1) a-tocopherol (2) a-tocotrienol (3) /3-tocopherol (4) y-tocopherol (5) /3-tocotrienol (6) y-tocotrienol (7) 5-tocopherol (8) 5-tocotrienol. (B) Saponified rice bran sample. Chromatographic conditions as in (A) except for mobile phase isooctane/ethyl acetate/2,2-dimethoxypropane (98.15 0.9 0.85 0.1). (From Ref. 228. AOCS Press.)...

H Indyk. The photoinduced reduction and simultaneous fluorescence detection of vitamin K, with HPLC. J Micronutr Anal 4 61-70, 1988. 

Table 1 HPLC Methods for Quantitating Total Vitamin C in Foods (C,8 Columns Fluorescence Detection)... 

Recently developed HPLC methods determine thiamine either alone or concomitantly with other vitamins. Tables 6-10 review HPLC methods, published from 1992 to 1997, for the determination of total thiamine. All but one method (79) uses fluorescence detection of the thiochrome derivative. Those methods that determine thiamine simultaneously with other B vitamins are reviewed in Sec. XI of this chapter. 

KY Dodson, ER Young, A-GM Soliman. Determination of total vitamin C in various food matrixes by liquid chromatography and fluorescence detection. J AOAC Int 75 887-891, 1992. 

The second major, and growing, route is the use of HPLC and either UV or fluorescence detection. Here vitamins are extracted from the product and then assayed using HPLC. This is another very specialised area and is often best left to an expert laboratory, as there may be problems with interferences and losses due to such factors as oxidation during extraction. 

Chromatographic methods including thin-layer, hplc, and gc methods have been developed. In addition to developments in the types of columns and eluents for hplc appHcations, a significant amount of work has been done in the kinds of detection methods for the vitamin. These detection methods include direct detection by uv, fluorescence after post-column reduction of the quinone to the hydroquinone, and electrochemical detection. Quantitative gc methods have been developed for the vitamin but have found limited appHcations. However, gc methods coupled with highly sensitive detection methods such as gc/ms do represent a powerful analytical tool (20). 

Plasma PLP levels have been used frequently as an indicator of vitamin status. Vitamin compciunds can be separated from each other and measured individually by high-pressure liquid chromatography (UPLC), where, after separation, the vitamins are detected by fluorescence (Tsuge, 1997). Great care needs tt> be taken... 

Determination of four tocopherols and four tocotrienols in vegetable oils and fats by the official American Oil Chemists Society method is based on separation by normal-phase HPLC and fluorescence detection (AOCS, 1990). Oil samples are dissolved in hexane, whereas margarines and other fats containing vitamer esters need a cold saponification step to liberate the vitamers. The American Association of Cereal Chemists has a method to analyze vitamin E in various foods. This method (AACC, 1997) is applicable to a vitamin E range of 1 x 10" - 100%, and it includes hot saponification and separation by reversed-phase HPLC. Results are calculated as a-tocopherol acetate. The Royal Society of Chemistry has approved a method to analyze vitamin E in animal feedstuffs by normal-phase HPLC after the vitamers have been liberated by hot saponification (Analytical Methods Committee, 1990). 

Fluorescence detection is often used where no other property of the solute (e.g. UV of RI detection) is convenient and can be either an intrinsic property of the solute itself or a derivatised form of the solute. Solution studies have indicated that the sensitivity of detection can be increased by up to three orders of magnitude over UV. This has increased the popularity of post-column fluorescence detection methods for may compounds, including physiological fluids, catecholamines, and other polyamines. A popular use of fluorescence detection is in peptide chemistry where no convenient intrinsic chro-mophore is present. Derivatising agents such as orthophthalaldehyde and fluorescamine are used extensively in both pre- and post-column systems allowing detection of low picomole quantities (Chapter 11). In addition, detection can be performed using the intrinsic fluorescence of many compounds such as steroids, vitamins, and nucleotides. 

Since FMN can be chromatographed on DEAE-Sephadex the newer hydrophihc ion-exchangers (TSK DEAE or Mono Q) may offer some advantages. FMN may be purified by affinity chromatography using apoflavodoxin immobihsed on conventional supports. The intrinsic absorption of the various species of vitamin B2 faciUtates detection by monitoring UV absorbance at 280 nm however, more commonly fluorescence detection is used (excitation at 450 nm and emission at 520 nm). 

Reports on the applications of h.p.l.c. to specific problems include an efficient separation of the reduction products of progesterone, of the conjugates of natural bile acids, and of 2-hydroxy- and 2-methoxy-oestrogens ( catechol oestrogens). Fluorescence detection is reported to be some 500 times more sensitive than u.v. absorption for h.p.l.c. of oestriol. The h.p.l.c. behaviour of compounds in the vitamin D series appears to be correlated with the degree of molecular planarity. " A first report on the use of cholesteric liquid crystals as stationary phases for h.p.l.c. shows promise. Various cholesteryl esters coated on or bonded to Corasil II showed increased capacity factors (k ) when steroids were chromatographed, and permitted some useful separations. 

The interesting combination of capillary electrophoresis-X-ray fluorescence detection was recently described as an element-specific detector [136]. A special plastic cell, which was both X-ray transparent and did not produce any interfering emission, was used. Vitamin B-12 (cyanocobalamin) and the cyclohexanediaminotetraacetic acid complexes of iron, zinc, cobalt, and copper were separated. Although the detection limits found were in the nanogram range, the authors estimate that with optimization that 2 or 3 orders of magnitude more sensitivity is likely. 

Thiamine (vitamin Bj) occurs in foods in free and bound forms, the free form predominates in cereals and plants, whereas the pyrophosphate ester is the main form in animal products. Acid hydrolysis is required to release thiamine from the food matrix. Enzymatic hydrolysis is then needed to convert phosphate esters to thiamine. Prior to CE analysis it is necessary to clean up samples by using ethanol to precipitate protein and by passing through an ion-exchange resin. Thiamine has been determined in meat and milk samples using MEKC with ultraviolet (UV) detection at 254 nm, obtaining comparable sensitivity to that achieved by HPLC using an ion-pair reversed-phase column with postcolumn derivat-ization and fluorescence detection. 

Fluorescence spectroscopy plays an important function in modern food analysis as can be seen from its wide use in the determination of numerous food components, contaminants, additives, and adulterants. This technique has made available very sensitive and selective methods that satisfy the requirements of food analysis, which are usually very complex, taking into account the large number of species to be determined, frequently at very low concentrations, and the wide variety of foodstuffs available. Initially, the use of fluorescence spectroscopy in food analysis was limited to the determination of species with intrinsic fluorescence (e.g., vitamins, aflatoxins, and some polycyclic aromatic hydrocarbons (PAHs)), but now it is widely applied to nonfluorescent species, using several physicochemical means such as chemical or photochemical derivatization reactions. Numerous techniques involve fluorescence detection in liquid chromatography (LC), frequently using pre- or postcolumn derivatization. In addition to conventional fluorime-try, which is commonly chosen for this purpose, other fluorimetric techniques such as laser-induced... 

Vitamin K Vitamin K exhibits an important anti-hemorrhagic activity, which has increased the interest in developing analytical methods to determine its content in foods. The sample preparation includes various steps such as enzymatic hydrolysis and cleanup using SPE cartridges. Normal-phase LC and RP-LC have been used to separate vitamin K in conjunction with UV detection at 270 nm fluorescence detection can be used only after phylloquinone has been converted to the corresponding hydroquinone after electrochemical or chemical reduction. 

Vitamin B2 Food contains three B2 vitamers, riboflavin and its two coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, which are the predominant vitamers in foods and are usually bound to proteins. Their analysis usually takes place after extraction with dilute mineral acids with or without enzymatic hydrolysis of the coenzymes (which is necessary to convert all forms to riboflavin and to quantify them as total riboflavin). The extracts may be purified using SPE with Cig cartridges. All the operations performed prior to analysis need to be done under subdued lighting to avoid decomposition of riboflavin upon exposure to light. RP chromatography with Cig columns is used along with fluorescence detection (excitation, 440 nm emission, 520 nm). 

Although vitamins can be determined, both qualitatively and quantitatively, by MS, routine analysis is usually best conducted by other means (e.g., HPLC with ultraviolet (UV) or fluorescence detection, immunoassay methods, or microbiological methods). Analytically, MS does have an important role as a reference technique, especially when used in isotope... 

The method of choice for the determination of most vitamins is HPLC due to its high separation capability, its mild analytical conditions, and the possibility to use various specifically adapted detection methods, e.g., LTV, fluorescence, or MS detection. All fat-soluble vitamins and most water-soluble vitamins have chromophores suitable for UV detection. Separation of vitamers and stereoisomers can be achieved. If a higher sensitivity is required HPLC with fluorescence detection can be used, either directly (e.g., vitamins A and E) or after derivatization (e.g., thiamine). A further improvement in sensitivity and specificity has been achieved by introducing HPLC with mass spectrometric detection in vitamin analysis. Due to the structural information retrievable, e.g., molecular mass, fragmentation pattern, this is the method of choice for analysis of samples with complex mixtures or low vitamin concentrations. Examples for the use of HPLC-MS in vitamin analysis include the determination of 25-hydroxy-D3 and pantothenic acid. However, one drawback of mass spectrometry is the need for an isotopically labeled reference compound for reliable quantification. Due to the structural complexity of many vitamins, these reference compounds are often expensive and difficult to synthesize. An interesting unique application is the determination of vitamin B12 by HPLC-IPC-MS, which is possible due to its cobalt content. 

UV absorbance detection has been most widely used for vitamin A analysis. However, because retinol and retinyl esters are highly fluorescent, detection limits of one order of magnitude better than in assays with UV detection can be obtained using fluorescence detection. Also, electrochemical detection is a valuable alternative to UV and fluorescence detection provided the eluent contains water to incorporate essential electrolytes. Another detector for LC is the mass spectrometer. The LC-MS approach has also been applied to the analysis of vitamin A and its metabolites. 

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