Vitamin absorbance detection

Fig. 7 Comparative HPLC separations of a standard solution of six vitamins using (A) 250 X 4.0-mm-ID standard-bore and (B) 250 X 2.0-mm-ID narrow-bore columns. Stationary phase (both columns), 5-/tm Nu-cleosil-120-5 C8 (octyl) mobile phase, methanol/water (92 8). Flow rate (A) 0.7 ml/min, (B) 0.2 ml/min. Injection volume, 1 fi 1. Wavelength-programmed absorbance detection. Peaks (1) retinol (2) retinyl acetate (3) vitamin D3 (4) a-tocopherol (5) a-tocopheryl acetate (6) retinyl palmitate. (From Ref. 108.)...

Fig. 12 Analytical HPLC of fractions from a saponified chicken sample isolated by semipreparative HPLC. (A) Vitamins D2 (internal standard) and D3. Tandem columns, Zorbax ODS + 5 /nm Vydac 201 TP54 Cl8 (250 X 4.6-mm ID) mobile phase, methanol/water (96 4), 1 ml/min absorbance detection, 264 nm. (B) 25-Hydroxyvitamin D2 (internal standard) and 25-hydroxyvitamin D3. Tandem columns, 5 /nm Spherisorb S5NH2 (250 X 4.6 mm) + 10 /nm /nPorasil (300 X 3.9 mm) mobile phase, hexane/2-propanol (97 3), 1 ml/min absorbance detection, 264 nm. (Reprinted in part with permission from Ref. 205. Copyright 1995, American Chemical Society.)...

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). 

Table 30 Capillary Electrophoresis Methods for Quantitating Vitamin C (UV Absorbance Detection)... 

For accurate determination of carotenoids, LC is the preferred technique and many, LC procedures have been published. Generally the sample is extracted into a solvent before being analyzed by reversed-phase LC with UV absorbance detection. These methods have the advantage of being able to detect and quantify individual carotenoids, allowing for more accurate calculation of vitamin A activity. 

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. 

The highest sensitivity and selectivity in vitamin E LC assays are obtained by using fluorescence or electrochemical detection. In the former, excitation at the low wavelength (205 nm) leads to improved detection limits but at the expense of selectivity, compared with the use of 295 nm. Electrochemical detection in the oxidation mode (amperometry or coulometry) is another factor 20 times more sensitive. In routine practice, however, most vitamin E assays employ the less sensitive absorbance detection at 292-295 nm (variable wavelength instrument) or 280 nm (fixed wavelength detectors). If retinol and carotenoids are included, a programmable multichannel detector, preferably a diode array instrument, is needed. As noted previously, combined LC assays for vitamins A, E, and carotenoids are now in common use for clinical chemistry and can measure about a dozen components within a 10 min run. The NIST and UK EQAS external quality assurance schemes permit interlaboratory comparisons of performance for these assays. 

Monferrer-Pons, L., Capella-Peiro, M.E., Gil-Agusti, M., and Esteve-Romero, J., 2003. Micellar liquid chromatography determination of B vitamins with direct injection and ultraviolet absorbance detection. Journal of Chromatography A. 984 223-231. 

E Wang, W Hou. Determination of water-soluble vitamins using high-performance liquid chromatography and electrochemical or absorbance detection. J Chromatogr 447 256-262, 1988. 

Stancher B, Zonta F. High performance liquid chromatographic analysis of riboflavin (vitamin B2) with visible absorbance detection in Italian cheeses. J Food Sci 1986 51(3) 857-8. 

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]. 

Pharmacokinetics Phytonadione is only absorbed from the Gl tract via intestinal lymphatics in the presence of bile salts. Although initially concentrated in the liver, vitamin K is rapidly metabolized, and very little tissue accumulation occurs. Parenteral phytonadione is generally detectable within 1 to 2 hours. Phytonadione usually controls hemorrhage within 3 to 6 hours. A normal prothrombin level may be obtained in 12 to 14 hours. Oral phytonadione exerts its effect in 6 to 10 hours. 

Radiation absorption monitoring of the column effluent at an appropriate wavelength provides the most versatile means of detection for the fat-soluble vitamins. Vitamins A, D, E, and K exhibit characteristic absorption spectra in the UV region, whereas the carotenoid pigments absorb light in the visible region. 

Thompson and Hatina (135) showed that the sensitivity of a fluorescence detector toward unesterified vitamin E compounds under normal-phase conditions was at least 10 times greater than that of a variable-wavelength absorbance detector. The relative fluorescence responses of the tocopherols at 290 nm (excitation) and 330 nm (emission), as measured by HPLC peak area, were a-T, 100 /3-T, 129 y-T, 110 and 5-T, 122. The fluorescence responses of the corresponding to-cotrienols were very similar to those of the tocopherols, and therefore tocotrienol standards were not needed for calibration purposes. The fluorescence detector also allows the simultaneous monitoring of ubiquinone derivatives for example ubiquinone-10 has been detected in tomato (136). 

Because the vitamins occur in food in trace quantities, detection sensitivity is often an issue. Ultraviolet absorbance is the most common detection method. Fluorescence and electrochemical detection are used in specific cases where physicochemical properties permit and where increased sensitivity and selectivity are desired. Refractive index is seldom used, due to its lack of specificity and sensitivity. 

The detection of the individual C vitamers is complicated by their distinctly different properties. Although AA and DHAA are both ultraviolet (UV) absorbers, the absorbance maximum of DHAA is between 210 and 230 nm (15,18,42,43). For practical detection purposes, this makes DHAA particularly susceptible to interferences from a number of naturally occurring food constituents and limits the choice of reagents and solvents. In contrast, AA exhibits a pH-dependent absorbance maximum of 245-265 nm, which makes UV absorbance an ideal choice for detection. On the strength of its reducing capacity, AA can be detected electrochemically, but DHAA is electrochemically inactive. Neither AA nor DHAA fluoresce naturally. However, DHAA readily forms a fluorescent quinoxaline derivative upon reaction with o-phenylenediamine. As a result, chemical derivatization is often used to achieve the sensitivity needed to detect the naturally occurring vitamin C in food. 

Figure 5.8 Separation of eleven water-soluble vitamins by MECC. Peaks 1, pyridoxamine 2, nicotinamide 3 pyridoxal 4, vitamin B6 5, vitamin B2 6, vitamin B12 7, vitamin B2 phosphate 8, pyridoxamine 5 -phosphate 9, niacin 10, vitamin Bi 11, pyridoxal 5 -phosphate. Conditions buffer, 50 mM SDS in 20 mAf phosphate-borate buffer, pH 9.0 applied voltage, 20 kV detection, UV absorbance at 210 nm. (Reprinted from Ref. 20 with permission.)...

Second the test is repeated with intrinsic factor added to the oral dose. The radioactive vitamin B is now absorbed in pernicious anaemia (but not in intestinal malabsorption) and is detected in plasma and urine. Both stages of the test are needed to maximise reliability of diagnosis of pernicious anaemia. 

Fluorescence detectors are used in the detection of e.g., aflatoxins, polynuclear aromatics, certain vitamins and derivatised amino acids. Fluorescence (molecules that absorb and subsequently re-emit radiation) is a more selective mode of detection than absorbance. It is often a phenomenon... 

---