Vitamin samples

Figure 2 shows emission spectra of a solution containing thiamine and riboflavin (vitamin B2) taken with the IDA at various times. Curve A is the spectrum of the original vitamin sample solution acidified to pH 2 with Hg + added. Under computer control this initial spectrum is acquired in 2 s and then pH 12.2 buffer is injected into the sample cell to raise the reaction mixture pH to initiate the thiochrome reaction. Curves B, C, and D are the emission spectra obtained at 16, 40, and 60 s after initiation of the reaction with a 2 s integration time. These spectra are now used to illustrate a number of important points. 

Vitha, M. F. Carr, P. W. A Laboratory Exercise in Statistical Analysis of Data, /. Chem. Educ. 1997, 74, 998-1000. Students determine the average weight of vitamin E pills using several different methods (one at a time, in sets of ten pills, and in sets of 100 pills). The data collected by the class are pooled together, plotted as histograms, and compared with results predicted by a normal distribution. The histograms and standard deviations for the pooled data also show the effect of sample size on the standard error of the mean. 

Many pharmaceutical compounds contain chromophores that make them suitable for analysis by UV/Vis absorption. Products that have been analyzed in this fashion include antibiotics, hormones, vitamins, and analgesics. One example of the use of UV absorption is in determining the purity of aspirin tablets, for which the active ingredient is acetylsalicylic acid. Salicylic acid, which is produced by the hydrolysis of acetylsalicylic acid, is an undesirable impurity in aspirin tablets, and should not be present at more than 0.01% w/w. Samples can be screened for unacceptable levels of salicylic acid by monitoring the absorbance at a wavelength of... 

Samples of urine are analyzed for riboflavin before and after taking a vitamin tablet containing riboflavin. Concentrations are determined using external standards or by the method of standard additions, fluorescence is monitored at 525 nm using an excitation wavelength of 280 nm. 

The elution order for neutral species in MEKC depends on the extent to which they partition into the micelles. Hydrophilic neutrals are insoluble in the micelle s hydrophobic inner environment and elute as a single band as they would in CZE. Neutral solutes that are extremely hydrophobic are completely soluble in the micelle, eluting with the micelles as a single band. Those neutral species that exist in a partition equilibrium between the buffer solution and the micelles elute between the completely hydrophilic and completely hydrophobic neutrals. Those neutral species favoring the buffer solution elute before those favoring the micelles. Micellar electrokinetic chromatography has been used to separate a wide variety of samples, including mixtures of pharmaceutical compounds, vitamins, and explosives. 

Procedure. A vitamin B complex tablet Is crushed and placed In a beaker with 20.00 mL of a 50% v/v methanol solution that Is 20 mM In sodium tetraborate and contains 100.0 ppm of o-ethoxybenzamIde. After mixing for 2 min to ensure that the B vitamins are dissolved, a 5.00-mL portion Is passed through a 0.45- xm filter to remove Insoluble binders. An approximately 4-nL sample Is loaded Into a 50- xm Internal diameter capillary column. For CZE the capillary column contains a 20 mM pH 9 sodium tetraborate/sodlum dIhydrogen phosphate buffer. For MEKC the buffer Is also 150 mM In sodium dodecylsulfate. A 40-kV/m electric field Is used to effect both the CZE and MEKC separations. 

Bohman and colleagues described a reverse-phase HPLC method for the quantitative analysis of vitamin A in food using the method of standard additions. In a typical example, a 10.067-g sample of cereal is placed in a 250-mL Erlenmeyer flask along with 1 g of sodium ascorbate,... 

The vitamin B12 content of a multivitamin tablet is determined by dissolving ten tablets in water. The dissolved tablets are transferred to a 100-mL volumetric flask and diluted to volume. A 50.00-mL portion is removed and treated with 0.500 mg of radioactive vitamin B12 having an activity of 572 cpm. After homogenization, the vitamin B12 in the sample is isolated and purified, producing 18.6 mg with an activity of 361 cpm. Calculate the average concentration of vitamin B12 in the tablet (in milligrams per tablet). 

Pyridine is also sold as a 1° grade, which means that the boiling poiat range of 98% of the sample will fall ia a 1°C range which iacludes the normal boiling poiat of (1) (115.3 0.1°C). Niacia (27) and niacinamide (26), equivalent forms of vitamin B, are generally sold under a US. Pharmacopeia (USP) specification (78). They are also sold as a feed-grade supplement (see Vitamins). 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

Spectrophotometric deterrnination at 550 nm is relatively insensitive and is useful for the deterrnination of vitamin B 2 in high potency products such as premixes. Thin-layer chromatography and open-column chromatography have been appHed to both the direct assay of cobalamins and to the fractionation and removal of interfering substances from sample extracts prior to microbiological or radioassay. Atomic absorption spectrophotometry of cobalt has been proposed for the deterrnination of vitamin B 2 in dry feeds. Chemical methods based on the estimation of cyanide or the presence of 5,6-dimethylben2irnida2ole in the vitamin B 2 molecule have not been widely used. 

Various aspects of the chromatography of vitamin B 2 and related corrinoids have been reviewed (59). A high performance Hquid chromatographic (hplc) method is reported to require a sample containing 20—100 p.g cyanocobalamin and is suitable for premixes, raw material, and pharmaceutical products (60). 

In 1949 the World Health Organization adopted the biological activity of 1 mg of an oil solution containing 0.025 p.g of crystalline D as the analytical standard for vitamin D. This standard was discontinued in 1972. USP uses crystalline cholecalciferol as a standard (80). Samples of reference standard may be purchased from U.S. Pharmacopeia Convention, Inc., Reference Standards Order Department, 12601, Twinbrook Parkway, Rockville, Maryland 20852. One international unit of vitamin D activity is that activity demonstrated by 0.025 ]1 of pure crystalline (7 -vitamin D. One gram of vitamin D3 is equivalent to 40 x 10 lU or USP units. The international chick unit (ICU) is identical to the USP unit. 

The standard chemical and biological methods of analysis are those accepted by the JnitedStates Pharmacopeia XXIII as well as the ones accepted by the AO AC in 1995 (81—84). The USP method involves saponification of the sample (dry concentrate, premix, powder, capsule, tablet, or aqueous suspension) with aqueous alcohoHc KOH solvent extraction solvent removal chromatographic separation of vitamin D from extraneous ingredients and colormetric deterrnination with antimony trichloride and comparison with a solution of USP cholecalciferol reference standard. 

Preferably, high pressure Hquid chromatography (hplc) is used to separate the active pre- and cis-isomers of vitamin D from other isomers and allows their analysis by comparison with the chromatograph of a sample of pure reference i j -vitainin D, which is equiUbrated to a mixture of pre- and cis-isomers (82,84,85). This method is more sensitive and provides information on isomer distribution as well as the active pre- and cis-isomer content of a vitamin D sample. It is appHcable to most forms of vitamin D, including the more dilute formulations, ie, multivitamin preparations containing at least 1 lU/g (AOAC Methods 979.24 980.26 981.17 982.29 985.27) (82). The practical problem of isolation of the vitamin material from interfering and extraneous components is the limiting factor in the assay of low level formulations. 

Physical Methods. Vitamins D2 and D exhibit uv absorption curves that have a maximum at 264 nm and an (absorbance) of 450—490 at 1% concentration (Table 8). The various isomers of vitamin D exhibit characteristically different uv absorption curves. Mixtures of the isomers are difficult to distinguish. However, when chromatographicaHy separated by hplc, the peaks can be identified by stop-flow techniques based on uv absorption scanning or by photodiodearray spectroscopy. The combination of elution time and characteristic uv absorption curves can be used to identify the isomers present in a sample of vitamin D. 

Table 12. Vitamin Content of Various Samples of Cocoa Beans and Chocolate Products, Whole Weight Basis, mg/100 g...

FIGURE 7.17 Separation of a complex mixture on Fractogel EMD BioSEC (S) with a column dimension of 1000 X 50 mm (Superformance glass column). The sample contained ferritin (I), immunoglobulin G (2), transferrin (3), ovalbumin (4), myoglobin (5), aprotinin (6), and vitamin B, (7). Five milliliters of the mixture was injected onto the column at a flow rate of 3 ml/min (eluent 20 mAI sodium phosphate buffer, 0.1 M NaCI, pH 7.2). 

Gel permeation ehromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard referenee material (SRM) samples of a fortified eoeonut oil (SRM 1563) and a eod liver oil (SRM 1588). The on-line GPC/normal-phase proeedure eliminated the long and laborious extraetion proeedure of isolating vitamins from the oil matrix. In faet, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC eolumns used for the vitamin determinations were a 10 p.m polystyrene/divinylbenzene gel eolumn and a semipreparative aminoeyano eolumn, with hexane, methylene ehloride and methyl tert-butyl ether being employed as solvent. 

Problem 14,14 A knowledge of molar absorpiivities is particularly important in biochemistry, where UV spectroscopy can provide an extremely sensitive method of analysis. For example, imagine that you wanted to determine the concentration of vitamin A in a sample. If pure vitamin A has Amax = 325 (e = 50,100), what is the vitamin A i concentration in a sample whose absorbance at 325 nm is A = 0.735 in a cell with 1 a pathlength of 1.00 cm ... 

Suppose a sample of vitamin C is known to contain 1.29 X 1024 hydrogen atoms (as well as other kinds of atoms). What is the chemical amount (in moles) of hydrogen atoms in the sample ... 

Suppose that an analytical laboratory reported a composition of 40.9% carbon, 4.58% hydrogen, and 54.5% oxygen for a sample of vitamin C. In what atom ratios are the elements present in vitamin C ... 

SOLUTION We consider a sample of exactly 100 g, then convert the masses into I amounts in moles by dividing the mass percentage for each element by the element s molar mass. The mass of carbon in a sample of vitamin C of mass 100 g is 40.9 g. Because the molar mass of carbon is 12.01 g-moD1,... 

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