Cyanocobalamin analysis

Another problem associated with saturation analysis is that abnormally low results may be obtained unless cyanide is present when the vitamin is freed from its binder. It appears that forms other than cyanocobalamin are difficult to separate completely from the binding protein. Early studies that failed to recognize this not infrequently found that results from patients with pernicious anemia gave negative values (R9). A recent study by Brown et al. (B6) examined the effect of varying the concentration of cyanide used in the test. They found that an excess of cyanide resulted in a significant increase in apparent vitamin B12 levels in sera from patients who were deficient in the vitamin, but it had little effect on sera from normal patients. They found the mean of 12 vitamin B12-deficient sera to be 49 ng/liter when 3 mg/liter of cyanide was used in the extraction mixture, 104 ng/liter in the presence of 30 mg/liter of cyanide, and 196 ng/liter when 300 mg/liter of cyanide was used. The authors emphasized that cyanide was necessary to convert all of the several forms of vitamin B12 present in serum to cyanocobalamin but warned that the concentration should not be greater than 5 mg/liter. 

The incubation mixture contained in a Anal volume of 100 /iL 400 fiM DL-homocysteine, 500 (iM ( )-L-A/5-methyltetrahydrofolate, 50 fiM cyanocobalamin, 300 fiM 5-adenosylmethionine, 125 mM 2-mercaptoethanol, 20 fiM L-norvaline, 50 mM potassium phosphate buffer (pH 7.4), and 50 /xL of liver or cell extract. The incubation mixture was immediately flushed with nitrogen and overlayered with 50 /tL of bis(3,5,5-trimethylcyclohexyl)-phthalate. The incubation, carried out at 37°C in the dark, was stopped by the addition of 10 / L of 4 TV perchloric acid. The acid was then neutralized by addition of 10 / L of 4 TV KOH containing 3.3 M potassium bicarbonate. After centrifugation, 90 /iL of supernate was mixed with 175 fiL of o-phthaldialdehyde reagent (prepared by mixing 1 mL of 56 mM o-phthaldialdehyde in methanol with 9 mL of 0.1 M sodium borate buffer, pH 9.5, then adding 40 fiL of 2-mercaptoethanol). After 2 minutes at 23°C, 220 /xL of this mixture was used for HPLC analysis. The assay is linear for at least 2 hours. 

The structure of vitamin B12, called cyanocobalamin, is shown in Figure 8.13 and is a water-soluble vitamin. It has a molecular weight of 1355 and has a complex chemical structure. The molecule is hydrophilic and is not easily extractable into organic solvents. Thus, SPE is an excellent way to isolate it from aqueous solutions followed by HPLC analysis. It is isolated by reversed-, phase sorption onto a C-18 sorbent. 

The presence of the benzimidazole system in a natural product is most striking in the case of vitamin Bi2 (cyanocobalamin). It was isolated from liver extracts and from the fungus Streptomyces griseus. It is an antipemicious anaemia factor. Elucidation of its complex structure was achieved by X-ray analysis (Crawfoot-Hodgkin 1957). 5,6-Dimethylbenzimidazole is bonded via the atom N-I to D-ribose as an A -glycoside N-3 is linked to a cobalt ion which is situated in the centre of a corrin system (see p 489). 

Gradient reversed phase HPLC has also been used in the analysis of cobamides present in Methanobacterium bryantii (Whitman and Wolfe, 1984). After initial purification by isocratic reversed phase HPLC at neutral pH the extracts were subjected to gradient HPLC using a C g stationary phase with an initial mobile phase of 100 mM lithium chloride-methanol (76 24) and a final mobile phase of 100 mM lithium chloride-methanol (52 48). Detection by UV absorbance at 254 nm allowed quantitation of as little as 1 pmol of cyanocobalamin. 

Allerhand (70) has reported the C NMR spectrum of aquocyanocobyric acid. Many of the carbon resonances of each of the two isomers (aquocyano-cyanoaquo) are distinct. Allerhand and Doddrell (71) have also utilized partially relaxed FT NMR to assign a large number of the resonances of cyanocobalamin, coenzyme B-12 and cobinamidedicyanide. The C spectra clearly demonstrate the greater resolution and simplicity of analysis of C over iH NMR. 

We have developed a method for simultaneous analysis of thiamine hydrochloride, pyridoxine hydrochloride and cyanocobalamin in pharmaceuticals and dietary supplements (Marszall et al. 2005) and in fortified food (Lebiedzinska and MarszaH 2006) using HPLC-ED. Vitamins were determined in their free forms, so an extraction step from fortified fruit juice was performed prior to the chromatographic isolations. The extraction procedure was based on a study by Ndaw el al. (2000). The enzymatic digestion prior to the separation and quantification step made it possible to release the vitamins bound to proteins or sugars and converted vitamin esters to free forms thus we were able to obtain the total vitamin contents of the fruit juices. The supernatants were adjusted to pH 4.5 with 2.5 M sodium acetate and a single extraction procedure for all vitamins was carried out using mixture of the enzymes, papain and diastase (Lebiedzinska and MarszaH 2006). 

In our study (Lebiedzihska et al. 2007), the extraction procedure used for determination of the vitamers Bg and B12 was based on a combination of acid digestion and enzymatic hydrolysis according to a study by Esteve et al. (1998) and recommended by AOAC s Ojficial Methods of Analysis to release vitamins from food followed by FIPLC analysis. Vitamins Bg and B12 were detected following HPLC separation with coulometric detection. Our experiments performed with standards i.e. methyl-, hydroxyl-, adenosyl- and cyanocobalamin) clearly indicated the same peak area response at the same potentials and retention times. 

Using a single LC-MS/MS method to quantify all WSVs at such different levels of concentrations is very difficult. However, quantitation of all the WSVs except cyanocobalamin in a single chromatographic run has been achieved (Chen and Wolf 2007 Chen et al. 2009). Compromises have to be made to accommodate WSVs at different concentration levels. For multi WSV analysis using LC-MS or LC-MS/MS in multivitamin dietary supplements, the instrument may need to be intentionally tuned to lower the sensitivity for pantothenic acid to accommodate higher concentrations if folic acid and biotin need to be analysed together (Chen and Wolf 2007). 

Enzymatic analysis of the E. coli methionine-synthesizing system has progressed. E. coli 121-176, which requires methionine or Bi2, yielded an extract whose synthesis of methionine from homocysteine plus serine was increased six- to eightfold by cyanocobalamin (Helleiner et al., 1958). The compound formed by mixing equimolar HCHO and FH4 acted directly as donor of the Ci unit to homocysteine. The system requires (Kisliuk and Woods, 1958a) DPN, ATP, Mg++, hexose diphosphate, and inorganic P. 

Chem. Descrip. Soy protein cone., zinc oxide, niacinamide, ferrous suifate, copper giuconate, vitamin A paimitate, caicium pantothenate, thiamine mononitrate, pyri-doxine hydrochloride, riboflavin, and cyanocobalamin Chem. Analysis 6% max. moisture 20% total dietary fiber 290 calories/100 g CAS 68153-28-6 1314-13-2 98-92-0 7720-78-7 527-09-3 79-81-2 137-08-6 532-43-4 58-56-0 83-88-5 68-19-9 EINECS/ELINCS 232-720-8 215-222-5 202-713-4 231-753-5 208-408-2 201-228-5 205-278-9 208-537-4 200-386-2 201-507-1 200-680-0... 

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