Chromatography folates

Although affinity chromatography has not been used directly as an analytical method, it may be modified in the future to produce a viable technique. Leucovorin has been used as an effective spacer in obtaining active samples of dihydrofolate reductase.79 If the enzyme could be immobilized without losing its activity, perhaps it could be used to separate folates. 

Since the 1970s numerous HPLC methods using lEC, RP and ion-pair chromatography have been proposed. In the last years, RP chromatography has become the most used method, thanks to its simplicity, sensitivity, and compatibility with different detection techniques. The stationary phases usually used are C18 or phenyl-bonded silica-based phases. More recently, alternative stationary phases, such as polar-embedded, polar endcapped, and perfluorinated phases, have been successfully tested for folate analysis [577]. The mobile phase is usually a mixture of phosphate or acetate buffer and acetonitrile or methanol. 

Freisleben, A., Sehieberle, P, Ryehlik, M. (2003 May). Specific and sensitive quantification of folate vitamers in foods by stable isotope dilution assays using high-performance liquid chromatography-tandem mass speetrometry. Anal. Bioanal. Chem., 376 2), 149-156. 

Kariluoto, M. S., Vahteristo, L. T, Piironen, V. 1. (2001). Applicability of microbiological assay and affinity chromatography purification followed by high-performance liquid chromatography (HPLC) in studying folate contents in rye. J. Sci. Food Agric., 81, 938-942. 

Stabler SP, Marcell PD, Podell ER, Allen RH, Savage DG, Lindenbaum J (1988) Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromatography-mass spectrometry. J Clin Invest 81 466-474... 

A folate-binding protein (FBP) has been isolated from cow s milk by affinity chromatography on sepharose to which folate has been attached (Salter et al. 1972). Cow s milk contains -8 mg FBP per liter. The protein has a molecular weight of 35,000. Cow s milk has also been shown to bind vitamin Bi2, but the protein responsible has not been isolated from this source, although it has been obtained from the milks of other species (Burger and Allen 1974). 

Shane. B. "High Performance I JquiU Chromatography of Folates. Am.Clin. Nine. 35. 599 (19821. 

CM Pfeiffer, LM Rogers, JF Gregory. Determination of folate in cereal-grain food products using trienzyme extraction and combined affinity and reversed-phase liquid chromatography. J Agric Food Chem 45 407-413, 1997. 

E Seyoum, J Selhub. Combined affinity and ion pair column chromatographies for the analysis of food folate. J Nutr Biochem 4 488-494, 1993. 

J Selhub. Determination of tissue folate composition by affinity chromatography followed by high-pressure ion pair liquid chromatography. Anal Biochem 182 84-93,1989. 

LT Vahteristo, V Ollilainen, PE Koivistoinen, P Varo. Improvements in the analysis of reduced folate monoglutamates and folic acid in food by high-performance liquid chromatography. J Agric Food Chem 44 477-482, 1996. 

Indirect indicators of vitamin B12 deficiency include measurements of the metabolites homocysteine and methylmalonic acid (MMA) in serum and MMA in urine (see the Biochemical Perspectives section). Whereas the serum homocysteine concentration increases during folate or vitamin B12 deficiencies, the serum and urine MMA concentrations increase only in vitamin B12 deficiency. Therefore, MMA determinations can be used to differentiate vitamin B12 deficiency from folate deficiency. The normal concentration of MMA in serum ranges from 0.08 to 0.28 pmol/L. MMA is quantified using gas-liquid chromatography and mass spectrometry. Elevated concentrations of MMA and homocysteine in serum may precede the development of hematological abnormalities and reductions in serum vitamin B12 concentrations. One should be aware that other conditions, including renal in sufficiency and inborn errors of metabolism, can also result in elevated serum levels of MMA. 

Brown, J.P. Davidson, G.E. Scott, J.M. Thin-layer chromatography of peteroglutamates and related compounds. Application to transport and metabolism of reduced folates in blood. J. Chromatogr. 1973, 79, 195-207. 

Affinity chromatography of Ruta graveolens C>-methyltransferases 336 Affinity chromatographic purification of folate-binding protein in 355 cow s whey... 

The above explanation of the action of sulfonamides, namely enzyme inhibition, seems to account for most of the therapeutic action of these drugs. However, in some experiments the sulfonamide was made to unite with the pteridine intermediate (9.21). Thus, Brown s enzyme, in a cell-free system containing the sulfonamide, was incubated for 2 hours the resulting inhibition was irreversible by j AB, an effect which does not occur in growing cells (G. M. Brown, 1962). In pursuit of this effect, folate-synthesizing enzymes from . coli were made to convert sulfamethoxazole (9.11) to the pteroic acid analogue A -3-(5-methylisoxazolyl)-A -(7,8-dihydro-6-pterinylmethyl)sulfanilamide, which was isolated by chromatography and found identical with a synthetic specimen (Bock a/., 1974). 

The main folate vitamers occurring in fruits and vegetables are different from those found in animal and cereal products [16]. There is also evidence that the vitamers have different bioavailability [17] and stability [18]. A clear need existed for an analytical method that would be capable of independently quantifying each specific folate vitamer. Liquid chromatography (LC) seemed an ideal and logical technology for this purpose. 

The first liquid chromatography-mass spectrometry (LC-MS) method was developed by Stokes and Webb (1999). Here, folate was separated using reversed-phase chromatography, detected in a single ion monitoring (SIM) mode and quantified using external standards [25]. These authors successfully identified and determined folic acid, tetrahydrofolate, 5-methyltetrahydrofolate, and formyltetrahydrofolate in multivitamins and foods. 

Ruggeri, S., Vahteristo, L. T., Aguzzi, A., Finglas, P., and Camovale, E. 1999. Determination of folate vitamers in food and in Italian reference diet by high-performance liquid chromatography. J. Chromatogr. A. 855 237-245. 

Stokes, P. and Webb, K. 1999. Analysis of some folate monoglutamates by high-performance hquid chromatography-mass spectrometry. I. J. Chromatogr. A. 864 59-67. 

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