1

A LITERATURE REVIEW ON

HPLC METHODS FOR THE DETERMINATION OF SYNTHETIC FOLIC ACID IN FORTIFIED CEREAL FLOURS

INDEPENDENT STUDY
NFS 591

Submitted to: Dr. PADMANABAN KRISHNAN

By SOWMYA MARKA 08-08-2011

Abstract: In 1998, the United States has introduced a mandatory fortification of enriched cereal-grain products with folic acid. In the first few years after the initial mandate on January 1, the statistical rate of NTDs dropped by 25% in the U.S. according to the Center for Disease Control and Prevention. Ironically, synthetic folic acid supplement has more bioavailability than natural sources of folic acid. A robust, instrumental procedure that will determine synthetic folic acid is of great importance in food analysis. This review will focus on need for fortification of cereal flours with folic acid and techniques to determine folic acid in them. More specifically, this review will be restricted to methods that involve high performance liquid chromatography (HPLC) coupled with various detectors. Key words: Synthetic Folic Acid, Fortification, Cereal Flours, Techniques, HPLC

INTRODUCTION:

Vitamin B9 is chemically known as folic acid. It is one of the metabolically active compounds and is commonly referred to as folates. It is an essential water soluble vitamin helps the body to convert carbohydrates into glucose to produce energy. Folates differ according to their bioavailability and biological activity. In the body the liver converts the folates in N5 methyltetrahydrofolate. This methyltetrahydrofolate is the major circulating form in the blood which is responsible for the one-carbon transfer enzymatic reactions (homocysteine to yield methionine), including the DNA synthesis [6].The deficiency may cause megaloblastic anemia, and affect mental and emotional health, along with other biological effects. Deficiency of folic acid during pregnancy can increase the risk for neural tube birth defects including cleft palate, spina bifida, and brain damage. The chemical name for folic acid is pteroylglutamic acid and it is one of a family of chemically similar substances known as folates, the term folate(s) includes: Folic acid, a synthetic folate which is used in supplements and food fortification. Naturally occurring folates which are found in foods (dietary folates) [17]

Dietary Sources
Dietary sources of folate include liver, yeast extract, certain fruits ( particularly oranges) and vegetables (e.g. sprouts, asparagus, spinach in particular, and other green vegetables in general), fortified foods such as most bread and most brands of breakfast cereal. According to the National Food Survey in the UK diet the main sources are vegetables and cereals together providing about two-thirds of average intake (average intake is about 250g/day) [16].

Need for Fortification

Folates differ according to their bioavailability and biological activity. Folic acid is the most stable and bioavailable form of folate and the form used most often in food supplements and food fortification. It is rarely found naturally in food. Naturally occurring folates in reduced form are heat labile and readily destroyed by oxidation. Folates have a poorer bioavailability than folic acid, 50-66% compared with 85-100% respectively. In addition, food preparation and processing can destroy up to 100% of naturally occurring folate, as it is sensitive to light and air but especially heat. Fortification of food with vitamins is intended to compensate for the loss of these compounds due to the heat treatment to which they are subjected during manufacture. As such, both supplementation and fortification of foods is likely to be a more reliable means of increasing folate in body tissue levels and human folate status than dietary forms [17]

Folate Deficiency
A deficiency of folate can occur when an increased need for folate is not matched by an increased intake, when dietary folate intake does not meet recommended needs, and when folate loss increases. Medications that interfere with the metabolism of folate may also increase the need for this vitamin and risk of deficiency. [14] Medical conditions that increase the need for folate or result in increased loss of folate include:

pregnancy and lactation (breastfeeding) alcohol abuse malabsorption kidney dialysis liver disease certain anemias

NEED OF FOLIC ACID:

It acts as a coenzyme in the metabolism of amino acids and nucleic acids which are the precursors for DNA It plays an essential part in the production of purines and pyrimidines that make up DNA. This makes it a critical nutrient in relation to cell division and repair of genetic material It primes the homocysteine molecule for methylation It lowers homocysteine in the blood (together with vitamin B6 and B12) through regeneration of methionine. The methyl group donated in this process is taken up by the substance, S-adenosyl methionine (SAMe), which in turn participates in various chemical reactions throughout the body, including the synthesis of adrenaline, creatine and melatonin [17].

ADVANTAGES OF FOLIC ACID:

Folic acid reduces the risk of neural tube defects (eg, spina bifida, anencephole) in babies.[3] Evidence from observational studies suggests a protective effect of increasing folate intake on CVD risk[4]. However, randomised controlled trials have not demonstrated a beneficial effect of folic acid on CVD risk in people who have established cardiovascular disease.[5,6] Evidence from prospective studies in humans suggests a trend towards a protective effect of folate intake on colon cancer risk.[7] Some animal studies suggest that folic acid may inhibit tumour development in normal tissues, but promote the progression of established cancer. Data from observational studies [8, 9] suggests that cognitive impairment and Alzheimers disease is associated with poor folate status. A recent double-blind RCT in 818 men and women aged 5070 years found that folic acid 800 g daily for 3 years significantly improved domains of cognitive function including memory, information processing speed and sensorimotor speed compared with placebo.[10] As part of the same trial, folic acid was also found to slow age-related decline in hearing.

Figure 1: A synthetic process for folic acid Some studies have demonstrated a link between depression and low folate status.[11] 2,4,5-Triamino-6-hydroxypyrimidine sulfate (THP) synthesized from the reduction of 2,4Diamino-5- isonitroso-6-hydroxypyrimidine (DNHP) is an important precursor for the synthesis of folic acid and acyclovir.1 The synthetic process for folic acid is shown in Figure 1. For quality control of folic acid products, it is beneficial to determine the presence of THP, DNHP, and their degradation products. Folic acid analysis on reversed phase silica columns (for example, C18 and C8 columns) by high-performance liquid chromatography (HPLC) has been reported (Alaburda et., al, 2008, Breithaupt et., al, 2001). However, THP and DNHP are highly polar compounds with poor retention on C18 and C8 stationary phases (Hu, S.B. et., al, 2004).

Recommended Levels OF Folic Acid:

In 1996, the U.S. Department of Health and Human Service through the Food and Drug Administration (FDA) specified that grain products required to be fortified with folic levels ranging from 0.43 mg to 1.4 mg per pound (from 0.95 to 3.10 g g-1) of the product. [6]

The current US Public Health Service and the current Institute of Medicine (IOM) recommendations are that all women of reproductive age should consume 400 g of synthetic folic acid daily to prevent these birth defects. There is evidence to suggest that the median folic acid consumption of women of reproductive age may be as much as 200 g of synthetic folic acid per day post fortification, which means that most women are not getting the 400 g that is recommended. Increasing the concentration of folic acid required in enriched grains would increase the proportion of women who would consume the recommended amount of folic acid and increase the prevention of folic acidpreventable neural tube defects.[15]

TOXICITY
Intake of supplemental folic acid should not exceed 1,000 micrograms (g) per day to prevent folic acid from triggering symptoms of vitamin B12 deficiency. Folic acid supplements can correct the anemia associated with vitamin B12 deficiency. Unfortunately, folic acid will not correct changes in the nervous system that result from vitamin B12 deficiency. Permanent nerve damage can occur if vitamin B12 deficiency is not treated. [13]

OBJECTIVES:
To prepare literature review on fortification of enriched cereal flours with synthetic folic acid. To prepare literature review on determination of folic acid by HPLC method in last ten years.

LITERATURE REVIEW:
The prime objective of this literature review study is to focus on determination of folic acid by HPLC method. The determination of B vitamins in various samples is rather difficult due to the chemical instability and complexity of the matrices in which they usually exist. Vitamins are most often determined in the free form, which involves hydrolysis of the phosphorylated forms and/or those bound to proteins (and optionally glycosylated) during the extraction step performed prior to the chromatographic separation. The extraction procedure was considered to have the greatest impact on analytical results. During the last decade, the use of a trienzyme treatment method has been developed for more efficient extraction of folates from food than the conventional methods. The most popular method for their determination is chromatographic techniques belongs high-performance liquid chromatography (HPLC).

DETERMINATION OF FOLIC ACID BY HPLC:

Lebiedzin and his team made study on determination of folic acid by HPLC method. They used reversed-phase high-performance liquid chromatography, coupled with coulometric electrochemical detection, and were successfully applied for the quantification of added folic acid (FA) in fortified fruit juices and cereal products. They found good separation of the 5-HCOH4 folate and folic acid in cereal samples. They rapidly measured the retention time of vitamins by isocratic elution using 40 mM sodium phosphate dibasic, heptahydrate buffer, and 8% acetonitrile (v/v) (0.9 mL/min, pH 5.5) as mobile phase with the Supelco LC 18 column 5 m (25 cm 4.6 mm). They measured Folate concentrations using a trienzyme (hog kidney folate conjugase, -amylase, and protease) folate extraction method.

The determination of B vitamins in various samples is rather difficult due to the chemical instability and complexity of the matrices in which they usually exist. Vitamins are most often determined in the free form, which involves hydrolysis of the phosphorylated forms and/or those bound to proteins (and optionally glycosylated) during the extraction step performed prior to the chromatographic separation. This method includes combination of traditional folate conjugase

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method with protease and amylase enzymes followed by heat extraction (Hyun and Tamura 2005). Heudi and his team made study on folic acid separation by HPLC method (2005). In which they made modification by coupling HPLC with ultraviolet-visible absorbance. Alternative methods of HPLC are based on redox reaction with electrochemical detectors such as coulochemic instruments (Flangan et al. 2005). Their determination by ionpairing chromatography with reversed phase is the most frequent method (Breithaupt, 2001).The B vitamins are easily oxidized or reduced with online working electrode in electrochemical cell. The high sensitivity and lower limits of detection are obtained using the electrochemical detection allowing the analysis of folate from distribution in red blood cells and lymphocytes (Bagley and Selhub 2000). This work presents the application of several procedures including a trienzyme extraction and reversed-phase HPLC with coulometric electrochemical detection for the quantification of folic acid and 5-HCO-H4 folate in fortified food products.

Elolo and his team studied the effect of folic acid on fortified bread dough in bread making at different production stages by reverse phase ion-pair HPLC. In their study they combined reverse phase ion-pair HPLC with UV and fluorometric detection. They found that Sample extraction required -amylase and rat plasma deconjugase digestion, and sample preparation required purification by solid-phase extraction. Added folic acid was measured by monitoring UV absorption at 280 nm. They designed the study to use HPLC measurement for monitoring different stages of bread making for added folic acid and some native folates, and determining the retention and distribution of these folates among flour, dough, and bread. In their analysis they found reversed phase ion pair HPLC combined with UV and fluorometric detection provided and alternative approach for separating, identifying, and quantitating added folic acid and native folates in flour and bread.

Rosalia and team worked on quantitative estimation of folic acid in enriched cereal grain products. The purpose of their study was to separate and quantify folic acid and 5methyltetrahydrofolate, the most prominent naturally occurring folate in fortified foods, with a reliable and robust method. They separated folate using heat extraction process. They used trienzyme treatment (a-amylase, rat plasma conjugase, and protease) applied to the extracts

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followed by purification by affinity chromatography. Folic acid and 5-methyltetrahydrofolate were separated and quantified by reversed-phase HPLC with fluorescence and UV detection. A gradient elution with phosphate buffer and acetonitrile was used to separate the different forms of folates. They found a linear response in a range of 0.13 mmol/L and 0.01250.25 mmol/L for folic acid and 5-methyltetrahydrofolate, respectively. They found the ranges were similar to the expected levels in the samples. The CV of the peak areas of folic acid and 5methyltetrahydrofolate for 5 commercial wheat flour samples extracted and run separately on the same day was 2.0 and 5.7% and, run over 5 consecutive days, was 7.2 and 7.3%, respectively.

Helio and his team worked on quantification of folic acid in wheat flour by HPLC method with tandem mass spectrometry. To monitor the fortification level of folic acid (vitamin B9) in wheat flour samples, they developed and validated a simple and fast analytical method using the liquid chromatography with tandem triple quadrupole mass spectrometry (LC-MS/MS) technique. They presented an alternative and effective method to analyze folic acid which was an important dietary nutrient, in wheat flour. The use of LC-ESI/MS/MS technique allowed the determination of this compound at the required set limits (sensitivity), without matrix interferences (selective), time consuming (rapid) and tedious sample preparation.

Janete and his team also worked on determination of folic acid in fortified wheat flours. They used FA extraction with tetraborate and trichloroacetic acid buffer solution and purification by solid-phase extraction with strong anion-exchange cartridges. They concluded a reversedphase HPLC separation with ultraviolet detection at 280nm is a reliable method to determine FA in fortified wheat flour. This procedure includes an extraction step with the SAX cartridge, which provides selectivity for FA quantification. The results on the ELISA were also in accordance with those obtained on HPLC method and MA.

Liisa and his team worked on Improvements in the Analysis of Reduced Folate Monoglutamates and Folic Acid in Food by High-Performance Liquid Chromatography. They used reversed-phase high-performance liquid chromatographic method with fluorescence and ultraviolet detection was used to evaluate the different steps in folate analysis to detect and quantify the most abundant folate forms naturally present in foods. In this study Rapid heat

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extraction by microwave heating and an enzymatic deconjugation with hog kidney conjugase and chicken pancreas was presented. The extracts were purified with strong anion-exchange cartridges before injection. The combined use of ascorbic acid and mercaptoethanol in the extraction step noticeably improved the stability of tetrahydrofolate, making it also possible to analyze some of the most labile folate vitamers. Tetrahydrofolate was also shown to be easily degraded during the deconjugation step. The compounds 5-methyltetrahydrofolate and, to a lesser extent, 5-formyltetrahydrofolates were also found as important vitamers in foods.

EXPERIMENTAL PROCEDURES
SAMPLING:
In their study Rosalia Poo and his group used samples of enriched cereal-grain products and other products fortified with folic acid. Commercial wheat flour used to determine recovery and precision. Rat plasma was used as a source of folate conjugase

In a study they used sponge and proofed dough pinched from the respective loaves immediately after fermentation and frozen before drying. The samples were freeze dried followed by grinding with Kunkel grinder ( model A-10, Tekmar Co., Cincinnati, OH) to pass through a 40-mesh sieve, and stored frozen until analysis was done. Few portions of the samples were air-oven dried at 130 C for 1 hr for the moisture determination (E LOLO S ET., AL, 2001).
STANDARD SOLUTIONS AND REAGENTS:

Following samples were used ELOLO S AND HIS TEAM Tetrahydrofolic acid (THF), 5-Methyl-Tetrahydrofolic acid (5-CH3-THF) 5-Formyl-Tetrahydrofolic Acid (5-CHO-THF) 10-formyl-folic acid (10-CHO-FA) Folic acid (FA) Stock folate standard solution Working solution of folic acid -Amylase

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Rosalia Poo and his group used following reagents: Folic acid and 5-methyltetrahydrofolate Standard stock solutions

Janete Alaburda and his group used following material and reagents: SPE columns (SAXstronganion-exchange/quartenary amine, 500 mg/3 ml) ELISA kit FA standard was obtained from DSM Nutritional Products

METHODS:
Extraction of folic acid Purification of folic acid Chromatography

Extraction of folic acid:

Elolo S and his group took flour and freeze-dried dough and bread (5 g) bread samples. They were homogenized by stirring for 1 hr in 50 mL of 0.1M K2HPO4 (pH 89) containing 0.1% ascorbate (w/v) and 0.1% MCE. The pH of the slurry was adjusted to 6.6 Phosphoric acid was used to adjust the slurry pH, followed by -amylase treatment (1 hr) at 65C for hydrolysis of starch. After the -amylase digestion the extract was cooled and treated with rat plasma suspension at a concentration of 0.5 mL/10 mL of extract. The mixture was incubated for 3 hr at 37C for deconjugation of polyglutamylfolates into monoglutamylfolates. After incubation, the extract was heated in boiling water for 5 min, and then centrifuged for 20 min at 5,000 g. The volume of the resulting supernatant was adjusted to 50 mL when necessary, and immediately stored at low temperature (20C), or an aliquot was immediately filtered and injected for the determination of added folic acid, or submitted to the purification procedure for the determination of native folic acid derivatives
(E LOLO S ET., AL , 2001).

In their experiment Rosalia Poo and his group took thawed samples and they suspended (1 g/10 mL final volume) in a 0.026 mol/L Tris-HCl extraction buffer (pH 7.4) containing 1%

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(w: v) sodium ascorbate and 0.02 mCi/L [3H] folic acid tracer (specific activity: 69 Ci/mmol; Movareck Biochemicals). The samples were capped and heated in an autoclave for 15 min at 120 C (1.034 bars), cooled in an ice bath, and then homogenized with a polytron homogenizer for 30 s at medium setting (Brinkmann Instruments). The homogenates were subjected to a modification of the trienzyme treatment method. The homogenates were incubated for 4 h at 37_C with 1.25 mL of a-amylase solution and 0.2 mL of rat plasma folate conjugase. Subsequently, the samples were treated with 1 mL protease for an additional 1 h at 37_C, then heated for 5 min in a boiling water bath, cooled in ice, and centrifuged for 20 min at 36,0003g and 4 C. The supernatants were filtered through a microfilter (Millex-AA, 0.8 mm, Millipore) and radioactivity in an aliquot of the sample was measured.

In their study Janete Alaburda and his team performed three different extraction techniques were tested, using phosphate buffer solution pH 7.0 and 9.0 and tetraborate buffer solution pH 8.5 with SPE extraction. Five grams of wheat flour sample were extracted with 50mL of extraction phosphate buffer pH 7.0, 9.0 and TCA buffer pH 8.5 (0.25 mol and 0.37 mol 1 dibasic sodium phosphate

1 monobasic potassium phosphate) (Lima et al., 2004). The mixture was shaken

for 30 min in a rotational shaker, and centrifuged at 3500 rpm for 15 min. The supernatant was filtered through a 0.45 mm nylon membrane (Advantec MFS, Inc.) before chromatography analysis.

Purification of folic acid:

For the sample clean up Elolo S and his group used disposable strong anion-exchange (SAX) cartridges (Quaternary amine [N+], Baker 7091-3). The cartridges (3-mL SPE column, 500 mg) were conditioned by wetting with 3 mL of hexane, followed by 3 mL of methanol, and then equilibrated with 5 mL of 0.1M K2HPO4 buffer (pH 89) containing 0.1% (w/v) ascorbic acid and 0.1% MCE. A portion of the extract (3 mL) was diluted with phosphate buffer (13 mL) before loading onto a cartridge, with an elution rate of 0.6 m /min. Analytes were eluted with at least 3 mL of 0.1M sodium acetate (pH 4.5) containing 5% (w/v) Na2HPO4 (HPLC grade), 0.1% (w/v) ascorbic acid, and 0.1% MCE. The eluent was filtered through a 0.45-m microporous filter before injection.

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In their experiment Rosalia Poo and his group sample extracts were applied onto a 0.6 mL (bed volume) folate binding protein (FBP)Affigel 10 (Bio- Rad Laboratories) affinity columns as previously described. The columns were washed sequentially with 5 mL of 1 mol/L potassium phosphate buffer (pH 7.0), 5 mL of water, and 3 mL of water. Bound folates were eluted from the columns with 2.5 mL of 20 mmol/L trifluoracetic containing 10 mmol/L dithioerythritol. The acid eluate was promptly neutralized with 40 mL of 1 mol/L piperazine, and a 50 mL aliquot was used for tritium counting to assess folate recovery. The FBP affinity columns had a binding capacity 4 mg of folate. In their study Janete Alaburda and his team used strong anionic exchange (SAX) cartridge was conditioned with 5mL of methanol followed by 5mL of water, before applying the sample (Johansson et al., 2002). An analytical aliquot of 2.5mL supernatant was transferred to the cartridge. The vacuum was adjusted to elute the sample at about 0.5 drop s 1. When the entire extract was eluted, the column was rinsed with 5mL of water and completely drained after the last washing step. FA was eluted with acetate-phosphate buffer (pH 4.5, 0.1 mol 1 sodium

acetate, adjusted with acetic acid (AA) containing 5%dibasic potassium phosphate) directly into a 5mL volumetric flask. The eluate was filtered through a 0.45 mm nylon membrane (Advantec MFS, Inc.) before chromatography analysis.

HPLC Chromatography:
In their experiment Elolo S and his group performed reversed-phased ion-pair HPLC using a system consisting of two pumps: one low-pressure mixing pump (Hitachi model L-6200) and one model L-6000 solvent metering pump. Added folic acid was determined by a Hitachi L-4000 UV detector operating at 280 nm, and a Waters model 474 scanning fluorescence detector set at an excitation wavelength of 290 nm and emission wavelength of 350 or 450 nm was used in monitoring endogenous folates. The injection was made with a Rheodyne (Cotati, CA) loop type injection valve (20 ). A Brownlee (30 mm 2.1 mm i.d.) guard column with 5 m ODS packing (Varian Chromatography Systems, Walnut Creek, CA) was installed before a Microsorb-MV C18 analytical column (150 4.6 mm i.d., 3 m particle diameter). The mobile phase was composed of 24% methanol (v/v) in aqueous potassium phosphate buffer (3.5 mM KH2PO4 and 3.2 mM K2HPO4), pH 6.8, containing 5 mM tetrabutylammonium dihydrogen phosphate (Sigma) as an ion-pairing agent. Isocratic elution at ambient temperature

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was used, and the measurement of added folic acid was made at a flow rate of 1 mL/min with the UV detector sensitivity set at 0.01 absorbance units full scale. The determination of native folates required a flow rate of 1.3 mL/min, with the fluorescence detector gain set at 100. Post column pH adjustment of the eluent stream was achieved upstream of the detector by pumping 0.2 mL/min of 0.5% (v/v) aqueous phosphoric acid through a reaction tee. Chromatograms were recorded, and peak areas and heights quantitated using a Hitachi model D-2500 chromatointegrator. The sensitivity to- noise ratio of the recorder was 4.

In their experiment Rosalia Poo and his group used a reversed-phase gradient HPLC method was performed. The folates were separated on an ODS-Hypersil (5 mm, 4.63250mmi.d.) analytical column (Keystone Scientific). A flow rate of 1 mL/min was used. The mobile phase program consisted of 3 min with 100% A (28 mmol/L dibasic potassium phosphate and 60 mmol/L phosphoric acid in water) followed by a linear gradient of 10 min to 70% A:30% B (28 mmol/L dibasic potassium phosphate and 60 mmol/L phosphoric acid in 200 mL/L acetonitrile and 800 mL/L water). A second linear gradient from 70% A:30% B to 45% A:55% B was then run over the next 17 min, followed by a third linear gradient to 43% A:57% B over the next 15 min. At 45 min, the column was equilibrated for 5 min in the initial conditions and another sample analysis could be initiated immediately. The absorbance of folic acid was monitored with a diode array detector set at 280 nm, and of 5-methyltetrahydrofolate with a fluorescence detector set at 295 nm excitation and 360 nm emission wavelengths.

In their study Janete Alaburda and his team Determination of FA was carried out in a Shimadzu 10AD-Vp high-performance liquid chromatography (HPLC) system equipped with a multi solvent pumping system (Shimadzu Pump LC- 10 AD-VP, Kyoto, Japan), a degasser (Shimadzu DGU-14 A, Kyoto, Japan), an auto-sampler (Shimadzu Pump FCV- 10 AD-VP, Kyoto, Japan), a column oven (Shimadzu CTO-10 AS-VP, Kyoto, Japan), computer software (Shimadzu Class VP version 6.02, Kyoto, Japan), and an ultraviolet detector (Shimadzu SPD-10 AV-VP, Kyoto, Japan). FA was separated with a Supelcosil LC 18 reversedphase column (Supelco) (250mm_4.6mm, 5 mm) protected with a guard column Phenomenex C18 (4mm_3.0mm, 5 mm). The column temperature was maintained at 35 C. Gradient elution with ACN and AA 1% (pH 2.8) was used to separate FA. The flow rate was 0.7mLmin_1. The gradient was started at 8%

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(v/v) ACN, and after 10 min the ACN proportion was linearly raised to 26%, it was isocratically maintained for 5 min. Thereafter, ACN concentration was linearly raised to 50% within 6 min, and isocratically maintained for 9 min. ACN proportion came back to 8% after 5 min, and the column was restabilized for 10 min before the next injection. FA was detected by a UV detector at a wavelength of 280 nm.

RESULTS AND DISCUSSION:

Elolo S and his group experiment about stability and distribution of added folic acid determination by HPLC in bread making sensitivity were observed in folic acid determination. The reverse phase chromatography provided enough separation even low level of folic acid during the process. Chromatograms of bread (Fig. 2) showed a near baseline resolution, making it unnecessary to conduct any sample purification. Changes that occurred in the added folic acid level during bread processing are summarized in Table I.

Figure 2: Chromatographic separation of added folic acid in bread.

Another important observation is that this study has confirmedthe fact that folic acid molecules do not fluoresce without derivatization. Folic acid is probably not a significant naturally occurring form of the vitamin, but it is often found in small quantities as an oxidation product of THF. It was detected some native folic acid and stated that this might be an oxidation product of THF. By using UV detection, also measured native folic acid in unfortified bread samples, but they claimed that no significant inter-conversions or oxidation occurred during their

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analysis. Therefore, it is not clear whether folic acid occurs naturally in cereal products. More studies are needed to clarify this aspect.

Rosalia Poo and his group made representative chromatograms of 5-methyltetrahydro folate and folic acid standards are shown in Figure 3. using UV absorption at 280 nm for folic acid determination and fluorescence spectroscopy for the determination of 5-methyltetrahydrofolate. This different modality of spectroscopic detection stems from the high levels of folic acid in fortified foods that allow UV detection. This different modality of spectroscopic detection stems from the high levels of folic acid in fortified foods that allow UV detection. The advantage of this method is that it is highly specific, and any peak activity emerging from the analytical column, irrespective of whether it is determined byUV absorption, fluorimetry, or electrochemical activity, represents folate activity. They found although the values from the HPLC were, on average, 17% higher than those derived from the traditional microbiological assay, the difference was constant across the range of folate values, thus allowing for good prediction. Figure 3:

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In their evaluation of the method Janete Alaburda and his team found UV detector response at 280nm was linear for FA from 6.0 to 600 mgL_1. Least-square regression curve analyses of data from ten calibrators resulted in excellent straight-line fits at the concentration examined with the calibration curve described by equation A = 487.24.AF998.17 (r2 = 0.9999, where A is the peak area and FA the folic acid concentration). Detection (LOD) and quantification (LOQ) limits for the HPLC method were 0.06 and 0.19 g g_1, respectively. The accuracy of the HPLC method with tetraborate TCA buffer solution and SAX clean-up for extraction was determined using fortified wheat flour at 0.5, 1.5, and 3.0 g g_1 (Table ). Table 2:

CONCLUSION:
Reversed-phase ion-pair HPLC combined with UV and fluorometric detection provided an alternative approach for separating, identifying, and quantitating added folic acid and native folates in cereal based product. Trienzyme deconjugation, extraction procedure, reversed-phase HPLC separation) provides sufficient selectivity to resolve folic acid. Coupling this separation with coulometric detection provides a suitable instrumental method for determining folic acid in food samples. The simple mobile phase and the isocratic elution used to separate folic acid and folate yielded low detection limits, good sensitivity, and resolution within a minimum analysis time of 16 min.

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REFERENCES:
1. Ball, G.F. M. 2006. Vitamins in Foods. Analysis, Bioavailability, and Stability. Taylor & Francis, Boca Raton, FL. [BOOK] 2. Bailey, L.B. 2005. Do low doses of folic acid result in maximum lowering of homocysteine? Am. J. Clin. Nutr. 82:717718. 3. Alaburda, J.; de Almeida, A.P.; Shundo, L.; Ruvieri, V.; Sabino, M. Determination of Folic Acid in Fortified Wheat Flours. J. Food Composition and Analysis 2008, 21(4), 336342. 4. Hu, S.B.; Yang, G.L.; Yin, G.F.; Wang, L.J.; Li, Z.W.; Liu, H.Y.; Yang, G.L.; Chen, Y. Separation and Determination of Folic Acid and Related Compounds with HighPerformance Liquid Chromatography. Chinese J. Anal. Lab. 2004, 23(11), 3335. 5. A. Lebiedzin ska, M. Dabrowska, and P. Szefer, High-Performance Liquid Chromatography Method for the Determination of Folic Acid in Fortified Food Products, Toxicology Mechanisms and Methods, 18:463467, 2008. 6. Helio A. Martins-Jnior, Alexandre Y. Wang, Janete Alabourda, Maria A. F. Pires, Oscar B. Vega and Daniel T. Lebre, Validated Method to Quantify Folic Acid in Wheat Flour Samples using Liquid Chromatography - Tandem Mass Spectrometry, J. Braz. Chem. Soc., Vol. 19, No. 5, 971-977, 2008. 7. Janete Alaburda_, Adriana P. de Almeida, Luzia Shundo, Valter Ruvieri, Myrna Sabino, Determination of folic acid in fortified wheat flours, Received 26 April 2007; received in revised form 29 October 2007; accepted 27 December 2007. 8. Flangan, R.J., Perrett, D., and Whelpton, R. 2005. Electrochemical Detection in HPLC. Analysis of drugs and poisons (RSC Chromatography Monographs), Royal Society of Chemistry, Cambridge. [BOOK] 9. Bagley, P.M., and Selhub, J. 2000. Analysis of folate form distribution by affinity followed by reversed-phase chromatography with electrochemical detection. Clin. Chem. 46:404411. 10. Elolo S. Osseyi, Randy L. Wehling, and Julie A. Albrecht, HPLC Determination of Stability and Distribution of Added Folic Acid and Some Endogenous Folates During Bread making, Cereal Chem. 78(4):375378. 11. Rosalia Po o-Prieto, David B. Haytowitz, Joanne M. Holden, Gail Rogers, Silvina F. Choumenkovitch, Paul F. Jacques, and Jacob Selhub, Use of the Affinity/HPLC Method for Quantitative Estimation of Folic Acid in Enriched Cereal-Grain Products, The Journal of Nutrition, 136: 30793083, 2006. 12. Lima, J.A., Catharino, R.R., Godoy, H.T., 2004. HPLC methodology for folic determination in enriched wheat flour and bread. Tecnica Molitoria International 55, 151158.

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13. Institute of Medicine. Food and Nutrition Board. Dietary Reference Intakes: Thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. National Academy Press. Washington, DC, 1998 14. http://ods.od.nih.gov/factsheets/folate/ 15. Robert L. Brent and Godfrey P. Oakley, The Folate Debate, Jr Pediatrics. 2006;117;1418-1419 16. Judy Buttriss, (2000) Folic Acid and Health. British Nutrition Foundation Nutrition Bulletin, 25, 67-68 17. http://www.hsis.org/pressoffice/ebulletinPDF/folicacid08.pdf