HPLC Methods for Folic Acid
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Transcript of HPLC Methods for Folic Acid
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
2
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 NTD’s 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
3
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
250µg/day) [16].
4
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
5
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
Alzheimer’s disease is associated with poor folate status. A recent double-blind RCT in
818 men and women aged 50–70 years found that folic acid 800 μg 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.
6
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 2,4-
Diamino-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 μg 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 µg 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 µg of synthetic folic
acid per day post fortification, which means that most women are not getting the 400 µg 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 acid–preventable neural tube defects.[15]
7
TOXICITY
Intake of supplemental folic acid should not exceed 1,000 micrograms (μg) 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]
8
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.
9
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-HCO-
H4 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 μm
(25 cm × 4.6 mm). They measured Folate concentrations using a trienzyme (hog kidney folate
conjugase, α-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
10
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 α-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 5-
methyltetrahydrofolate, 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
11
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 0.1–3 mmol/L and 0.0125–0.25 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 5-
methyltetrahydrofolate 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 reversed-
phase 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
12
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 (ELOLO 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 α-Amylase
Rosalia Poo and his group used following reagents:
13
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 8–9) 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 α-amylase treatment (1 hr) at 65°C for hydrolysis of
starch. After the α-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
37°C 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 (–20°C), 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 (ELOLO 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%
(w: v) sodium ascorbate and 0.02 mCi/L [3H] folic acid tracer (specific activity: 69 Ci/mmol;
14
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 L S 1 dibasic sodium phosphate
and 0.37 mol L S 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 8–9) containing 0.1% (w/v) ascorbic acid
and 0.1% MCE. A portion of the extract (3 mL) was diluted with phosphate buffer (1–3 mL)
before loading onto a cartridge, with an elution rate of ≈0.6 mL/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-μm
microporous filter before injection.
15
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 S1. 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 L S 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 μL). A Brownlee (30 mm × 2.1 mm i.d.) guard column with 5 μm ODS
packing (Varian Chromatography Systems, Walnut Creek, CA) was installed before a
Microsorb-MV C18 analytical column (150 × 4.6 mm i.d., 3 μm 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
16
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 chromato-
integrator. 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%
17
(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
18
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:
19
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.AF–998.17 (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 µg 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 µg 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.
20
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. 82:717–718.
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