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HPLC ANALYSIS OF VITAMIN B1, B2, B3, B6, B9, B12 AND
VITAMIN C IN VARIOUS FOOD MATRICES
Jessy van Wyka*, Larry Dolleyb & Ndumiso Mshicilelib
a
Department of Food Technology, Cape Peninsula University of Technology, P.O. Box 1906,
Bellville 7535, South Africa, [email protected]
b
Agrifood Technology Station, Cape Peninsula University of Technology, P.O. Box 1906,
Bellville 7535, South Africa, [email protected]; [email protected]
ABSTRACT
Various methods had been described for the analysis of vitamins in food matrices, with more
and more of these including the use of HPLC to measure the levels of these micronutrients in
foodstuffs. The renewed interest in rapid and accurate quantification of micronutrients in
foodstuffs is due to more stringent requirements by food regulatory agencies around the
world. Legislation now demands that the nutrition information displayed on food labels be
backed up by reliable results obtained using validated analyses. Three challenges are
common in terms of quantifying vitamins in food matrices: 1) extraction techniques that are
sufficiently effective to liberate the various forms of the vitamin from each unique matrix, 2)
ensuring that labile forms of the vitamin are protected against degeneration by light and/or air
(oxygen) for a sufficiently long period to afford accurate quantification and 3) obtaining an
analytical method with sufficient sensitivity, selectivity, accuracy and precision, with cost
and time also being considerations . The chapter dealt with these aspects concerning vitamin
B1, B2, B3, B6, B9, B12 and vitamin C. Extraction procedures were described, as well as
typical HPLC methods and recent improvements in this field.
INTRODUCTION
The family of water-soluble vitamins (WSVs) comprises nine vitamins, namely thiamine
(vitamin B1), riboflavin (vitamin B2), niacin / nicotinic acid / nicotinamide (vitamin B3),
pantothenic acid (vitamin B5), pyridoxine / pyridoxal (vitamin B6), biotin (vitamin B7 or B8
(as D-(þ)-biotin)), folic acid (vitamin B9), cyanocobalamin (vitamin B12) and L-ascorbic acid /
L-dehydroascorbic acid
(vitamin C) (Heudi, 2012; Nohr & Biesalski, 2009).
Like fat-soluble vitamins, WSVs are essential components in the human diet since they
play a vital role in optimal health owing to their involvement in fundamental functions of the
body, such as growth and metabolism (Groff et al., 1995; Kim, 2011). Their importance in
preventing deficiency diseases is well-established, with mounting evidence of the
prophylactic role of many of these vitamins protecting against other forms of disease, such as
elevated maternal folate intake resulting in reduced incidence of neural tube defects (NTDs)
(Berry et al., 2010; Quinlivan & Gregory, 1997) and vitamin B12 and folate supplementation
normalizing blood homocysteine concentrations, thus minimizing a risk factor for heart
disease (Groff et al., 1995) and other chronic diseases, such as cancer and age-related
dementia (Iyer & Tomar, 2009; Kim, 2007; Russell, 2012). As a result of the crucial role that
vitamins play in optimum nutrition, disease-prevention and as therapeutic aids, many dietary
supplements (also known as nutrition or food supplements) are available (Heudi, 2012), while
mandatory fortification of specified foodstuffs are enforced by around 75 governments
around the globe (Samaniego-Vaesken et al., 2013). Hence, there is a global thrust to develop
accurate and precise analytical techniques for compliance monitoring and also for
establishing reliable information for nutrient databases (Blake, 2007) and food labels
(Russell, 2012).
The WSVs are a group of biomolecules with considerable variation as concerns their
molecular weight, structure, chemical properties and biological activity (Groff et al., 1995;
Russell, 2012). In addition, in biological samples they can occur as a number of closely
related compounds, or vitamers (examples are pyridoxine, pyridoxal or pyridoxamin), while
these can be present either in free or phosphorylated form (such as riboflavin-5’adenosyldiphosphate (FAD)) or non-covalently, but strongly, bound to polysaccharides and
proteins (Ball, 2006; Heudi, 2012). The diversity in chemical reactivity or sensitivity to
degradation, as well as the necessity to release the bound forms of the various WSV species
highlight the challenges in terms of quantitative liberation of the analytes via effective sample
extraction, particularly for methods aimed at simultaneous quantification of various WSVs in
non-fortified food matrices (Ball, 2006; Heudi, 2012). Generally, extraction processes include
acid or alkaline hydrolysis of the sample (Ball, 2006), heat denaturation of proteins, often by
autoclaving (Van Wyk & Britz, 2010; 2012), deproteinization with trichloroacetic acid or
similar agent (Ball, 2006) and digestion with appropriate enzymes (Lim et al., 1998; Pfeiffer
et al., 2010).
While the extraction process is of critical importance, it is also vital to apply an
analytical method that is accurate, precise, selective and sensitive, as well as rapid. Hence,
while the time-consuming microbiological assays were the gold standard for many years
(Blake, 2007), they have largely been replaced by other methods that produce results more
swiftly (Ball, 2006; Heudi, 2012). Due to the hydrophobic, non-volatile character of the
WSVs, they are most suitable for reversed phase high performance liquid chromatography
(RP-HPLC) methods (Russell, 2012), which feature prominently among the afore-mentioned
rapid methods (Ball, 2006; Heudi, 2012). The first generation of the LC methods used UV
detection most frequently, followed by fluorescence and electrochemical detection methods
(Russell, 2012). More recent developments are the use of liquid chromatography-tandem
mass spectrometry (LC-MS/MS) (Ball, 2006; Heudi, 2012; Chandra-Hioe et al., 2011;
Freisleben et al., 2003) and even ultraperformance liquid chromatography-tandem mass
spectrometry (UPLC-MS/MS) coupled with isotope dilution MS. An electron-spray
ionisation (ESI) interface facilitates the exploitation of the high selectivity of MS detection
with the improved sensitivity when operating in multiple reactions monitoring (MRM) mode.
The application of isotope dilution mass spectrometry (IDMS) facilitates the simultaneous
determination of various WSVs (Heudi, 2012), including different vitamers (folates), with
enhanced quantitative accuracy (Chandra-Hioe et al., 2011), while UPLC effects very high
resolution separations and fast separations (Edelmann et al., 2012; Chandra-Hioe et al., 2011;
Heudi, 2012) due to the high pressure (up to 1 000 atm) and small stationery phase particles
(<2 µm) (Russell, 2012). However, the cost and complexity of operating the equipment based
on MS detection (Iyer & Tomar, 2009: Pfeiffer et al., 2010), with still higher costs applying
to stable isotope labelling (SIL) and greater complexity to IDMS (Heudi, 2012), put it beyond
the reach of the average routine analytical or research laboratory.
The aim of this chapter is to focus on the extraction of selected water-soluble vitamins
in food matrices, followed by quantification using HPLC methods. Since several recent
reviews have been published (Heudi, 2012; Kumar et al., 2010; Plonka et al., 2012; Russell,
2012), including an excellent treatise on method validation (Russell, 2012), in most cases the
narrative will concentrate on the period after 2010. The exceptions will be folate and vitamin
B12 where a more comprehensive description includes the work performed in our own
laboratories, these two vitamins being a key element in one of our research focus areas (Van
Wyk, 2002; Van Wyk & Britz, 2010; Van Wyk et al., 2011; Van Wyk & Britz, 2012). At the
end of the chapter, a summary of a selection of the most recent methods is presented in Table
1, with the information arranged in alphabetical order based on food matrix. Since food
analytical methods are often based on methods originally developed for non-food matrices,
some of these methods are also included at the end of the table to provide a complete picture
in terms of the available methodology.
In addition, a brief generic description, structures, nutritional and physiological
importance is also covered for each vitamin. As much as this includes a description of typical
deficiency diseases in each case, it is important to note that in most modern societies, singlenutrient deficiencies are rare. Generally, a diet sub-optimal in one vitamin would be suboptimal in others. In addition, the metabolism of some vitamins are interconnected, such as
the interrelationship between vitamins B9 and B12 in terms of megaloblastic anaemia, while
vitamins B6, B9 and B12 are involved in homocysteine synthetic and degradative reactions
(Rivlin, 2007), preventing hyperhomocysteinemia (Shaik & Gan, 2013). Moreover, vitamin
B6 plays a role in the synthesis of niacin from tryptophan, thus forming the basis of vitamin
B6 deficiency potentially causing secondary niacin deficiency (Rivlin, 2007).
VITAMIN B1 (THIAMINE)
Generic description and structures – Vitamin B1
Vitamin B1 in the form of its pyrophosphate acts as a coenzyme for reactions involving the
transfer of activated aldehyde groups. Other forms of this vitamin are thiamine itself,
thiamine monophosphate, thiamine triphosphate (Russell, 2012) and protein-bound thiamine
(Bucholz et al., 2012). A deficiency of this vitamin results in the development of beri-beri.
The latter deficiency disease is characterized by neurological and cardiac aberrations
resulting in amnesia, hence the suggestion that it can be used to treat Alzheimer’s disease. A
range of other symptoms include muscle weakness, lack of coordination and, in severe cases,
death (Akyilmaz et al., 2006; Harper, 2006).
The structures of various forms of vitamin B1 are depicted in Figure 1. More thiamine
is found in plant products while thiamine pyrophosphate is predominant in muscle foods
where pork is the exception (Russell, 2012). Reactions in which thiamine vitamers participate
are best described by the main enzymes which use it as a coenzyme. These are pyruvate
dehydrogenase, transketolase and α-ketoglutarate dehydrogenase (Belitz & Grosch, 1999).
OH
N
N+
S
ClCl-
N
NH3+
Figure 1. Vitamin B1 structure (thiamine).
Nutritional/Physiological importance and Dietary sources – Vitamin B1
Thiamine, together with its counterpart riboflavin (see later), are two major water-soluble
vitamins used in fortification of food products (Boyaci et al., 2012). As a coenzyme, it plays
a role in the mechanism for the decarboxylation of α-keto acids. Through mediation or
participation in these reactions, the deficiency symptoms are generated when thiamine is not
available. In developed countries there are marginal deficiencies of this vitamin (Nohr &
Biesalski, 2010), but severe deficiencies, resulting in brain damage are still common among
certain population groups such as children and the aged (Harper, 2006).
The main dietary sources of thiamine are meat (Lombardi-Boccia, 2005), the pericarp
and germ of cereals (Belitz & Grosch, 1999), baked products, legumes, nuts yeast and organrich meats (Russell, 2012), milk, oats, rye, sunflower, lentils, broccoli and potatoes (Nohr &
Biesalski, 2010).
Assay methodology for Vitamin B1 (vitamins B2, B3 and B6) – General
Due to the similarities among the assay techniques, the following discussion grouped
vitamins B1 to B6 together. Microbiological assays exist for all of these vitamins, but the
extraction and assay procedures are tedious and time-consuming. Other methods that are used
are: HPLC, capillary electrophoresis (thiamine); HPLC, direct fluorometric, electrophoretic
and biosensor-based methods (riboflavin); HPLC and colorimetric methods (niacin); and
HPLC (pyridoxine) (Da Silva et al., 2013; Heudi et al., 2005; Gao et al., 2008; Hidiroglou et
al., 2008; Sikorska, 2007). A recent paper describes the simultaneous quantification of
thiamine, nicotinic acid, niacin, pyridoxine and even vitamin C in a yeast supplement, beer
and dietary supplements using capillary electrophoresis with diode array detection (CE-DAD)
(Mazina & Gorbatsova, 2010).
There are very few papers between 2010 and now dealing with single-vitamin
analytical techniques. The majority of recent analytical work conducted with respect to the Bgroup vitamins has been related to multi-vitamin analysis. Examples of this is seen in the
following articles viz. Aslan et al. (2013), De Arruda et al. (2012), Hucker et al. (2012), Jin et
al. (2012), Leόn-Ruiz et al. (2013), Rudenko & Kartsova (2010), San José et al. (2012),
(Table 1), Hälvin et al. (2013), Zand et al. (2012) amongst others. However, there are
established methods and ongoing research into single-vitamin assays (Gratacόs-Cubarsí et al.
2011, Yoshida et al. 2012).
There are standard methods for the analysis of the vitamins as a group and for B1 in
particular such as those of the American Association of Cereal Chemists International e.g.
thiochrome method, fluorometric method, HPLC and microbiological methods. Biosensorbased methods (Akyilmaz et al., 2006) and a method using adsorptive stripping voltammetry
(Tyszczuk-Rotko, 2012) are also available. Microbiological methods are still the standard
against which to reference (Chen et al., 2009).
However, most of the methodology being used is based on HPLC modifications of
established methods e.g. thiochrome. Table 1 summarizes a number of HPLC-based methods
based on the matrix involved, most of which again is based on multi-vitamin analysis with a
few exceptions. In terms of the methods summarized for vitamins B1 to B6 in the table, the
literature (with a few exceptions) was only polled back to 2010. The work by Russell (2012)
can be considered a baseline upon which this writing is built. It elegantly captures the state of
the analytical field with an observation that the thiochrome-based method is well-matured to
the extent very little new comes out in terms of its basic principles and further development.
One area where development does take place is in terms of hardware e.g. the move from
HPLC to UHPLC with its quicker run times and better resolution (Di Stefano et al. 2012).
In general, based on the observations in Table 1 regarding the B-group vitamins, the
following position emerges in terms of analytical regimes.
After reducing the sample (milling, homogenization, cutting, shearing), an acid
treatment at elevated temperature (sometimes autoclaving) is usually employed to free the
vitamers from the sample (Boyaci et al., 2012; Ersoy & Özeren, 2009). This may include
enzyme treatment e.g. taka-diastase (Khair-un-Nisa et al., 2010) or Clara-diastase (Bui &
Small, 2009) and other steps such as sonication (Santos et al., 2012) toward releasing
vitamers bound to protein. Other enzymes that may be employed include papain and αamylase (Chandra-Hioe et al., 2011; San José et al., 2012). To assist with this the sample
may also be de-proteinized by trichloroacetic acid treatment or by using other precipitating
agents (Bucholtz, at al., 2012). In some cases liquid-liquid separation of vitamins may be
employed (Giorgi et al., 2012). In most cases buffers are employed as part of the suspending
agent. Where dry samples are required after preparation, drying is conducted using a stream
of nitrogen (Grataćos-Cubarśi et al., 2011).
In the case of vitamins B1 and B2, derivitization to a stable thiochrome is performed
using alkaline ferricyanide ((Boyaci et al., 2012) or cyanogen bromide (Bucholtz et al.,
2012). The latter is not common due to the toxicity of cyanogen bromide.
All other standard HPLC procedures, including micro-filtration, are employed in the
general analytical scheme with permutations based on the nature of the required output e.g.
different types of micro-filters with different porosities.
General hardware required for the run included branded equipment, the most important
of which is the column and column conditions employed. This ranges from the most common
i.e. reversed phase C18 HPLC columns at ambient or at 30oC (Bui & Small, 2009; San José et
al., 2012) to UHPLC columns with higher resolution and shorter run-times (Mihhalevski et
al., 2013) to a hydrophilic interaction chromatography (HILIC) column at ambient
temperature (Grataćos-Cubarśi et al., 2011).
In terms of elution, the generic methods are isocratic and gradient elution including
both concentration differences as well as, in one case, flow rate differences.
Detection was mostly UV (single fixed wavelength or DAD at a specific set of
wavelengths) or fluorescence at appropriate excitation and emission wavelengths pertinent to
each study reported. In some cases, a mass spectrometer was also employed.
For any analytical technique, one of the more important steps that dictate the outcomes
and quality of results is sample preparation, in particular extraction of the analytes. In this
case the analyte(s) is a vitamin (with possibly more than one vitamer) with its own degree of
stability and sensitivity to different reagents. Thiamine itself is sensitive to pH (stable at pH
2.0 to 4.00 and highly unstable at alkaline pH), unstable in solution, easily oxidized and can
be degraded by UV light (Russell, 2012; Santos et al., 2012).
A more detailed summary of extractions and analytical work is given in the next section
per vitamin and is common to a range of vitamins where multi-vitamin analysis is conducted.
HPLC assay methodology for Vitamin B1
For cereal grains extraction of the vitamin may be conducted by placing a sample in cold 4%
trichloroacetic acid to be milled or homogenized before filtering, derivatizing (in this case
using cyanogen bromide) after which the pH is adjusted to 10 (the thiochrome formed
fluoresces strongly). Further treatment may involve cooling, centrifugation and SPE
(Bucholtz et al, 2012).
A more environmentally friendly derivatizing agent is alkaline ferricyanide. In this case
the tricholoracetic acid mixture is centrifuged after which the supernatant is treated further
(Hucker et al., 2012). At this point the sample can be injected directly onto a column to
determine riboflavin while a second supernatant stream is further treated with alkaline
potassium ferricyanide, degassed, vortexed and neutralized and then filtered prior to injection
onto the column. Fluorescence detection iss done at excitation/emission wavelengths of
360/245 nm for thiamine.
For fruit and vegetable matrices the fruit is peeled and extracted in methanol,
centrifuged and filtered. The sample can then be clarified at this point followed by SPE using
different solvents to extract at least three different vitamin fractions eventually allowing for
analysis of ascorbic acid, thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxine and
folic acid using DAD at appropriate wavelengths (Plonka et al., 2012).
For complex cereal foods a sample may be mixed in 0.05M H2SO4 with sodium acetate,
papain, diastase and amylase and incubated at 37oC to release vitamins from other cereal
components. Both thiamine and riboflavin may be determined using UV detection at 268 nm
(San José et al., 2012). However, the detection method of choice is fluorescence.
VITAMIN B2 (RIBOFLAVIN)
Generic description and structures – Vitamin B2
Various forms of vitamin B2 occur, namely riboflavin, FMN (flavine mononucleotide), FAD
(flavine adenine dinucleotide) and lumiflavin. The structure of riboflavin is depicted in
Figure 2. This vitamin is quite stable to heat processing but is extremely sensitive to UV
light, thus necessitating protection from light when the existing pool in a matrix is to be kept
stable. This is further related to light barriers in packaging e.g. dark bottles, opaque materials
and light-tight seals. The effect of light in the 420–560 nm range cleaves ribitol from the
parent structure leading to the formation of lumiflavin (Belitz & Grosch, 1999). This is also
related to analytical challenges associated with losses during sample preparation (Ciulu et al.,
2011).
CH2OH
HOCH
CHOH
HOCH
CH2
H3C
N
H3C
N
N
O
NH
O
Figure 2. Vitamin B2 (riboflavin) structure.
Based on its role in the generic reactions involving decarboxylation, hydroxylation and
reduction of oxygen to hydrogen peroxide amongst others, it plays a role in a wide range of
biochemical reactions and pathways. This is done via FAD and FMN. These include the
electron transport chain, Krebs cycle, oxidation of amino acids and the replenishment of
reduced glutathione which has a protective effect against oxidation in cells (Russell, 2012;
Nohr & Biesalski, 2010).
Nutritional/Physiological importance and Dietary sources – Vitamin B2
Riboflavin (7,8-dimethyl-10-(1’-D-ribityl)isoalloxazine) is the counterpart to thiamine used
in the fortification of food products (Boyaci et al. 2012). It is associated with FMN and FAD.
These coenzymes are involved in reactions requiring electron carriers e.g. hydroxylation and
decarboxylation, and have a wide variety of roles to play in general metabolism (Nohr &
Biesalski, 2010). Vitamin B2 is not associated with any specific deficiency symptom since it
is a very stable pool in the body (Belitz & Grosch, 1999; Nohr & Biesalski, 2010). The main
dietary source is muscle products as well as liver and other organs, milk and eggs (Russell,
2012), wheat germ, yeast, soya beans and mackerel (Nohr & Biesalski, 2010).
Assay methodology for Vitamin B2 – General
Apart from microbiological assay and HPLC methods, analytical techniques for vitamin B2
include
fluorescence,
chemiluminescence,
capillary
electrophoresis
and
spectro-
electrochemical and electrochemical methods. Electrochemical methods had been found to
show sensitivity and selectivity for the determination of riboflavin in multivitamin
preparations and foods like milk, cereal and beer (Safavi et al., 2010).
HPLC assay methodology for Vitamin B2
There is a great deal of overlap between the extraction and determination of thiamine and
riboflavin as is noted in the previous section on thiamine. The standard extraction techniques
together with the added value of enzymatic and derivatizing techniques apply toward
detection by fluorescence due to the thiochrome formed. However, based on advances in
technology regarding analytical hardware, there is a trend toward mass spectroscopy as an
added technique toward identifying and quantifying analytes, including riboflavin. However,
due to the costs and challenges of mass spectrometry and the sensitivity and selectivity of
fluorescence detection, methods based on thiochrome generation are still favoured (Hälcin et
al., 2013).
The simultaneous determination of thiamine and riboflavin in malt may be used as an
example of a typical experimental regime (Hucker et al., 2012) as was previously described.
A further method for the determination of riboflavin and thiamine is described by Santos et
al. (2012) where green, leafy vegetables are freeze-dried, milled, extracted with 10 mM
ammonium acetate in methanol containing an antioxidant (butylated hydroxytoluene) and
then sonicated. After centrifuging at 14 000 g for 15 minutes and filtering through a 0.45 µm
nylon filter, it was concentrated in a nitrogen stream prior to injection and detection using
DAD in the 200 to 680 nm range. This allows determination of a number of other watersoluble vitamins as well.
The methods available all have different permutations which involve enzymic digestion,
use of opaque flasks, trichloroacetic acid protein precipitation and inactivation of interfering
enzymes by acid treatment (Englberger et al., 2010). Choices may be made based on cost,
speed and sensitivity for each specific user and application. Co-enzymes need to be extracted
in the pH 5.0–7.0 range due to instability outside of the range. This may be done using an
acetonitrile/phosphate buffer mixture (Russell, 2012).
VITAMIN B3 (NIACIN)
Generic description and structures – Vitamin B3
The structures of two forms of vitamin B3 are depicted in Figure 3. There are more vitamers
than that represented graphically below (Mihhalevski et al., 2013). This vitamin is quite
stable to different processing conditions. Its active forms (NAD and NADP) are involved in
oxidation reduction reactions, acting as electron carriers. Similar to riboflavin and its
coenzyme forms, niacin is indirectly involved in a wide range of central biochemical
reactions.
O
COOH
C
N
Nicotinic acid
NH2
N
Nicotinamide
Figure 3. Vitamin B3 structures.
Nutritional/Physiological importance and Dietary sources – Vitamin B3
Niacin (nicotinic acid amide) is better known in the form of nicotinamide adenine
dinucleotide (NAD) or its phosphorylated form i.e. nicotinamide adenine dinucleotide
phosphate (NADP). A lowered NAD or NADP level is an indicator of deficiency, the
symptoms of which are pellagra (skin, digestive and nervous disorders) (Belitz & Grosch,
1999). The main dietary source is muscle products as well as plant products, being more bioavailable in meat and in milk (Nohr & Biesalski, 2010).
HPLC assay methodology for Vitamin B3
There is a further deal of overlap between the extraction and determination of the B vitamins,
including thiamine, riboflavin and niacin. However, the many different vitamers of this
vitamin do provide challenges when conducting quantitative determinations in different food
matrices. The different vitamers are those mentioned previously viz. nicotinamide, nicotinic
acid, nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate.
Further vitamers are nicotinic acid dinucleotide, nicotinic acid dinucleotide phosphate, Nribosyl nicotinamide and N-ribosyl nicotinic acid (Mihhalevski et al., 2013).
These vitamers (as with other vitamins) are usually integrated into the food matrix,
including binding to protein or other structural components, in some cases being unavailable
to extraction procedures and quantitation (Russell, 2012; Mihhalevski et al., 2013; Plonka et
al., 2012). This, together with poorly defined vitamers in terms of what exactly is being
extracted, makes for some uncertainty in terms of niacin analysis.
Santos et al. (2012) have developed a method for the simultaneous determination of
water- and fat-soluble vitamins in leafy vegetables using LC-MS/MS and LC-DAD, the
former for analyzing the water-soluble components. Sample extraction from green, leafy
vegetables included protection from light and by maintaining samples on ice. In this instance
samples were extracted into 10 mM ammonium acetate/methanol 50/50 v/v containing
butylated hydroxytoluene as an antioxidant (Santos et al., 2012). Hippuric acid was used as a
standard. The samples were shaken and then sonicated for 15 minutes with the temperature
not going above 25oC. The centrifuged sample (14 000 g for 12 minutes) was then passed
through a 0.45 µm nylon filter, concentrated to remove methanol in a nitrogen stream and
injected onto an HPLC-MS/MS system to determine water-soluble vitamins. The detection
system was a DAD and MS via an electrospray interface. The column was an ACE-100 C18
(100 X 2.1 mm i.d., 3 µm particle size). Separation of vitamins in a single run was achieved
using a complex gradient with 3 mobile phases viz. A: 10 mM ammonium acetate (pH 4.5),
B: 0.1% acetic acid and C: 0.3% acetic acid. The method was validated and then used to
study vitamins (including nicotinamide) in the vegetables as a function of storage time.
The above method did not specify SPE even though it is usually standard practice to do
so. In addition, the use of enzymes or other protein precipitating agents were also not used for
this particular sample matrix. The latter, together with heating, would be suited to other
matrices such as cereals.
Leόn-Ruiz et al. (2013) conducted studies on honey using a simpler system and
included the determination of nicotinic acid and nicotinamide. The vitamins all occur in
solution in honey and are thus more easily extracted via dilution of honey in water followed
by filtration through a 0.45 µm nylon filter. Isocratic elution was conducted using a mobile
phase containing 0.01 % sulphuric acid and 2% methanol (v/v) at pH 3.5 and a column
temperature of 25oC.
This gave both high sensitivity and low limits of detection. A
µBondapak C18 column (150 X 3.9 mm, 10 µm) was used.
VITAMIN B6 (PYRIDOXINE)
Generic description and structures – Vitamin B6
The structures of various forms of vitamin B6 are depicted in Figure 4. While these include
pyridoxine, pyridoxal, pyridoxamine, vitamers that are not depicted are pyridoxine
phosphate, pyridoxal phosphate, pyridoxamine phosphate and 5’-O-β-D-glucopyranosyl
pyridoxine (Mihhalevski et al., 2013). As much as it is considered relatively stable as a group
of vitamers, pyridoxine itself can be affected in highly processed foods. As with other groups
of vitamers, different forms are either free or bound to different elements of food matrices.
Due to this, hydrolysis or SPE is necessary prior to HPLC analysis (Zand et al., 2012).
CHO
CH2OH
OH
HOH2C
N
OH
HOH2C
N
CH3
Pyridoxine
CH2NH2
OH
HOH2C
CH3
Pyridoxal
N
CH3
Pyridoxamine
Figure 4. Vitamin B6 structures.
Nutritional/Physiological importance and Dietary sources – Vitamin B6
Pyridoxine (vitamin B6) is the precursor for pyridoxal phosphate. The latter is a prosthetic
group for a group of enzymes. The latter includes amino acid decarboxylases, racemases,
dehydrases, transferases and synthases (Belitz & Grosch, 1999; Zand et al., 2012). Ingestion
is usually in the form of pyridoxal or pyridoxamine. The most stable form is pyridoxal.
This vitamin is found in most food products and also, due to its stability, is often used
for fortifying food products (Russell, 2012). It is also much more bio-available in animal than
in plant sources. A deficiency of this vitamin (or its vitamers) is protein metabolism-related,
e.g. hemoglobin synthesis. It also plays a role in homocysteine synthetic and degradative
reactions (Rivlin, 2007)
HPLC assay methodology for Vitamin B6
Zand et al. (2012) developed a UHPLC-DAD method for determining riboflavin and
pyridoxine together with a parallel LC-MS analysis using the same solvents. This was
considered advantageous in terms of one method being used to validate another.
A simple sample preparation process included different baby food samples which were
homogenized and diluted with 1% acetic acid and incubated at 70oC for 40 minutes, cooled
and made to volume with acetic acid. After centrifuging at 8 000 g for 10 minutes at 5oC, the
supernatants were passed through a 0.45 µm filter prior to injection. The system consisted of
a UHPLC system and rapid resolution high definition C18 column (2.1 X 50 mm, 1.8 µm
particle size, RRHD Eclipse Plus). The flow rate was 0.5 mL/ minute with A = water:acetic
acid (99:1) and B = acetonitrile:acetic acid (99:1). The gradient started at A:B = 90:10 to
reach 10:90 after 3 minutes (20 µL injection volume). Pyridoxine was measured at 280 nm.
The LC-MS version was by the same authors using a C18 Ultrasphere Behcam (4.6 mm
X 25 cm, 5 µm particle size) column coupled to a mass spectrometer with electrospray
ionization interface. Conditions were the same as for the UHPLC system. This allows then
for validation of the UHPLC method and for the determination of other water-soluble species
in the samples.
Other multivitamin methods include that by Santos et al. (2012) and Jin et al. (2012)
which include the determination of B6 vitamers.
Improvements and recent developments: Vitamin B6 methods
One of the recent developments was in terms of microbiological assay (MA), where a
colorimetric microbial viability assay based on the reduction of a tetrazolium salt was used to
determine vitamin B6 in a wide range of foods and beverages with satisfactory accuracy and
within 24 hours, as opposed to the 2 days required for the conventional MA (Tsukatani et al.,
2011).
With respect to HPLC methods, Hälvin et al. (2013) developed an analytical system
using nutritional yeast as a food model to conduct LC-MS/TOF to determine B complex
vitamins since they considered that HPLC methods employing UV or fluorescence detection
did not always have enough sensitivity and selectivity if proper clean-up parameters are not
observed. In this instance, isotope-labeled standards were employed to compensate for losses
during sample preparation and for matrix effects during MS and MS/MS analysis.
Finely ground yeast was weighed into flasks to which were added labeled vitamin
standards and 0.05 M ammonium format buffer to volume. This was centrifuged (14 000
rpm, 5 minutes at room temperature) and passed through a 0.2 µm PTFE filter prior to
injection. All analyses were conducted using an ACQUITY UPLC® with a time of flight
mass spectrometer. An ACQUITY UPLC HSS C18 (2.1 X 150 mm, 1.8 µm) column was
used with two eluents viz. A: water + 0.1% formic acid and B: acetonitrile + 0.1% formic
acid. The gradient was run at 0–3 minutes100% A, 3–8.5 minutes (A:B = 80:20), 8.5–10
minutes (A:B = 5:95) and 10–15 minutes (100% A). The flow rate was 0.25 mL/minutes,
temperature of 25oC and a sample injection volume of 5 µL.
This method was proven to work without pre-concentration for determination of the B
group vitamins, including that of pyridoxine. Challenges were encountered, including the
need to prepare additional labeled vitamers viz. pantheine, N-ribosyl nicotinamide and Nribosyl nicotinic acid. A second challenge was the selection of enzyme preparations to
minimize the inclusion of interfering agents in these preparations with respect to MS-TOF.
VITAMIN B9 (FOLATE)
Generic description and structures – Vitamin B9
Pteroyl-L-glutamic acid (PGA or PteGlu) is the synthetic form of the vitamin that is most
commonly known as folic acid, while “folate” is the collective name for a wide range of PGA
derivatives, some of which are depicted in Figure 5. As the most stable vitamer, folic acid is
the preparation of choice for inclusion in fortified food products and in dietary supplements
(Kim, 2011; Pfeiffer et al., 2010; Shane, 2010). The folates that occur in nature are the 7,8-
dihydro- and 5,6,7,8-tetrahydro-reduced forms of PGA, resulting in Dihydrofolate (DHF) and
tetrahydrofolate (THF). Substitution at the N-5 and/or N-10-position with a one-carbon
adduct generates 5-methyl-THF, 5-formyl-THF, 5-formino-THF, 10-formyl-THF, 5,10methylene-THF and 5,10-methenyl-THF (Fig. 5). Although only the monoglumate form is
metabolically active, approximately 80% of all native folates are polyglutamates, with 5–8
conjugated glutamate units most abundant (Bailey, 2006; McGuire, 2006; Russell, 2012;
Vahteristo & Finglas, 2000), while a chain length of 2–14 conjugated glutamates has been
reported (Garrat et al., 2005). In vivo, monoglutamates, notably 5-CH3-THF, is the transport
form of folate, while the storage form in tissues like the liver (the main storage organ), is
polyglutamates (Alegría et al., 2008; Shane, 2010).
O
OH
4a
N3
N
O
6
5
CH2
N
9
2
1
H2C
O
H
4
8a
N
OH
H NH
10
O
7
8
H
N
R
Pterin
p -Aminobenzoic
acid
L - glutamic acid
Pteroic acid
Pteroylmonoglutamic acid (PGA or PteGlu)
H
N
4a
CH2
6
5
N
9
8a
7
8
R
4a
6
5
CH2
H
7
8a
7,8-Dihydropteroylmonoglutamic acid (DHF)
R
10
H
8
H
N
H
H
N
9
10
H
N
H
H
N
5,6,7,8-Tetrahydropteroylmonoglutamic acid (THF)
CHO
4a
N
5
H
4a
6
CH2
H
9
7
8a
N
R
7
H
R
10
H
8
H
N
10-Formyl-THF (10-CHO-THF)
NH
CH
CH3
N
H
N
CH2
H
5
7
8a
N
9
H
5-Formyl-THF (5-CHO-THF)
4a
CH2
H
8a
H
N
6
5
10
H
8
CHO
N
9
R
10
H
N
6
5
CH2
H
H
8
N
9
R
10
7
8a
H
N
4a
H
H
8
H
N
H
5-Methyl-THF (5-CH3 -THF)
N
CH2
5-Formimino-THF (5-CH=NH-THF)
CH
R
N
R
+
N
4a
N
6
5
H
7
8a
4a
6
5
H
H
5,10-Methylene-THF (5,10-CH3 -THF)
Figure 5. Vitamin B9 (folate) structures.
7
8a
9
H
8
N
CH2
H
9
H
8
N
CH2
H
H
5,10-Methenyl-THF (5,10=CH-THF)
Nutritional/Physiological importance – Vitamin B9
The nutritional importance of folate lies in its vital role in one-carbon metabolism. In the
body, various THF derivatives serve as co-factors which transfer one-carbon units in
numerous biosynthetic, catabolic and interconversion reactions. These include amino acid
synthesis (synthesis of serine, glycine, re-methylation of homocysteine to methionine),
degradation of histidine and purine and pyrimidine synthesis (Groff et al., 1995; Bailey,
2007; McGuire, 2006). The latter is critical for DNA synthesis and repair, for all cell
replication, including normal foetal development (Groff et al., 1995; Iyer & Tomar, 2009;
McGuire, 2005; Stover, 2010). Persistent low dietary folate intake results in deficiency with
bone marrow cells becoming megaloblastic and manifestation of anaemia. The formation of
the large immature erythrocytes which are characteristic of megaloblastic anaemia is the
consequence of impaired DNA synthesis (Groff et al., 1995; MacGuire, 2006). Folate
administration as therapeutic treatment against megaloblastic anaemia has been practiced for
almost seven decades (Bailey, 2007; Ye et al., 2008). More recently, it was discovered that
increased periconceptual folate consumption significantly reduces the incidence of birth
defects (Hobbs et al., 2010; Oakley, 2009), including Down syndrome, orofacial clefts and
congenital heart defects (Hobbs et al., 2010; Russell, 2012; Wallis et al., 2010) and, in
particular, neural tube defects (NTDs), with anencephaly and spina bifida the most common
and severe (Hobbs et al., 2010; Oakley, 2009). This precipitated mandatory folate
fortification on a global scale. The USA was the first to introduce such programme, targeting
grain flour and other cereal products (Berry et al., 2010), with other countries, including
Canada, Mexico, Hungary (Gregory, 2004; Oakley, 2009), Australia (Chandra-Hioe et al.,
2011) and South Africa (Anonymous, 2003) following suit. Countries where mandatory
fortification programmes have been implemented reported that the intended effect of
reducing NTDs had been achieved (Berry et al., 2010; De Wals et al., 2007; Gregory, 2004).
An association was indicated between methylenetetrahydrofolate reductase (MTHFR)
polymorphisms and the incidence of NTDs (Bailey et al., 2002; Trembath et al., 1999) and
the risk for Down Syndrome (Wu et al., 2013), especially if coupled with reduced folate
and/or cobalamin status (Brouns et al., 2008). Current research on the role of folate in health
promotion centres on the finding that similar single-nucleotide polymorphisms in folate
transport or metabolism genes have been implicated as risk factors for many age-related
chronic diseases associated with debility and mortality. These include cardiovascular
diseases, neurodegenerative disorders (such as stroke, Parkinson’s and Alzheimer’s disease),
macular degeneration and various cancers (Bailey et al., 2002; Caudill, 2004; Shaik & Gan,
2013; Ye et al., 2008). By interacting with the proteins encoded by the variant genes, folate
can reduce disease risk (Ye et al., 2008). Protection against cardiovascular disease (Iyer &
Tomar, 2009; Stover, 2010), as well as a range of vascular, neurological, ocular, renal and
pulmonary abnormalities is also linked to its role in preventing hyperhomocysteinemia (i.e.
elevated levels of homocysteine in the serum) (Caudill, 2004; Bottiglieri & Reynolds, 2010;
Russell, 2012; Shaik & Gan, 2013). While McGuire (2006) described the causal relationship
between folate deficiency and DNA damage in eukaryotes (including humans) leading to
chromosomal abnormalities associated with oncogenesis of leukemias, lymphomas and
tumors, epidemiological studies produced compelling evidence indicating that increased
folate intakes may reduce the risk of colorectal cancer (Iyer & Tomar, 2009; Kim, 2007).
Further research is required to confirm that folate plays a prophylactic or preventative role in
osteoporosis, psychiatric illness, depression and senile dementia (Russell, 2012).
Another aspect of the increasing interest in folate in terms of health promotion is the
recent discovery that it may play an important role as an antioxidant in vivo, both by
preventing the adverse effect of reactive oxygen species (ROS), as well as by inhibiting lipid
peroxidation (Merola et al., 2013). There is a tantalizing possibility that this is linked to one
of the consequences of folate and vitamin B12 deficiency, hyperhomocysteinemia. It was
suggested that hyperhomocysteinemia may be accompanied by increased ROS, elicited by
homocysteine. When folate (vitamin B9), pyridoxine (vitamin B6), betaine and vitamin B12
were combined as supplements in a protein restricted diet administered to homocystinuric
patients (a genetic disease characterised by hyperhomocysteinemia), the treatment resulted in
a significant reduction of indicators of oxidative stress, such as malondialdehyde (Vanzin et
al., 2011). The antioxidant potency of folate, pyridoxine (and thiamine) was also reported by
Gliszczyńska-Świgło, 2006).
Dietary sources – Vitamin B9
Good food sources of folates include fortified cereals (Russell, 2012), spinach, broccoli, peas,
(Vahteristo et al., 1997b), liver and kidneys (Kim, 2011; Vahteristo et al., 1996a), egg yolk,
yoghurt (Vahteristo et al., 1997a), lima beans, mushrooms (Groff et al., 1995) and orange
juice (Ye et al., 2008).
In cows’ milk, 90–95% occurs as 5-CH3-THF (Ye et al., 2008), while 5-CHO-THF is
the dominant folate in fermented milk products (Van Wyk & Britz, 2012). In fruit (bananas,
oranges and strawberries) and vegetables (spinach, lettuce, cabbage, peas and beetroot) 5CH3-THF is predominant (Jastrebova et al, 2003; Vahteristo et al., 1997b), while THF
dominates in liver tissue (Garratt et al., 2005; Vahteristo et al., 1996a) and in fresh fish
(Vahteristo et al., 1997a). The major folate forms in cereals are 5-CHO-THF, 10-CHO-THF
and 5-CH3-THF, with 5-CHO-THF dominating in bread, while 5-CH3-THF dominates in rice
(De Brouwer et al., 2008) and also in baker’s yeast, followed by THF (Patring et al., 2009).
However, the bioavailability of native dietary folates average only 50% (Gregory, 2004),
with polyglutamates being 70–80% bioavailable compared to monoglutamate forms (Bailey,
1988).
Digestibility is also affected by the matrix (Bailey, 1988). While protein in general
binds folate (De Souza & Eitenmiller, 1990; Strålsjö et al., 2002), folate binding protein
strongly and specifically binds folate in biological fluids like milk (Ball, 2006; Indyk, 2010;
Shane, 2010; Witthöft et al., 1999), but heating at 100oC for 5 minutes denatures the binding
proteins (Lim et al., 1998), liberating the folates. Most native folates occur as
folylpolyglutamates that are unable to traverse the cell membrane (Shane, 1982; 2010) and
are likely to be retained intracellularly. However, digestive processes (in vivo) (Iyer &
Tomar, 2009), or heating at 100oC for 6 minutes (in vitro) releases intracellular folates
(Shane, 1982; 2010).
Assay methodology for Vitamin B9 – General
The increasing significance of folates in health and disease, including the global wave of
mandatory folate fortification programmes, prompted by its proven role in protecting against
NTDs, has also focused attention on the need for accurate folate assay methods in order to
establish a credible database of folate levels in dietary sources high in native folates, as well
as in foods fortified with folates (Arcot & Shrestha, 2005; Rader et al., 1998). Methods to
quantify folates in biological samples in general and in food, in particular, are complicated
due to the numerous forms of native folates, their instability, the complexity of food matrices
and the relatively low concentration of the analytes (Arcot & Shrestha, 2005).
Folate analytical methods were developed over the last 50 years, with the
microbiological assay (MA) being the first. It was also the standard reference method for
determining folates with biological activity for decades and still finds wide application in
routine folate assays in food (Ball, 2006; Pfeiffer et al., 2010; Kim, 2011). The preferred
organism when applying the MA to food samples is Lactobacillus rhamnosus (ATCC 7469)
(or the chloramphenicol-resistant strain (ATCC 27773; NCIB 10463)), rather than
Enterococcus hirae (ATCC 8043) since the latter does not respond to many folate forms, a
notable exclusion being 5-CH3-THF, one of the most abundant folate forms in foods
(Kariluoto et al., 2001; Rader et al., 1998). The principle of the turbidimetric MA is the
specific growth-dependence of L. rhamnosus on folate. Hence, the growth response
is
directly proportional to the amount of folate in the medium. While the MA does not require
sophisticated instrumentation, is relatively economical and has high sensitivity (quantitation
of folate concentrations as low as 0.12 ng/mL is possible), its precision is inferior to other
methods, it is labour intensive, even with semi-automated procedures and results are only
available after 2–5 d. Moreover, it measures only total folates and shows a lower growth
response when PGA is used as the calibrator, compared to 5-CH3-THF, resulting in an
underestimation of the folate concentration to some extent (Pfeiffer et al., 2010; Rader et al.,
1998). Until recently, the MA was the only method given official status by the AOAC (Iyer
& Tomar, 2009), but two new methods were recently awarded “AOAC First Action Official
MethodSM” status. AOAC 2011.05 is entitled “An Optical Biosensor Assay for the
Determination of Folate in Milk and Nutritional Dairy Products” and is based on a surface
plasmon resonance (SPR) optical biosensor. AOAC 2011.06 (“Total Folates in Various
Foods by Trienzyme Extraction and UPLC-MS/MS Quantitation) uses UPLC-MS/MS to
measure total folates of seven folate forms (Sullivan, 2012).
Protein-binding assays to determine folates were developed during the 1970–1980s as
more rapid and simpler alternatives to the MA. These methods were developed for clinical
laboratories and started out as immunoassays using folate-specific antibodies as the folate
binder. In modern protein-binding assays, primarily folate binding protein (FBP) is used,
either in competitive binding assays with radiolabelled tracer folate (radioassays) or
automated protein-binding assays (using chemiluminescence as detection) or enzyme
immunoassays (typically using fluorescence detection). Two major limitations impede the
suitability of protein-binding assays to food samples, namely the questionable accuracy when
mixtures of folates are present and the limited dynamic range (typically extending to only 20
ng/mL serum folate), often coupled with inaccurate dilution linearity (Pfeiffer et al., 2010).
However, the accuracy of an enzyme linked immunosorbent assay (ELISA) was established
when comparing folate levels in fortified wheat flours analysed using MA v HPLC v ELISA
(Alaburda et al., 2008). Satisfactory results were obtained when using an optical biosensor
assay utilising folate binding protein to measure folates in milk and milk-based paediatric
formulae (Indyk, 2010), meat homogenate, skim milk, breakfast cereals, broccoli, egg yolk
and other food matrices (Indyk & Woollard, 2013).
HPLC methods for the determination of folates in foodstuffs are numerous (Doherty &
Beecher, 2003; Hefni et al., 2010; Lebiedzińska et al., 2008; Ndaw et al., 2001; Puthesseri et
al., 2013; Samaniego-Vaesken et al., 2013; Vahteristo et al., 1997a and b; Van Wyk & Britz,
2012; Wawire et al., 2012; Yazynina, et al., 2008). The water-soluble nature as well as
variations in hydrophobicity and ionic properties of the folate vitamers enable their separation
by ion exchange or reversed-phase liquid chromatography (Gregory, 1984). UV (as variable
wavelength or diode array), electrochemical (in the oxidative mode) and fluorescence
detection methods are the most common (Gregory et al., 1984; Hefni et al., 2010;
Lebiedzińska et al., 2008; Vahteristo et al., 1997a and b; Van Wyk & Britz, 2012; Wawire et
al., 2012) and are equally sensitive (Ball, 2006). The latter applies to the reduced forms of
folate which shows native fluorescence that is pH dependent, with pH 2.2–2.3 the best
compromise relative to the individual optima (Ball, 2006; Gounelle et al., 1989; Russell,
2012; Van Wyk & Britz, 2012). Inter-laboratory studies confirmed that fluorescence
detection is superior to UV absorbance detection, due to the greater sensitivity displayed by
fluorescence detection (Russell, 2012). The poor inter-laboratory agreement in terms of the
results for folate vitamers in foodstuffs, other than 5-CH3-THF (Finglas et al., 1999), was
attributed to the relative instability of the other reduced folate vitamers (Vahteristo et al.,
1996b), while Puwastien et al. (2005) attributed it to lack of uniformity/standardisation in
terms of methods of folate extraction and detection, as well as the paucity of reliable
reference materials. However, intra- and inter-laboratory studies confirm the superior
precision of LC methods, compared to the MA: coefficients of variation (CV) of up to
35%,with a mean value of 17.5% reported for inter-laboratory assays (MA) (Finglas et al.,
1999; MacLean et al., 2010; Puwastien et al., 2005), while the corresponding mean for HPLC
assays was 14.5%. For intra-laboratory studies, the CVs were 8.9% (MA) and 5.6% (HPLC)
(Finglas et al., 1999).
Assays performed using LC systems where detection is achieved using mass
spectrometry (LC-MS and LC-MS/MS) (Patring, et al., 2009; de Brouwer et al., 2008) are
adjudged the most selective, specific and sensitive (Pfeiffer et al., 2010). Quantitative
accuracy can be further improved by using isotope dilution MS with (13C5) 5-CH3-THF used
as the Internal Standard (IS) in an UPLC-MS/MS system (Chandra-Hioe et al., 2011).
HPLC assay methodology for Vitamin B9
In general, extraction procedures are aimed at liberating protein-bound folates and
deconjugating folylpolyglutamates. Hence, the procedure involves suspension in a suitable
buffer and then a heat treatment to break up particles, denature proteins and gelatinize starch
(Ball, 2006; Lim et al., 1998). Microwave heating was found to increase the measurable
folates, compared to conventional heating. This is ascribed to the rapidity of the microwave
heating, preventing folate losses due to the activity of endogenous enzymes (Puthesseri et al.,
2013). Heating is followed by followed by enzymatic deconjugation (Van Wyk & Britz,
2012). The enzyme preparations used to achieve deconjugation contain folate conjugase, the
common name for pteroylpolyglutamate hydrolase or folylpoly--glutamyl carboxypeptidase
(EC 3.4.22.12) (Rader et al., 1998). Various deconjugase preparations are available, each
named for the source that it originated from, encompassing a variety of mammalian tissues or
fluids, including human and porcine jejunal tissue and pancreatic fluid (Ball, 2006), chicken
pancreas, hog kidneys and rat and human plasma, as well as vegetable extracts (cabbage)
(Eitenmiller & Landen, 1999). Most conjugase perparations yield monoglutamates, but
chicken pancreas
conjugase
produces
diglutamyl
folates.
Since
HPLC
methods
predominantly require complete deconjugation, it is essential to use either hog kidney, human
or rat plasma conjugase to produce monoglutamates (Strålsjö et al., 2002; Ye et al., 2008).
Several researchers employed the trienzyme extraction method and reported that it
enhanced the yield of measureable folate significantly (Bui & Small, 2007; Johnston et al.,
2002a; 2002b; Kim, 2011; Lim et al., 1998; Pfeiffer et al., 2010; Rader et al., 1998; Shrestha
et al., 2000). However, this method also has its detractors whom reported that the reactions
with the -amylase and protease are time-consuming, that the analyst often faces the
dilemma of which pH to select due to the substantial differences in pH optima for the three
different enzymes (Ball, 2006), that the method sometimes has no effect on folate levels
(Ndaw et al., 2001; Strålsjö et al., 2002) and may result in folate losses, compared to the
single-enzyme methods (Yazynina et al., 2008). Dienzyme methods have been used to good
effect. For example, α-amylase in a starchy matrix (Chandra-Hioe et al., 2011; Hefni et al.,
2010; Vahteristo et al., 1997b) and protease in high-protein foods and dairy products (De
Souza & Eitenmiller, 1990) achieved enhanced liberation of the folates bound to
polysaccharides and proteins, respectively, followed by reaction with a suitable folate
deconjugase (Chandra-Hioe et al., 2011; De Souza & Eitenmiller, 1990). Moreover,
combining CP conjugase, which possesses significant amylolytic (Pedersen, 1988) and
proteolytic activity (Strålsjö et al., 2002), and HK conjugase, complete folate extraction and
conjugation without long incubation times was attained (Vahteristo et al., 1996; Van Wyk &
Britz, 2012). However, the commercial supply of CP conjugase has been erratic in recent
times and although Soongsongkiat et al. (2010) reported satisfactory results with a
lyophilised crude CP extract prepared by them, in our laboratory we were unable to source
raw material that had been produced under consistently controlled conditions to ensure a
preparation of consistent quality. As a result, we are presently doing a comparative
investigation to ascertain the effectiveness of various dienzyme and trienzyme extraction and
deconjugation regimes (results unpublished).
In solution, all folates are susceptible to oxidative degradation, which is exacerbated by
oxygen, heat, metal ions, such as cupric and ferric ions (Russell, 2012) and especially light
(Garrat et al., 2005; Kim, 2011). Moreover, the stability of the various folate forms varies
(Garrat et al., 2005), dependent on their composition, particularly the state of reduction and
substitution at the N-5 or N-10 position. Reduced forms are more labile, with THF the least
stable and substitution at the N-5 position increasing stability (Russel, 2012). The order of
stability is: 5-CHO-THF > 5-CH3-THF > 10-CHO-THF > THF (Witthöft et al., 1999).
Hence, the extraction is always conducted in the presence of at least ascorbic acid as
antioxidant, but often with mercapto-ethanol as well, and under subdued light (Pfeiffer et al.,
2010; Van Wyk & Britz, 2012).
Sample clean-up is often considered essential to ensure reliability of the results.
Affinity chromatography with folate-binding protein is used effectively to this end, as well as
reversed-phase (phenyl) or strong-ion exchange (SAX) solid phase extraction (Ball, 2006;
Jastrebova et al., 2003; Johansson et al., 2008; Kariluoto et al., 2001 Kim, 2011; Vahteristo
& Finglas, 2000; Van Wyk & Britz, 2012).
The following is a typical extraction, purification and HPLC assay protocol that is used
in our laboratories as reported in Van Wyk & Britz (2012) with some modifications, as
applied to various dairy products. Ten mL phosphate extraction buffer was added to 2 mL or
2 g sample, followed by homogenisation under a constant flow of nitrogen. The homogenate
was flushed with nitrogen and heated in a microwave oven at 75% power (610 kW) for 1
minute. The tube was then placed in a boiling waterbath at 100oC for 10 minutes to release
protein-bound folates. After cooling and centrifugation, decanting the supernatant into clean
centrifuge tubes, pH of the extract was then adjusted to 7.0 using NaOH. 25 mg α-amylase
was added per sample and dispersed, flushed with nitrogen, capped and incubated at 37oC for
4 h, followed by addition and dispersion of 25 mg protease and incubation at 37oC for 4 h.
The extracts were then transferred to a boiling waterbath (100oC) for 5 minutes to inactivate
the enzymes, followed by cooling and then the addition of 40 mg lyophilised Human plasma
(HP) conjugase, and incubation as before. Extracts were again heated at 100oC for 5 minutes
to inactivate the enzymes, followed by rapid cooling and centrifugation.
Deconjugation efficiency in each sample type was tested by adding 110.4 nmol of
PteGlu3 to the sample extract prior to the addition of the enzymes. Control samples, where
110.4 nmol of PteGlu3 was added after inactivation of the deconjugase enzymes, were also
assayed. In the sample treated with the HP deconjugase, the absence of the PteGlu3, having
been replaced by a PteGlu peak, testified to complete deconjugation of the Pte-triglutamate.
The folate extracts were effectively purified using quaternary amine-based strong anion
exchange solid phase extraction (SPE) columns (Strata SAX/3 mL/500 mg) (Phenomenex,
Torrance, California).
Oxidative degeneration of the labile reduced folates induced by light and oxygen was
minimised by expedient handling of all samples and standards under subdued light (gold
fluorescent), the use of opaque containers or amber glassware, inclusion of antioxidants into
solutions and flushing with nitrogen gas.
An Agilent 1100 quaternary HPLC system (Agilent Technologies, Waldbron,
Germany), with a scanning fluorescence detector with excitation and emission wavelengths
set at 290 and 356 nm was used. Folic acid and PteGlu3 were detected with the diode array
UV detector at a wavelength range of 270–300 nm.
Two analytical columns (3 µm Luna-C18 column (Phenomenex, Torrance, California)),
followed by a 3.5 µm Zorbax SB-C18 column (Agilent Technologies, Waldbron, Germany)
were used. The column temperature was maintained at 30 ± 1oC during separation of the
vitamers by means of gradient elution with acetonitrile and a 30 mM potassium phosphate
buffer (pH 2.2) at a flow rate of 0.45 mL/min. The gradient was started at 5% acetonitrile and
then linearly increased to 13% over 18 min, increased to 25% acetonitrile over the next 7
min, followed by recycling to initial conditions before the following injection.
The limits of detection (LOD) and limits of quantitation (LOQ) for the different folate
vitamers differ and so do the percentage recovery (quantitative validity or accuracy), but
these, as well as the folate levels determined in the matrices were in agreement with other
authors (Van Wyk & Britz, 2012).
It has become the norm to use Standard Certified Reference Materials (SRMs) as a
quality control tool to confirm the accuracy of the assay, while Certified Reference Materials
are used to validate the method (Johansson et al., 2008; Phillips et al., 2006; 2011; Rader et
al., 1998; Samaniego-Vaesken et al., 2013). Examples are SRM 1849, a milk-based
infant/adult nutritional powder with reference values defined in terms of folic acid (Anon.,
2012) and CRM 421 (spray-dried milk powder). During the study in our laboratories
described above, CRM 421 was not available, having been discontinued by the supplier, the
Institute for Reference Materials and Measurements (Geel, Belgium). A substitute was
obtained by combining a food grade folic acid fortificant (DSM Nutritional Products, Isando,
SA) with fat free milk powder (Elite, Clover, Roodepoort, SA) to yield a folate concentration
of 100 mg per 100 g. The results of our study agreed with the theoretical value, as well as
with the results obtained when a commercial accredited laboratory analysed a duplicate
sample (Van Wyk & Britz, 2012).
In addition, the method was validated against results of an MA. Congruent with several
other researchers (Alaburda et al., 2008; Doherty & Beecher, 2003; Edelmann et al., 2012;
Puthusseri et al., 2013), we found that the HPLC results were in agreement (p < 0.05) with
the results of the MA (Van Wyk & Britz, 2012).
Improvements and recent developments – Vitamin B9 methods
A UPLC-DAD/Fluorescence method was employed to assay folates in oats. Contrary to
HPLC methods applied to cereals, the UPLC method showed a high correlation with the MA.
The method enabled the determination of seven different folate monoglutamates in the
samples, with satisfactory precision and accuracy and substantially faster separation
(Edelmann et al., 2012).
Garrat et al. (2005) assayed the folate content in several food matrices, namely
vegetables (including spinach, cauliflower and peas), wheat and liver tissue. They overcame
the problems posed by the customary intricate and laborious extraction and deconjugation
methods by applying a simple extraction technique based on snap-freezing tissue in liquid
nitrogen, followed by homogenization in an ice cold 95% methanol / phosphate buffer (75
mM K2PO4, 0.4 M ascorbic acid, 0.8% MCE, pH 6.0), followed by centrifugation and
filtration. They used an internal standard mixture which included polyglutamates. Assays
were performed using LC-MS/MS (liquid chromatography/negative ion electrospray
ionization tandem mass spectrometry). Apart from the fact that they could not distinguish
between 5-formyl-tetrahydrofolyl- and 10-formyl-tetrahydrofolyl-polyglutamates, they
obtained full quantitative analysis for 16 folates, including folylpolyglutamates where the
chain contained less than eight residues. Semi-quantitative analysis was possible for folates
with up to eight conjugated glutamates and qualitative results were obtained for
folylpolyglumates with up to 14 conjugated glutamates.
Pawlosky et al. (2003) reported that acceptable precision and accuracy was
demonstrated when doing a comparative study between folates determined in various food
matrices and CRMs using HPLC-Fluorescence v a stable isotope LC-MS method with ESI.
However, there are clear advantages in using LC-MS methods, when directly compared to
HPLC-Fluorescence methods. Not only did the stable isotope LC-MS method allow
quantification of 5-CHO-THF, but it also yielded folate values that were 67% higher than the
HPLC-Fluorescence method, confirming the greater sensitivity of the former (Freisleben et
al., 2003). A further demonstration of the superior selectivity of MS detection is the use of 5
kDa cut-off ultrafiltration that was sufficient in terms of sample purification, in contrast to the
affinity binding or SPE purification methods that are required for LC-UV/fluorescence
detection (Russell, 2012). Further LC-MS methods are listed in Table 1 (Chandra-Hioe et al.,
2011; De Brouwer et al., 2008; Delchier et al., 2013; Young et al., 2011). However, as
mentioned before, the cost and complexity of the MS-based methods limit their wide-spread
application, even though they are superior in terms of sensitivity, selectivity and accuracy
(Russell, 2012).
VITAMIN B12 (COBALAMIN)
Generic description and structures – Vitamin B12
The term vitamin B12 is routinely used as a generic descriptor for all cobalamins that have
biochemical functions in the human (Eitenmiller et al., 2008), including their role in
antipernicious anaemia activity (Ball, 2006).
The structures of various vitamin B12 (cobalamin) derivatives are depicted in Figure 6.
Described as an octahedral cobalt complex, the structure consists of an equatorial chiral
ligand called corrin (the tetrapyrrole macrocycle), a lower axial ligand and various upper
axial
ligands.
The
lower
ligand,
comprising
a
rare
α-ribofuranoside
of
5,6-
dimethylbenzimidazole, is attached to a corrin ring side chain, forming a “nucleotide loop”
and coordinates to the cobalt via the N-3 atom of the imidazole. The upper ligand varies from
cyanide to 5’-deoxyadenosine (Fig. 6) (Chandra & Brown, 2008; Randaccio et al., 2006).
Nutritional/Physiological importance – Vitamin B12
Cyanocobalamin (CNCbl) is the most stable form of the vitamin and hence the form used in
dietary supplements and food fortification (Ball, 2006; Kim, 2011). OHCbl is also used in
this role (Eitenmiller et al., 2008). CNCbl is not biologically active and needs to be
enzymatically
modified
(Wong,
1989)
to
methylcobalamin
(MeCbl)
or
5’-
Deoxyadenosylcobalamin (co-enzyme B12, AdoCbl) which are important cofactors for
methionine synthase (MeCbl) and L-methylmalonyl-CoA mutase as well as leucine aminomutase (AdoCbl) (Randaccio et al., 2006; Russell, 2012). Eitenmiller et al. (2008) tabulated
the complete set of vitamin B12-dependent enzymes.
The remethylation of methionine by 5-CH3-THF:homocysteine methyl transferase (EC
2.1.1.13) occurs via the transfer of the methyl group to the cobalamin co-enzyme and then to
homocysteine, forming methionine and regenerated THF (Bailey, 2006; Ball, 2006),
illustrating the close relationship between folate and vitamin B12 deficiencies. As with folate,
vitamin B12 deficiency results in impaired DNA synthesis, resulting in megaloblastic anaemia
which is clinically indistinguishable from that due to folate deficiency. The vitamin B12induced anaemia is known as pernicious or Addisonian anaemia (Ball, 2006).
CH3
CH3
R'
R
R
H3C
R'
X
N
N
Co+
N
N
CH3
R
CH3
CH3
H3C
CH3
R'
R = CH2CONH2
R' = CH2CH2CONH2
X
X
X
X
X
X
X
= CN = Cyanocobalamin
= OH = Hydroxocobalamin
= H2O = Aquacobalamin
= CH3 = Methylcobalamin
= SO3 = Sulphitocobalamin
= NO = Nitrosocobalamin
= 5'-Deoxyadenosine
= 5'-Deoxyadenosylcobalamin (AdoCbl)
= Co-enzyme B12
HN
O
OH OH
O
O-
O
H
P
O
OH
N
CH3
CH2
O
N
H
H
O
CH2OH
N
H
CH3
H
N
N
N
NH2
5'-Deoxyadenosine
Figure 6. Vitamin B12 (cobalamin) structures.
Other important roles of vitamin B12 in normal metabolism include synthesis of DNA,
proteins, phospholipids, creatine and neurotransmitters. Apart from the haematological
effects, vitamin B12 deficiency also results in neurological changes, caused by the inability to
synthesise the lipid component of myelin. This leads to general demyelination of nerve tissue.
The first symptoms are peripheral neuropathy with the feet and fingers are first to be affected,
then the spinal cord, culminating in brain damage if not treated (Parker-Williams, 2013;
Russell, 2012). Hence, the concern that high folate intakes may mask the haematological
effects of vitamin B12 deficiency, thus allowing the progression of neuropsychiatric damage,
lies in the fact that advanced neurological damage cannot be reversed (Ball, 2006; Groff et
al., 1995; Russell, 2012). Cerebellar ataxia (Morita et al., 2003), triggering seizures in
epileptics (Thiel & Fowkes, 2004) and reversible involuntary movements in adults (Celik et
al., 2003) are further conditions ascribed to vitamin B12 deficiency.
Vitamin B12 is also associated with the same chronic diseases as folate, due to the coincidences in their biological functions (Russell, 2012). Hence, subnormal vitamin B12 levels
have been linked to reversible dementia (Tucker et al., 1996), while low folate and vitamin
B12 levels have been implicated in psychiatric diseases, such as bipolar disorder (Ozbek et al.,
2008). Optimum vitamin B12 intake may also play an ameliorating role in depression, senile
dementia (Eitenmiller et al., 2008), Alzheimer’s disease, cardiovascular disease, breast
cancer, hearing loss, osteoporosis, NTDs (Green & Miller, 2007) and multiple sclerosis
(Kocer et al., 2009). Furthermore, not only were low vitamin B12 levels recorded in HIVinfected patients (Hepburn et al., 2004), but adequate vitamin B12 may slow the onset of Aids
in HIV-positive individuals (Green & Miller, 2007).
Dietary sources – Vitamin B12
The vitamers that predominate in foodstuffs include AdoCbl in animal liver (Metzler, 1977),
with hydroxocobalamin (OHCbl), AdoCbl and MeCbl dominant in animal tissue in general
(Alegría et al., 2008; Kumar et al., 2010) and hydroxocobalamin (OHCbl) or aquacobalamin
(H2Ocbl) are the forms most prevalent in milk (Ball, 2006; Kumar et al., 2010). The native
vitamin B12 vitamers present in food are normally bound to proteins in the matrix, while
certain foods contain specific vitamin B12-binding protein reducing the bioavailability. In
contrast to folate, milk does not contain vitamin B12 binding protein, while raw egg yolk and
egg white do. Heating denatures the protein and releases the vitamin B12 (Eitenmiller et al.,
2008; Russell, 2012).
All native vitamin B12 in the human diet is originally synthesised by microorganisms,
either in the rumen and intestinal tract of animals or in soil and water, which is then
assimilated by animals (Ball, 2006; Eitenmiller et al., 2008; Kim, 2011; Nohr & Biesalsky,
2009). Commercial production of vitamin B12 also exploits biosynthetic processes, since
chemical synthesis is extremely complex (Murooka et al., 2005). The micro-organisms most
widely used in these fermentations include Pseudomonas denitrificans (Mazumder et al.,
1987), methanogens (Methanosarcina barkeri, Methanobacterium spp.) and Bacillus spp.
(Mazumder et al., 1987; Yang et al., 2004) and Propionibacterium spp. (Gardner &
Champagne, 2005), the preferred organisms for vitamin B12 production (Murooka et al.,
2005).
The most important dietary sources of vitamin B12 are meat and animal organ meats,
particularly liver (Alegría et al., 2008; Van Heerden et al., 2007; Schönfeldt & Gibson,
2008). Ruminant meat accounts for two-thirds of the daily intake of vitamin B12 in the human
diet (Alegría et al., 2008). Shellfish, dairy products, eggs and fortified cereal products are
also dietary sources (Russell, 2012).
Assay methodology for Vitamin B12 – General
Vitamin B12 assay techniques include microbiological assays (MA), HPLC analyses,
radioisotope dilution assay (RIDA), electrothermal atomic absorption spectrometry, capillary
electrophoresis, biomolecular interaction analysis (BIA), chemiluminescence techniques
(Alegría et al., 2008; Ball, 2006; Cheung et al., 2009; Gao et al. 2008; Kim, 2011;
Markopoulou et al., 2002), fluorescence resonance energy transfer (FRET) combined with
flow-injection analysis (FIA) (Xu et al., 2008) as well the more novel approach of a dipstickbased immunochemiluminescence biosensor used for energy drinks (Selvakumar & Thakur,
2011; 2012). Of the abovementioned methods, only the MA, HPLC, RIDA and
chemiluminescence methods have been used successfully to quantify vitamin B12 in foods
(Eitenmiller et al., 2008; Kumar et al., 2010; Selvakumar & Thakur, 2012).
Until recently the MA was the only official AOAC method and is still widely used to
determine vitamin B12 in food (Ball, 2006; Eitenmiller et al., 2008; Kim, 2011), but is very
protracted (Alegría et al., 2008). The organism most frequently used for vitamin B12
determination in foods is Lactobacillus delbrueckii ssp. Lactis (ATCC 7830). The growth
response (measured titrimetrically) (Van Wyk & Britz, 2010) is similar for most forms of
cobalamin (CN-, NO-, OH-, SO3Cbl). However, AdoCbl produces a greater and MeCbl a
lesser response, but exposure of the samples to fluorescent light accomplishes the complete
conversion of MeCbl and AdoCbl to OHCbl (Kim, 2011).
Alternatives to the MA were recently adopted as official methods by the AOAC. An
SPR optical biosensor protein-binding assay for vitamin B12 in milk products (Official
method 2011.16) and three HPLC methods to determine vitamin B12 in food products were
adopted. The HPLC methods include “Determination of Vitamin B12 in food products by
Liquid Chromatography/UV detection with Immunoaffinity extraction: Single laboratory
validation” (Campos-Giménez et al., 2008) (Official method 2011.08), “Determination of
vitamin B12 in baby food (milk formulas) using HPLC after purification on an
immunoaffinity column, cleanup and LC-UV quantitation” (Official method 2011.09) and
“Determination of vitamin B12 by HPLC” (Official method 2011.10) (Sullivan, 2012).
Several researchers employed HPLC methods to determine vitamin B12 in multivitamin and
mineral tablets. These matrices are relatively simple, compared to foodstuffs, making
extraction and resolution of the vitamin simple in comparison (Markopoulou et al., 2002).
Although vitamin B12 levels in foods are often too low for detection using HPLC methods
(Ball, 2006), reversed-phase HPLC methods with immunoaffinity extraction (Heudi et al.,
2006) and UV detection (Heudi et al., 2006; Van Wyk & Britz, 2010; Van Wyk et al., 2011),
and coulometric electrochemical detection (Lebiedzińska et al., 2007) have been used
effectively to determine vitamin B12 in dairy products (Van Wyk & Britz, 2010), petfood and
various infant formulae (Heudi et al., 2006), fruit juice and various seafood (Lebiedzińska et
al., 2007).
HPLC assay methodology for Vitamin B12
Extraction of cobalamins from tissues or fluids is a prerequisite for all assays. The ideal
process achieves full recovery of the cobalamins, while preventing degradation to
noncobalamin corrinoids (Gimsing & Beck, 1986). Using phosphate-buffers containing
sodium metabisulphate or acetate-buffers containing KCN, combined with a heat treatment,
liberates the vitamin B12 from the matrix, converts all forms of vitamin B12 to the more stable
sulphitocobalamin or cyanocobalamin, respectively, as well as achieves protein denaturation
(Alegría et al., 2008; Ball, 2006; Kim, 2011; Russell, 2012; Van Wyk, 2002). In high-protein
foods, like meat and dairy products, heat-induced or protease-catalysed protein denaturation
is essential, since both vitamin B12 and folate are protein-bound in these products (Alegría et
al., 2008; Deeth & Tamime, 1981; Kumar et al., 2010).
Cobalamins are stable to thermal processing, with cyanocobalamin the most stable.
However, strong acid and alkaline conditions, oxidizing agents and intense visible light
inactivate the vitamin (Ball, 2006; Kim, 2011). Hence, during extraction and subsequent
sample handling, it is advisable to avoid any of the above. Light exposure is minimized by
working under subdued light and using amber glassware (Van Wyk & Britz, 2010).
Purification of extracted cobalamins will achieve removal of compounds that may
interfere with the resolution of cobalamins, as is present in most biological samples.
Purification of the extracted cobalamins should ideally remove all these interfering
substances, in such a manner that the extracted cobalamins react similar to standards during
the analysis. Removal of these substances is based on the amphoteric nature of cobalamins,
i.e. they possess both polar and non-polar groups. Hence, in aqueous solutions, cobalamins
are absorbed to nonpolar materials such as silanized silica gel, Amberlite XAD-2, or alkylbonded phase columns such as C8 and C18 reverse phase columns, while water or weak
organic solvents wash off less hydrophobic compounds. When followed by application of
higher concentrations of organic solvents, yet not concentrated enough to elute compounds
that are more hydrophobic than cobalamins, complete elution of purified cobalamins is
achieved (Gimsing & Beck, 1986; Blanche et al., 1990; Dalbacke & Dahlquist, 1991).
Immunoaffinity, ion exchange chromatography (Eitenmiller et al., 2008), or SPE methods
can also be used to purify and/or concentrate CNCbl in extracts (Van Wyk & Britz, 2010).
RP-LC on C18 columns with gradient elution of the analyte(s) is the most common
approach. The mobile phase consists of water or buffers and methanol or acetonitrile. Since
all vitamin B12 absorb UV light, detection based on absorbance in the UV–Visual range with
absorbance maxima from 264–551 nm, with the absorbance maxima of CNCbl 278, 361 and
551 nm (Eitenmiller et al., 2008). CNCbl is most often detected at 361 (Russell, 2012; Van
Wyk & Britz, 2010) or 550 nm (Russell, 2012). UV photodiode array detectors are preferred
to single lamp instruments.
Some of the most recent methods are described in Table 1 (Campos-Giménez et al.,
2008; Guggisberg et al., 2012; Heudi et al., 2006; Lebiedzińska et al., 2007), including
multivitamin (Luo et al., 2006) and human serum assays (Shaik & Gan, 2013). Details of the
method used in our laboratories (Van Wyk & Britz, 2010, with modifications) follow.
Samples were extracted using a 0.31 M KCN-acetate buffer at pH 4.5. The sample and
buffer were combined in a 4:10 ratio, autoclaved at 121oC for 25 min, cooled and filtered into
amber vials. Work with OHCbl standards showed that all OHCbl is converted to CNCbl
during this extraction, so it is reasonable that all other cobalamins are converted to CNCbl
(Van Wyk, 2002).
The HPLC system described in the previous section was used. Separation was achieved
using a Phenomenex Luna (150 X 4.6 mm, 5 µm) column, with the column temperature at
30oC and the flow rate at 0.05 mL/min. Gradient elution was used: A = Acetonitrile; and B =
10 mM phosphate buffer (pH 2.2). Starting with 5% A: 95% B initial conditions, the gradient
is increased to 15%A:85%B (0–10 min), then 40%A:60%B (10–13 min), 15%A:85%B (13–
15 min), 10%A:90%B (15–20 min) and then 5%A:95%B (20–25 min), with 5 min post run
time at initial conditions.
Detection was achieved using a diode array detector at 360.4 nm (sample) and 100 nm
(reference). The LOD and LOQ were 0.002 and 0.005 µg/mL sample and all measures of
sensitivity, selectivity, precision and accuracy were satisfactory. However, sensitivity was
enhanced even more by using SPE columns to purify (Chromabond SB/3 mL/500 mg,
Machery-Nagel) and concentrate (Chromabond C18ec/6 mL/1000 mg column, MacheryNagel) the extracts. Moreover, the HPLC results were validated against the microbiological
assay (Van Wyk & Britz, 2012).
Improvements and recent developments – Vitamin B12 methods
HPLC analysis of vitamin B12 in foodstuffs had recently received attention from the AOAC,
resulting in the adoption of three official methods (Sullivan, 2012), as described in a previous
section. LC-MS methods are not much in evidence, but those that have been reported (Luo et
al., 2006 – also see Table 1), indicate their potential in terms application of soft ionization
techniques suitable to thermolabile and nonvolatile compounds to improve the selectivity and
sensitivity of vitamin B12 analyses (Eitenmiller et al., 2008).
VITAMIN C (ASCORBIC ACID)
Generic description and structures – Vitamin C
Vitamin C, also referred to as L-ascorbic acid belongs to a group of vitamins called watersoluble vitamins. Its systematic name is 5(R)-5-[(1S)-1,2-Dihydroxyethyl]-3,4-dihydroxy-
2(5H)-furanone. It occurs naturally in food as L-ascorbic acid (AA) and as its oxidised
product, dehydro- L-ascorbic acid (DHAA) (Figure 7) (Eitenmiller, 2008; Lee, 2004).
Humans must obtain vitamin C through their diet. Some other mammals have the ability to
produce their own vitamin C supply by converting it from glucose using the enzyme Lgulono-γ-lactone oxidase (Basu, 1996). L-ascorbic acid has a stereoisomer, isoascorbic acid
(IAA), which can only be synthetically produced. IAA has its oxidised product called
dehydroisoascorbic acid (DHIAA).
HO H
HOH2C
HO H
HOH2C
O
O
O
HO
Ascorbic acid
OH
O
O
O
Dehydroascorbic acid
Figure 7. Vitamin C structures.
Nutritional/Physiological importance and Dietary sources
Vitamin C is present in fresh fruit, vegetables, juices and organ meats such as liver (Combs,
1992; Jurczuk, 2005). Since vitamin C is highly unstable it can be used as an indicator of the
quality of foodstuffs and fruit beverages during manufacturing and storage. It can be
degraded by enzymes, oxygen, heat, light and metal ions (Bhagavan et al., 2001).
Vitamin C is well known for its antioxidant properties as it assists the body in
contesting viral infection, bacterial infections and toxicity. It is required for the synthesis of
collagen, an important structural component of tendons, bones, teeth, blood vessels and
muscles. Vitamin C enhances iron adsorption and regenerates other antioxidants such as
vitamin E (Brody, 1994). Vitamin C also plays a role in wound healing (Fox, 1989). A
deficiency in vitamin C causes bruising, bleeding, dry skin and depression (Olson, 1999).
These are all symptoms of a deficiency-induced potentially fatal skin disease called
scurvy. The symptoms are all related to diminished levels of collagen in bones, blood vessels
and connective tissue. Over-consumption of vitamin C may cause a vitamin B12 deficiency
(Herbert & Jacob, 1974). It also acts as an antioxidant and protects carbohydrates, fats,
proteins and nucleic acids (DNA and RNA) from damage induced by free radicals and other
reactive species. (Bhagavan et. al, 2001; Cathcart et al., 1991).
Assay methodology for vitamin C – General
Many analytical methods have been reported for the determination of vitamin C (L-ascorbic
acid) such as spectrophotometric, cyclic voltammetry (Güçlü, et al., 2005; Pisoschi, et al.,
2008) as well as enzymatic methods (Akyilmaz, 1999). Before the development of high
performance liquid chromatography (HPLC) as a method of analysis, one of the most
commonly used methods was titration, which is based on the reduction of 2,6dichlorophenolindophenol by ascorbic acid (Anon., 1999). It has since been modified (Matei
et al., 2004) and is used in the determination of vitamin C not only in fruit, vegetables and
other food products but also in pharmaceutical products (Hernandez et al., 2006). The
volumetric techniques have limited use as they can only be applied to samples not containing
other reducing agents (O’Connell, 2001).
With the development of HPLC, many HPLC based methods proved to be selective and
specific to vitamin C determination (Iwase & Ono, 1998; Kall & Anderson, 1999; Oliviera &
Watson, 2000). These methods also allow simultaneous determination of L-ascorbic (AA) and
dehydro-ascorbic acid (DHAA) (Nisperos-Carriedo et al., 1992), and separation of ascorbic
acid and its stereoisomer, isoascorbic acid (IAA) (Kall & Anderson, 1999). Russell (2012)
covers HPLC methods employed by various researchers and selected methods were
summarised in Table 1.
HPLC assay methodology for Vitamin C
The extraction of vitamin C in various matrices requires procedures that limit or prevent its
degradation as it is labile. Most extraction methods tend to use metaphosphoric acid as an
extraction solution as it precipitates protein from the samples and thus preventing its
interference with the results and degradation from enzymes and metal induced oxidation
(Davey et al., 2000; Eitenmiller, 2008). Based on a specific matrix, different concentrations
of metaphosphoric acid combined with acetic acid, sulfuric acid, ethylenediaminetetra-acetic
acid (EDTA), acetone or ethanol can be used at acidic pH to prevent factors that contribute to
instability of vitamin C (Ball, 2006; Kall & Andersen, 1999; Nyyssönen, et al., 2000). The
recommended AOAC 967.22 method for extraction of vitamin C in most food matrices uses
the metaphosphoric acid/glacial acetic acid extractant (Anon., 1993). More extraction
protocols based on matrix type are listed in Table 1. The stability of vitamin C can further be
increased by using low actinic glassware or bubbling inert gas through the extraction process.
Amongst the most reported HPLC methods, reversed-phase is the most commonly used
by means of C18 stationary phase column with 5 µm particle size and polar mobile phase with
diode array detector (DAD) or ultraviolet (UV) absorbance detection as indicated in Table 1.
The advantage with diode array detection is that it combines absorbance detection and peak
purity. The methods report absorbance wavelengths that range between 210 nm and 265 nm
(Shafqat et al., 2012; Tarrago-Trani et al., 2012; Valls et al., 2002). Electrochemical
monitoring and fluorescence detection can also be utilised after chemical derivatisation to a
fluorescent compound (Vanderslice & Higgs, 1984; Bognár & Daood, 2000).
Iwase (2000) and Rizzolo (2002) reported HPLC methods with electrochemical
detection as more selective and sensitive for vitamin C analysis in foodstuffs and biological
fluids. Refractive Index detection has been reported by Doner (1981) to be non-selective,
with low sensitivity and not suitable for accurate measurement of small amounts of Ldehydroascorbic acid and other oxidation products. The vitamer and its oxidised products are
commonly eluted isocratically with varied flow rates, or with gradient elution to improve
resolution of the peaks. Mass spectrometer (MS) detection of Vitamin C has been reported
but is limited to multivitamin assays (Russell, 2012). However, Garrido Frenich et al. (2005)
reported the HPLC analysis of vitamin C in tomatoes, mango and kiwi fruit with MS
detection.
Table 1
Water-soluble Vitamin Estimation by HPLC
Matrix
Food Matrices
Bread,
sourdough
Bread, white,
wholemeal and
multigrain
Analyte
Extraction
Column
Elution
Detection
Ref.
Thiamine
Riboflavin
Nicotinic acid
Nicotinamide
Pantothenic acid
Pyridoxal
Pyridoxine
Incubation of freezedried
samples
with
o
agitation at 37 C for 18
hours; centrifuge 14 00
rpm; filter 0.2 µm
Acquity UPLC HSS C18 1.8 µm (2.1 X 150
mm)
A: Water + 0.1% formic acid
and B: Acetonitrile + 0.1%
formic acid gradient at 2.5
mL/min and 5 µL injection (0-3
min 100% A, 3-8.5 min 80% A
and 20% B, 8.5-10 min 5% A
and 95% B, 10-15 min 100%
A)
LCT Premier
XE ESI
TOF MS (Waters)
Mihhalevski et
al. (2013)
Folic Acid (FA) and
5-Methyl-THF
(5MTHF)
Phosphate buffer (0.1 M,
pH 6.1) with 2% (w/v)
Na-ascorbate and 0.1%
(w/v) mercapto-propanol.
13
13
C5 FA and C5 5MTHF
added
to
extraction
buffer.
Homogenised samples
treated with α-amylase to
hydrolyse
starch,
followed
by
deconjugation with rat
serum
SPE and ultrafiltration
used for sample cleanup
and concentration
Acquity UPLC HSS C18 1.8 µm (2.1 X 150
mm)
A: 0.1% Formic acid in Milli-Q
water and B: acetonitrile. 200
µL/min and 20 µL injection (00.2 min 99.5% A, 0.2-2.2 min
65% A and 35% B, 2.2-3.0 min
30% A and 70% B, 3.0-3.2 min
30% A and 70% B, recycled to
initial conditions. Total run time
6 min
Thermo TSQ Quantum
Access
tandem
quadrupole MS (ThermoScientific) operated in
positive ion ESI mode
Chandra-Hioe
et al. (2011)
TM
Matrix
Analyte
Extraction
Column
Cereal grains
Thiamine and its
phosphate esters
4 g samples were milled
and extracted with cold
4%
aqueous
trichloroacetic acid with
stirring. This centrifuged
at 1 500 g for 5 min and
supernatant
filtered
through fine glass wool
(3 X). To 2 mL a 1 mL
volume of 3% aqueous
cyanogen bromide was
added and the pH
adjusted to 10 with 1 M
NaOH. For thiochrome
reaction, it was left in
dark for 25 min on ice,
pH
adjusted
to
7,
centrifuged at 16 000 g
for 2 min and subjected
to solid phase extraction
(SPE)
C-18
connected
column
Corn extrudates
(fortified)
Thiamine
Riboflavin
50 mL 0.1 N H2SO4
added to 1 g extrudate,
boiled for 30 min with
vortexing,
cooled,
takadiastase in 2.5 M Na
acetate added, incubated
o
(4 h in 37 C waterbath).
Diluted to 100 mL with
water, filtered through
Whatman 541 paper.
Riboflavin:
Filtered
through 0.2 µm nylon
filter before HPLC.
Thiamine: 5 mL filtrate
plus 1 mL of 0.18 M
potassium ferricyanide in
3.5 M NaOH. Filtered
through 0.2 µm nylon
filter before HPLC
µ-Bondapak
column (3.9
mm, 10 µm)
Elution
Detection
Ref.
column,
to
NH2
Gradient elution gave reversed
elution order due to the two
columns linked (according to
Poel et al., 2009)
Fluorescence detection
Bucholtz et al.
(2012)
C18
X 300
Injection volume = 20 µL. Flow
rate = 1 mL/min. Temperature
o
= 30 C. Isocratic elution with
methanol: water = 30:70 v/v
Fluorescence
with
excitation wavelength =
365 nm and emission
wavelength = 435 nm for
Thiamine and 450 nm and
510 nm for riboflavin
Boyaci et al.
(2012)
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Complex cereal
foods
Thiamine
Riboflavin
15 g cereal in 90 mL 0.1
N H2SO4, adjusted to pH
4.5 with 2.5 M sodium
acetate which was added
papain, diastase and αo
amylase, incubated 37 C
overnight, made to 100
mL with water, filtered
through Whatman 40
paper and through 0.45
µm
polyvinylidene
fluoride membrane
Purospher STAR RP18e (250 X 4 mm, 5
µm particle size)
Eluent: 12.5 mM sodium
acetate in methanol / water
(25/75) + 2.4 mM sodium
heptanesulphonate. Flow rate
= 0.9 mL/min
UV-Vis at 268 nm
San José et
al. (2012)
Cooked meals,
with cereals,
vegetables and
legumes.
Milk and fruit
Folates (five
vitamers)
Samples homogenised
and freeze-dried. To 0.51 g 10 mL HEPES-CHES
buffer,
pH
7.85,
containing
2%
Naascorbate and 0.01 M 2mercaptoethanol. Placed
in waterbath for 10 min at
o
100 C, cooled.
Trienzyme
extraction
with a protease, αamylase and human
plasma (various pH and
o
times, all at 37 C),
followed by heating for
o
10 min at 100 C, cooling
and centrifugation.
Purification achieved on
SAX SPE cartridges.
Final eluate reduced to
dryness
in
vacuum
centrifuge
and
resuspended in 100 µL
0.1% formic acid
C18
RP
column
(Zorbax Eclipse, 150 X
2.1 mm, 5 µm particle
size)
A: Aqueous formic acid (0.1%),
B: Acetonitrile
Elution:
0-4 min: Isocratic at 7% B
4-16 min: Linear gradient: 30%
B
16-10 min: Linear gradient: 7%
B
Flow rate: 0.2 mL/min
Quantification
achieved
using selected monitoring
(SR) MS.
System: HPLC coupled to
ion trap MS via an ESI
interface.
MS operated in positive
ion mode. Quantification
13
of folates using
Clabelled I.S.
Vishnumohan
et al. (2013)
®
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Dates
Vitamin B1
Vitamin B2
Vitamin B3
Vitamin B5
Vitamin B6
Vitamin B9
Vitamin B12
Samples homogenised,
25 mL buffer (Na-hexane
sulphonate + KH2PO4 +
DI
water
+
trimethylamine) added,
heated
in
shaking
waterbath for 40 min at
o
70 C, cooled, filtered and
diluted
Waters Symmetry C18
(250 X 4.6 mm, 5 µm
particle size)
A: 940 mL DI water + 5 mL
triethylamine + H3PO4 to pH
3.0;
B: Methanol
Isocratic
elution
with
buffer:methanol at 96:4.
Flow rate: 1 mL/min
UV, using two channels
simultaneously at 210 nm,
a bandwidth of 5 nm and
another wavelength of 280
nm
Aslam et al.
(2013)
Dried bee pollen
Thiamine
Riboflavin,
Nicotinic acid
Nicotinamide
Pyridoxol
Pyridoxal
Pyridoxamine
Simultaneous extraction
of 5 g pollen with 50 mL
0.1 M HCl in boiling bath
for 30 min. pH adjusted
to 4.6 with 2.5 M sodium
acetate
followed
by
adding 0.5 g fungal
diastase, incubating 2 h
o
at 42 C in water bath,
cooling, diluted to 100
mL
with
water,
homogenizing
and
filtering through paper
and 0.45 µm prior to
injection
C18
reversed-phase
column (250 mm X 4.6
mm, 5 µm particle
size) with pre-column
(5 µm/10 mm X 4.6
mm)
Thiamine and riboflavin: Flow
rate = 1 mL/min, injection
volume = 20 µL. Mobile phase:
10 mM phosphate buffer (pH
7.2) and dimethylformamide
(85:15).
Niacin vitamers: Flow rate =
1.5 mL/min, injection volume =
20
µL.
Mobile
phase:
phosphate / copper sulphate /
hydrogen
peroxide
buffer
complex with UV treatment in
the loop.
Vitamin B6 vitamers: Flow rate
= 0.6 mL/min, injection volume
= 20 µL. Mobile phase: 39 mM
phosphate buffer (pH 2.5) and
ion pair (PIC 7) and acetonitrile
(96:4)
Fluorescence
detection
(excitation/emission)
for
thiamine (368/440 nm),
riboflavin (450/530 nm),
niacin and niacinamide
(322/380 nm) and B6
vitamers (296/390 nm)
De Arruda et
al. (2013)
Matrix
Analyte
Dry-cured
sausages
Thiamine
Elution
Detection
Ref.
Frozen samples milled
with bowl cutter, 1 g;
incubated for 30 min at
o
100 C in 9 mL water and
this treated with 2.5 M
Na-acetate and 10%
taka-diastase overnight
o
at 37 C, centrifuged at
o
4 C at 4 500g for 5 min,
supernatant diluted with
water; pH adjusted to 6.0
with Na-acetate. Loaded
®
onto pre-treated Owasis
WCX cartridge, thiamine
washed off sequentially,
evaporated to dryness
o
under nitrogen at 25 C,
re-dissolved in mobile
phase A and filtered
through 0.45 µm PTFE
filter
Feed, pre-mixes
and supplements
Fennel
Extraction
Column
Luna HILIC (150 mm
X 3.0 mm i.d., 3 µm
particle size) column at
ambient temperature
Gradient elution at 0.5 mL/min
using (A): acetonitrile:50mM
ammonium acetate pH 5.8
(90:10
v/v)
and
(B):
acetonitrile:10mM ammonium
acetate pH 5.8 (50:50 v/v) by
varying B from 0 to 10% in 8
min. Injection volume = 40 µL
UV detection at 270 nm
and spectral trace from
220 nm to 420 nm
GrataćosCubarśi et al.
(2011)
Thiamine
Riboflavin
Pyridoxine
Nicotinic acid
nicotinamide
Extraction with 0.01 M
o
HCL at 40 C for 10 min,
centrifuged at 8 000 rpm
for 10 min followed by
SPE
using
various
adsorbents followed by
filtration
through
a
microfilter
Supelco C18 column
(250 X 4.6 mm); 5 µm
particle size
Gradient elution at 1 mL/min
using (A) 11mM phosphate pH
2.5 buffer containing 4%
acetonitrile and (B) acetonitrile
(permutations
available in
original article).
UV at 260 nm and 280 nm
Rudenko
Kartsova
(2010)
Ascorbic acid
Homogenise with 0.2%
potassium metabisulfite.
Dilute with 20 mM
phosphate buffer. Keep
in the dark for 5 min and
filter (0.2 µm). Flush with
nitrogen and protect from
light
Precolumn: Aqua C18
(Phenomenex),
Analytical: Aqua C18
(250 X 4.6 mm, 5 µm,
Phenomenex)
Isocratic: 20 mM phosphate
buffer, pH 2.14 at 1 mL/min
UV absorbance: 240 nm
Galgano et al.
(2002)
®
&
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Fish
Thiamine
Riboflavin
Niacin
Pyridoxine
Weighed into 0.1 M HCl
and
autoclaved.
pH
adjusted 60 6.5 and 4.5,
made to volume with
water
and
filtered
through
paper.
Any
turbidity removed by
centrifugation at 6 000
rpm for 10 min and then
passed through 0.45 µm
filter
C18 Omnisphere
(250 X 4.6 mm)
5
Flow rate = 1 mL/min. Injection
volume = 20 µL. Isocratic
elution with phosphate buffer:
methanol = 100:36. Run time =
22 min
UV at 254 nm
Ersoy &
Özeren
(2009)
Fish
Thiamine
Riboflavin
Niacin
Pyridoxine
30g fish was extracted
with 60 mL extraction
solution (10 mL acetic
acid
and
50
mL
acetonitrile in 1 L water),
boiled in an ultrasonic
o
bath at 70 C for 30 min
and cooled. This was
filtered through Whatman
paper and a 0.45 µm
filter
Lichospher 60 RPselect B (5 µm)
LiChroCART
250-4
HPLC cartridge
Flow rate = 1 mL/min. Injection
volume = 20 µL.. Isocratic
elution with buffer containing
phosphate,
hexanesulfonic
acid and trimethylamine at pH
2.4–2.5
UV detection at as follows:
0–6.5 min. at 264 nm for
niacin, 6.5–20 min at 6.4–
20 min for riboflavin and
pyridoxine and 20–35 min
for thiamine
Erkan et al.
(2010)
Food Supplement
(athlete)
Total vitamin C as
ascorbic acid
Mix sample with 0.2%
phosphoric
acid
containing
20µM
Lo
methionine at 5 C; filter
(0.45 µm).Protect from
light
Vydac 201-HS C18, 10
µm, 250 X 4.6 mm
Isocratic: 0.2% phosphoric
acid, pH 2.1, 0.4 mL/min. 5µl
injection volume
Electrochemistry: glassy
carbon working electrode,
+400 mV vs. Ag/AgCl
reference electrode
Iwase (2000)
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Food products
and multivitaminmineral tablets
Vitamin B12
50
mM
Na-acetate
buffer, pH 4.0 + 1%
NaCN + α-amylase +
pepsin; digest for 3 h at
o
37 C; cool, adjust pH to
4.8; Heat for 35 min at
o
100 C;
Cool,
dilute,
centrifuge and filter (0.45
µm
membrane).
Ginsenoside used as I.S.
Xterra MS C18 column
(150 mm X 3.9 mm), 5
µm
Gradient,
water/acetonitrile.
Flow rate 1 mL/min
ESI-MS, positive ion mode
Luo et
(2006)
Fruit juices
(apple), fortified
fruit juices,
salmon, oysters,
scallop
Pyridoxamine
Pyridoxal
Pyridoxine
Thiamine
Cyanocobalamin
Variable based on matrix
and analytes, in general:
acid
digestion
and
enzymatic
hydrolysis
(papain and diastase),
followed
by
enzyme
inactivation and filtration
into amber glass.
For
cyanocobalamin
extraction,
the
acid
digestion was replaced
with autoclaving after
addition of a NaCNcontaining acetate buffer
at pH 4.0
Supelco C18 column
(250 X 4.6 mm); 5 µm
particle size
Isocratic elution with methanolphosphate buffer (10:90) and
0.018 M trimethylamine at pH
3.55.
Flow rate 1 mL/min
Electrochemical
(EC)
detector Coulochem II
with dual analytical cell
and guard cell connected
in-line before the injection
port. Detector response
set to give full scale at 1
µA and 50 µA current
output
received
from
analytical cell. Peak area
of the EC signal at the
porous graphite electrode
used
for
quantitative
analysis
Lebiedzińska
et al. (2007)
al.
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Fruit drinks,
fortified fruit
drinks, juices an d
cereal products
Folates
Phosphate buffer (pH
6.8)
+
sample;
homogenized; 10 min at
o
100 C,
cooling,
pH
adjusted to 4.9; trienzyme treatment (Hog
Kidney
conjugase);
o
heating 5 min at 100 C to
inactivate
enzyme;
cooling,
centrifugation,
filatration (Whatman no.
o
1), storage at -20 C;
filtration through 0.22 µm
pore size prior to HPLC.
Protection against light;
subdued light
LC 18 column (250
mm X 4.6 mm), 5 µm,
Supelco, Inc.
40 mM sodium phosphate
dibasic heptahydrate and 8%
acetonitrile (v/v), adjusted to
pH 5.5 with 85% phosphoric
acid.
o
Column temperature: 25 C
Flow rate 0.9 mL/min
Electrochemical detector
Coulochem II with dual
analytical cell and guard
cell
connected
in-line
before the injection port.
Detector response set to
give full scale at 1 µA and
50 µA current output
received from analytical
cell.
For
obtaining
optimum
detection,
electrode potential set at
0.85 V (guard cell), 0.20 V
(electrode E1) and 0.80 V
(electrode E2)
Lebiedzińska
et al. (2008)
Fruit (fresh
tomatoes)
Ascorbic acid (AA),
pentanophenone
(internal standard)
Homogenize with 6%
HPO3,
mix aliquot of slurry with
MeOH, add 0.05%
pentanophenone
(internal
standard), centrifuge,
dilute
with MeOH containing
0.0015 M pyrogallol
Precolumn:
Optimal
ODS-H C18 (20 X 4.6
mm, 5 µm, Capital
HPLC).
Analytical:
Optimal ODS-H C18
(250 X 4.6 mm, 5 µm,
o
Capital HPLC) at 25 C.
MeOH/H2O/MeCN (60 þ 40 þ
1) containing 0.5 mM tridecyl
ammonium formate, adjusted
to pH 4.25
UV absorbance: 247 nm
Russell
(1986)
Fruit, dog rose
Ascorbic acid
Freeze in liquid nitrogen
and homogenise in 5%
o
MPA, centrifuge at 4 C.
Dilute with water and
filter (0.45 µm)
Precolumn:
Inertsil
ODS-3 C18 (10 X 3
mm, 5 µm). Analytical:
Inertsil ODS-3 C18
(150 X 3mm, 5 µm; GL
Sciences)
Isocratic: Methanol + 0.25%
MPA at 1 mL/min. Injection
volume: 20 µl)
UV absorbance: 245 nm
Nojavan et al.
(2008)
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Fruit and
vegetables
(Tomatoes,
mango, kiwi fruit)
Ascorbic acid
Homogenise sample in
methanol + 3% MPA
containing 8% acetic acid
(1 + 5, v/v), filter, make
to volume with 0.1%
acetic acid. Protect from
light during extraction
Injection volume = 10 μL
Analytical column:
Symmetry C18 (75 X
4.6 mm, 3.5 μm,
Waters) + Atlantis
dC18 (150 X 2.0 mm,
5 μm, Waters) in
o
series; 30 C
Isocratic: 0.005% acetic acid in
methanol + 0.05% acetic acid
(70 + 30, v/v)
Flow rate: 0.3 mL/min
Mass spectrometry: single
quadrupole
ESI
in
negative ion mode
Garrido
Frenich et al.
(2005)
Fortified juices
and cereals.
Folic acid
Juice centrifuged, filtered
through nylon membrane
filter.
Cereals ground in mortar
and pestle, solution of DI
water,
NH4-formate,
methotrexate, ascorbate
and NH3 added. After
sonication, centrifugation
and membrane filtration
HPLC-UV:
Diamond
TM
Hydride
column (75
X 4.6 mm, 4.2 µm
particle size).
LC-MS:
Diamond
TM
Hydride column (150
X 2.1 mm, 4.0 µm
particle size)
A: DI water + 10 mM NH4formate
B: 90:10 acetonitrile: DI water
+ 10 mM NH4-formate
Gradient elution:
0-5 min: 90% B
5-9 min: 50% B
9-10 min: 100% B.
Flow rate: 1 mL/min
HPLC-UV: UV at 384 nm.
LC-MS: MSD-TOF with
dual
sprayer
ESI,
operated in negative ion
mode
Young et al.
(2011)
Fruit and
vegetables
Thiamine
Riboflavin
Niacinamide
Pyridoxine
Folic acid
Material cut into small
pieces
and
heat
extracted in 0.1 M HCl
and filtered through 0.45
µm filter
Supelco
Discovery
C18 column (25 cm X
0.45 internal diameter).
Isocratic elution with 50 nM
phosphate
and
methanol
(70:30) at 1 mL flow rate with
10 µL injection volume
UV at 254 nm
Ismail et al.
(2013).
Fruit juice, infant
formula, peas,
pears
Total vitamin C as
ascorbic acid
Fruit juice: Dilute with
water, add 1 mg DTT.
Hold for more than 2 h,
filter (0.45 µm).
Fruit and
processed
food: Homogenise with
water add 1 mg DDT,
filter after 2 h. Add
5%TCA
to
protein
containing samples filter
or centrifuge. Filter again
after 2 h
Polymeric silica-based
C18 (250 X 4 mm, 5
µm)
Isocratic: 0.1% DTT in 0.5%
potassium phosphate buffer,
pH 2.5. 0.5–1.0 mL/min
UV at 254 nm
Brause, et al.
(2003)
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Fruit juices,
packed
Ascorbic acid
5 mL juice + 5 mL mobile
phase (20 mM K2PO4 /
MeOH), Centrifuge at
5000 rpm for 5 min, filter
through 0.45 µm PVDF
Millipore filters
ODS-3 C18 column
Flow rate at 1 mL/min. Mobile
phase: 20% MeOH + 80%
buffer
UV-Vis detector (Hitachi),
wavelength at 240 nm.
Shafqat et al.
(2012)
Green, leafy
vegetables
Thiamine
Riboflavin
Nicotinamide
Pantothenic acid
Pyridoxine
Folic acid
Ascorbic acid
(and fat-soluble
vitamins)
0.25 g freeze-dried and
milled
sample
was
extracted with 16 mL 10
mM
ammonium
acetate/methanol (50:50
v/v) containing
0.1%
BHT, shaken and then
sonicated for 15 min.
o
below
25 C.
The
centrifuged at 14 000 g
for
15
min
and
supernatant
passed
through a 0.45 µm nylon
filter. 1 mL supernatant
was concentrated in a
nitrogen stream and then
injected to determine
water-soluble vitamins
ACE-100 C18 column
(100 X 2.1 mm, 3 µm
particle size)
HPLC-MS/MS
(via
ESI
interface). Injection volume =
10 µL and flow rate was 0.2
mL/min. Gradient extraction
using 3 buffers viz. 10mM
ammonium acetate (pH 4.5),
methanol containing 0.1%
acetic acid and methanol with
0.3% acetic acid
DAD recording spectra
from 200 to 680 nm
Santos et al.
(2012)
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Green vegetables
Folates
To 1-2 g ground sample,
10 mL MES buffer
added. I.S. mix contained
four deuterated vitamers.
Samples boiled for 10
o
min at 100 C, cooled,
Chicken pancreas + Rat
serum
conjugase
preparations added and
incubated overnight at
o
37 C
Deconjugated samples
heated for 10 min at
o
100 C,
cooled
and
centrifuged.
Purification performed on
SAX SPE cartridges
Phenomenex
Hyper
Clone
BDS
C18
column (150 X 3.2
mm, 3 µm particle
size)
A: 10 mL/L acetic acid; B:
Acetonitrile, acidified with 10
mL/L acetic acid.
Gradient elution:
0-2 min: 2% B; 98% A
2-7 min: 10% B; 90% A
7-10 min: 10% B; 90% A
10-18 min: 15% B; 85% A
18-20 min: 100% B
20-21 min: 100% B
22-23 min: 2% B; 98% A
Flow rate: 0.2 mL/min
HPLC coupled with triple
quadrupole MS, operated
in positive ESI mode using
MRM
Delchier et al.
(2013)
Green tea
extracts with
folate
Folate as 5CH3THF
Extracts + folate added
to 0.04 M BrittonRobinson buffer at pH
5.5 in water
Agilent Zorbax SB-C18
(150 X 4.8 mm, 1.8 µm
particle size)
Linear gradient elution (details
not published), A: 0.2% formic
acid in water; B: 0.1% formic
acid in acetonitrile
Flow rate 1 mL/min
HPLC-DAD at 230 nm.
LC-MS Single quadrupole
MS with API-ES source (in
positive ion mode)
Rozoy et al.
(2013)
Honey
Ascorbic acid
Thiamine
Nicotinamide
Nicotinic acid
Pantothenic acid
Pyridoxine
Riboflavin
Various – see original
article
µBondapack C18 10
µm(10 X 3.9 mm), one
of which was saturated
in CTAB (hexadecyl
trimethyl-ammonium
bromide)
Second version with
surfactant
Many permutations of gradient
elution
Programmable
UV/Vis
detector and fluorescence
detector
Leόn-Ruiz. Et
al. (2013)
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Honey
Riboflavin
Nicotinic acid
Pantothenic acid
Folic acid
Ascorbic acid
10
mL
honey
homgenized in 10 mL
water to which added 1
mL 2 M NaOH and 12.5
mL phosphate buffer (pH
5.5). Filtered through
0.45
µm
PVDF
membrane and stored.
Reversed
column
phase
Multi-step gradient elution
using TFA (0.025%) and
acetonitrile
UV detection: 254 nm for
nicotinic acid and 210 nm
for the rest
Ciulu et
(2011)
Malt
Thiamine
Riboflavin
Milled samples (0.5 g)
were vortexed in 5 mL
0.5 M
trichloroacetic
acid, agitated for 15 min,
centrifuged at 2 000g
and the supernatant
removed to a clean tune.
1 mL of 10 mM
phosphate was added
and the pH adjusted to
6.5 – 6.6 with 2 M KOH,
diluted to 10 mL with
buffer
and
filtered
through
0.45
µm
regenerated
cellulose
filter.
For
Riboflavin,
samples were injected at
this stage. For thiamine,
300
µL
alkaline
potassium
ferricyanide
was added to 1 mL
degassed
sample,
vortexed for 15 seconds
and neutralized with 600
µL 1.33 M phosphoric
acid and filtered through
0.45 µm regenerated
cellulose filter
Varian Pursuit C18
(250 mm X 4.6 mm)
with
C18
guard
cartridge (4 X 3.0 mm)
Gradient elution with A: 10 mM
phosphate,
pH
6.5;
B:
methanol.
Gradient
programme: 0–0.5 min with
A:B = 95:5, 0.5–10 min at
63:35, 10–15 min at 63:35,
15–16 at 95:5
Fluorescence
detection
with excitation emission
being 360/425 nm for
thiamine and 270/516 nm
for riboflavin
Hucker et al.
(2012)
al.
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Meat (cooked
sausages)
Ascorbic acid
Homogenise with 20 mM
monosodium
Lglutamate, pH 2.1; filter
through 0.45 µm
Spherisorb NH2 (250
X 4.0 mm), 5 µm
o
(Teknokroma) at 35 C
Isocratic: Acetonitrile + 20mM
phosphate buffer, pH 3.6
(60+40, v/v) at 1 mL/min
UV absorbance, 248 nm
Valls et
(2002)
Meat products
(salami,
sausages, bacon,
ham)
Vitamin B12
To 30 g sample 50 mL
mM Na-acetate buffer
(pH 4.0), 2 g pepsin, 0.5
g α-amylase and 2 mL
KCN
(1%)
added.
Homogenisation
with
Polytron, incubated for
o
30 min at 37 C, then
heated for further 30 min
o
at 100 C. After cooling,
centrifugation
and
filtration, sample loaded
onto
immunoaffinity
column.
Vitamin
B12
eluted with methanol.
Eluate is concentrated to
o
dryness at 60 C, then
reconstituted to 300 µL
with mobile phase
Nucleosil RP C18 HD
100 column (250 X 4.6
mm, 5 µm particle
size)
Water/acetonitrile (87:13) with
0.025% TFA (isocratic elution)
Flow rate: 1.2 mL/min
UV at 361 nm
Guggisberg et
al. (2012)
Milk, human
Total vitamin C as
AA, DHAA by
difference
Total vitamin C: Reduce
DHAA to AA with 100
mM DTT, keep in the
dark for 15 min, add
0.56% MPA, centrifuge
o
at 10 C.
Ascorbic acid: Mix 0.56%
o
MPA, centrifuge at 10 C.
Injection volume = 50 μL
Precolumn:
Tracer
C18, 5 μm (Tracer
Analytica).
Analytical:
Tracer
Spherisorb ODS C18
(250 x 4.6 mm, 5 μm,
Tracer
Analytica),
o
25 C
Isocratic:
Methanol + 0.1% acetic acid (5
+ 95, v/v)
0.7 mL/min
UV at 254 nm
Romeu-Nadal
et al. (2006)
al.,
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Milk
Thiamine
Riboflavin
Niacin
Pyridoxine
Folic acid
Vitamin extraction by
adding 0.1 M HCl and
digesting while mixing in
boiling batch, cooling,
adjusting pH to 4.0 – 4.5
with 2.5 M sodium
acetate. Digestion with
10% taka-diastase for 3
o
hrs at 45 – 50 C, mixing
every 30 min. Cooled,
filtered, diluted and then
passed through 0.2 µm
filter
Zorbax SB-C8 (4.6 X
150 mm, 5 nm)
column) at ambient
temperature
Gradient elution at 1.2 mL/min
using (A) 50 mM phosphate
buffer in water and (B) 50 mM
phosphate buffer in methanol
(10:90). Started run on A and
increased B from zero to 70%
within 18 min
UV at 245 nm
Khair-un-Nisa
et al. (2010)
Milk powder SRM
1846, milk
powder, infant
formula,
breakfast cereal,
orange juice,
fortified soup
Total vitamin C as
ascorbic acid
Sample with no starch:
Mix with TCEP solution.
Dilute with 1%TCA, filter
and dilute with mobile
phase. Samples with
starch: Mix with 10mg
takadiastate,
incubate
o
(42 C) for 30 min, add
1%TCA and filter. Dilute
with mobile phase
Analytical
column:
LiChrospher
RP-18
(250 X 4.6 mm, 5 µm)
Isocratic: Acetonitrile + 25 mM
acetate-phosphate buffer, pH
5.4, containing 10.17 mM
deccylamine and 0.17 mM
TCEP at 1 mL/min
UV at 265 nm
Fontannaz et
al. (2006)
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Milk-based infant
formula
(powdered),
infant cereals
with milk and
fruit, infant
cereals without
milk, milk-based
infant formula,
hypoallergenic
infant formula,
milk powder SRM
1846, meat SRM
1546, milk
powder CRM 421
Total and free
vitamin B12 as
CNCbl
Free vitamin B12: 50 mM
Na-acetate buffer, pH 4.0
+ 0.016% NaCN + αamylase; digest for 30
o
min at 37 C; cool, adjust
pH to 4.8; reflux under N2
o
for 35 min at 100 C to
convert all vitamers to
CNCbl; Cool, dilute and
filter.
Total vitamin B12: 50 mM
Na-acetate buffer, pH 4.0
+ 0.016% NaCN + αamylase + pepsin; digest
o
for 3 h at 37 C; cool,
adjust pH to 4.8; reflux
under N2 for 35 min at
o
100 C to convert all
vitamers to CNCbl; Cool,
dilute and filter
Ace 3 AQ C18 (150 mm
X 3.9 mm), 3 µm
A: Acetonitrile
B: 0.025% aqueous TFA
0-3.5 min: 0% A + 100%
(isocratic)
3.5-11 min: 25% A + 75%
(linear gradient)
11-19 min: 35% A + 65%
(linear gradient)
19-20 min: 10% A + 90%
(linear gradient)
20-26 min: 0% A + 100%
(linear gradient)
26-30 min: 0% A + 100%
(isocratic)
Flow rate 0.25 mL/min
UV absorbance: 361 nm
(DAD)
Heudi et al.
(2006)
B
B
B
B
B
B
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Milk- and soybased infant
formula, cereals,
cocoa beverages
Total and free
vitamin B12 as
CNCbl
All extracts (Total and
free vitamin B12): 1%
NaCN
+
α-amylase;
incubate for 30 min at 40
o
± 5 C; add 20 mM Naacetate, pH 4; hold for 35
o
min at 100 C to convert
all vitamers to CNCbl;
Cool, dilute and filter;
clean up filtrate on
commercial
B12
immunoaffinity column,
elute
CNCbl
with
methanol;
evaporate
eluate
under
N2;
reconstitute residue in
0.025%
TFA
in
acetonitrile + 0.025%
aqueous TFA (10 + 90
v/v)
Work under subdued
light in low actinic glass
ware
Nucleosil 100-3 C18
HD (150 mm X 3.9
mm), 3 µm, MacheryNagel or Ace 3 AQ C18
(150 mm X 3.9 mm), 3
µm, Ace
A: 0.025% TFA in acetonitrile
B: 0.025% aqueous TFA
0 min: 10% A + 90% B
(isocratic)
0-0.5 min: 10% A + 90% B
(isocratic)
0.5-4 min: 25% A + 75% B
(linear gradient)
4-5 min: 90% A + 10% B
(linear gradient)
5-9 min: 90% A + 10% B
(isocratic)
9-11 min: 10% A + 90% B
(linear gradient)
11-16 min: 10% A + 90% B
(isocratic)
Flow rate 0.25 mL/min
UV absorbance: 361 nm
(DAD)
CamposGiménez
al. (2008)
Noodles
Riboflavin
6
g
noodles
homogenized in 30 mL
1.1 M H2SO4 for 2 min
and autoclaved for 15
o
min at 121 C and then
cooled and pH adjusted
to 4.5 using sodium
acetate.
Riboflavin was extracted
using Clara-diastase for
o
90 min at 45 C acidified
using H2SO4, made to
volume, filtered through
paper and then through
1.2 µm membrane
Spherisorb ODS 2 C18
Column (250 X 4.6
mm, 0.5 µm) with
matching
guard
cartridge
Methanol-water (15:85) with
water
containing
1hexsulphonic acid (0.005 M),
glacial acetic acid (2.4%) and
triethylamine
(0.5%).
o
Temperature = 30 C. Flow rate
= 1 mL/min. Injection volume =
25 µL
UV detector at 268 nm
and 35 min run time
Bui & Small
(2009)
et
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Rice
Folic acid (FA)
THF
+
5,10-CH THF
5-CHOTHF
10-CHOFA
50 mM phosphate buffer
+ 1% ascorbic acid +
0.5% dithiothreitol, pH +
IS. Added to 1 g rice,
homogenised,
boiled,
cooled. 1.5 mL aliquot +
10 µL α-amylase; 10 min
at RT. 150 µL protease.
o
Incubation at 37 C for 1
h; 10 min boiling to
inactivate; 100 µL rat
o
serum, incubated at 37 C
for 2 h. Boiling to
inactivate
enzyme,
centrifugation
and
ultrafiltration (12 000x g
for 30 min on 5 kDa
Millipore filter). Subdued
light throughout
Polaris 3 µm C18-A
column (150 X 4.6
mm)
A: (0.1% formic acid in water);
B: (0.1% formic acid in
Acetonitrile (acetonitrile).
0-2.5 min: 95% A (isocratic);
2.5-3,5 min 85% A, 15% B;
3.5-5.5 min 75% A, 25% B;
5.5-10.5 min 100% B
Flow rate 0.8 mL/min
Applied Biosystems API
4000 tandem quadrupole
MS operated in positive
ion ESI mode.
Detection carried out in
MRM mode
De Brouwer et
al. (2008)
Rice bran powder
Thiamine
Riboflavin
Niacin
Pyridoxine
Cobalamin
Extraction
heated
ultrasonic
system
in
water and then passed
through 0.45 µm filter
Reversed phase C18
column (YMC, 250 mm
X 10 mm)
Flow rate = 1 mL/min. Injection
volume = 20 µL. Gradient
elution
using
A:
99.8%
methanol and B: 0.2% acetic
acid in water. Ratio change
from 0:100 to 25:75 in 20 min
with flow rate change from 1 to
0.7 mL/min at 8 min
UV at 260nm.
Chen et
(2011)
al.
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Tea, nutritional
supplement,
dried Lycium
barbarum fruit
Total vitamin C as
AA in the presence
of related
compounds
Dried fruit: Homogenize
fruit with 200 mg/L DTT
in acetonitrile + water (30
+ 70, v/v) to reduce
DHAA to AA; centrifuge;
dilute supernatant with
200 mg/L DTT in
acetonitrile + 66.7 mM
ammonium acetate (85 +
15, v/v)
Other samples: Dilute
with 200 mg/L DTT in
acetonitrile + 66.7 mM
ammonium acetate (85 +
15, v/v) to reduce DHAA
to AA
Keep samples at ≤4°C
during extraction.
Autosampler at 4°C
Injection volume = 10 μL
Analytical:
Inertsil Diol (250 X 4.6
mm, 5 μm; GL
Sciences); 40°C
Isocratic:
Acetonitrile + 66.7 mM
ammonium acetate (85 + 15,
v/v)
Flow rate: 0.7 mL/min
UV absorbance: 260 nm
External standardization
Linearity: 1–50 μg/mL AA
(r = 0.9996)
Precision: Intra-day CV =
1.0–2.8% for AA in
standards and control
samples (n = 6); inter-day
CV = 1.0–2.0% for AA in
standards and control
samples (n = 3)
Accuracy: 92% AA
recovery from standard
solutions and control
samples
LoD = 300 ng/mL AA
Tai & Gohda
(2007)
Various Matrices
(e.g. fruits and
vegetables, dry
foods etc.)
Ascorbic acid
2 g sample + 8 mL
extraction buffer (5%
MPA/1 mM EDTA/5 mM
TCEP, pH 1.8) mix and
centrifuge. Supernatant
filtered through 0.45 µm
PVDF membrane
Phenomenex Synergi
4 µm Hydro-RP (250 X
4.6 mm)
Mobile Phase: 0.05% (w/v)
aqueous formic acid or 0.02%
aqueous ortho-phosphoric acid
(w/v). Flow rate at 0.4 mL/min,
0.7 mL/min or 1 mL/min
DAD
(Perkin
Elmer),
wavelength at 254 nm
Tarrago-Trani
et al. (2012)
Wide range of
food matrices
where
polyphenols may
or may not occur
Thiamine
Various – see original
article
L-column ODS (250 X
4.6 mm i.d., 5 µm
particle
size).
o
Temperature = 40 C
Injection volume = 20 µL. Flow
rate = 1 mL/min. Isocratic
elution
for
post-column
derivitization. Mobile phase =
[0.01 M sodium phosphate,
015 M NaClO4 buffer pH 2.2]methanol = 95:5 v/v. Reaction
reagent = 0.91 M K3Fe(CN)63.75 M NaOH
Fluorescence
detection
excitation wavelength =
375 nm and emission
wavelength = 440nm
Yashida et al.
(2012)
Matrix
Analyte
Non-food matrices
Ampoules
Thiamine
Pyridoxine
Lidocaine
Extraction
Ampoule contents
diluted to 100 mL
with 0.1 mL 0.1 M
shaken for 10 min
diluted to range
Column
and
with
HCl,
and
Elution
Detection
Ref.
UPLC: Acuity UPLC
BEH C18 column (50
X 2.1 mm i.d., 1.7 µm
particle size
®
Gradient elution with methanol
and HCl at 0.6 mL/min
UV detection at 220 nm
and data treatment
Dinç (2012)
Human serum
Homocysteine
Vitamin B12
Vitamin B6
Vitamin B9
Not included
LiChrocart
cartridge (150
4.6
mm
diameter)
Purospher
endcapped
(both 5 µm)
C18
mm X
internal
with
RP-18
column
Methanol
and
1-heptane
sulphonic acid sodium salt
(33:67) + 0.5% triethylamine
(pH 2.3). Flow rate 0.5 mL.min.
o
Column temperature 28 C
DAD at 210 nm
Shaik & Gan,
(2013)
Vitamin mixture
Thiamine
Riboflavin
Pyridoxine
Folic acid
Cobalamin
Biotin
Dissolved in methanol
and passed through 0.2
µm PTFE filter
RP-WAX Column (125
X 4 mm, 5 µm particle
size)
Injection volume = 10 µL. Flow
rate = 1 mL/min
IUV at 270 nm
Dabre et al.
(2011)
Vitamin/Mineral
tablets
Ascorbic acid
Thiamine (B1)
Niacinamide (B3)
Pantothenic acid
(B5)
Pyridoxine (B6)
Riboflavin (B2)
Folic acid (B9)
Powdered
tablets
suspended in diluting
solutions as per article,
sonicate for 25 min at
o
25 C and filter through
0.2 µm PTFE membrane
Alltima C18 column
(250 mm X 4.6 mm
i.d., 5 µm, ambient
temperature
Injection volume 20 µL. Mobile
phase 50 mM ammonium
dihydrogen phosphate and
acetonitrile delivered at 0.5
mL/min. Set at 95:5 for or
85:15 depending on vitamers
being investigated
DAD with 275 nm or 280
nm depending on vitamers
Jin
et
(2012)
al.
Matrix
Analyte
Extraction
Column
Elution
Detection
Ref.
Vitamin capsules
(special study of
design space)
Thiamine
Riboflavin
Pantothenic acid
Pyridoxine
Cobalamin
Capsule
content
dissolved in 25 mL
25mM phosphate buffer
(pH 2.6) and filtered
through 0.45 µm filter
Hypersil Gold C18
column (250 X 4.6
mm, 3 µm)
Methanol (100%)
UV at 254 nm
Wagdy et al.
(2013)
AA = L-Ascorbic Acid
DAD: Diode array detector
DTT = 1, 4-dithiothreitel
EDTA =ethylenediaminetetraacetate
ESI: Electrospray Ionization
I.S. = Internal standard
MPA = metaphosphoric acid
MS: Mass Spectrometer
SPE: Solid phase extraction
TOF: Time of Flight
TFA: Trifluoroacetic acid
TCA =trichloroacetic acid
TCEP = Tris[2-carboxyethyl]phosphine
U(H)PLC: Ultra Performance Liquid Chromatography
REFERENCES
Akyilmaz, E. & Dinçkaya, E. (1999). A new enzyme electrode based on ascorbate oxidase
immobilized in gelatin for specific determination of L-ascorbic acid. Talanta, 50, 87–
93.
Akyilmaz, E. Yasa, I & Dinçkaya, E. (2006). Whole cell immobilized amperometric
biosensor based on Saccharomyces cerevisiae for selective determination of vitamin B1
(thiamine). Analytical Biochemistry, 354, 78–84.
Alaburda, J. De Almeida, A. P. Shundo, L. Ruveiri, V. & Sabino, M. (2008). Determination
of folic acid in fortified wheat flours. Journal of Food Composition and Analysis, 21,
336–342.
Alegría, A. Barberá, R., Lagarda, M. J. & Farré, R. (2008). Vitamins. In L. M. L. Nollet & F.
Toldrá (Eds.), Handbook of Processed Meats and Poultry Analysis, (1st edition, pp.
291–326). Boca Raton, Forida, USA: CRC Press (Taylor & Francis Group, LLC).
Anonymous. (1993). AOAC International, Report on the AOAC International Task Force on
Methods for Nutrient Labeling Analyses, Journal of the Association of Official
Analytical Chemists International, 76: 180A.
Anonymous. Method 967.21. AOAC, Official Methods of Analyis. (16th ed., 5th Rev.)
Gaithersburg, MD: AOAC, 1999.
Anonymous. (2003). Regulations relating to the fortification of certain foodstuffs, GNR
2003/2003. Foodstuffs, Cosmetics and Disinfectants Act and Regulations. Act no. 54 of
1972. Department of Health. Johannesburg, South Africa: Lex Patria Publishers.
Anonymous. (2012). AOAC SMPR 2011.006. Standard method performance requirements
for folate in infant formula and adult/pediatric nutritional formula. Appendix 1: AOAC
SMPR 2011.006: Journal of AOAC International, 95, 1.
Arcot, J. & Shrestha, A (2005). Folate: methods of analysis. Trends in Food Science &
Technology, 16, 253–266.
Aslam, J. Khan, S. H. & Khan, S. A. (2013). Quantification of water soluble vitamins in six
date palm (Phoenix dactylifera L.) cultivar’s fruits growing in Dubai, United Arab
Emirates, though high performance liquid chromatography. Journal of Saudi Chemical
Society, 17, 9–16.
Bailey, L. B. (1988). Factors affecting folate bioavailability. Food Technology, 42, 206–210,
212, 238.
Bailey, L. B. (2007). Folic acid. In J. Zempleni, R. B. Rucker, D. B. McCormick & J. W.
Suttie (Eds.), Handbook of Vitamins, (4th edition, pp. 385–413), Boca Raton, Forida,
USA:
CRC
Press
(Taylor
&
Francis
Group,
LLC.
http://www.foodnetbase.com/books/5534/4022_C012.pdf (accessed November 15,
2012).
Bailey, L. B. Duhaney, R. L. Maneval, D. R. Kauwell, G. P. A. Quinlivan, E. P., Davis, S. R.
Cuadras, A. Hutson, A. D. & Gregory, J. F. (2002). Vitamin B12 status is inversely
associated with plasma homocysteine in young woman with C677T and/or A1298C
methylenetetrahydrofolate reductase polymorphisms. Journal of Nutrition, 132, 1872–
1878.
Ball, G. F. M. Vitamins in Foods, Analysis, Bioavailability and Stability. Boca Raton,
Florida, USA: CRC Press (Taylor & Francis Group, LLC), 2006.
Basu, T. K. & Dickerson, J. W. T. Vitamins in Human Health and Disease. (Pp. 125–147).
Oxford, UK: Cab International, 1996.
Berry, R. J. Mulinare, J. Hamner, H. C. (2010). Folate acid fortification. Neural tube defect
reduction – A global perspective. In L. B. Bailey (Ed.), Folate in Health and Disease,
(2nd edition, pp. 179–204). Boca Raton, Forida, USA: CRC Press (Taylor & Francis
Group, LLC).
Belitz, H-D. Grosch, W. Food Chemistry. (2nd Edition, Pp. 383–387). Berlin, New York,
London, Tokyo: Springer, 1999.
Bhagavan, N.V. Medical Biochemistry. (4th edition). Amsterdam, The Netherlands: Elsevier,
2001.
Blake, C. J. (2007). Analytical procedures for water-soluble vitamins in foods and dietary
supplements: a review. Analytical and Bioanalytical Chemistry, 389, 63–76.
Blanche, F. Thibaut, D. Couder, M. & Muller, J.-C. (1990). Identification and quantitation of
corrinoid precursors of cobalamin from Pseudomonas denitrificans by HighPerformance Liquid Chromatography. Analytical Biochemistry, 189, 24–29.
Bognár, A. & Daood, H. G. (2000). Simple in-line postcolumn oxidation and derivatization
for the simultaneous analysis of ascorbic and dehydroascorbic acids in foods. Journal
of Chromatographic Science A, 38, 162–168.
Bottiglieri, T. & Reynolds, E. (2010). Folate and neurological disease. Basic mechanisms. In
L. B. Bailey (Ed.), Folate in Health and Disease, (2nd edition, pp. 355–380). Boca
Raton, Forida, USA: CRC Press (Taylor & Francis Group, LLC).
Boyaci, B.B. Han, J. Masatcioglu, M.T.,Yalcin, E. Celik, S. Ryu, G. & Koksel, H. (2012).
Effects of cold extrusion process on thiamine and riboflavin contents of fortified corn
extrudates. Food Chemistry, 132, 2165–2170.
Brause, A. R. Woollard, D. C. & Indyk, H. E. (2003). Determination of total vitamin C in
fruit juices and related products by liquid chromatography: Interlaboratory study.
Journal of the Association of Official Analytical Chemists International, 86, 367–374.
Brody, T. Nutritional Biochemistry. (Pp. 450–459). San Diego, CA: Academic Press, 1994.
Brouns, R. Ursem, N. Lindemans, J. Hop, W. Pluijm, S. Steegers, E. & Steegers-Theunissen,
R. (2008). Polymorphisms in genes related to folate and cobalamin metabolism and the
associations with complex birth defects. Prenatal Diagnosis, 28, 485–493.
Bucholtz, M. Drotleff, A. M. & Ternes, W. (2012). Thiamin (vitamin B1) and thiamin
phosphate esters in five cereal grains during maturation. Journal of Cereal Science, 56,
109–114.
Bui, L. T. T. & Small, D. M. (2007). Folate in Asian noodles: I. Microbiological analysis and
the use of enzymes. Journal of Food Science, 72, C276–C282.
Bui, L. T. T. & Small, D. M. (2009). Riboflavin in Asian noodles: The impact of processing,
storage and the efficacy of fortification of three product styles. Food Chemistry, 114,
1477–1483.
Campos-Giménez, E. Fontannaz, P. Trisconi, M-J. Kilinc, T. Gimenez, C. & Andrieux, P.
(2008). Determination of Vitamin B12 in food products by Liquid Chromatography/UV
detection with Immunoaffinity extraction: Single laboratory validation Journal of
AOAC International, 91, 786–793.
Cathcart, R. F. (1991). A unique function for ascorbate. Medical Hypotheses, 35, 32–37.
Caudill, M. (2004). The role of folate in reducing chronic and developmental disease risk: an
overview. Journal of Food Science, 69, SNQ55–SNQ58.
Celik, M. Barkut, I. K. Öncel, C. & Forta, H. (2003). Involuntary movements associated with
vitamin B12 deficiency, Parkinsonism Relative Disorders, 10, 55–57.
Chandra, T. & Brown, K. L. (2008). Vitamin B12 and α-ribonucleosides. Tetrahedron, 64, 9–
38.
Chandra-Hioe, M. V. Bucknall, M. P. & Arcot, J. Folate analysis in foods by UPLC-MS/MS:
development and validation of a novel, high throughput quantitative assay; folate levels
determined in fortified Australian breads. Annals of Bionanalytical Chemistry, 401,
1035–1042.
Chen, P. Atkinson, R. & Wolf, W. R. (2009). Single-laboratory validation of a highperformance liquid chromatographic diode array detector-fluorescence detector/mass
spectrometric method for simultaneous determination of water-soluble vitamins in
multivitamin dietary tablets. Journal of the Association of Official Analytical Chemists
International, 92, 680–687.
Chen, C-H. Yang, Y-H, Shen, C-T, Lai, S-M, Chang, C-M. J. & Shieh, C-J. (2011). Recovery
of vitamins B from supercritical carbon dioxide-defatted rice bran powder using
ultrasound water extraction. Journal of the Taiwan Institute of Chemical Engineers, 42,
124–128.
Cheung, R. H. F. Hughes, J. G. Marriott, P. J. & Small, D. M. (2009). Investigation of folic
acid stability in fortified Asian noodles. Food Chemistry, 112, 507–514.
Ciulu, M. Solinas, S. Floris, I. Panzanelli, A. Pilo, M. I. Piu, P. C. Spano, N. & Sanna, G.
(2011). RP_HPLC determination of water-soluble vitamins in honey. Talanta, 83, 924–
929.
Combs Jr., G. F. The Vitamins: Fundamental Aspects in Nutrition and Health. (Pp. 4–6, 24–
25 and 223–249). San Diego, CA: Academic Press, 1992.
Dabre, R. Azad, N. Schwämmle, A. Lämmerhofer, M. & Lindner, W. (2011). Simultaneous
separation and analysis of water- and fat-soluble vitamins on multi-modal reversedphase weak anion exchange material by HPLC-UV. Journal of Separation Science, 34,
761–772.
Dalbacke, J. & Dahlquist, I. (1991). Determination of vitamin B12 in multivitaminmultimineral tablets by high-performance liquid chromatography after solid-phase
extraction. Journal of Chromatography, 541, 383–392.
Da Silva, D. C. Visentainer, J. V. De Souza, N. E. & Oliviera, C. C. (2013). Micellar
electrokinetic chromatography method for determination of the ten water-soluble
vitamins in food supplements. Food Analytical Methods, Published on-line: 01 March
2013 (doi: 10.1007/s12161-013), http://www.springerlink.de/content/120914 (accessed
March 15, 2013).
Davey, M. W. Van Montagu, M. Inzé, D. Sanmartin, M. Kanellis, A. & Smirnoff, N. (2000).
Plant L-ascorbic acid: Chemistry, function, metabolism, bioavailability and effects of
processing. Journal of the Science of Food and Agriculture, 80, 825–860.
De Arruda, V. A. S. Pereira, A. A. S. de Freitas, A. S. Barth, O. M. & Almeida-Muradian, L.
B. (2013). Dried bee pollen: B complex vitamins, physicochemical and botanical
composition. Journal of Food Composition and Analysis, 29, 100–105.
De Brouwer, V. Storozhenko, S. Van De Steene, J. C. Wille, S. M. R. Stove, C. P. Van Der
Straeten, D. & Lambert, W. E. (2008). Optimisation and validation of a liquid
chromatography-tandem mass spectrometry method for folates in rice. Journal of
Chromatography A, 1215, 125–132.
Deeth, H. C. & Tamime, A. Y. (1981). Yoghurt: nutritive and therapeutic aspects. Journal of
Food Protection, 44, 78–86.
Delchier, N. Ringling, C. Le Grandois, J. Aoudé-Werner, D. Galland, R. Georgé, S. Rychlik,
M. & Renard, C. M. G. C. (2013). Effects of industrial processing on folate content in
green vegetables. Food Chemistry, 139, 815–824.
De Souza, S. Eitenmiller, R. (1990). Effects of different enzyme treatments on extraction of
various folates from foods prior to microbiological assay and radioassay. Journal of
Micronutrient Analysis, 7, 37–57.
De Wals, P. Tairou, F. Van Allen, M. I. Uh, S. Lowry, R. B. Sibbald, B. Evans, J. A. Van der
Hof, M. C. Zimmer, P. Crowley, M. Fernandez, B. Lee, N. S. & Niyonsenga, T. (2007).
Reduction in neural-tube defects after folic acid fortification in Canada. New England
Journal of Medicine, 357, 135–142.
Dinç, E. (2012). Fractional-Continuous Wavelet Transforms and Ultra-Performance Liquid
Chromatography for the Multicomponent Analysis of a Ternary Mixture Containing
Thiamine, Pyridoxine, and Lidocaine in Ampules. Journal of AOAC International, 95,
903–912.
Di Stefano, V. Avellone, G. Bongiorno, D. Cunsolo, V. Muccilli, V. Sforza, S. Dossena, A,
Drahos, L. & Vékey, K. (2012). Applications of liquid chromatography-mass
spectrometry for food analysis. Journal of Chromatography A, 1259, 74–85.
Doherty, R. F. & Beecher, G. R. (2003). A method for the analysis of natural and synthetic
folate in foods. Journal of Agricultural and Food Chemistry, 51, 354–361.
Doner, L. W. & Hicks, K.B. (1981). High-performance liquid chromatographic separation of
ascorbic acid, erythorbic acid, dehydroascorbic acid, dehydroerythorbic acid,
diketogulonic acid, and diketogluconic acid. Analytical Biochemistry, 115, 225–230.
Edelmann, M. Kariluoto, S. Nystrӧm, L. & Piironen, V. Folate in oats and its milling
fractions. Food Chemistry, 135, 1938–1947.
Eitenmiller, R. R. & Landen, W. O. Folate. Vitamin Analysis for the Health and Food
Sciences. eBook ISBN: 978-1-4200-5016-5, Boca Raton, Florida, USA: CRC Press
LLC,
1999.
http://www.crcnetbase.com/doi/pdf/10.1201/9781420050165.ch11
(accessed 5 December 2012).
Eitenmiller, R. R. & Landen, W. O. Vitamin B12. Vitamin Analysis for the Health and Food
Sciences. 2nd ed., pp. 507–534, Boca Raton, Florida, USA: CRC Press LLC, 2008.
http://www.crcnetbase.com/doi/pdf/10.1201/9781420009750.ch11
December 2012).
(accessed
5
Eitenmiller, R. R. & Landen, W. O. Vitamin C. Vitamin Analysis for the Health and Food
Sciences. 2nd ed., pp. 507–534, Boca Raton, Florida, USA: CRC Press LLC, 2008.
http://www.crcnetbase.com/doi/pdf/10.1201/9781420009750.ch5 (accessed 15 January
2013).
Englberger, L. Lyons, G. Foley, W. Daniells, J. Aalbersberg, B. Dolodolotawake, U. Watoto,
C. Iramu, E. Taki, B. Wehi, F. Warito, P. & Taylor, M. (2010). Carotenoid and
riboflavin content of banana cultivars from Marika, Solomon Islands. Journal of Food
Composition and Analysis, 23, 624–632.
Erkan, N. Selçuk, A. & Özden, Ö. (2010). Amino acid and vitamin composition of raw and
cooked horse mackerel. Food Analytical Methods, 3, 269–275.
Esroy, B. & Özeren, A. (2009). The effect of cooking on mineral and vitamin contents of
African catfish. Food Chemistry, 115, 419–422.
Farah, F. Talpur, F. N. & Memon, A.N. (2013). Determination of water soluble vitamin in
fruits and vegetables marketed in Sindh Pakistan. Pakistan Journal of Nutrition, 12,
197–199.
Finglas, P. M. Wigertz, K. Vahteristo, L. Witthӧft, C. Southon, S. & de Froidmont-Gӧrtz, I.
(1999). Standardisation of HPLC techniques for the determination of naturallyoccurring folates in food. Food Chemistry, 64, 245–255.
Fontannaz, P. Kilinc, T. & Heudi, O. (2006). HPLC-UV determination of total vitamin C in a
wide range of fortified food products. Food Chemistry, 94, 626–631.
Freisleben, A. Schieberle, P. & Rychlik, M. (2003). Comparison of folate quantification in
foods by high-performance liquid chromatography – fluorescence detection to that by
stable isotope dilution assays using high-performance liquid chromatography – tandem
mass spectrometry. Analytical Biochemistry, 315, 247–255.
Gao, Y. Fuo, F. Gokavi, S. Chow, A. Sheng, Q. & Guo, M. (2008). Quantification of watersoluble vitamins in milk-based infant formulae using biosensor-based assays. Food
Chemistry, 110, 769–776.
Galgano, E. Favati, E. Lapelosa, L. Albanese, D. & Montanari, L. (2002). Effect of chilling
on the vitamin C content of fennel during storage. Italian Journal of Food Science, 14,
167.
Gardner, N. & Champagne, C. P. (2005). Production of Propionibacterium shermanii
biomass and vitamin B12 on spent media. Journal of Applied Microbiology, 99, 1365–
2672.
Garrat, L. C. Ortori, C. A. Tucker, G. A. Sablitzky, F. Bennett, M. J. & Barrett, D. A. (2005).
Comprehensive metabolic profiling of mono- and polyglutamated folates and their
precursors in plant and animal tissue using liquid chromatography/negative ion
electrospray ionization tandem mass spectrometry. Rapid Communications in Mass
Spectrometry, 19, 2390–2398.
Garrido Frenich, A. Hernandez Torres, M. E. Belmonte Vega, A. Marinez Vidal, J. L. &
Plaza Bolanos, P. (2005). Determination of ascorbic acid and carotenoids in food
commodities by liquid chromatography with mass spectrometry detection. Journal of
Agricultural and Food Chemistry, 53, 7371–7376.
Gimsing, P. & Beck, W. S. (1986). Determination of cobalamins in biological material.
Methods Enzymology, 123, 3–49.
Giorgi, M. G. Howland, K. Martin C. & Bonner, A. B. (2012). A Novel HPLC Method for
the Concurrent Analysis and Quantitation of Seven Water-Soluble Vitamins in
Biological Fluids (Plasma and Urine): A Validation Study and Application. The
Scientific
World
Journal,
10.1100/2012/359721, 1–8.
Volume
2012,
Article
ID
359721,
doi:
Gliszczyńska-Świgło, A. (2006). Antioxidant activity of water soluble vitamins in the TEAC
(trolox equivalent antioxidant capacity) and the FRAP (ferric reducing antioxidant
power) assays. Food Chemistry, 96, 131–136.
Grataćos-Cubarśi, M. Sárraga, C. Clariana, M. Regueiro, J. A. & Castellari, M. (2011).
Analysis of vitamin B1 in dry-cured sausages by hydrophilic interaction liquid
chromatography (HILIC) and diode array detection. Meat Science, 87, 234–238.
Green, R. & Miller, J. W. (2007). Vitamin B12. In J. Zempleni, R. B. Rucker, J. W. Suttie &
D. B. McCormick (Eds.), Handbook of Vitamins, (4th edition, pp. 414–458), Boca
Raton,
Forida,
USA:
CRC
Press
(Taylor
&
Francis
Group,
LLC.
http://www.foodnetbase.com/books/5534/4022_C013.pdf (accessed November 15,
2012).
Gregory, J. F. (2004). Dietary folate in a changing environment: bioavailability, fortification,
and requirements. Journal of Food Science, 69, SNQ59–SNQ60.
Gounelle, J-C. Ladjumi, H. & Prognon, P. (1989). A rapid and specific extraction procedure
for folates determination in rat liver and analysis by high-performance liquid
chromatography with fluorometric detection. Analytical Biochemistry, 176, 406–411.
Groff, J. L. Gropper, S. S. & Hunt, S. M. Advanced Nutrition and Human Metabolism. 2nd
Edition. Minneapolis/St. Paul New York Los Angeles San Francisco: West Publishing
Company, 1995.
Güçlü, K. Sozgen, K. Tutem, E. Ozyurek, M. and Apak, R. (2005). Spectrophotometric
determination of ascorbic acid using copper(II)-neocuproine reagent in beverages and
pharmaceuticals, Talanta, 65, 1226–232.
Guggisberg, D. Risse, M. C. & Hadorn, R. (2012). Determination of vitamin B12 in meat
products by RP-HPLC after enrichment and purification on an immunoaffinity column.
Meat Science, 90, 279–283.
Hälvin, K. Paalme, T. & Nisamedtinov, I. (2013). Comparison of different extraction
methods for simultaneous determination of B complex vitamins in nutritional yeast
using LC-MS-TOF and stable isotope dilutions assay. Analytical and Bioanalytical
Chemistry, 405, 1213–1222.
Harper, C. (2006). Thiamine (vitamin B1) deficiency and associated brain damage is still
common throughout the world and prevention is simple and safe! European Journal of
Neurology, 13, 1078–1082.
Hefni, M. Öhrvik, V. Tabekha, M. & Witthöft. C. (2010). Folate content in foods commonly
consumed in Egypt. Food Chemistry, 121, 540–545.
Herbert, V. & Jacob, E. (1974). Destruction of vitamin B12 by ascorbic acid. Journal of
American Medical Association, 230, 241–246.
Hepburn, M. J. Dyal, K. Runser, L. A. Barfield, R. L. Hepburn, L. M. & Fraser, S. L. (2004)
Low serum vitamin B12 levels in an outpatient HIV-infected population. International
Journal of Sexually Transmitted Disease & AIDS, 15, 127–133.
Hernández, Y. Lobo, M. G. & Gonzalez, M. (2006). Determination of vitamin C in tropical
fruits: a comparative evaluation of methods. Food Chemistry, 96, 654–664.
Heudi, O. Kilinç, T. & Fontannaz, P. (2005). Separation of water-soluble vitamins by
reversed-phase high performance liquid chromatography with ultra-violet detection:
Application to polyvitaminated premixes. Journal of Chromatography A, 1170, 49–56.
Heudi, O. Kilinç, T. Fontannaz, P. & Marley, E. (2006). Determination of vitamin B12 in food
products and in premixes by reversed-phase high performance liquid chromatography
and immunoaffinity extraction. Journal of Chromatography A, 1173, 63–68.
Heudi, O. (2012). Methods for the simultaneous quantitative analysis of water-soluble
vitamins in food products. In L. M. L. Nollet & F. Toldrá (Eds.), Handbook of Analysis
of Active Compounds in Functional Foods, (1st edition, pp. 149–165). Boca Raton,
Forida, USA: CRC Press.
Hidiroglou, N. Peace, R. W. Jee, P. Leggee, D. & Kuhnlein, H. (2008). Levels of folate,
pyridoxine, niacin and riboflavin in traditional foods in Canadian Artic indigenous
peoples. Journal of Food Composition and Analysis, 21, 474–480.
Hobbs, C. A. Shaw, G. M. Werler, M. M. & Mosley, B. (2010). Folate status and birth defect
risk. Epidemiological perspective. In L. B. Bailey (Ed.), Folate in Health and Disease,
(2nd edition, pp. 133–153). Boca Raton, Forida, USA: CRC Press (Taylor & Francis
Group, LLC).
Hucker, B. Wakeling L. & Vriesekoop, F. (2102). Investigations into the thiamine and
riboflavin content of malt and the effects of malting and roasting on their final content.
Journal of Cereal Science, 56, 300–306.
Indyk, H. E. (2010). The determination of folic acid in milk and paediatric formulae by
optical biosensor assay utilising folate binding protein. International Dairy Journal, 20,
106–112.
Indyk, H. E. & Woollard, D. C. (2013). Single laboratory validation of an optical biosensor
assay method for the determination of folate in foods. Journal of Food Composition
and Analysis, 29, 87–93.
Iwase, H. (2000). Use of nucleic acids in the mobile phase for the determination of ascorbic
acid in foods by high performance liquid chromatography with electrochemical
detection, Journal of Chromatography A, 881, 327–330.
Iwase, H. & Ono, I. (1998). Determination of ascorbic acid in food by column liquid
chromatography with electrochemical detection using eluent for pre-run sample
stabilization. Journal of Chromatography A, 806, 361–364.
Iyer, R. & Tomar, S. K. (2009). Folate: a functional food constituent. Journal of Food
Science, 74, R114–R122.
Jastrebova, J. Witthӧft, C. Grahn, A. Svensson, U. & Jägerstad, M. (2003). HPLC
determination of folates in raw and processed beetroots. Analytical, Nutritional and
Clinical Methods, 80, 579–588.
Jin, P. Xia, L. Li, Z. Che, N. Zou, D. & Hu, X. (2012). Rapid determination of thiamine,
riboflavin, niacinamide, pantothenic acid, pyridoxine, folic acid and ascorbic acid in
vitamins with minerals tablets by high-performance liquid chromatography with diode
array detector. Journal of Pharmaceutical and Biomedical Analysis, 70, 151–157.
Johansson, M. Furuhagen, C. Frølich, & W. Jägerstad, M. (2008). Folate content in frozen
vegetarian ready meals and folate retention after different heating methods.
Lebensmittel Wissenschaft und Technologie, 41, 528–536.
Johnston, K. E. DiRienzo, D. B. & Tamura, T. (2002a). Folate content of dairy products
measured by microbiological assay with trienzyme treatment. Journal of Food Science,
67, 817–820.
Johnston, K. E. Lofgren, P. A. & Tamura, T. (2002b). Folate concentrations measured by
trienzyme extraction method. Food Research International, 35, 565–569.
Jurczuk, M. Moniuszko-Jakoniuk, J. Brzóska, M. M. & Roszczenko, A. (2005). Vitamins E
and C Concentrations in the Liver and Kidney of Rats Exposed to Cadmium and
Ethanol. Polish Journal of Environmental Studies, 14, 599–604.
Kall, M. A. & Andersen, C. (1999). Improved method for simultaneous determination of
ascorbic acid and dehydro-ascorbic acid in food and biological samples. Journal of
Chromatography B, 730, 101–111.
Kariluoto, M. S. Vahteristo, L. T. & Piironen, V. I. (2001). Applicability of microbiological
assay and affinity chromatography purification followed by high-performance liquid
chromatography (HPLC) in studying folate contents in rye. Journal of Agricultural and
Food Science, 81, 938–942.
Khair-un-Nisa, A. Tarar, O.M. Ali, S.A. Jamil, K. & Begum, A. (2010). Study to evaluate the
impact of heat treatment of water soluble vitamins in milk. Journal of the Pakistani
Medical Association, 60, 909–912.
Kim, Y. I. (2007). Folate and colorectal cancer: an evidence-based critical review. Molecular
Nutrition and Food Research, 51, 267–292.
Kim, Y. (2011). Vitamins. In F. Toldrá (Ed.), Handbook of the Analysis of Edible Animal Byproducts, (1st edition, pp. 161–182). Boca Raton, Forida, USA: CRC Press.
Kocer, B. Engur, S, Ak, F & Yilmaz, M. (2009). Serum vitamin B12, folate and homocysteine
levels and their association with clinical and electrophysical parameters in multiple
sclerosis. Journal of Clinical Neuroscience, 16, 399–403.
Kumar, S. S. Chouhan, R. S. & Thakur, M. S. (2010). Trends in analysis of vitamin B12.
Analytical Biochemistry, 398, 139–149.
Lebiedzińska, A. Marszałł, M. L. Kuta, J. & Szefer, P. (2007). Reversed-phase highperformance liquid chromatography method with coulometric electrochemical and
ultraviolet detection for the quantification of vitamins B1 (thiamine). B6 (pyridoxamine,
pyridoxal and pyridoxine) and B12 in animal and plant foods. Journal of
Chromatography A, 1173, 71–80.
Lebiedzińska, A. Dąbrowska, M. Szefer, P. & Marszałł M (2008). High-performance liquid
chromatography method for the determination of folic acid in fortified food products.
Toxicology Mechanisms and Methods, 18, 463–467.
Lee,
J.
Koo,
N.
&
Min,
D.
B.
(2004).
Reactive
oxygen
species,
antioxidativenutraceuticals, Comprehensive Reviews in Food Safety, 3, 21–33.
aging,
Leόn-Ruiz, V. Vera, S. González-Porto, A. & Andrés, M. (2012). Analysis of Water-Soluble
Vitamins in Honey by Isocratic RP-HPLC. Food Analytical Methods, 6, 488–496.
Li, K-T. Liu, D-H. Li, Y-L. Chu, J. Wang, Y-H. Zhuang, Y-P. & Zhang, S-L. (2008).
Improved large-scale production of vitamin B12 by Pseudomonas denitrificans with
betaine feeding. Bioresource Technology, 99, 8516–8520.
Lim, H-S. Mackey, A. D. Tamura, T. Wong, S.C. & Picciano, M. F. (1998). Measurable
human milk folate is increased by treatment with -amylase and protease in addition to
folate conjugase. Food Chemistry, 63, 401–407.
Lombardi-Boccia, G. Lanzi, S. & Aguzzie, A. (2005). Aspects of meat quality: trace elements
and B vitamins in raw and cooked meats. Journal of Food Composition and Analysis,
18, 39–46.
Luo, X. Chen, B. Ding, L. Tang, F. & Yao, S. (2006). HPLC-ESI analysis of vitamin B12 in
food products and in multivitamins-multimineral tablets. Analytica Chimica Acta, 562,
185–189.
MacLean, W. C. Van Dael, P. Clemens, R. Davies, J. Underwood, E. O’Risky, L. Rooney, D.
& Schrijver, J. (2010). Upper levels of nutrients in infant formulas: comparison of
analytical data with the revised Codex infant formula standard. Journal of Food
Composition and Analysis, 23, 44–53.
Markopoulou, C. K. Kagkadis, K. A. Koundourellis, J. E. (2002). An optimized method for
the simultaneous determination of vitamins B1, B6, B12, in multivitamin tables by high
performance liquid chromatography, Journal of Pharmaceutical and Biomedical
Analysis, 30, 1403–1410.
Matei, N, Magearu, V. Birghila, S. & Dobrinas, S. (2004). The determination of vitamin C
from sweet cherries and cherries. Revista de Chimie, 55, 294–296.
Mazina, J. & Gorbatsova, J. (2010). Sample preparation for CE-DAD analysis of the water
soluble vitamins in food products. Procedia Chemistry, 2, 46–53.
Mazumder, T. P. Nishio, N. Fukuzaki, S. & Nagai, S. (1987). Production of extracellular
vitamin B12 compounds from methanol by Methanosarcina barkeri. Applied
Microbiology and Biotechnology, 26, 511–516.
McGuire, J. J. (2006). Folic acid, folates and cancer. In A. B. Awad & P. G. Bradford (Eds.),
Nutrition and Cancer Prevention, (DOI: 10.1201/9781420026399.ch8), Boca Raton,
Forida, USA: CRC Press (Taylor & Francis Group, LLC).
Merola, N.Alonso, F. J. G. Ros, G. & Cáston, M. J. P. (2013). Antioxidant activity
comparison between [6S]-5-methyltetrahydrofolic acid calcium salt and the related
racemate form. Food Chemistry, 136, 984–988.
Metzler, D.E. Biochemistry. The Chemical Reactions of Living Cells. London: Academic
Press, Inc., 1977.
Mihhalevski, A. Nisamednitov, L. Hälvin, K. Ošeka, A. & Paalme, T. (2013). Stability of Bcomplex vitamins and dietary fibre during rye sourdough bread production. Journal of
Cereal Science, 57, 30–38.
Morita, S. Miwa, H. Kihira, T. & Kondo, T. (2003). Cerebrellar ataxia and
leukoencephalopathy associated with cobalamin deficiency. Journal of Neurological
Science, 216, 183–184.
Murooka, Y. Piao, Y. Kiatpapan, P. & Yamashita, M. (2005). Production of tetrapyrrole
compounds and vitamin B12 using genetically engineering of Propionibacterium
freudenreichii. An overview. Lait, 85, 9–22.
Ndaw, S. Bergaentzlé, M. Aoudé-Werner, D. Lahély, S. & Hasselmann, C (2001).
Determination of folates in foods by high-performance liquid chromatography with
fluorescence detection after precolumn conversion to 5-methyltetrahydrofolates.
Journal of Chromatography A, 928, 77–90.
Nisperos-Carriedo, M. O. Buslig, B. S. & Shaw, P. E. (1992). Simultaneous detection of
dehydro-ascorbic and some organics acids in fruits and vegetables by HPLC. Journal of
Agricultural and Food Chemistry, 40, 1127–1130.
Nohr, D. & Biesalski, H. K. (2009). Vitamins in milk and dairy products: B-group vitamins.
In P. L. H. McSweeney & P. F. Fox (Eds.), Advanced Dairy Chemistry, Volume 3:
Lactose, Water, Salts and Minor Constituents. (pp. 591–630). New York: Springer
Science + Business Media, LLC.
Nojavan, S. Khalilian, F. Kiaie, F. M. Rahimi, A. Arabanian, A. & Chalavi, S. (2008).
Extraction and quantitative determination of ascorbic acid during different maturity
stages of Rosa canina L. fruit. Journal of Food Composition and Analysis, 21, 300–305.
Nyyssönen, K., Salone, J. T. & Parviainen, M. T. Ascorbic acid. (2000). In A. P. Leenheer,
W. E. Lambert & J. F. Van Bocxlaer (Eds.), Modern Chromatographic Analysis of
Vitamins. (3rd edition, pp. 271–300). New York: Marcel Dekker.
Oakley, G. P. (2009). The scientific basis for eliminating folic acid-preventable spina bifida:
a modern miracle from epidemiology. Annals of Epidemiology, 19, 226–230.
O’Connell, P. J. Gormally, C. Pravda, M. & Guilbault, G. G. (2001). Development of an
amperometric L-ascorbic acid (Vitamin C) sensor based on electropolymerised aniline
for pharmaceutical and food analysis. Analytica Chimica Acta, 431, 239–247.
Olson, R. E: Water soluble vitamins. (1999). In Principles of Pharmacology. Edited by P. L.
Munson, R. A. Mueller & G. R. Bresse. (Chapter 59). NewYork: Chapman and Hall.
Oliviera, E. J. & Watson, D. G. (2001). Chromatographic techniques for determination of
putative dietary anticancer compounds in biological fluids. Journal of Chromatography
B, 764, 3–25.
Ozbek, Z. Kucukali, C. I. Ozkok, E. Orhan, N. Aydin, M. Kilic, G. Sazci, A. & Kara, I.
(2008). Effect of the methylenetetrahydrofolate reductase gene polymorphisms on
homocysteine, folate and vitamin B12 in patients with bipolar disorder and relatives.
Progress in Neuropsychopharmacology and Biological Psychiatry, 32, 1331–1337.
Parker-Williams, E. J. (2013). Investigation and management of anaemia. Medicine, 41, 212–
218.
Patring, J. Wandel, M. Jägerstad, M. & Frølich, W. (2009). Folate content of Norwegian and
Swedish flours and bread analysed by use of liquid chromatography-mass spectrometry.
Journal of Food Composition and Analysis, 22, 649–656.
Pawlosky, R. J. Flanagan, V. P. & Doherty, R. F. (2003). A mass spectrometric validated
high-performance liquid chromatography procedure for the determination of folates in
foods. Journal of Agricultural and Food Chemistry, 51, 3726–3730.
Pedersen, J. C. (1988). Comparison of y-glutamyl hydrolase (conjugase; EC 3.4.22.12) and
amylase treatment procedures in the microbiological assay for food folates. British
Journal of Nutrition, 59, 261–271.
Pfeiffer, C. M. Fazili, Z. & Zhang, M. (2010). Folate analytical methodology. In L. B. Bailey
(Ed.), Folate in Health and Disease, (2nd edition, pp. 517–574). Boca Raton, Forida,
USA: CRC Press (Taylor & Francis Group, LLC).
Phillips, K. M. Ruggio, D. M. Ashraf-Khorassani, M. & Haytowitz, D. B. (2006). Folate
composition of 10 types of mushrooms determined by liquid chromatography–mass
spectrometry. Food Chemistry, 129, 630–636.
Phillips, K. M. Ruggio, D. M. & Haytowitz, D. B. (2011). Difference in folate content of
green
and red
sweet peppers
(Capsicum
annuum) determined by
liquid
chromatography–mass spectrometry. Journal of Agricultural and Food Chemistry, 54,
9998–10002.
Pisoschi, A. M. Danet, A. F. & Kalinowski, S. (2008). Ascorbic Acid Determination in
Commercial Fruit Juice Samples by Cyclic Voltammetry. Journal of Automated and
Management
in
Chemistry,
Published
on-line
29
March
2009,
doi:
10.1155/2008/937651, 2008: 937651, 1–8.
Plonka, J, Toczek A. and Tomczyk, V. (2012). Multivitamin Analysis of Fruits, FruitVegetable Juices, and Diet Supplements. Food Analytical Methods, 5, 1167–1176.
Poel, C. Bäckermann, S. & Ternes, S. (2009). Degradation and conversion of thiamin and
thiamin phosphate esters in fresh stored pork and in raw sausages. Meat Science, 83,
506–510.
Puthesseri, B. Divya, P. Lokesh, V. & Neelwarne, B. (2013). Salicylic acid-induced
elicitation of folates in coriander (Coriandrum sativum L.) improves bioaccessibility
and reduces pro-oxidant status. Food Chemistry, 136, 569–575.
Puwastien, P. Pinprapai, N. Judprasong, K. & Tamura, T. (2005). International interlaboratory analyses of food folate. Journal of Food Composition and Analysis, 18, 387–
397.
Quinlivan, E. P. & Gregory, J. F. (2007). Reassessing folic consumption patterns in the
United States (1999–2004): potential effect on neural tube defects and overexposure to
folate. American Journal of Clinical Nutrition, 86, 1773–1779.
Rader, J. I. Weaver, C. M. & Angyal, G. (1998). Use of a microbiological assay with trienzyme extraction for measurement of pre-fortification levels of folates in enriched
cereal-grain products. Food Chemistry, 62, 451–465.
Randaccio, L. Geremia, S. Nardin, G. & Wuergess, J. (2006). X-ray structural chemistry of
cobalamins. Coordination Chemistry Reviews, 250, 1332–1350.
Rivlin, R. S. (2007). Vitamin deficiencies. In C. D. Berdanier, E. B. Feldman & J. Dwyer
(Eds.), Handbook of Nutrition and Food, (2nd edition, pp. 177–292). Boca Raton,
Forida, USA: CRC Press (Taylor & Francis Group, LLC).
Rizzolo, A. Brambilla, A. Valscchi, S. & Eccher-Zerbini, P. (2002). Evaluation of sampling
and extraction procedures for the analysis of ascorbic acid from pear fruit tissue. Food
Chemistry, 79, 141–144.
Romeu-Nadal, M. Morera-Pons, S. Castellote, A. I. & López-Sabater, M. C. (2006). Rapid
high-perfomance liquid chromatographic method for Vitamin C determination in
human milk versus an enzymatic method. Journal of Chromatography B, 830, 41–46.
Rozoy, E. Araya-Farias, M. Simard, S. Kitts, D. Lessard, J. & Bazinet, L. (2013). Redox
properties of catechins and enriched green tea extracts effectively preserve
L-5-
methyltetrahydrofolate: assessment using cyclic voltammetry analysis. Food Chemistry,
138, 1982–1991.
Rudenko, A.O. & Kartsova, L.A. (2010). Dtermination of Water-Soluble Vitamin B and
Vitamin C in Combined Feed, Premixes, and Biologically Active Supplements by
Reversed-Phase HPLC. Journal of Analytical Chemistry, 65, 71–76.
Russell, L.F. (1986). High performance liquid chromatographic determination of vitamin C in
fresh tomatoes. Journal of Food Science, 51, 1567–1568.
Russell, L. F. (2012). Water soluble vitamins. In L. M. L. Nollet & F. Toldrá (Eds.), Food
Analysis by HPLC, (3rd edition, pp. 325–442). Boca Raton, Forida, USA: CRC Press.
Safavi, A. Maleki, N. Ershandifar, H. & Tajabadi, F. (2010). Development of a sensitive and
selective riboflavin sensor based on carbon ionic liquid electrode. Analytica Chimica
Acta, 674, 176–181.
San José, R. Fernández-Ruiz, V. Cámara, M & Sánchez-Mata, M.C. (2012). Simultaneous
determination of vitamin B1 abd B2 in complex cereal foods, by reverse-phase isocratic
HPLC-UV. (2012). Journal of Cereal Science, 55, 293–299.
Santos, J, Mendiola, J. A. Oliviera, M. B. P. P. Ibánez, E. & Herrero, M. (2012). Sequential
determination of fat- and water-soluble vitamins in green leafy vegetables during
storage. Journal of Chromatography A, 1261, 179–188.
Schönfeldt, H. C. & Gibson, N. (2008). Changes in the nutrient quality of meat in an obesity
context. Meat Science, 80, 20–27.
Selvakumar, L. S. & Thakur, M. S. (2011). Competitive immunoassay for analysis of vitamin
B12. Analytical Biochemistry, 418, 238–246.
Selvakumar, L. S. & Thakur, M. S. (2012). Dipstick based immunochemiluminescence
biosensor for the analysis of vitamin B12 in energy drinks: A novel approach. Analytica
Chimica Acta, 722, 107–113.
Shafqat, Ullah Hussain, A. Ali, J. Khalikurrehman & Asad, U. (2012). A simple and rapid
HPLC method for analysis of Vitamin C in local packed juices of Pakistan. MiddleEast Journal of Scientific Research, 12, 1085–1091.
Shaik, M. M. & Gan, S. H. (2013). Rapid resolution liquid chromatography method
development and validation for simultaneous determination of homocysteine, vitamins
B6, B9 and B12 in human serum. Indian Journal of Pharmacology, 45, 159–167.
Shane, B. (1982). High performance liquid chromatography of folates: identification of poly-glutamate chain lengths of labelled and unlabelled folates. American Journal of
Clinical Nutrition, 35, 599–608.
Shane, B. (2010). Folate chemistry and metabolism. In L. B. Bailey (Ed.), Folate in Health
and Disease, (2nd edition, pp. 1–24). Boca Raton, Forida, USA: CRC Press.
Shrestha, A. K. Arcot, J. & Paterson, J. (2000). Folate assay of foods by traditional and trienzyme treatments using cryoprotected Lactobacillus caseii. Food Chemistry, 71, 545–
552.
Sikorska, E. (2007). Analysis of vitamin B2 using front-face intrinsic beer fluorescence.
European Food Research and Technology, 225, 43–48.
Soongsongkiat, M. Puwastien, P. Jittinandana, S. Dee-Uam, A. & Sungpuag, P. (2010).
Testing of folate conjugase from chicken pancreas vs. commercial enzyme and
studying the effect of cooking on folate retention in Thai foods. Journal of Food
Composition and Analysis, 23,681–688.
Stover, P. J. (2010). Folate biochemical pathways and their regulation. In L. B. Bailey (Ed.),
Folate in Health and Disease, (2nd edition, pp. 49–74). Boca Raton, Forida, USA:
CRC Press (Taylor & Francis Group, LLC).
Strålsjö, L. Arkbåge, K. Witthöft, C. & Jägerstad, M. (2002). Evaluation of a radioproteinbinding assay (RPBA) for folate analysis in berries and milk. Food Chemistry, 79, 525–
534.
Sullivan, S. (2012). Infant formula and adult/pediatric nutritional methods approved First
action using the AOAC voluntary consensus standards process. Journal of AOAC
International, 95, 1–4.
Tai, A. & Gohda, E. (2007). Determination of ascorbic acid and its related compounds in
foods and beverages by hydrophilic interaction liquid chromatography. Journal of
Chromatography B, 853: 214–220.
Tarrago-Trani, M. T. Phillips, K. M. & Cotty, M. (2012). Matrix-specific method validation
for quantitative analysis of vitamin C in diverse foods. Journal of Food Composition
and Analysis, 26, 12–25.
Thiel, R. J. & Fowkes, S. W. (2004). Down syndrome and epilepsy: a nutritional connection.
Medical Hypotheses, 62, 35–44.
Trembath, D., Sherbondy, A. L. Vandyke, D. C. Shaw, G. M. Todoroff, K. Lammer, E. J.
Finnell, R. H. Marker, S. Lerner, G. & Murray, J. C. (1999). Analysis of select folate
pathway genes, PAX3, and human T in a Midwestern neural tube defect population.
Teratology, 59, 331–341.
Tsukatani, T. Suenega, H. Ishiyama, M. Ezoe, T. & Matsumoto, K. (2011). Determination of
water-soluble vitamins using a colorimetric microbial viability assay based on the
reduction of water-soluble tetrazolium salts. Food Chemistry, 127, 711–715.
Tyszczuk-Rotko, K. (2012). New voltammetric procedure for determination of thiamine in
commercially available juices and pharmaceutical formulation using a lead film
electrode. Food Chemistry, 134, 1239–1243.
Vahteristo, L. & Finglas, P. M. (2000). Chromatographic determination of folates. In A. P.
Leenheer, W. E. Lambert & J. F. Van Bocxlaer (Eds.), Modern Chromatographic
Analysis of Vitamins. (3rd edition, pp. 301–323). New York: Marcel Dekker.
http://www.foodnetbase.com/books/1672/DKE416_Ch06.pdf (accessed January 21,
2013).
Vahteristo, L. T. Ollilainen, V. Koivistoinen, P. E. & Varo, P. (1996a). Improvements in the
analysis of reduced folate monoglutamates and folic acid in food by HighPerformance Liquid Chromatography. Journal of Agricultural and Food Chemistry,
44, 477–482.
Vahteristo, L. T. Ollilainen, V. & Varo, P. (1996b). HPLC determination of folate in liver
and liver products. Journal of Food Science, 61, 524–526.
Vahteristo, L. T. Ollilainen, V. & Varo. P. (1997a). Liquid chromatographic determination of
folate monoglutamates in fish, meat, egg and dairy products consumed in Finland.
Journal of AOAC International, 80, 373–378.
Vahteristo, L. T. Lehikoinen, K. Ollilainen, V. & Varo, P. (1997b). Application of an HPLC
assay for the determination of folate derivatives in some vegetables, fruits and berries
consumed in Finland. Food Chemistry, 59, 589–597.
Valls, F. Sancho, M. T. Fernandez-Muino, M. A. Alonso-Torre, S. & Checa, M. A. (2002).
High presssure liquid chromatographic determination of ascorbic acid in cooked
sausages. Journal of Food Protein, 65, 1771–1774.
Vanderslice, T. J. & Higgs, D. J. (1984). HPLC analysis with fluorometric detection of
vitamin C in food samples. Journal of Chromatographic Science, 22, 485–489.
Van Heerden, S. M. Schӧnfeldt, H. C. Kruger, R. & Smith, M. F. (2007). The nutrient
composition of South African lamb (A2 grade). Journal of Food Composition and
Analysis, 20, 671–680.
Van Wyk, J. (2002). An HPLC evaluation of vitamin B12 and folate synthesis by the
Propionibacterium freudenreichii group. Doctoral Dissertation, University of
Stellenbosch, Stellenbosch, South Africa.
Van Wyk, J. & Britz, T. J. (2010). A rapid HPLC method for the extraction and
quantification of vitamin B12 in dairy products and cultures of Propionibacterium
freudenreichi. Dairy Science and Technology, 90, 509–520.
Van Wyk, J. Witthuhn, R. C. & Britz, T. J. (2011). Optimisation of vitamin B12 and folate
production by Propionibacterium freudenreichii strains in kefir. International Dairy
Journal, 21, 69–74.
Van Wyk, J. & Britz, T. J. (2012). A rapid high-performance liquid chromatography (HPLC)
method for the extraction and quantification of folates in dairy products and cultures of
Propionibacterium freudenreichii. African Journal of Biotechnology, 11, 2087–2098.
Vanzin, C. S. Bianchi, G. B. Sitta, A. Wayhs, C. A. Y. Perreira, I. N. Rockenbach, F. Garcia,
S. C. De Souza Wyse, A. T. Schwartz, I. V. D. Wajner, M. & Vargas, C. R. (2011).
Experimental evidence of oxidative stress in plasma of homocystinuric patients: A
possible role for homocysteine. Molecular Genetics and Metabolism, 104, 112–117.
Vishnumohan, S. Arcot, J. & Pickford, R. (2011). Naturally-occurring folates in foods:
Method development and analysis using liquid chromatography–tandem mass
spectrometry (LC-MS/MS). Food Chemistry, 125, 736–742.
Wallis, D. Ballard, J. L. Shaw, G. M. Lammer, E. J. & Finnell, R. H. (2010). Folate-related
birth defects. Embryonic consequences of abnormal folate transport and metabolism. In
L. B. Bailey (Ed.), Folate in Health and Disease, (2nd edition, pp. 155–178). Boca
Raton, Forida, USA: CRC Press (Taylor & Francis Group, LLC).
Wagdy, H. A. Hanafi, R. S. El-Nashar, R. M. & Aboul-Ein, H. Y. (2013). Determination of
the design space of the HPLC analysis of water soluble vitamins. Journal of Separation
Science, Accepted article (doi: 10.1002/jssc.201300082), 1–19.
Wawire, M. Oey, I. Mathooko, F. M. Njoroge, C. K. Shitanda, D Sila D. & Hendrickx, M.
(2012). Effect of harvest age and thermal processing on poly-γ-glutamate folates and
minerals in African cowpea leaves (Vigna unguiculata). Journal of Food Composition
and Analysis, 25, 160–165.
Witthöft, C. M. Forssén, K., Johannesson, L. & Jägerstad, M. (1999). Folates – food sources,
analyses, retention and bioavailability. Scandanavian Journal of Nutrition, 43, 138–
146.
Wong, D. W. S. Mechanism and Theory in Food Chemistry. Pp. 353–355. New York:
Nostrand Reinhold, 1989.
Wu, X. Wang, X. Chan, Y. Jia, S. Luo, Y. & Tang, W. (2013). Folate metabolism gene
polymorphisms MTHFR C677T and A1298C and risk for Down syndrome offspring: a
meta-analysis. European Journal of Obstetrics & Gynecology and Reproductive
Biology, 167, 154–159.
Xu, H. Li, Y. Liu, C. Wu, Q. Zhao, Y. Lu, L. & Tang H. (2008). Fluorescence resonance
energy transfer between acridine orange and rhodamine 6G and its analytical
application for B12 with flow-injection laser-induced fluorescence detection. Talanta,
77, 176–181.
Yang, Y. Zhang, Z. Lu, J. & Maekawa, T. (2004). Continuous methane fermentation and the
production of vitamin B12 in a fixed-bed reactor packed with loofah. Bioresource
Technology, 92, 285–290.
Yazynina, E. Johansson, M. Jägerstad, M. & Jastrebova, J. (2008). Low folate content in
gluten-free cereal products and their main ingredients. Food Chemistry, 111, 236–
242.
Ye, L. Eitenmiller, R. R. & Landen, W. O. (2008). Vitamin Analysis for the Health and Food
Sciences, 2nd Edition. Boca Raton, Florida, USA: CRC Press.
Yoshida, M. Hishiyama, T. Ogawara, M. Fuse, K., Mori, M. Igarashi, T. & Yaniguchi, M.
(2012). A novel method for determining vitamin B1 in a wide variety of foodstuffs with
or without polyphenols. Food Chemistry, 135, 238–2392.
Zand, N. Chowdry, B. Z. Pullen F. S. Snowden, M. J. & Tetteh, J. (2012). Simultaneous
determination of riboflavin and pyridoxine by UHPLC/LC-MS in UK commercial
infant food products. Food Chemistry, 135, 2743–2749.