Journal of Cereal Science 55 (2012) 293e299

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Journal of Cereal Science

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Simultaneous determination of vitamin B1 and B2 in complex cereal foods,

by reverse phase isocratic HPLC-UV
R. San Jos Rodriguez, V. Fernndez-Ruiz, M. Cmara, M.C. Snchez-Mata*
Departamento de Nutricin y Bromatologa II, Facultad de Farmacia, Universidad Complutense de Madrid, Pza. Ramn y Cajal s/n, E-28040 Madrid, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 20 July 2011
Received in revised form
11 December 2011
Accepted 15 December 2011

The evaluation of nutritional or functional components in grain products is an important feature for the
industry, especially when recent regulations require a correct nutrition labelling, valid during all the shelf
life of the product. For that reason, industry usually makes many efforts to develop simple and reliable
analytical methods that can be easily applied in any quality control laboratories for routine analysis.
Spectrouorimetric analysis of thiamine and riboavin are sensitive, but need specic equipment. A few
HPLC-UV methods have been described but they are less sensitive, and present difculties due to
interfering compounds, particularly in complex food matrixes, as grains and derivatives.
A combination of extraction and separation systems, that allows enough sensitivity, precision and
accuracy for the analysis of vitamin B1 and B2 in complex cereal food products, by isocratic UV-HPLC, in
a single wavelength simultaneous separation is presented, with the advantage of using low-cost
equipment requirements, simple sample pre-treatment and short time. The achievement of this goal
has involved the optimization of compatible extraction and measurement protocols for cereal matrices,
comparing seven different separation conditions and six extraction/clarication matrices analysis. The
selected method was comparatively validated and compared to reference AOAC spectrouorimetric
methods, providing comparable linearity and accuracy, with better specicity and precision parameters,
as well as practical applicability.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Vitamin B1
Vitamin B2
HPLC
Cereal products

1. Introduction
Cereals have been traditionally used as basic foods; however in
recent years, their consumption has declined due to new life styles.
As the necessity of their recovery in the diet is recognized, some
new cereal products have been developed by the industry, adapted
to different uses and providing nutritional and functional benets
to the organism, including the intake of energy from complex
carbohydrates, as well as the presence of dietary bre and micronutrients as minerals and vitamins.
Thiamine and riboavin are two vitamins involved in different
processes in the human body, such as glucose metabolism, nervous
transmission, replication of genes, development of foetal tissues
and biosynthesis of corticoids, among others (Illera Martn et al.,
2000). These two compounds are unstable to heat, light and
other factors, so the technological processes in cereal grains can

* Corresponding author. Tel./fax: 34 91 394 17 99.

E-mail addresses: rsanjose@aemps.es (R. San Jos Rodriguez), vfernand@
farm.ucm.es (V. Fernndez-Ruiz), mcamara@farm.ucm.es (M. Cmara), cortesm@
farm.ucm.es (M.C. Snchez-Mata).
0733-5210/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2011.12.011

partially destroy these compounds. For that reason, many industrial

cereal products are enriched in B group vitamins to recover their
levels. The evaluation of nutritional or functional components in
grain products is an important feature for the industry, in relation
to nal product quality. The recent regulations regarding food
nutrition labelling, introduced by the governments and regulatory
bodies around the world, require a guaranteed content of nutrients
and other bioactive compounds in food products during all their
shelf life. Particularly, European Regulation (EC) No 1925/2006 of
the European Parliament and of the Council of 20 December 2006,
on the addition of vitamins and minerals and of certain other
substances to foods, establishes that not only the added fraction,
but the total sum of endogenous (that could need enzymatic
extraction) and exogenous vitamins, should be given on the label of
the food products. This is the reason why the majority of methods
are targeting the determination of total vitamin content, and has
led to a growing need for cheap, cost-effective, rapid and reliable
analytical monitoring methods for the determination of nutrients
and vitamins and other nutrients in food products, that can be
easily applied in any laboratories for routine analysis (Heudi et al.,
2005; Zafra-Gmez et al., 2006). Many analytical methods have
been proposed for thiamine and riboavine analysis. The choice of

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R. San Jos Rodriguez et al. / Journal of Cereal Science 55 (2012) 293e299

one method usually depends on the accuracy and sensitivity

required and the interferences encountered in the sample matrix.
Both vitamin B1 and B2 can be easily determined in pharmaceutical
products using HPLC-UV methods; but for foods, more specic
techniques of analysis should be applied, due to the presence of
a high number of interfering compounds, and different food
matrixes (Lynch and Young, 2000). For this purpose, chromatographic techniques with ion-exchange or silica columns are of
interest to separate vitamins prior to analysis by reverse-phase
HPLC or other methods. Most authors use reverse-phase HPLC,
with a C18 column and methanol/water as the eluent, although
some authors also have reported the use of ion-exchange columns
(Hilker and Clifford, 1982) or amide-based columns (Vias et al.,
2003). The presence of buffers is often useful to adjust pH in the
solvent and get more reproducible conditions, and the addition of
an ion-pair reagent is also applied to improve resolution of the
peaks (Ayi et al., 1985; Finglas and Faulks, 1984; Ndaw et al., 2000;
Van Niekerk, 1988).
Spectrouorimetric determination, either directly (AOAC, 2011)
or coupled to HPLC separation (Arella et al., 1996; Kyritsi et al., 2011;
Ndaw et al., 2000; Reyes and Subryan, 1989; Watada and Tran,
1985) is one of the most recommended methods for the determination of these vitamins in foodstufs. Flavines as riboavin, FAD and
FMN present native uorescence, either in a neutral isoxazoline or
oxidated form. However, the reduced forms do not show uorescence and their binding to proteins also reduces the uorescent
signal. The oxidized derivative of thiamine (thiocrome) is uorescent in alkaline medium, so for the HPLC-uorimetric method,
either a pre- or post-column derivatization is needed (Lynch and
Young, 2000; Ohta et al., 1993).
Spectrouorimetric analysis of vitamin B1 and B2 has the
advantage of being extremely sensitive, but requires specic
equipment (spectrouorimeter or spectrouorimetric detector
for HPLC) that is not always available for many laboratories
(Mondragn-Portocarrero et al., 2011). Some HPLC-UV methods
have been described for food products (Albal-Hurtado et al., 1997;
Kamman et al., 1980; Nicholas and Pfender, 1990; Vidal-Valverde
and Reche, 1990), but often have the disadvantages of being less
sensitive, and with a limited application to various food samples,
due to the presence of many interfering compounds absorbing light
in the UV range.
To solve these problems, careful hydrolysis and purication
procedures have to be applied and also the chromatographic
conditions have to be improved. In some cases, this induces very
long retention times in the chromatograms (40 min or more), which
can be accompanied by poor sensitivity and characteristics of the
peaks. HPLC-UV analysis of vitamins B1 and B2 requires high amount
of sample to make possible the detection of both compounds. In
other cases, UV detection is accompanied by a more sensitive and
specic technique, as the use of gradients, different wavelengths
programs, Diode Array, Coulometric or Mass Spectrometry Detection (Albal-Hurtado et al., 1997; Aranda and Morlock, 2006; Engel
et al., 2010; Heudi et al., 2005; Mandal et al., 2009; Marsza11 et al.,
2005; Vias et al., 2003; Zafra-Gmez et al., 2006), or other techniques such as capillary electrophoresis, biosensors or chemiluminescence (Bai et al., 2008; Gao et al., 2008), which often makes
necessary the availability of highly complex equipment.
Grains and their derivatives are food matrixes that show special
difculties for this kind of analysis, mainly due to two reasons: rst,
they are starchy products and often gel when using high amount of
sample and hot extraction; and second, they have a lot of interfering compounds, especially when analysing industrial complex
cereal mixtures including many different ingredients. For that
reason, only a few methods use HPLC-UV, and they often require
additional strategies to avoid the problems mentioned above.

Due to the signicant challenges that must be faced when

a specic equipment is not available for the analysis of vitamins B1
and B2 in cereal food products, the contribution of this work has
been the establishment of a protocol that combines compatible
extraction and separation systems, to get enough sensitivity,
precision and accuracy for the analysis of vitamin B1 and B2 in
a very complex food matrix, with clean, well-resolved analyte
peaks, in a single wavelength simultaneous separation, using very
simple and easily available equipment.
2. Experimental
2.1. Reagents and samples
Standards of thiamine and riboavin, as well as enzymes
(diastase, alpha-amylase, beta-amylase, acid phosphatase, papain
and pepsin) and other common enzymes were purchased from
Sigma (St. Louis, USA). HPLC grade solvents were purchased from
Symta (Madrid, Spain). Commercial complex cereal products, consisting of breakfast cereal food (containing rice, oat, whole wheat,
wheat our and wheat bran) with enrichment levels of 2e2.7 mg/
100 g of vitamins B1 and B2, were purchased from local markets.
2.2. Instrumentation
A PerkineElmer LS-3 Fluorescence Spectrophotometer (Massachusetts, USA) was used for this study. The HPLC apparatus consisted of a PU II isocratic pumping system; a Jasco (Tokyo, Japan)
AS-1555 autosampler; an ERC-Gecko-2000 (Riemerling, Germany)
column heater and a Spectra Series UV100 UVeVis detector
(Micron Analitica, S.A., Madrid, Spain). For data processing and
analysis, Biocrom 2000 3.0 version software (Micron Analitica, S.A.,
Madrid, Spain) was used. The analytical column was a PurospherSTAR RP-18e (250  4 mm), 5 mm pore size, purchased from Merck
(Darmstad, Germany).
2.3. Sample preparation
Vitamins B1 and B2 are usually bound to macromolecules as
proteins and carbohydrates, as well as in phosphorylated forms,
less frequent in vegetal origin tissues (Ball, 1994). For this reason,
acidic hydrolysis (with HCl or H2SO4 together with heating at
60e150  C for 10e30 min), enzymatic hydrolysis (with at least
diastase and protease, and in some cases phosphatase, at about
37  C overnight), or both, has to be applied to the samples in order
to extract these vitamins (Arella et al., 1996; Augustin et al., 1985;
Matallana Gonzlez et al., 1998; Ollilainen et al., 1993).
According to these previous studies, the most suitable extraction procedure for vitamin B1 and B2 in this kind of samples were
chosen through different acid and enzymatic hydrolysis (with
overnight incubation at 37  C) assays in order to separate both
vitamins from other compounds in the seeds (mainly proteins and
carbohydrates) (Table 1). Some extracts were concentrated after
incubation, using a Savant Speed Vac PD121P concentrator.
2.4. Chromatographic conditions
According to the literature, different solvent systems with
methanol, water and acetate buffer have been used for the separation of both vitamins in an endcapped-C18 column, with UV
detection (268 nm) (Ndaw et al., 2000; Ottaway, 1993; VidalValverde and Reche, 1990). The use of ion-pair reagents (sodium
hexanesulfonate or heptanesulfonate) were tried for the separation
of both compounds from other interfering substances in the

R. San Jos Rodriguez et al. / Journal of Cereal Science 55 (2012) 293e299

295

Table 1
Different conditions assayed for the optimization of the analytical procedure.
Extraction assays
Protocol
number

Acid hydrolysis

Heat

pH

Enzymes

Time, temperature

1
2

0.1 M HCl
e

100  C, 30 min
e

2.5 M AcNa / 4.5

4.5 (5 mM AcNa buffer)

18 h, 37  C
18 h, 37  C

3
4

e
50 mM HCl

e
e

4.5 (5 mM AcNa buffer)

a) 2.5 M AcNa / 2
b) 25 M AcNa / 4.5

0.1 N H2SO4

2.5 M AcNa / 4.5

0.1 N H2SO4

2.5 M AcNa / 4.5

Diastase 500 mg
Diastase 500 mg
Papain 500 mg
Acid phosphatase 10 mg
a) Pepsin 100 mg
b) Acid phosphatase 10 mg
a-amylase 40 mg
b-amylase 0.75 mg
Diastase 500 mg
a-amylase 500 mg
Diastase 500 mg
a-amylase 500 mg
Papain 500 mg

18 h, 37  C
a) 4 h, 37
b) 18 h, 37  C

18 h, 37  C
18 h, 37  C

Mobile phase assays

Solvent
system

Methanol:H2O

Na hexane-sulphonate

Na heptane-sulphonate

Na acetate

tR (min) vitamin B1

tR (min) vitamin B2

A
B
C
D
E
F
G

50:50
50:50
25:75
25:75
25:75
25:75
25:75

5 mM
e
e
e
e
e
e

e
5 mM
2.5 mM
1 mM
2.5 mM
2.5 mM
2.5 mM

e
e
e
e
12.5 mM
25 mM
50 mM

3.20
5.56
14.14
22.44
8.25
7.26
6.56

3.37
4.13
23.13
25.06
18.67
19.92
20.64

tR time of retention.

extracts (Albal-Hurtado et al., 1997; Arella et al., 1996). The

conditions assayed are presented in Table 1.
2.5. Reference spectrouorimetric methods
The developed HPLC method was compared to the spectrouorimetry, as AOAC reference methods (Horwitz and Latimer,
2005), using the same extraction conditions:
These methods were as follows:
- Vitamin B1: 5 mL of ltered extract was added with 3 mL of
0.3 mg/mL K3Fe(CN)6, in 15% NaOH and 4 mL of 25% KCl for the
alkaline oxidation of thiamine to its uorescent form (thiocrom).
This compound was extracted with isobutanol, and the uorescent signal in the alcoholic medium was quickly measured at
365/435 nm. For each sample, a blank assay was carried out, with
the addition of 3 mL of 15% NaOH instead of K3Fe(CN)6 solution.
The 100% uorescent signal was adjusted with a quinine
sulphate solution, and calibration curves were made with
solutions of thiamine chlorhydrate diluted in an alcoholeacidic
medium, as reported by AOAC method number 953.17.
- Vitamin B2: 10 mL of ltered extract of the samples were
oxidized with 0.5 mL of KMnO4, in the presence of glacial acetic
acid. After 2 min, the excess of KMnO4 was reduced with H2O2
until discolouration. Fluorescence was measured at 440/
565 nm, and extracts in which riboavin was reduced with
20 mg de Na2S2O4 were used as blanks. Calibration curves were
made with solutions of riboavin in 0.02 N acetic acid, as
reported by AOAC (method number 970.65).
3. Results and discussion
Different extraction and HPLC measurement protocols were
assayed in order to get the best extraction efciency and separation
conditions in the complex samples analysed.

3.1. Extraction procedure selection

For extractions assays, the proportion of sample weight and
extraction medium were about 15 g/100 mL, although these
proportions were adjusted to get enough amount of vitamins, and
improve the handling of the samples.
From all the extraction assays (Table 1), protocols number 1
(HCl heat diastase) and number 2 (diastase papain) did not
provide a complete extraction of vitamin B1, only being detected
the vitamin B2. Besides, in protocol number 1, the use of heat in this
kind of sample gave rise to great difculties for the extraction
process, since starch formed a gel, and for this reason, it was not
possible to use a high amount of sample, as would be required by
the low sensitivity of UV detection. As has been previously reported, heat extraction can also partially destroy thiamine, especially
in its free form and for this reason, Ball (1998) recommended a low
temperature acidic hydrolysis in vegetal products rich in free
thiamine. For all these reasons, heat treatment was discarded in
this study.
Protocols 3 and 4 (using an enzyme cocktail with acid
phosphatase, alpha- and beta-amylase and a protease) provided
a high amount of interference in the chromatograms, due to
all the enzymes used. Hasselmann et al. (1989) obtained
a complete dephosphorylation of vitamins B1 and B2 using
takadyastase beta-amylase, even better than using a phosphatase. According to Ndaw et al. (2000), there is no need of
phosphatase for vegetal origin products, since phosphorylated
forms of these vitamins are not abundant (5% of total vitamins
in wheat our) and they usually have some natural phosphatase
activity; besides, the use of some amylases can provide also
some residual protease and phosphatase activity (Bognr and
Ollilainen, 1997). These authors also suggested that acidic
hydrolysis can be avoided for thiamine and riboavin extraction
in food samples when an enzymatic cocktail with enough diastase and protease activity is used, which was also demonstrated by Ndaw et al. (2000).

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R. San Jos Rodriguez et al. / Journal of Cereal Science 55 (2012) 293e299

On the basis of those previous ndings, phosphatase was discarded from the enzyme cocktail, and only diastases and proteases
were used. Besides, low amount of samples and the avoidance of
heat are desirable for the vitamin B1 and B2 extraction in starchy
samples such as cereals and derivatives. The concentration of the
extracts after the extraction avoids using high amount of samples,
However, it was not a good alternative, because high concentrations of interfering substances also appeared.
Some authors (Van Niekerk,1988) recommended the use of H2SO4
for this kind of analysis. Protocols 5 and 6 applied acid hydrolysis with
H2SO4 diastase and alpha-amylase, with better results for the
extraction procedure and detection of vitamins. The use of different
types of diastases has led to good results in thiamine and riboavin
extraction of different foodstuffs (Ndaw et al., 2000). These authors
recommended acid extraction only for starchy samples, due to the
improvement in the ltration process, and indicated the possible
substitution of acid hydrolysis by amylase activity for this purpose.
These authors also suggested that the presence of protease activity in
some diastase enzymes is often enough for a total extraction of
vitamins, even without acid hydrolysis.
As there can be great differences regarding the activity of
different diastases, in the present study, the addition of papain to
the extraction medium (H2SO4 diastase and alpha-amylase) was
considered useful and provided a better clean-up of some interfering substances, in agreement with Russel (1996) who recommended the use of papain for thiamine analysis, due to its high
protease activity. The combination papain diastase has been
successfully applied in previous studies, for vitamin B group
hydrolysis in other types of vegetal products (Snchez-Mata et al.,

2003). In these conditions, some variations were introduced to

avoid interferences of some protein compounds in the extracts,
based on Nicholas and Pfender (1990): addition of Na acetate,
change of pH to 1.7e2 and after to 4e4.2, and purication through
Sep Pack C18 cartridge. These strategies did not improve the
extraction and resolution of the vitamins studied.
3.2. Optimization of HPLC method
From all the HPLC conditions assayed (Table 1), the A solvent
system (methanol/water 50/50 with sodium hexanesulphonate)
resulted in coelution of thiamine and riboavin. However
the change of the ion-pair reagent to sodium heptanesulphonate
(B solvent system number) permitted the separation of both vitamins. Increasing the water content in the mobile phase resulted in
much longer retention time for riboavin and better separation
between both compounds and the interferences. The less the
amount of sodium heptanesulphonate, the longer the retention time
of both vitamins (solvent systems BeD), and its concentration was
xed at 2.5 mM, as it provides better resolution between the peaks.
Some problems related to poor reproducibility of retention time
of thiamine were detected using these conditions. As has been
previously reported by Vidal-Valverde and Reche (1990) and
Nicholas and Pfender (1990), thiamine is very sensitive to pH
changes in the mobile phase. In the assays performed in this study,
the inclusion of sodium acetate in the eluent provided a xed pH
which improved the reproducibility of the retention time of
vitamin B1. When increasing the concentration of sodium acetate in
the mobile phase between 12.5 and 50 mM (solvent systems EeG),

Fig. 1. Chromatograms of a) standards of thiamine and riboavin; b) commercial cereal mixture sample; and c) chromatographic parameters of the peaks. Chromatographic
conditions: PurospherSTAR RP-18e (250  4 mm), 5 mm pore size (Merck); mobile phase methanol/water (25:75 v/v) 2.5 mM sodium heptanosulfonate and 12.5 mM sodium
acetate; ldetection 268 nm.

R. San Jos Rodriguez et al. / Journal of Cereal Science 55 (2012) 293e299

297

Table 2
Linearity of HPLC and spectrouorimetric methods.
Intercept (X  SD)
Vitamin B1
HPLC
Spectrouorimetry
Vitamin B2
HPLC
Spectrouorimetry

Slope (X  SD)

r2

Response factors
(X  SD)

RSD (%)

8633.9  3323.0
6.5  12.3

4872.1  120.7
909.5  16.8

0.9978
0.9986

99.57%
99.73%

4642.0  159.2
966.1  42.0

3.43
4.35

7856.4  4533.1
2.4  1.3

11404.0  261.3
166.1  4.4

0.9981
0.9985

99.63%
99.71%

10800.9  516.8
156.7  10.9

4.78
6.93

X Mean value (n 3); SD Standard deviation (n  1); RSD relative standard deviation.

shorter retention times for thiamine and longer for riboavin were
obtained. The best conditions were those of solvent system E,
which provided retention times of 8  0.3 min for thiamine and
18  0.3 min for riboavin (Fig. 1), and therefore good resolution
and retention times long enough to separate vitamins from other
interferences with total runs no longer than 20 min. Therefore, the
mobile phase selected for analysis was: 12.5 mM sodium acetate in
a mixture of methanol/water 25/75 2.5 mM sodium heptanesulphonate, at a ow rate of 0.9 mL/min.
In these conditions, the suitability of both peaks was evaluated
by the calculation of the following chromatographic parameters
(Hsu and Chien, 1994):

(PVDF) membrane (Millipore, Bedford, USA). This extract

was injected in the chromatographic system, using a PurospherSTAR RP-18e (250  4 mm, 5 mm) column (Merck) and 12.5 mM
sodium acetate in a mixture of methanol/water 25/75 2.5 mM
sodium heptanesulphonate, as eluant at a ow rate of 0.9 mL/min
and ldetection 268 nm.
The identity of the peaks obtained was conrmed by the addition of standards to the extracts, and excellent results were
obtained using these extraction conditions, with no interference
with the chromatographic peaks and the quantication of the
expected amount of vitamins.

N 16tR =w2

(1)

3.4. Validation of HPLC method

Hp L=N

(2)

k0 tR =tRsolv:  1

(3)

T W0:05 =2f

(4)

R 2 tR2  tR1



W1=22  W1=21

(5)

where N is the number of theoretical plates; Hp is the height of each

plate; tR, tR1 and tR2 are the retention times of each compound; tRsolv:
is the dead time; w is the width of the peak at the baseline; W0.05 is
the width of the peak at 5% of the peak height; W1/2 (1) and W1/2 (2)
are the width of the peak at half-height for each compound; L is the
length of the column; k0 is the retention factor; T is the tailing
factor; f is the distance between the perpendicular dropped from
the peak maximum and the leading edge of the peak at 5% of the
peak height; and R is the resolution factor.
In the applied conditions, good characteristics for the chromatographic peaks were obtained (Fig. 1), with high column
performance, calculated as the apparent number of theoretical
plates and low value of Hp. Good retention factors (between 1 and
5), were obtained for both thiamine and riboavin, which showed
symmetric peaks (tailing factor between 0.8 and 1.5), as recommended by Hsu and Chien (1994), Skoog et al. (2001) and British
Pharmacopoeia (2004). The resolution factor was above the
minimum 1.5 recommended by these authors, due to the long
separation between the peaks.
3.3. Final analytical procedure
Considering all previous assay results, the nal selected
extraction method was as follows: 15 g of cereal sample was mixed
with 90 mL 0.1 N H2SO4, adjusted to pH 4.5 with 2.5 M Na acetate
and added with papain, diastase and alpha-amylase (500 mg each).
Samples were incubated at 37  C overnight and made up to 100 mL
with distilled water, ltered through Whatman 40 paper (Maidstone, Kent, England) and through 0.45 mm polyvinylidene uoride

The developed HPLC method was validated for enriched cereal

products. Linearity, precision, accuracy and limit of detection (LOD)
for thiamine and riboavin were evaluated compared to the spectrouorimetric standard method (Tables 2e5).
3.4.1. Linearity
Triplicate calibration curves of thiamine and riboavin were
performed from standards (in 10 different concentrations between
0.05 and 75 mg/mL), and linear calibration curves were obtained for
a concentration range including those of the samples analysed. The
linearity of the method was conrmed by regression statistics.
As can be deduced from the calibration parameters (Table 2),
determination coefcients were always above 99.57%, being very
close using both methods, for either vitamin B1 or B2. The RSD of
the slopes were always below 2.4%, and those of the response
factors were not higher than 6.9%.
3.4.2. Precision
Precision of the instrumental technique was evaluated, analysing 6 standards of both vitamins in the same day (repeatability)
and in different days (reproducibility), in amounts of 0.05e0.1 mg/
mL for spectrouorimetry, and 20e40 mg/mL for HPLC. For the
whole procedure including sample extraction and instrumental
analysis, 6 equal cereal samples were analysed by both methods,
also in the same and in different days.
All the RSD obtained in this study (Table 3) were below the limits
of 11%, considered as maximum for substances around 1 mg/mL
according to AOAC (1993). Slight variations in HPLC repeatability are

Table 3
Precision assays (% RSD).
HPLC

Standards repeatability
Samples repeatability
Standards reproducibility
Samples reproducibility

Spectrouorimetry

Vitamin B1

Vitamin B2

Vitamin B1

Vitamin B2

2.84
4.78
0.353
10.17

4.46
4.88
4.6
2.67

4.86
3.29
7.43
5.16

6.44
1.85
2.38
7.36

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R. San Jos Rodriguez et al. / Journal of Cereal Science 55 (2012) 293e299

Table 4
Assays for recovery percents.

HPLC

Spectrouorimetry

CL 1
CL 2
Mean
CL 1
CL 2
Mean

Vitamin B1

Vitamin B2

X  SD (%)

X  SD (%)

95.82
85.96
93.32
101.62
94.13
97.87








9.18
17.27
12.8
15.50
13.12
19.3

100.33
109.37
104.85
111.06
107.04
109.45








10.58
11.22
11.6
18.67
17.23
15.6

X mean value (n 3); SD standard deviation (n  1); CL Concentration level.

due to small volume injection or integration variations, and were

comparable to those obtained using spectrouorimetry.
3.4.3. Accuracy assays
Accuracy and matrix effects of both methods were evaluated by
the recovery percents. Two addition levels (between 15 and 200 mg
for vitamin B1 and between 30 and 400 mg for vitamin B2) of
vitamin B1 and B2 standards were added to complex cereal samples
(Table 4). Triplicate analyses were performed for each addition
level, as well as for samples without addition, using both methods.
Mean recovery percents for the HPLC method ranged between
85.9% and 109.4%. All these values are in the interval accepted by
AOAC (1993) for substances around 10 ppm (80e110%), as it is the
case for these vitamins. Non-statistical differences (p < 0.05) were
detected for the recovery values using both analytical methods, but
RSD were lower for recovery percents in HPLC than the spectrouorimetric method.
No statistically signicant differences (p < 0.05) were found
from the comparison of the results obtained for analysis of vitamin
B1 and B2 in complex cereal commercial samples (p < 0.05) using
both methods (Table 5), so HPLC can be used for vitamin B1 and B2
analysis in complex cereal samples, with no loss of accuracy with
respect to the AOAC recommended method, and lower RSD for
recovery percents in HPLC. Both, the results obtained from HPLC
and spectrouorimetry were coincident, but different from the
level of enrichment indicated in labelling. In the case of vitamin B1,
the levels found by both methods were half of the added level,
which could be indicative of the instability of thiamine during the
shelf life of the product. In the case of vitamin B2, the analytical
levels found were coincident or higher than the addition level,
meaning that the endogenous content of riboavin may not have
been taken into account in the labelling (especially in sample
number 2, which included oat and wheat bran). These facts conrm
the necessity of accessible methodology to assess the nal vitamin
contents in enriched food products, for labelling purposes, in
agreement with ofcial regulations.
3.4.4. Limits of detection
The sensitivity of the method was evaluated as the limit of
detection estimated following ICH Guidelines (1997). For HPLC
Table 5
Analysis of cereal samples using HPLC and spectrouorimteric methods.

Vitamin B1
Complex cereal
Complex cereal
Vitamin B2
Complex cereal
Complex cereal

Enrichment
level
(mg/100 g)

HPLC
X  SD
(mg/100 g)

Spectrouorimetry
X  SD (mg/100 g)

mixture 1
mixture 2

2.3
2.3

1.14  0.20
1.30  0.29

1.26  0.06
0.90  0.22

mixture 1
mixture 2

2.7
2.0

2.75  0.07
3.57  0.10

2.85  0.20
3.48  0.10

X mean value (n 3); SD standard deviation (n  1).

analytical methods, the limit of detection (LOD) can be dened as

the concentration resulting from a signal/noise ratio of 3. The LOD
obtained for the HPLC method were 62.8 ng for vitamin B1 and
21.9 ng for vitamin B2, corresponding to 0.628 mg/100 g and
0.219 mg/100 g, respectively in the cereal samples.
For spectrouorimetry, the LOD can be calculated as:

LD 3S=m

(6)

where S is the standard deviation of the intercept, and m is the

slope of the calibration curve. Using this equation, the LOD for
spectrouorimetry is 0.0406 mg/mL (vitamin B1) and 0.025 mg/mL
(vitamin B2), corresponding to 0.203 mg/100 g and 0.125 mg/100 g,
respectively in the cereal samples.
As expected, spectrouorimetry is more sensitive than HPLC,
which however was sensitive enough to analyse this kind of cereal
sample. According to the assays performed and the results found,
the developed protocol was suitable for grains and derivatives
analysis (whole grains, ours, complex mixtures) in terms of no
signicant matrices effects, with the only limitation of the LOD
and LOQ.
4. Conclusion
The proposed method provides comparable linearity and accuracy as the reference spectrouorimetric method, being less sensitive but more precise for vitamin B1 and B2 analysis on different
complex cereal samples. It also has the advantages of the rapid and
simultaneous determination of both vitamins in a single chromatographic run (same wavelength), being less time consuming and
avoiding the use of as many reagents as ofcial methods, with a quite
simple sample preparation, and easily available equipment (isocratic
HPLC-UV). The election of the analytical method depends on the
equipment available and the sensitivity requirements of the analysis
and, in this sense, the developed HPLC method can be of great
interest for the analysis of complex enriched cereal products over
0.63 mg/100 g in vitamin B1 and 0.22 mg/100 g in vitamin B2, which
are subject to special labelling regulations.
Acknowledgements
This work has been funded by University Complutense of
Madrid (PR78/02-11037), and a grant for R. San Jose Rodriguez was
provided by the Spanish Government.
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