ACTA CHROMATOGRAPHICA, NO. 15, 2005
DETERMINATION OF SEVEN WATER-SOLUBLE
VITAMINS IN TARHANA, A TRADITIONAL TURKISH
CEREAL FOOD, BY HIGH-PERFORMANCE
LIQUID CHROMATOGRAPHY
R. Ekinci and Ç. Kadakal*
Pamukkale University, College of Engineering, Department of Food Engineering,
Çamlık, Denizli, 20020, Turkey
ABSTRACT
A reversed-phase high-performance liquid chromatographic proce-
dure has been developed for determination of water-soluble vitamins (as-
corbic acid, niacin, panthothenic acid (vitamin B5), pyridoxine (vitamin
B6), thiamine (vitamin B1), folic acid, and riboflavin (vitamin B2)) in tar-
hana samples, a traditional Turkish cereal food. Sample-treatment entails
SPE with Sep-Pak C18 (500-mg) cartridges. The water-soluble vitamins
were analyzed by HPLC on a Discovery C-18 150 mm × 4.6 mm column
with 0.1 mol L−1 KH2PO4 (pH 7.0)–methanol, 90:10, as mobile phase (0.7
mL min−1) in isocratic mode. Identification of compounds was achieved
by comparing their retention times and UV spectra with those of standards
stored in a data bank. The detection limits ranged from 0.1 to 0.5 mg L−1.
The accuracy of the method was tested by measuring average recovery;
values ranged between 96.51 and 99.40%.
INTRODUCTION
Tarhana, a traditional Turkish food, is prepared by mixing wheat
flour, yoghurt, yeast and a variety of cooked vegetables (tomatoes, onions,
green pepper etc.), salt and spices (mint, paprika) followed by fermenta-
tion for one to seven days [1]. In the central and eastern part of the Turkey,
one or more ingredients such as milk, soybean, lentil, chickpea, corn flour,
and egg are also added. [1,2]. Tarhana has an acidic and sour taste with a
strong yeast flavour and also a good source of proteins, vitamins, and mi-
nerals and is therefore used largely for feeding children and elderly people
[1,3]. Vitamins are a broad group of organic compounds that are minor, but
essential, constituents of food required for normal growth, self-maintenan-
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ce, and functioning of human and animal bodies. These compounds can be
classified in two main groups – water-soluble and fat-soluble vitamins.
Among the B group of water-soluble vitamins, both thiamine (B1) and py-
ridoxine (B6) are important. They play different specific and vital functions
in metabolism, and their lack or excess produces specific diseases. Vita-
mins are relatively unstable and can be lost during processing and storage
of food [4,5]. Because of the critical role of vitamins in nutrition and their
relative instability, qualitative and quantitative analyses are important issues
and a challenging task for food manufacturers. HPLC is the preferred tech-
nique for vitamin separation, because of its high selectivity [6]. In addition,
solid-phase extraction is necessary before HPLC to remove interfering com-
ponents [7].
The literature contains several methods, including microbiological
assay, for determination of vitamins in tarhana [8,9] but there is no report
of simultaneous determination of water-soluble vitamins in tarhana. In this
paper, a method for simultaneous determination of seven water-soluble vi-
tamins by HPLC with diode-array detection is reported. The proposed me-
thod is simple and rapid and it is possible to identify and simultaneously
determine seven water-soluble vitamins in less than 25 min with only one
injection.
EXPERIMENTAL
Materials
The tarhana samples studied were obtained from local markets (An-
kara, Turkey).
Equipment
Liquid chromatography was performed with a Shimadzu (Kyoto, Ja-
pan) system consisting of a column oven (model CTO-10ASVP), a UV–vi-
sible diode-array detector (model SPD-M10 Avp), a degasser (model DGU
14A), and a liquid chromatography pump (model LC-10AT-VP); Shimadzu
software was used to calculate peak areas. The sample (20 µL) was injec-
ted into the HPLC with a syringe (Hamilton, Reno, NV, USA). The HPLC
column used was a reversed-phase Discovery C18 (150 mm × 4.6 mm,
5 µm; #504955) from Supelco (Bellefonte, PA, USA).
Reagents
Methanol (HPLC grade) and K2HPO4 (extra pure) were obtained
from Merck (Darmstadt, Germany). Water used in all the experiments was
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doubly distilled and deionised. The vitamin standards (ascorbic acid, niacin,
pantothenic acid, pyridoxine, thiamine, folic acid, and riboflavin) were of
analytical-reagent grade from Sigma (Sigma–Aldrich, Deisenhofen-Germa-
ny) and were not further purified. Stock and standard solutions of water-
soluble vitamins were prepared in mobile phase. Five different concentra-
tions of each standard were used to prepare the calibration plot. These so-
lutions were sonicated and stored in dark glass flasks, to protect them from
light, and kept under refrigeration. A calibration plot was prepared for each
vitamin. Correlation coefficients for ascorbic acid, niacin, pantothenic acid,
pyridoxine, thiamine, folic acid, and riboflavin on the basis of plots of con-
centration (µg mL−1) against peak area (mAU) were found to be >0.999.
Sample Preparation (Solid-Phase Extraction, SPE)
Tarhana consists of many components that cause chromatographic
interferences with vitamins. For this reason the sample treatment proposed
consists of SPE with Sep-Pak C18 (500 mg) cartridges that enable separa-
tion of water-soluble vitamins and remove most of the interfering compo-
nents. Four parts of deionised water (20 g) were added into one part of tar-
hana (5 g) (dilution factor, F, = 5). The mixture was homogenized using a
homogenizer at medium speed for 1 min. The homogenized samples were
centrifuged for 10 min at 14 × 103g (Sigma, Bioblock Scientific 2-16). The
SPE method of Cho et al. [7] was used for the extraction of water-soluble
vitamins. The stationary phase was flushed with 10 mL methanol and 10
mL water adjusted to pH 4.2 to activate the stationary phase. Homogeni-
zed and centrifuged tarhana (10 mL) was the loaded. Acidified water was
prepared by adding a 0.005 M HCl solution drop by drop with stirring
until the pH reached a predetermined value. The sample was eluted with
5 mL water (pH 4.2) then 10 mL methanol at a flow rate of 1 mL min−1.
The eluent was collected in a bottle and evaporated to dryness. The residue
was dissolved in mobile phase. Before HPLC analysis all samples were
filtered through 0.45 µm pore size FP 30/45 CA-S filters (Schleicher and
Schuell, Darmstadt, Germany) at 7 bar max. Samples (20 µL) of solutions
of the water-soluble vitamins were injected into the HPLC column.
Methods
The column eluate was monitored with a photodiode-array detector
at 265 nm for vitamin C, 234 nm for thiamine, 266 nm for riboflavin, 324
nm for pyridoxine, 282 nm for folic acid, 204 nm for biotin, 261 nm for
niacin, and 204 nm for pantothenic acid. The mobile phase was filtered
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through a 0.45-µm membrane and degassed by sonication before use. The
mobile phase was 0.1 mol L−1 KH2PO4 (pH 7)–methanol, 90:10. The flow-
rate was 0.7 mL min−1. The column was operated at room temperature (25°C).
Chromatographic peak data were integrated up to 39 min. Identification of
compounds was achieved by comparing their retention times and UV spec-
tra with those of standards stored in a data bank. Concentrations of the wa-
ter-soluble vitamins were calculated from integrated areas of the sample
and the corresponding standards.
Recovery of Water-Soluble Vitamins
Tarhana samples containing known amounts of ascorbic acid, nia-
cin, panthothenic acid pyridoxine, thiamine, folic acid, and riboflavin were
spiked with two addition levels of standard ascorbic acid, niacin, pantho-
thenic acid, pyridoxine, thiamine, folic acid, and riboflavin to determine the
recovery. Six determinations were performed for each level of addition.
RESULTS AND DISCUSSION
The isocratic separation published by Supelco [10] was modified
by increasing the concentration of KH2PO4 from 0.05 mol L−1 to 0.1 mol
L−1, reducing the solvent flow rate from 1.0 to 0.7 mL min−1, reducing the
column temperature from 35 to 25°C and setting detection at 265 nm for
vitamin C, 234 nm for thiamine, 266 nm for riboflavin, 324 nm for pyrido-
xine, 282 nm for folic acid, 204 nm for biotin, 261 nm for niacin, and 204
nm for pantothenic acid. In addition, KH2PO4 content was reduced from
99% to 90%, and so methanol content was increased from 1% to 10%.
To the best of our knowledge there are no published research stu-
dies about HPLC determination of water-soluble vitamins in tarhana. We
analyzed tarhana samples using a Discovery C-18 reversed phase HPLC
column, 15 cm × 4.6 mm i.d., 5 µm particles, to determine the water-so-
luble vitamins. Because each of these water-soluble vitamins has its own
maximum absorbance at a different wavelength, the detection wavelengths
were set at the wavelength of maximum absorption for each vitamin to ob-
tain higher sensitivity.
For separation and determination of water-soluble vitamins in tar-
hana, the best results were obtained with 0.1 mol L−1 KH2PO4 (pH 7)–
methanol mobile phases. When the proportion of methanol was more than
10%, the vitamins ascorbic acid, niacin, pantothenic acid, pyridoxine, thia-
mine, and folic acid eluted in less than 14 min (within 5 min) but resolu-
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tion was very poor. In contrast, riboflavin eluted at 32 min and resolution
of riboflavin was very good only when the proportion of methanol was
more than 10%. We used a mobile phase containing 10% methanol, which
increased the total analysis time but enabled better resolution than other
compositions. Also, riboflavin was detected when the mobile phase contai-
ned 10% methanol. Thus, only riboflavin can be detected in tarhana samples
when the proportion of methanol was more than 10%. On the other hand,
ascorbic acid, niacin, pantothenic acid, pyridoxine, thiamine, folic acid, and
riboflavin eluted and were detected well when the mobile phase contained
10% methanol.
The pH of the KH2PO4 was a critical factor for separation of the vi-
tamins. KH2PO4 at pH from 3 to 7 was studied and all water-soluble vita-
mins were well resolved only at pH 7.0. When the pH of KH2PO4 was diffe-
rent from 7, however, the resolution was poor because ascorbic acid, niacin,
pantothenic acid, pyridoxine, thiamine, and folic acid eluted within 5.0 min.
Changing the temperature from 20 to 35°C reduced the retention
times slightly but had no effect on the quantitative results.
Figure 1 shows the chromatogram obtained from tarhana by use of
KH2PO4 (pH 7)–methanol, 80:20, as the mobile phase. Resolution is poor
Fig. 1
Separation of water-soluble vitamins by isocratic elution with KH2PO4 (pH 7)–methanol,
80:20, as mobile phase.
except for riboflavin when the methanol content of the mobile phase in-
creased from 10% to 20%. Figure 2 shows a typical chromatogram obtai-
ned from a tarhana sample when the 0.1 mol L−1 KH2PO4 (pH 7)–metha-
nol, 90:10, was used as mobile phase. As shown in Fig. 2, good separation
can be achieved in 37 min. It is apparent from the chromatogram that as-
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corbic acid, niacin, pantothenic acid, pyridoxine, thiamine, folic acid, and
riboflavin are separated well. Identification of the compounds was achieved
by comparing their retention times and UV spectra with those of standards
stored in a data bank.
Fig. 2
Separation of water-soluble vitamins by isocratic elution with KH2PO4 (pH 7)–methanol,
90:10, as mobile phase. Peaks: 1. ascorbic acid, 2. niacin, 3. pantothenic acid, 4. pyrido-
xine, 5. thiamine, 6. folic acid, 7. riboflavin
Analytical Characteristics of the HPLC Method
Linearity and Detection Limits
As seen from Table I, an r value >0.9985 was obtained for all vita-
mins. Coefficients of determination (r2) were greater than 99.67% for all vi-
tamins. The detection limits (S/N = 3) [11] ranged from 0.1 to 0.5 mg L−1.
Table I
Linearity of standard curves and detection limits for the seven water-soluble vitamins
Vitamin Linear range (mg L−1) R r
2 Detection limit (mg L−1)
Ascorbic acid 5.0–200.0 0.9992 99.90 0.1
Niacin 10.0–200.0 0.9996 99.93 0.1
Panthothenic acid 1.0–30.0 0.9989 99.83 0.5
Pyridoxine 0.5–30.0 0.9990 99.82 0.1
Thiamine 1.0–50.0 0.9985 99.69 0.5
Folic acid 0.5–20.0 0.9992 99.67 0.1
Riboflavin 1.0–40.0 0.9998 99.94 0.2
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Precision
Six determinations of the same tarhana sample were performed using
the same reagents and apparatus to evaluate the method precision in tarha-
na. The precision of the method for determination of seven water-soluble
vitamins in tarhana is shown in Table II.
Table II
Precision of method for determination of water-soluble vitamins in tarhana
Vitamin Mean ± S.D.a (mg kg−1) R.S.D. (%)
Ascorbic acid 17.8 ± 0.67 2.63
Niacin 12.4 ± 0.54 3.12
Panthothenic acid 5.3 ± 0.28 3.54
Pyridoxine 0.38 ± 0.08 5.62
Thiamine 4.7 ± 0.20 3.30
Folic acid 0.40 ± 0.09 4.98
Riboflavin 1.8 ± 0.17 4.41
aMean ± standard deviation
Recovery
Recovery was tested by the standard addition procedure. Two addi-
tion levels were used for each water-soluble vitamin in tarhana samples
(Table III). Six determinations were performed for each addition level. Mean
recoveries obtained were always satisfactory – higher than 99% for niacin,
higher than 98% for ascorbic acid, pantothenic acid, pyridoxine and thia-
mine, higher than 97% for riboflavin, and higher than 96% for folic acid.
The average recoveries of ascorbic acid, niacin, pantothenic acid, pyrido-
xine, thiamine, folic acid, and riboflavin in tarhana were 98.77%, 99.40%,
98.28%, 98.74%, 98.40%, 96.51%, and 97.96% respectively, for added
levels of two different concentrations.
CONCLUSION
A new method is proposed for separation and quantification of se-
ven water-soluble vitamins – ascorbic acid, niacin, panthothenic acid (vi-
tamin B5), pyridoxine (vitamin B6), thiamine (vitamin B1), folic acid, and
riboflavin (vitamin B2). SPE proved an effective tool for isolation of the se-
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Table III
Recovery of method for determination of water-soluble vitamins in tarhana
Content after addition
(mg kg−1) Recovery (%) Vitamin
Initial
content
(mg kg−1) Level Ia Level IIb Mean ± S.D.c R.S.D. (%)
Ascorbic acid 17.8 37.22 ± 0.28 57.36 ± 0.30 98.77 ± 0.62 0.75
Niacin 12.4 22.34 ± 0.20 32.17 ± 0.19 99.40 ± 0.54 0.60
Panthothenic
acid 5.3 10.05 ± 0.16 15.11 ± 0.14 98.28 ± 0.60 0.74
Pyridoxine 0.38 0.86 ± 0.08 1.37 ± 0.10 98.74 ± 0.71 1.1
Thiamine 4.7 9.49 ± 0.12 14.67 ± 0.13 98.40 ± 0.52 0.76
Folic acid 0.40 0.86 ± 0.05 1.36 ± 0.10 96.51 ± 1.08 1.33
Riboflavin 1.8 3.72 ± 0.10 5.69 ± 0.11 97.96 ± 0.85 0.96
a20 mg for ascorbic acid, 10 mg for niacin, 5 mg for pantothenic acid and thiamine, 0.5 mg
for pyridoxine and folic acid and 2 mg for riboflavin
b40 mg for ascorbic acid, 20 mg for niacin, 10 mg for pantothenic acid and thiamine, 1 mg
for pyridoxine and folic acid and 4 mg for riboflavin
cMean ± standard deviation
ven water-soluble vitamins and HPLC enabled rapid, accurate, and reliab-
le determination of the compounds with recoveries ranging from 96.51 to
99.40%. The isocratic mobile phase used for the vitamins resulted in high
detection limits and good resolution within a minimum analysis time of 37
min.
ACKNOWLEDGEMENT
The authors thank Professor Dr Sebahattin Nas, Food Engineering
Department, Pamukkale University, for the helpful discussions.
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