See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/237560812

Fast simultaneous determination of multiple water-soluble vitamins and

vitamin-like compounds in infant formula by UPLC-MS/MS

Article in Journal of Food Agriculture and Environment · April 2009

DOI: 10.17925/ENR.2009.04.01.88

CITATIONS READS

14 1,496

4 authors, including:

Hong Zhang Si Chen

Zhejiang Gongshang University Zhejiang Gongshang University
856 PUBLICATIONS 21,614 CITATIONS 4 PUBLICATIONS 80 CITATIONS

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Hong Zhang on 29 March 2014.

The user has requested enhancement of the downloaded file.

 WFL Publisher
Science and Technology

Meri-Rastilantie 3 B, FI-00980 Journal of Food, Agriculture & Environment Vol.7 (2) : 88-93. 2009 www.world-food.net
Helsinki, Finland
e-mail: info@world-food.net

Fast simultaneous determination of multiple water-soluble vitamins and vitamin-like

compounds in infant formula by UPLC-MS/MS
Hong Zhang 1*, Si Chen 1, Wenjuan Liao 1 and Yiping Ren 2
1
College of Food Science and Biological Engineering, Zhejiang Gongshang University, No. 149 Jiaogong Road, Hangzhou,
Zhejiang 310035, P.R. China. 2 Zhejiang Provincial Centre for Disease Prevention and Control, No. 630 Xincheng Road,
Hangzhou, Zhejiang 310051, P.R. China. *e-mail: zhanghong163@hotmail.com, sichen31@hotmail.com, chilly.333@163.com,
renyiping@263.net

Received 18 January 2009, accepted 14 April 2009.

Abstract
Fortification of infant formula provides the sole source of nutrition for bottle-fed baby; therefore, particular attention should be paid to ensure an
adequate and balanced intake of vitamins, which cannot be synthesized by the body, but essential for the normal growth and functioning of human
body. An Ultra Performance LC-Tandem Mass Spectrometry (UPLC-MS/MS) method was developed for fast simultaneous determination of 14
different water-soluble vitamins and vitamin-like compounds in infant formula, and methotrexate and nicotinamide (D4) were used as internal
standards. To achieve a high sensitivity, selectivity and accuracy for the analyte, the most crucial factors, such as mass spectrometry parameter,
column and mobile phase choosing were optimized. Identification and quantification were performed by using multiple reaction monitoring (MRM)
with one parent ion, one daughter ion for each analyte, and electrospray ionization in positive ion scan mode. A chromatographic separation within
10 minutes was achieved by using a BEH Shield RP18 (2.1 mm ×100 mm, 1.7 µm, Waters) column with a mobile phase program, consisting of
methanol and 10 mM aqueous ammonium acetate. Recoveries were 81.8-106.1%, the limits of detection were 0.0042-28.872 ng‚ml-1 (inject 10 µl),
with Relative Standard Deviation (RSD) of 1.17-5.09% for intra-day and 2.61-7.43% for inter-day. Different water-soluble vitamins and vitamin-like
compounds at a wide range of concentration level in 14 different commercial samples of infant formula, rice flour and wheat powder were tested using
developed method and the results showed that it has offered certain advantages compared to previous reports and provided important and reliable
information for government regulation, quality control and nutrition labeling.

Key words: UPLC-MS/MS, water-soluble vitamins, vitamin-like compounds, infant formula.

Introduction
Vitamins comprise a structurally heterogeneous group of
compounds, essential for the normal growth and functioning of
human body, which cannot be synthesized by the body and has
to be supplied in the diet. Early nutritional studies classified two
general groups of such compounds based on their solubility in
non-polar organic solvents as fat-soluble or water-soluble
vitamins 1. The latter (water-soluble vitamins) mainly include
vitamin B1 (thiamine), B2 (riboflavin), B6 (pyridoxine, pyridoxamine,
pyridoxal), B12 (cobalamin), C (ascorbic acid), pantothenic acid,
biotin, folic acid, nicotinic acid, niacinamide, L-carnitine, choline,
taurine, etc. They play different specific and vital functions in
metabolism, and their lack or excess produces specific
diseases2-3.
WHO statistical data revealed that only 35% of infants have
been breastfed during the first four months after birth in the
research of 94 countries from 1994 to 2000 4. Compared with the
adult requirements, there are additional needs for the normal
growth of infants. Therefore, water-soluble vitamins fortification
of certain food products, in particular infant formula which is the
sole source of nutrition, is needed to ensure an adequate and
balanced intake of vitamins. The Dietary Reference Intake (DRI)
for the year 2000 5 and the Food Additive Handbook 6 have issued
the reference value of infants’ daily requirement of vitamins and
additive amount of vitamins in infant formula, respectively.
However, loss of water-soluble vitamins often occurs during
inappropriate processing and storage. Quantification of these
vitamins in infant formula is required for the purposes of
government regulation, quality control and nutrition labeling.
These facts lead to a need for a reliable, rapid and sensitive
simultaneous determination of multiple water-soluble vitamins and
vitamin-like compounds in infant formula.
The most common officially accepted methods for water-soluble
vitamin analysis are often based on microbiological assays, which
are labor intensive and sometimes not completely specific 7-11.
High-performance liquid chromatography (HPLC) method, a better
choice for determining water-soluble vitamins, offers simplicity,
speed, sensitivity and specificity 12-18. However, much effort has
been focused on the RP-chromatography method 16, 17 which usually
was combined with complex and costly ion-pair reagent 18. The
electrophoresis method 19-22 and Fourier Transform Infrared

88 Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009

Spectroscopy (FTIR-ATR) method 23 are also used for determining
vitamins. The former is rapid, inexpensive, and has a promising
prospect of application; the latter is not very common. The methods
described above are mainly applied to determine high-vitamin
medicines and chemical reagents. The highly selective and
sensitive high-performance liquid chromatography-tandem mass
spectrometry (LC-MS/MS) method could allow the determination
of samples with low vitamin content. Nine water-soluble vitamins
in pasta have been quantified by LC-MS/MS method using a
specific reversed-phase C16 amide column, which took 14 minutes
to test per running 3. Chen et al.24 adopted HPLC/ESI-MS method
to determine 10 water-soluble vitamins and taurine simultaneously
in the multi-vitamin tablet, within 30 minutes per running.
There are more than ten kinds of water-soluble vitamins and
vitamin-like compounds as only micro-constitutes in infant formula
milk having the common property of being relatively unstable and
easily damaged, also they are present in distinct chemical
structures and properties which makes the efficient separation
extremely difficult. Another problem was that these components
do not exist in real samples with a uniform concentration range.
Their biopotencies and recommended daily allowances are widely
scattered. B12 and folic acid must therefore exist in the samples in
small quantities, while others are at higher levels 25. Also the
determination of multiple vitamins represents a complex analytical
problem for mass spectrometry due to its sensitivity to the serious
interfering caused by complicated infant formula matrixes
themselves. Therefore, a novel multi-vitamin analysis method using
ultra-performance liquid chromatography (UPLC) tandem mass
spectrometry was developed in this paper.
Experimental
Reagents and chemicals: HPLC-grade solvents (Merck & Co.,
INC, USA) were obtained from Huadong Shiji Chemicals,
ammonium acetate (99.4%), formic acid (88%), ammonia and
methotrexate (internal standard 99%) (Fluka, USA) from Guoyao
Chemicals. Thiamine, riboflavin, pantothenic acid, nicotinic acid,
nicotinamide, pyridoxine, pyridoxal, biotin, folic acid,
cyanocobalamin, ascorbic acid, carnitine, choline, taurine,
methotrexate and nicotinamide (D4) were obtained through Sigma-
Aldrich (St. Louis, MO, USA) and their purity were more than
98% according to the manufacturer. Water for HPLC was treated
with a Milli-Q water purification system (Millipore, MA, USA).
Some of infant formula milks were supplied from Dumex and Nestlé,
others were obtained from local supermarket.
Each standard of 0.5 mg was accurately weighed and diluted
with 10 ml water to prepare stock solutions; folic acid and
pantothenic acid required use of ammonia and riboflavin required
use of hydrochloric acid for complete dissolution. Ascorbic acid
was prepared daily, the other solutions were prepared weekly.
Methotrexate and nicotinamide (D4) working solutions were
diluted to 40 and 10 µg‚ml-1, respectively. The mixed solution
containing the 14 analytes was papered by pooling the appropriate
amount of the each standard solutions and diluting with 10 mM
aqueous ammonium acetate. All the solutions were preserved
under 0°C and avoided light.
Sample preprocessing: One gram of homogenized sample was
accurately weighed and placed into a 50 ml centrifuge tube. After
adding 500 µl of 40 ng‚ml-1 methotrexate, 500 µl of 10 µg‚ml-1
Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009
nicotinamide (D4) internal standard solutions and 9 ml of 10 mM
aqueous ammonium acetate, the mixture was vortex mixed for 1
min and extracted in an ultrasonic bath for 15 min. Subsequently
10 ml of chloroform was added, vortex mixed for 1 min and
centrifuged for 10 min at 10,000 rpm. The supernatant was collected,
and filtered through the 0.22 µm micropore membrane.
Apparatus: UPLC was performed on an ACQUITY UPLC system
(Waters Corp., Milford, MA, USA) equipped with a binary solvent
delivery system, an autosampler and a TUV detector.
Chromatography was performed on an ACQUITY UPLC BEH
Shield RP18 column (2.1 mm×100 mm, 1.7 µm, Waters) at a flow rate
of 0.2 ml‚min-1 and the injection volume was 10 µl. The mobile
phase was a multistep linear solvent gradient system consisting
of (A) 10 mM aqueous ammonium acetate and (B) methanol. The
elution profile was: 2.4 min 100% A, then the solvent B was
increased first to 11% in 2 min, thereafter to 30% in 0.2 min, to 32%
in 1.9 min and further increased to 100% in 0.2 min, stayed constant
till 7.0 min, and subsequently decreased to 0% B in 0.2 min and 2.8
min for equilibration.
The mass spectrometry was performed on a Micromass Quattro
Ultima TM Pt triple quadrupole mass spectrometer (Waters Corp.,
Milford, MA, USA). Column effluent was introduced into the
electrospray chamber with a 0.1 mm internal diameter fused silica
capillary. Electrospray ionization spray was positive ion scan mode,
capillary voltage was 3.50 kV, cone voltage was 55 V; source
temperature and de-solvent temperature were 120 and 350°C,
respectively. Nitrogen was used as sheath gas at 400 l‚hr-1 and as
the auxiliary gas at 50 l‚hr-1 and argon as the collision gas at
3.0×10-3 mbar. Identification and quantification were performed
by multiple reactions monitoring (MRM).
Results and Discussion
Optimization of MS/MS parameters: The optimization of
quantitative ion and impact energy are extremely important for
various vitamins determination by MRM. For vitamin B1, parent
ion at m/z 265.2 and two stable, abundant daughter ions at m/z
122.2 and m/z 144.2 were found by MS scan and daughter scan,
respectively. One more abundant daughter ion at m/z 122.2 was
selected as quantitative ion, the other one was selected as auxiliary
qualitative ion, and the former was the most intense using collision
energy at 8 eV. In the same way, quantitative ion, auxiliary
qualitative ion and optimum impact energy of the internal standards
and other compounds were chosen and are indicated in Table 1.
Chromatography optimization: Choosing a suitable
chromatographic column gives the possibility to perform various
water-soluble vitamins and vitamin-like compounds separation in
one run with better resolution. An Ultra Performance LC column
shows a smaller system volume and greater throughput to separate
more peaks in shorter run time, due to its smaller particles and
ultra-high pressure. ACQUITY UPLC BEH C18 column (2.1 mm
×100 mm 1.7, µm, Waters), ACQUITY UPLC BEH Shield RP18 column
(2.1 mm ×100 mm, 1.7 µm, Waters) and Antilants C18 column (2.1
mm × 150 mm, 5 µm, Waters) were selected to evaluate the column
performance on the basis of retention factor in this study. The
data of this comparison showed that BEH Shield RP18 column was
the most appropriate for the separation and this column was
selected for the present work.
89
 Table 1. LC-MS-MS parameters using MRM mode.
Auxiliary
Parent ion Daughter ion Collision Collision energy
Name qualitative ion
(m/z) (m/z) energy (eV) (eV)
(m/z)
Thiamine 265.2 122.2 8 144.2 8
Riboflavin 377.5 243.3 18 257.2 18
Pantothenic acid 219.9 89.9 12 202.0 11
Nicotinic acid 124.1 80.1 12 78.1 12
Nicotinamide 123.1 80.1 10 96.1 12
Pyridoxine 170.2 152.1 8 134.1 12
Pyridoxal 168.1 150.0 12 122.1 16
Biotin 244.9 226.9 10 172.0 19
Folic acid 442.2 295.1 10 176.9 16
Cyanocobalamin 678.9 a 147.0 30 359.2 20
Ascorbic acid 177.0 141.0 5 94.9 10
L-Carnitine 162.0 60.0 12 102.8 12
Choline 104.1 a 60.1 15 45.1 18
Taurine 126.0 107.8 8 44.0 10
Methotrexate 455.5 308.0 20 174.8 36
Nicotinamide (D4) 127.3 84.4 10 83.3 10
a. For cyanocobalamin and choline, the doubly charged ion ([M+2H]2+) at m/z 678.9 and [M-H2O]+ ion at m/z 104.1 were selected as parent ions, respectively.

A I The mobile phase has a significant effect on peak

B J
C K
Relative Abundance
shape, sensitivity and resolution. Identical injections
of standard mixture were carried out to evaluate the
signal-to-noise ratio and retention time of every
compound using different mobile phase composition:
acetonitrile or methanol with 0.1% (v/v) formic acid
aqueous solution and acetonitrile or methanol with 10
mM aqueous ammonium acetate. After using a series

of optimization, limit of detection, resolution and peak

D L
E M
F N
G O
H P
Relative Abundance
shape for specific compounds were not quite satisfied
with most of the mobile phase described, however,
methanol with 10 mM aqueous ammonium acetate gave
the best overall performance and was deemed to be
most suitable when all 14 compounds were considered.
Fig.1 indicates MRM and total ion current
chromatograms of water-soluble vitamins and vitamin-
like compounds standard under best program
condition.
Validation of the analytical procedure: The calibration
curves were obtained by plotting concentration against
peak area. For each analyte, a series of five concentration
points were prepared and each solution was injected
three times. Methotrexate was used as internal standard

Time (min) Time (min)

for riboflavin, cyanocobalamin, biotin and folic acid,
Q
while nicotinamide (D4) was used as internal standard
for the other compounds. The correlations (r2) for
calibration curve were all greater than 0.9989. Linear
range, limit of detection (LOD) and intra- and inter-day
Time (min)
precision of the vitamins are shown in Table 2. The
Figure 1. Multiple reaction monitoring (MRM) of: (A) Thiamine (m/z 265.2>122.2);
recoveries shown in Table 3 were evaluated at three
(B) Riboflavin (m/z 377.5>243.3); (C) Pantothenic acid (m/z 219.9>89.9); (D) levels. These results indicate that the method
Nicotinic acid (m/z 124.1>80.1); (E) Nicotinamide (m/z 123.1>80.1); (F) Pyridoxine developed was accurate, precise and reproducible.
(m/z 170.2>152.1); (G) Pyridoxal (m/z 168.1>150); (H) Biotin (m/z 244.9>226.9);
(I) Folic acid (m/z 442.2>295.1); (J) Cyanocobalamin (m/z 678.9>147); (K) Ascorbic
acid (m/z 177>141); (L) L-Carnitine (m/z 162>60); (M) Choline (m/z 104.1>60.1);
Sample test: To verify the applicability and reliability,
(N) Taurine (m/z 126>107.8); (O) Methotrexate (m/z 455.5>308); (P) Nicotinamide 14 different commercial samples of infant formula, rice
(D4) (m/z 127.3>84.4) and (Q) total ion current chromatograms of vitamins standard. flour and wheat powder were analyzed according to
Identification and quantification were performed by using MRM with one parent the method established, and the results are presented
ion, and one daughter ion for each analyte. in Table 4. Vitamin C was not detected from all the

90 Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009

Table 2. Linear range, LOD (S/N = 3) and intra and inter-day precision of the vitamins.
Vitamin Linear range LOD Intra-day variability Inter-day variability
(µg ‚l-1) (µg ‚l-1) R.S.D. (n = 11) (%) R.S.D. (n = 11) (%)
Thiamine 0.5-50 0.279 2.27 2.81
Riboflavin 1.0-100 0.0042 5.09 7.43
Pantothenic acid 0.5-50 0.153 3.11 5.23
Nicotinic acid 5-500 0.129 2.66 7.24
Nicotinamide 2.5-250 0.174 4.52 6.13
Pyridoxine 0.25-25 0.075 3.00 4.83
Pyridoxal 0.25-25 0.075 3.31 3.78
Biotin 0.25-25 0.150 3.80 6.45
Folic acid 1.0-100 0.045 2.27 3.42
Cyanocobalamin 2.5-250 0.036 2.70 3.55
Ascorbic acid 100-10,000 28.872 7.81 8.42
L-Carnitine 0.5-50 0.111 4.06 5.80
Choline 0.5-50 0.213 1.17 2.61
Taurine 100-10,000 22.140 3.18 3.70

Table 3. Recoveries obtained for each vitamin evaluated at three levels.

Vitamin Low concentration Medium concentration High concentration
Added Recovery Added Recovery Added Recovery
ng·ml-1 (%) ng·ml-1 (%) ng·ml-1 (%)
Thiamine 4 104.7 40 99.6 90 97.1
Riboflavin 8 98.8 80 117.4 180 90.3
Pantothenic acid 4 103.1 40 100.6 90 96.4
Nicotinic acid 40 89.7 400 87.5 900 103.1
Nicotinamide 20 86.8 200 94.3 450 89.8
Pyridoxine 2 105.6 20 96.0 45 94.4
Pyridoxal 2 100.5 20 97.5 45 95.3
Biotin 2 105.6 20 97.8 45 98.5
Folic acid 8 105.4 80 96.6 180 97.0
Cyanocobalamin 20 122.3 200 85.7 450 84.7
Ascorbic acid 800 75.8 8,000 60.9 18,000 77.1
L-Carnitine 4 103.7 40 90.6 90 89.7
Choline 4 85.3 40 74.8 90 62.1
Taurine 800 87.0 8,000 77.2 18,000 80.5

samples. Seven out of the 9 vitamins natural content in 5 different
pasta samples but only 4 vitamins in fortified pasta samples were
analyzed successfully by a LC-MS/MS method 3. The results
demonstrated that the proposed method in this paper is enough
sensitive and selective to be applied to the determination of these
compound at a wide range of concentration level in real samples.
The values for riboflavin and nicotinamide were 103 times higher
than the value for cyanocobalamin. Several compounds, like
nicotinic acid, biotin, L-carnitine and taurine, were found at
different levels in infant formula, rice flour and wheat powder. The
primary advantage of this method is that the shorter resolution
time benefits from the UPLC system and the selectivity of the
detector allows co-eluting compounds to be detected separately.
Thus, compared to previous reports 2-4, this UPLC- ESI-MS/MS
method offers lower LODs and faster determination for more
compounds within 10 minutes per running. Besides, this method
performed in positive ion scan mode no longer needs switching
continuously from positive mode to negative ion mode as the
method developed by Chen et al. 24. For these reasons, this method
provides a promising prospect of application for large-scale studies
and other kinds of foods, like cereals, pastas and functional
beverages.
Conclusions
An UPLC-MS/MS method has been developed for fast
simultaneous separation and determination of 14 different water-
soluble vitamins and vitamin-like compounds in infant formula,
rice flour and wheat powder at low level (ng‚ml-1). A simple sample
preprocessing compatible with MS was performed. This method
was used to analyze the commercial samples on the market, and
the results prove it is a promise in controlling the quality of the
infant formula and protecting healthy development of term infants.

Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009 91

 Table 4. Results of the LC-ESI-MS/MS determination of the compound contents in samples.
Sample Thiamine Riboflavin Pantothenic acid Nicotinic acid Nicotinamide Pyridoxine Pyridoxal
(mg/100 g) (mg/100 g) (mg/100 g) (µg/100 g) (mg/100 g) (mg/100 g) (µg/100 g)
Ma 1 0.289 3.750 1.094 1.200 1.253 0.162 14.686
M2 0.649 5.177 1.621 㧙 3.906 0.369 14.645
M3 0.338 6.353 1.184 1.390 3.999 0.254 15.009
M4 0.585 5.846 1.283 2.711 4.652 0.380 16.623
M5 0.663 3.139 1.227 61.134 3.551 0.247 15.967
M6 0.572 6.058 1.238 38.539 1.036 0.205 12.124
M7 0.658 4.682 1.451 71.057 1.323 0.279 13.391
M8 0.999 6.956 1.195 154.350 4.193 0.294 64.213
M9 0.853 13.312 1.253 529.761 0.098 0.171 15.421
M 10 0.566 13.797 0.491 409.900 0.122 0.546 15.177
Rb 1 1.700 16.322 2.822 913.296 0.068 0.157 3.056
R2 1.476 2.327 1.591 36.927 3.307 0.094 2.229
R3 0.777 11.093 0.724 215.111 0.074 0.434 4.090
Wc 1 1.502 4.974 1.272 67.623 3.820 0.141 15.766
Biotin Folic acid Cyanocobalamin Ascorbic acid L-Carnitine Choline Taurine
(µg/100 g) (mg/100 g) (µg/100 g) (mg/100 g) (µg/100 g) (µg/100 g) (µg/100 g)
Ma 1 76.230 0.224 9.542 㧙 132.123 356.414 85.348
M2 79.527 0.571 12.319 㧙 151.895 327.995 57.561
M3 103.874 0.328 11.389 㧙 143.081 331.862 54.554
M4 81.457 0.283 㧙 㧙 121.827 312.748 11.569
M5 97.282 0.081 3.749 㧙 93.844 252.772 㧙
M6 254.163 0.118 8.560 㧙 33.842 389.849 18.767
M7 147.240 0.191 9.945 㧙 71.893 414.743 13.788
M8 207.661 0.402 11.452 㧙 88.636 476.991 2375.696
M9 271.453 0.835 2.261 㧙 21.154 365.833 190.829
M 10 474.254 0.938 㧙 㧙 24.537 84.827 㧙
Rb 1 36.364 2.874 1.382 㧙 9.480 434.374 746.215
R2 208.498 0.104 5.426 㧙 3.388 253.439 35.888
R3 82.211 0.017 1.254 㧙 3.443 342.050 㧙
Wc 1 342.296 0.043 7.390 㧙 35.587 330.832 8.948
a. M = powdered milk, b. R = powdered rice, c. W = powdered wheat. -: analyte was not detected from the sample.

References
1
Zheng, J. and Chen, J.H. 2001. General Biochemistry (in Chinese).
Higher Education Press, Beijing, 211 p.
2
Sizer, F.S. and Whitney, E.N. 2000. Nutrition: Concepts and
Controversies. Wadsworth/Thomson Learning, Belmont, 210 p.
3
Leporati, A., Catellani, D., Suman, M., Andreoli, R., Manini, P. and
Niessen, W.M.A. 2005. Application of a liquid chromatography tandem
mass spectrometry method to the analysis of water-soluble vitamins
in Italian pasta. Anal. Chim. Acta 531:87-95.
4
Kou, M.Y. and Han, J.Q. 2005. Study on the quality standards and
development trend of infant formula milk. China Dairy 11:38.
5
Chinese Nutrition Society 2000. Chinese DRIs (in Chinese). China Light
Industry Publishing House, Beijing, 305 p.
6
China Food Additive Production and Application Industry Association
1996. Food Additives Handbook (in Chinese). China Light Industry
Publishing House, Beijing, 12 p.
7
Strohecker, R. and Henning, H. M. 1965. Vitamin Assay - Tested
Methods. Verlag Chemie GmbH., Weinheim, 34 p.
8
USP 1990. The United States Pharmacopeia/the National Formulary.
17th edn. USP Convention Inc., Rockville.
9
Tanner, J.T. and Barnett, S.A. 1986. Methods of analysis for infant
formula: Food and Drug Administration and Infant Formula Council
Collaborative Study, Phase III. J. Assoc. Off. Anal. Chem. 69:777-
785.
10
Tanner, J.T., Barnett, S.A. and Mountford, M.K. 1993. Analysis of
milk-based infant formula. Phase V. Vitamins A and E, folic acid, and
pantothenic acid. Food and Drug Administration, Infant Formula
Council: Collaborative study. J. AOAC Int. 76:399-413.
11
National Standards of P. R. China. General Test Method for Infant
Formula. Standard Number GB/T5413.14-1997.
12
Iwase, H. 2003. Routine high-performance liquid chromatographic
determination of ascorbic acid in foods using L-methionine for the pre-
analysis sample stabilization. Talanta 60:1011-1021.
13
Moreno, P. and Salvadó, V. 2000. Determination of eight water- and
fat-soluble vitamins in multi-vitamin pharmaceutical formulations by
high-performance liquid chromatography. J. Chromatogr. A. 870:207-
215.
14
Cho, C.M., Ko, J.H. and Cheong, W.J. 2000. Simultaneous determination
of water-soluble vitamins excreted in human urine after eating an
overdose of vitamin pills by a HPLC method coupled with a solid
phase extraction. Talanta 51:799-806.
15
García, L., Blázquez, S., San Andrés, M.P. and Vera, S. 2001.
Determination of thiamine, riboflavin and pyridoxine in pharmaceuticals
by synchronous fluorescence spectrometry in organized media. Anal.
Chim. Acta 434:193-199.
16
Höller, U., Brodhag, C., Knobel, A., Hofmann, P. and Spitzer, V. 2003.
Automated determination of selected water-soluble vitamins in tablets
using a bench-top robotic system coupled to reversed phase (RP-18)
HPLC with UV detection. J. Pharm. Biomed. Anal. 31:151-158.
17
Viñas, P., López-Erroz, C., Balsalobre, N. and Hernández-Córdoba,
M. 2003. Reversed-phase liquid chromatography on an amide
stationary phase for the determination of the B group vitamins in
baby foods. J. Chromatogr. A 1007:77-84.
18
Ivanovic, D., Popovic, A., Radulovic, D. and Medenica, M. 1999.
Reversed-phase ion-pair HPLC determination of some water-soluble

92 Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009

 vitamins in pharmaceuticals. J. Pharm. Biomed. Anal. 18:999-1004.
19
Buskov, S., Møller, P., Sørensen, H., Sørensen, J.C. and Sørensen, S.
1998. Determination of vitamins in food based on supercritical fluid
extraction prior to micellar electro kinetic capillary chromatographic
analyses of individual vitamins. J. Chromatogr. A 802:233-241.
20
Gomis, D.B., González, L.L. and Álvarez, D.G. 1999. Micellar
electrokinetic capillary chromatography analysis of water-soluble
vitamins. Anal. Chim. Acta 396:55-60.
21
Delgado-Zamarreño, M.M., González-Maza, I., Sánchez-Pérez, A. and
Carabias-Martínez, R. 2002. Separation and simultaneous
determination of water-soluble and fat-soluble vitamins by
electrokinetic capillary chromatography. J. Chromatogr. A 953:257-
262.
22
Shabangi, M. and Sutton, J.A. 2005. Separation of thiamin and its
phosphate esters by capillary zone electrophoresis and its application
to the analysis of water-soluble vitamins. J. Pharm. Biomed. Anal.
38:66-71.
23
Wojciechowski, C., Dupuy, N., Ta, C.D., Huvenneb, J.P. and Legrandb,
P. 1998. Quantitative analysis of water-soluble vitamins by ATR-
FTIR spectroscopy. Food Chem. 63:133-140.
24
Chen, Z., Chen, B. and Yao, S.Z. 2006. High-performance liquid
chromatography/electrospray ionization-mass spectrometry for
simultaneous determination of taurine and 10 water-soluble vitamins
in multivitamin tablets. Anal. Chim. Acta 569:169-175.
25
Dalbacke, J. and Dahlquist, I. 1991. Determination of vitamin B12 in
multivitamin-multimineral tablets by high-performance liquid
chromatography after solid-phase extraction. J. Chromatogr. 541:383-
392.

Journal of Food, Agriculture & Environment, Vol.7 (2), April 2009 93

View publication stats