ISSN: 0970-020 X

ORIENTAL JOURNAL OF CHEMISTRY CODEN: OJCHEG

An International Open Free Access, Peer Reviewed Research Journal
2016, Vol. 32, No. (2):
Pg. 947-953
www.orientjchem.org

Analysis of Water Soluble Vitamins (Thiamine, Nicotinamide

and Pyridoxine) in Fortified Infant Food Products by HPLC
NARJIS NAZ1, AIZA KASHIF1, WARDA SHEIKH1,
MATEEN ABBAS2 and ABDUL MUQEET KHAN2

Department of Chemistry, Lahore College for Women University, Lahore, Pakistan.

1

2
Department of Toxicology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
*Corresponding author Email: [email protected]

http://dx.doi.org/10.13005/ojc/320221

(Received: March 29, 2016; Accepted: April 11, 2016)

ABSTRACT

The present study provides information about the levels of fortification of three water soluble
vitamins i.e. thiamine (B1), nicotinamide (B3) and pyridoxine (B6) in a variety of foodstuffs include
milk products and cereals for young children. Food fortification is key implement for improving health
of the growing children. Twenty food samples were chosen for analysis because of their common
utilization in the local area. The vitamin concentrations were determined by high performance liquid
chromatography with C18 column with a gradient of mobile phase made of water and acetonitrile
and a diode array detector set at 280 nm. The thiamine content investigated in the samples ranging
from 268 µg/mL to 3 µg/ml, nicotinamide content was from 41 µg/ml to 1 µg/mL while the pyridoxine
level was in between 412 µg/mL to 20 µg/mL. Detection and Quantification of compounds were
attained by comparing their retention times with standard reference materials and on the basis the
off peak area match against those of a standard. The method used, offer excellent linearity with
r2 > 0.994, detection limits, reproducibility, and analyte recovery.

Keywords: Water soluble vitamins, Thiamine, Nicotinamide, Pridoxine,

High performance liquid chromatography, Food products.

INTRODUCTION with a proper and balanced diet4. Even in developed

countries, the vitamin deficiencies are amounting
Vitamins counting B 1 (thiamine), B 3 problem5. Foods that do not restrain ample amounts
(nicotinamide) and B6 (pyridoxine) is a diverse group of vitamins result in vitamin deficiency-related profuse
of organic nutrients1, vital for sustaining good health, health problems6 such as deficiency of vitamins B6
normal growth and for numerous physiological have recently been connected to cardiovascular
processes in humans especially in growing diseases7.The fructification and utilization of diverse
children2-3. These important constituents are supplied dietary supplements and fortified food products
948 NAZ et al., Orient. J. Chem., Vol. 32(2), 947-953 (2016)

are often recommended to overcome this dilemma acid, sodium salt monohydrate (C 7 H 15 NaO 3 S.
curiously in infants 8. Precise information on vitamin H2O) – 96% pure was provided by Fluke Chemika.
content in food products consumed frequently is Vitamin B1 (Thiamin-Hydrochloride - 99%) and
critical to assess for optimal nutrient intakes 9-12. vitamin B6 (Pyridoxine - 98%) was purchased from
So, these food products require consistent quality BDH Biochemicals, England, whereas vitamin B3
control analysis to ensure the need and protection (Nicotinamide – 98%) was from Alfa Aesar, UK.
of consumers13. The objective of this study was
to determine the quantity of dietary water soluble Twenty food products were purchased from
vitamins (thiamine, nicotinamide and pyridoxine) the local market depending on their consumption
in the selected fortified food products in order to for the infants and children’s. The food items bought
create awareness among the users. The detection of included milk products, Juices, biscuits and powdered
vitamins in food products was carried out by HPLC foods.
which is a very sensitive and selective technique for
the quantitative and qualitative analysis. A buffer solution was prepared by taking
1 gram of 1-heptane sulphonic acid, sodium salt in
EXPERIMNTAL a 1000 ml (1L) volumetric flask and dissolve with a
minimum amount of water. 10 ml of glacial acetic
Apparatus acid was added and finally the volume was made up
Analysis was carried out by using high to the mark with water. The standard solutions were
performance liquid chromatography instrument of prepared by taking the proper amount of a compound
Agilent 1100 series with a quaternary pump which (thiamin, nicotinamide and pyridoxine) and diluting it
includes vacuum degasser, Thermostatted column with the dilution medium that was prepared by mixing
compartment and Thermostatted Autosampler. A water, acetonitrile and glacial acetic acid in a ratio of
diode array detector was employed for detection 94 : 5 : 1.A series of dilutions, 100,200,300,400 and
purpose. The reverse-phase column Zorbax SB-C18 500 µg/mL were made for all the three compounds
4.6 × 75 mm was used for separation. from their respective 1000 µg/mL stock solutions.
These concentrations of standards are prepared
Chemicals/Reagents and solutions on the basis of the range required to contrive a
HPLC grade solvents were employed calibration curve. The mobile phase was prepared
throughout the analysis. Acetonitrile, methanol by adding buffer and acetonitrile in a ratio of 90 : 10
(Merck, UK) and glacial acetic acid were applied as v/v.
supplied by the manufacturers. 1-heptane sulphonic

Fig. 1: Standard Curve of thiamine (vitamin B1)

 NAZ et al., Orient. J. Chem., Vol. 32(2), 947-953 (2016) 949

Sample Preparation dissolved in 100 mL of buffer. The solution was put

The preparation of sample was done in in the shaker for 60 minutes for dissolution of the
three steps which are the following; food materials.

Buffer preparation Filtration

A buffer solution was prepared by taking 1 Afterwards the solution was filtered by
g of 1-heptane sulphonic acid sodium salt in a 1000 whatmann® filter paper so any hard suspended food
mL (1L) volumetric flask and dissolve with a minimum particles can be separated. The same solution was
amount of water. 10 mL of glacial acetic acid was again filtered through a Millipore filter of 0.22 µm
added and finally the volume was made up to the prior to the sample injection in the system.
mark with water.
Analytical Conditions for HPLC
Extraction Column = Zorbax SB-C18 4.6 × 75 mm
All the samples were homogenized in Particle size = 3.5 µm
a blender. 10 g of each sample were taken and Temperature of column compartment = 25 ºC

Fig. 2: Standard Curve of nicotinamide (vitamin B3)

Fig. 3: Standard Curve of pyridoxine (vitamin B6)

950 NAZ et al., Orient. J. Chem., Vol. 32(2), 947-953 (2016)

Sample Injection volume = 20 µL Detector = Diode array

Stop time = 20 minutes The Wavelength for detection = 280 nm
Mobile phase = Buffer (90) : Acetonitrile (10) [v/v] Vitamin Analysis By HPLC:
Flow rate = 1.2 mL/minute

Table 1. Statistical summary of data for HPLC

Analysis of vitamins

Parameters Thiamine Nicotinamide Pyridoxine

Mean 52 10 126
Median 36 (3-268) 09 (1-41) 63.5 (20-412)
(min-max)
Mode 48 10 412
Correlation 0.997 0.997 0.998
co-efficient (r)
Standard 66.01 10.22 133.44
Deviation
Intercept -1084.75 -330.508 -2160.87
y = ax+b 68983x - 1084.7 32034x - 330.51 91791x - 2160.9
Slope 68983 32033 91791

Table 2: The distribution of target The mobile phase was placed in the solvent
compounds concentrations in the reservoir and the pump was set at a flow rate 1.2 mL/
samples min and allowed to pump for 10 to 15 minutes before
the first analysis. The detector was switched on at
Thiamine Nicotinamide Pyridoxine least 30 minutes before analysis and was set at 280
(B1) (B3) (B6) nm wavelength. A 20 µL aliquot of sample solution
(µg/mL) (µg/mL) (µg/mL) was drawn into a clean HPLC injection syringe. The
sample was injected and the detector system was
S1 07 1.4 42 activated at the same time. After approximately 15
S2 36 01 52 to 20 seconds the injection valve is returned to the
S3 - 13 40 “load” position. The syringe and valve are rinsed
S4 90 10 101 or flushed with water for next sample analysis.
S5 03 11 412 The same method was used to inject the standard
S6 - 22 75 solutions and the rest of the samples. All the tests
S7 31 10 - were performed in triplicate. The studied compounds
S8 - 41 82 were documented by using retention time that
S9 48 19 41 matches against those of the calibration standards
S10 268 03 - while the quantification was achieved by means of
S11 56 10 211 peak area that match the standards.
S12 - - 219
S13 16 1.8 47 RESULT AND DISCUSSION
S14 48 2.3 20
S15 14 - 25 Young children and babies are mainly at
S16 89 09 120 threat of intensifying micronutrient deficiencies14-
S17 56 1.6 21
15
.Food fortification is important to avoid the
S18 11 - 330 problems associated with the deficiencies in which
S19 08 1.4 23 micronutrients are added to different food objects16-17.
S20 - 09 412 It is therefore of supreme meaning to know the sort
951 NAZ et al., Orient. J. Chem., Vol. 32(2), 947-953 (2016)

and quantity of micronutrients added to food products pyridoxine were linear with the regression co-efficient
especially for young children. So, the determination (R2) 0.996, 0.995 and 0.997 respectively as shown
of dietary water-soluble vitamins is conceded using in Figures 1-3.
high performance liquid chromatography to the
create awareness regarding the quantity of these The HPLC chromatograms were obtained
vitamins in diverse food product among common when the standards and the samples were analyzed
people. through the system (Figures 4-5). The studied
compounds were recognized by using retention
For calibration graphs, standard solutions of time that matches against those of the calibration
vitamins were made in the range of 100,200,300,400 standards while the quantification was achieved
and 500 µg /mL by the serial dilution to evaluate the by means of peak area that match the standards.
linear dynamic range. The calibration curves for all The retention times for the thiamine, pyridoxine
the three compounds, i.e thiamine, nicotinamide and and nicotinamide were 17.77,5.56 and 3.27 mins

Fig. 4: Chromatogram of Mixture of standard vitamins (1) [Nicotinamide (3.2min), (2) Pyridoxine
(5.5min) and (3) Thiamine (17.7min)

Fig. 5: Chromatogram of sample no. 5

952 NAZ et al., Orient. J. Chem., Vol. 32(2), 947-953 (2016)

Fig. 6: The distribution of target compounds in the samples

Fig. 7: Comparison of targeted compounds with the WHO recommendation

respectively. The nicotinamide was separated first, vitamins in the twenty samples of food products are
followed by pyridoxine and thiamine. Statistical shown in table 2 as well as in Figure 6.
summary of data for HPLC determination of vitamins
is mentioned in Table 1.Sample 10 shows the highest The results obtained from the experiment are
thiamine content (268 µg/mL) and sample 5 shows also compared with the World Health Organization’s
the lowest (3 µg/mL). Nicotinamide contents are high (WHO) recommended daily intake. The comparison
in sample 8 (41 µg/mL) and low in sample 2 (1 µg/ showed that the level of three vitamins in all the
mL) while pyridoxine contents are higher in samples samples was below the recommended intake as
5 and 20 (412 µg/mL) and lower in sample 14 (20 shown in Figure 7. A proper check is required on
µg/mL). The distribution and comparison of these these food products so that various diseases among
the young children’s and babies can be avoided.
953 NAZ et al., Orient. J. Chem., Vol. 32(2), 947-953 (2016)

CONCLUSION values and it is clinched that an appropriate check

and balance is required to control and pledge the
The level of three B-complex vitamins, quality of these types of fortified food items for babies.
Thiamine, Nicotinamide and Pyridoxine has been Also a more accurate endorsement for the vitamins
analyzed in a range of foodstuffs include milk should be followed to terminate having such a large
products and cereals for young children and babies variation as seen in this study.
by High performance liquid chromatography because
it enumerates the B-group vitamins concurrently in ACKNOWLEDGEMENT
food items within a single run. All the investigated
vitamins were separated completely under the 20 Authors are thankful to the University of
minutes run time. All the three vitamins studied in Veterinary Sciences (UVAS) and Central Lab of
food product samples are below the recommended Lahore college for Women University, Lahore for
providing Laboratory facilities.

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