ORIGINAL ARTICLE

Quantication of water soluble vitamins in six date palm

(Phoenix dactylifera L.) cultivars fruits growing in Dubai,
United Arab Emirates, through high performance liquid
chromatography
Junaid Aslam
a,c,
*
, Sheba Haque Khan
b
, Saeed Ahmad Khan
a
a
Dubai Pharmacy College, Al Muhasinah, P.O. Box 19099, Dubai, United Arab Emirates
b
G.F. College, Department of Chemistry, M.J.P. Rohilkhand University, Bareilly, Uttar Pradesh, India
c
Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110062, India
Received 19 January 2011; accepted 21 February 2011
Available online 24 February 2011
KEYWORDS
Arecaceae;
Date palm (Phoenix
dactylifera L.);
Water soluble vitamins;
High performance liquid
chromatography
Abstract In present investigation, a comparative analysis of water soluble vitamins viz., B1 (thia-
mine HCl), B2 (riboavin), B3 (nicotinamide), B5 (pantothenic acid), B6 (pyridoxine HCl), B9 (folic
acid), B12 (cyanocobalamin) was carried out in fruits (immature, semimature and mature) of six
date palm (Phoenix dactylifera L.) cultivars (Barhee, Khalasah, Muzati, Shishi, Zart,
Zardai) growing in United Arab Emirates (UAE) by high performance liquid chromatograph
(HPLC). The fruits were collected at three developing stages (immature, semimature and mature).
Quantitative analysis of water soluble vitamins yield showed a signicant variation within the dif-
ferent cultivars and the developing stages of date palm fruit. Vitamin B1, B3, B5, B6 were maximum
(lg/100 g f.w.) in Shishi, Zardai, Shishi and Muzati at their matured stage, however, vita-
min B2, B9, B12 were detected in immature fruit of Khalasah, Khalasah and Shishi culti-
vars. The vitamin production in fruits of different date palm cultivars was, therefore, developing
*
Corresponding author at: Department of Biotechnology, Jamia
Hamdard (Hamdard University), New Delhi 110062, India. Tel.: +91
99907 06557.
E-mail address: Junaidg1@gmail.com (J. Aslam).
1319-6103 2011 King Saud University. Production and hosting by
Elsevier B.V. All rights reserved.
Peer review under responsibility of King Saud University.
doi:10.1016/j.jscs.2011.02.015
Production and hosting by Elsevier
Journal of Saudi Chemical Society (2013) 17, 916
King Saud University
Journal of Saudi Chemical Society
www.ksu.edu.sa
www.sciencedirect.com
stage specic and cultivar dependent. The present study showed that the date palm fruit could be
used for human consumption with value addition of water soluble vitamins at their specic devel-
opmental stages.
2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
The date palm (Phoenix dactilyfera L.), species has 19 known
genetic relatives. The genus Phoenix belongs to the family
Arecaceae. The fruit is a Berry type (known also as Drupe)
with a single seed in each. Date palm is one of the most impor-
tant cash crops; it is especially grown in Arab region and the
area bordering the Mediterranean coast. It has been cultivated
since the prehistoric time in most of the Gulf, Asian and Afri-
can countries. More than 2000 date palm cultivars enlisted
through the world, but few of them have been screened for
their large agronomic performance and fruit quality (Al-Hooti
et al., 1997). Due to the nutritional value of the fruit; research-
ers have focused their attention on characterization of the
physico-chemical analysis of the important date palm cultivars
(Al-Shahib and Marshall, 2002; Mrabet et al., 2008). Increase
in date fruit production will, therefore, play a signicant role
worldwide in the improvement of nutritional status of people,
with special reference to calories and important minerals
(Ahmed et al., 1995). It is usually taken raw or with milk,
which makes it highly nutritious in most respects. Taking it
with curdled milk is also common. The seeds of the fruit,
roasted and ground into powder makes a beverage like coffee,
called date coffee. The date palm yields a sweet juice of high
food value. It can be taken fresh with great advantage or made
into a tasty country sugar. The juice can also be allowed to fer-
ment and made into alcohol. It provides natural sugar in the
form of glucose and fructose. This sugar is ready for immedi-
ate absorption and is, therefore, innitely superior to cane-
sugar. Dates are rich in sugar ranging from 65% to 80% on
dry weight basis mostly of inverted form (glucose and fruc-
tose). Fresh varieties have a higher content of inverted sugars;
semi dried varieties contain equal amounts of inverted and
sucrose, while dried varieties contain higher sucrose. Water
content is between 7% (dried) and 79% (fresh) depending on
the cultivars (Myhara et al., 1999; Al-Farsi et al., 2005).
Therefore, the moisture, total nitrogen, fat, ber, ash, tannins,
vitamin C, b-carotene and ten nutritionally important minerals
were highest in the early stage of development and decreased
during maturation. Date fruit, being exceptionally rich in
potassium and extremely low in sodium (Ahmed et al., 1995).
Dates are valuable as medicine for their tonic effect. Being
easily digested, they are very useful for supplying energy and
repairing waste. Milk in which fresh and cleaned dates have
been boiled proved very nourishing and proved as a restorative
drink for children and adults alike, especially during convales-
cence. Anumber of studies have been carried out to characterize
its chemical composition particularly minerals, polysaccha-
rides, carbohydrates in fresh date fruit (Fayadah and Al
Showiman, 1990; Al-Hooti et al., 1997;Al-Shahib and Marshall,
2002). It has been reported that the vitamins play a potent role
to reduce the damage carried out by the free radicals and check
degenerative disease (Jacab and Sotoudeh, 2002). In addition,
vitamin supplements showed the ergogenic and performance
enhancing effects (Clarkson, 1993; Zhao et al., 2004). Thus,
chemical and physical characterization has been carried out pre-
viously by many workers (Al-Juburi et al., 1994; Al-Hooti et al.,
1997; Attalla and Harraz, 1996; Mrabet et al., 2008), but there is
a distinct lack of information on water soluble vitamins during
the ripening stages of date palm fruit. Therefore, the present
investigation was undertaken to determine the presence of
water-soluble vitamins (Fig. 1) in fruits of selected date palm
cultivars at their developmental stages (immature, semimature
and mature) growing in Dubai, United Arab Emirates.
2. Experimental
2.1. Experimental material
The fruits of six different date palm (Phoenix dactylifera L.)
cultivars (Barhee, Khalasah, Muzati, Shishi, Zart,
Zardai) were collected at their developing stages (immature,
semimature and mature) from Al-Muhasinah-1, Dubai, Uni-
ted Arab Emirates.
2.2. Reagents and solvents
The reagents and chemicals were used in the present study
were of analytical grade viz., HPLC methanol (Merck), aceto-
nitrile HPLC (Merck), glacial acetic acid (BDH), triethylamine
(RDH), orthophosphoric acid (BDH), pic6 (hexane sulfonic
acid), sodium salt (Merck). The Millipore direct q system
based distilled and deionized water was used for the prepara-
tion of reagents and stock solution.
2.3. Standard preparation
The standard stock solution of the water soluble vitamins was
prepared as follows: (a) vitamin B1 (thiamine HCl) was pre-
pared by dissolving 26.7 mg of thiamine hydrochloride in
25 ml of double distilled water; (b) vitamin B2 (riboavin)
was prepared by dissolving 6.9 mg of riboavin in 100 ml of
extraction solution (the extraction solution has limit to dis-
solve 7 mg of riboavin); (c) vitamin B3 (nicotinamide) was
prepared by dissolving 41.5 mg of nicotinamide in 25 ml of
double distilled water; (d) vitamin B5 (calcium salt of panto-
thenic acid) was prepared by dissolving 21.4 mg of calcium
d-pantothenate in 25 ml of double distilled water; (e) vitamin
B6 (pyridoxine HCl) was prepared by dissolving 20.8 mg of
pyridoxine hydrochloride in 25 ml of double distilled water;
(f) vitamin B9 (folic acid) was prepared by dissolving
11.2 mg of folic acid in 25 ml of double distilled water; (g) vita-
min B12 (cyanocobalamin) was prepared by dissolving 19.8 mg
of cyanocobalamin in 25 mg of double distilled water.
2.4. Working standard
The working standards were prepared as follows: (i) B1,
1.068 mg/ml and 562 ll was taken in 10 ml volumetric ask,
10 J. Aslam et al.
(ii) B2, 0.069 mg/ml and 5.1 ml was taken in 10 ml volumetric
ask, (iii) B3, 1.66 mg/ml and 60.5 ll was taken in 10 ml volu-
metric ask, (iv) B5, 0.856 mg/ml and 292 ll was taken in
10 ml volumetric ask, (v) B6, standard 0.832 mg/ml and
72.2 ll was taken in 10 ml volumetric ask, (vi) B9, standard
0.072 mg/ml and 132 ll was taken in 10 ml volumetric ask,
(vii) and then B12, standard, 1.23 mg/ml and 142 ll was taken
in 10 ml volumetric ask and then all were nally mixed.
2.5. Preparation of buffer, solutions and samples
The buffer and sample preparation was carried out according
to our previous report (Junaid et al., 2008). In short, 1.08 g of
hexane sulfonic acid sodium salt and 1.36 g of potassium dihy-
drogen phosphate were dissolved in 940 ml of HPLC water
and 5 ml of triethylamine was added to it and the pH was ad-
justed to 3.0 with orthophosphoric acid. To prepare the mobile
phase, buffer and methanol were mixed with a ratio of 96:4
and ltered through 0.45 l membrane lter and degassed by
using helium gas. Extraction solution was made by mixing
50 ml of acetonitrile with 10 ml of glacial acetic acid and the
volume was nally made up to 1000 ml with double distilled
water. Ten grams of each sample was homogenized, weighed
and transferred into conical asks and 25 ml of extraction
solution was added, water bath was kept shaking at 70 C
for 40 min. Thereafter, the sample was cooled down, ltered
and nally the volume was made up to 50 ml with extraction
solution.
2.6. High performance liquid chromatography (HPLC)
The ve point calibration curve was prepared using mix stan-
dard in mobile phase. It was analyzed independently using
HPLC and a standard curve was plotted between the concen-
tration and the peak area. The injected quantities of the mix
standard showed a good linearity. The data of peak area vs.
the used standard vitamin concentrations were treated by lin-
ear least-square regression and the regression equation thus
Figure 1 Chemical structure of water soluble vitamins.
Quantication of water soluble vitamins in six date palm (Phoenix dactylifera L.) cultivars fruits 11
obtained from standard curve was used to quantify the water-
soluble vitamins in different date palm fruit samples. The col-
umn used for the analysis was waters symmetry C18
(4.6 250 mm 5 lm) with a linear gradient of buffer/methanol
(96:4) at a constant ow rate of 1 ml/min with 1800 pressure by
using waters pump (1515 isocratic). A UV (2487) detector was
employed for the detection of peaks, using two channels simul-
taneously at a wavelength of 210 nm, a bandwidth of 5 nm and
another wavelength of 280 nm.
2.7. Recovery study
The accuracy of the current method was conrmed in the sam-
ple using the recovery study. Pre-analyzed samples were spiked
with extra 50%, 100% and 150% of the standard vitamins; the
mixture of the standard was reanalyzed by adopting the pro-
posed method. The experiment was repeated four times and
the average recovered vitamins content was quantied using
regression equation, and the % recovery was calculated
accordingly.
2.8. Precision
The precision of the used method was obtained by determining
inter and intra-day variations in ve replicates of water-soluble
vitamins at two concentration levels (200400 ng per spot) and
the percent standard relative deviation (RSD) was calculated
accordingly.
2.9. Quantication of water soluble vitamins
Five replicates of each sample were analyzed using 60 ll. The
yield was calculated using regression equation of calibration
curve of each standard.
2.10. Statistic analysis
The signicant and non-signicant variations within water sol-
uble vitamin contents and the six date palm cultivars fruits
were analyzed using one-way analysis of variance (ANOVAs).
Values are means of ve replicates from two experiments, and
the presented mean values were separated using Duncans
Multiple Range Test (DMRT) at p 6 0.05.
3. Results and discussion
In the present study, water soluble vitamins viz., B1 (thiamine
HCl), B2 (riboavin), B3 (nicotinamide), B5 (pantothenic
acid), B6 (pyridoxine HCl), B9 (folic acid), B12 (cyanocobala-
min) were analyzed in fruits (immature, semimature, mature)
of six date palm (Phoenix dactylifera L.) cultivars (Barhee,
Khalasah, Muzati, Shishi, Zart, Zardai) growing
(Fig. 2) in Dubai, United Arab Emirates.
The solvent system (buffer/methanol, 96:4) used for the
analysis produced a sharp and compact peak of the studied
vitamins. The densitometric analysis of water soluble vitamins
was carried out at 210 and 280 nm absorbance mode, and the
regression analysis data showed a good linear relationship
(Table 1). The method used for the vitamin extraction and
subsequent estimation afforded recovery of 99.05100.11%.
The inter- and intra-variation of vitamins at two different con-
centration levels showed a low relative standard deviation
(0.901.02%). The yield of the water soluble vitamins in differ-
ent samples was analyzed from the regression equation using
value of area obtained from win-cat software.
Of the screened six date palm cultivars fruits; thiamine (B1)
was only detected in Khalasah, Shishi and Muzati. In
khalasah and Muzati cultivars, it was detected only in
semimature and mature fruit; however, in Shishi, it was
present in all the developmental stages of fruit. The maximum
thiamine content was noticed in Khalasah followed by Shi-
shi at mature level, thereafter; it started to decrease in semi-
mature and mature fruits. In Barhee, Zart and
Zardai; thiamine content was altogether absent (Fig. 3A).
Figure 2 Chromatogram showing baseline separation of water
soluble vitamins from date palm fruit samples: (1) cyanocobal-
amin (B12), (2) nicotinamide (B3), (3) pantothenic acid (B5), (4)
pyridoxine (B6), (5) thiamine (B1), (6) folic acid (B9), (7) riboavin
(B2).
Table 1 The important parameters of calibration curve.
Water
soluble
vitamins
y = ax + b r
2
Concentration
range (mg/ml)
Recovery Precision (R.S.D. (%)) Detection limit (mg l
1
)
B1 Y = 1.23e + 005x 1.17e + 004 0.9947 1.8620.8 0.70 2.43 0.1
B2 Y = 6.08e + 004x 4.59e + 005 0.9987 1.56320.01 0.62 3.08 0.2
B3 Y = 1.6e + 005x 2.37e + 004 0.9992 4.2052.0 0.78 3.64 0.5
B5 Y = 7.50e + 004x 1.13e + 005 0.9963 4.6950.4 1.2 5.58 0.1
B6 Y = 2.75e + 005x + 1.2e + 004 0.9921 1.5420.04 0.66 3.29 0.5
B9 Y = 1.31e + 005x 1.39e + 004 0.9987 2.5450.5 1.24 4.88 0.2
B12 Y = 5.07e + 004x 3.51e + 005 0.9945 1.8720.4 0.90 4.38 0.1
a slope; b intercept; r correlation coefcient.
12 J. Aslam et al.
0
2
4
6
8
10
12
14
Khalasah Shishi Muzati Barhee Zart Zardai
Cultivars
T
h
i
a
m
i
n
e

u
g
/
1
0
0

g

f
.
w
.
Immature Semimature Mature
a
b
c
a
b
c
a
d d b d d b d d b b b
(A)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Khalasah Shishi Muzati Barhee Zart Zardai
Cultivars
R
i
b
o
f
l
a
v
i
n

u
g
/
1
0
0

g

f
.
w
.
b a
a
c
b
a c
a
a d c a c a d a c d
(B)
0
5
10
15
20
25
30
35
40
45
50
Khalasah Shishi Muzati Barhee Zart Zardai
Cultivars
N
i
c
o
t
i
n
a
m
i
d
e

u
g
/
1
0
0

g

f
.
w
.
a
b b
c
c
a
a
b
c c
a
a
b
c
d d d d
(C)
0
1
2
3
4
5
6
7
8
9
Khalasah Shishi Muzati Barhee Zart Zardai
Cultivars
P
a
n
t
o
t
h
e
n
i
c

a
c
i
d

u
g
/
1
0
0
g

f
.
w
.
a
b
a
b
a
b c c b c c b
c c b c c
(D)
Figure 3 HPLC analysis of water soluble vitamins in fruits of different date palm cultivars (A) thiamine (B1), (B) riboavin (B2), (C)
nicotinamide (B3), (D) pantothenic acid (B5), (E) pyridoxine (B6), (F) folic acid (B9), (G) cyanocobalamin (B12).
Quantication of water soluble vitamins in six date palm (Phoenix dactylifera L.) cultivars fruits 13
A signicant variation in riboavin content was noticed
amongst the tested date palm fruits. The riboavin content
was detected only in Khalasah, Shishi and Barhee
fruits. In Khalasah and Shishi, it was isolated from semi-
mature and mature fruit; however, in Barhee, it was quanti-
ed only in semimature fruit. Among the Khalasah,
Shishi, and Barhee; the highest riboavin content was
quantied in Khalashs immature fruit followed by Barhee.
Riboavin was not detected in Muzati, Zart and
Zardai cultivars. Fig. 3B showed a comparative account of
the various level of the riboavin content at different fruit
developmental stages.
Nicotinamide (B3) was detected in Khalash, Muzati,
Barhee, Zart and Zardai cultivars. In Muzati, Bar-
hee, Zart and Zardai, nicotinamide content was isolated
in all the fruit developing stages; however, in Khalasah it
analyzed only in semimature and mature fruit. The maximum
vitamin content was noticed in mature fruit of Zardai culti-
var; hence, no signicant difference (p 6 0.05) was noticed in
Barhee, Zart and Zardai cultivars. Similarly, no signif-
icant variation in nicotinamide yield was noticed in semima-
ture and mature fruits of Khalasah and Muzati
cultivars. In Shishi, it was altogether absent. The maximum
yield of nicotinamide was detected in mature fruit of Zardai
followed by Barhee and Zart cultivars (Fig. 3C). The se-
quence of nicotinamide production at different developing
stages of fruit was as follows: mature, semimatute and
immature.
Pantothenic acid was detected only in two cultivars: (a)
Shishi and (b) Muzati. Although, in Muzati, it was iso-
lated only in the semi mature and mature fruits, while in the
immature, no signicant difference in the yield was, however,
0
0.5
1
1.5
2
2.5
3
3.5
4
Khalasah Shishi Muzati Barhee Zart Zardai
Cultivars
P
y
r
i
d
o
x
i
n
e

u
g
/
1
0
0
g

f
.
w
.
a
b
c
d
d
a
b
c
a
b
c
b b b
d
(E)
e e c
0
0.5
1
1.5
2
2.5
3
3.5
4
Khalasah Shishi Muzati Barhee Zart Zardai
Cultivars
F
o
l
i
c

a
c
i
d

u
g
/
1
0
0
g

f
.
w
.
a
b
a
a b
b
c c c c c c c c c
c c c
(F)
0
0.5
1
1.5
2
2.5
Khalasah Shishi Muzati Barhee Zart Zardai
Cultivars
C
y
n
o
c
o
b
a
l
a
m
i
n

u
g
/
1
0
0

g

f
.
w
.
a
b
a
b
b
b
ab
cd
d d
(G)
c c c e c c c c
Figure 3 (continued)
14 J. Aslam et al.
noticed. The highest production was noticed in the mature
fruit of Shishi cultivar. Fig. 3D showed pantothenic acid
production at different developing stages of date palm fruit.
Pyridoxine was quantied in most of the cultivars except
Barhee, whereas, the highest content was noticed in imma-
ture followed by semimature and mature fruits. The maximum
pyridoxine content was detected in immature Muzati fruit
followed by Shishi, Khalasah, Zart and Zardai culti-
vars. Similarly, in semimature fruits, the highest vitamin con-
tent was isolated from Muzati followed by Shishi,
Khalasah, Zardai and Zart. Hence, no signicant dif-
ference in pyridoxine content was detected in Khalasah
and Zardai semimature fruits. In mature fruits, pyridoxine
was also detected, but was in very low concentration. The
highest vitamin yield was noticed in Muzati mature fruit.
No signicant difference was observed in Khalasah,
Shishi, Zart and Zardai cultivar fruits (Fig. 3E). In
Barhee, it was altogether absent in all the tested fruit types.
Folic acid was detected only in Khalasah and Zardai
cultivars; hence, a signicant variation in content was noticed.
The maximum folic acid was detected in immature fruit of
Khalasah and semimature fruit of Zardai cultivars. It
was completely absent in Shishi, Muzati, Barhee and
Zart cultivars. Fig. 3F showed a comparative account of
the folic acid production at different developing stages of fruit.
The analysis of cyanocobalamin showed that it was present
in all the developing stages of Khalasah and Shishi fruits;
however, in Muzati it was detected only in semimature and
mature fruits. In Zart and Zardai, it was noticed only in
mature fruits. The highest yield of cyanocobalamin was
detected in the immature fruit of Shishi followed by
Khalasah. No signicant difference was found in the semi-
mature fruits of Khalasah and Muzati. Similarly, in
mature fruits of Khalasah and Shishi no signicant vari-
ation was noticed in yield. In addition, the content isolated
from mature fruits of Muzati, Zart and Zardai was
not signicantly different. Moreover, the cyanocobalamin con-
tent in immature and mature fruits of Barhee, Zart and
Zardai was altogether absent. Fig. 3G showed a compara-
tive account of the cyanocobalamin content at different
developing stages of date palm fruits.
We have screened water soluble vitamin content in fruits of
six date palm cultivars at their developing stages (immature,
semimature and mature). In present investigation, we found
that vitamin production was cultivar specic and fruit develop-
ing stage dependent. Vitamins B1, B3, B5, B6 were maximum
in mature fruits of Shishi, Zardai, Shishi, and Muzati
cultivars; however, B2, B9, B12 were found to be the highest
in Khalasah, Khalasah and Shishi at the immature stage
of developing fruits. The variation in vitamins yield noticed in
the present investigation might be due to the change in climatic
condition and the genetic pool of a particular type of the cul-
tivar, which directly inuenced metabolic pathways. The
occurrence of the studied vitamins with developing stages of
date palm fruit agreed with the previous nding in some other
plants, whereas, it was conrmed that the synthesis of end
products are associated with the degree of differentiation of
individual cell or tissues and environmental conditions
(Al-Shahib and Marshall, 2002; Pande et al., 2002; Ma et al.,
2006; Mrabet et al., 2008; Bhat et al., 2008; Junaid et al.,
2009, 2010a). The results observed in the present study are very
similar to the study that dealt with other active principle
(Datta and Srivastava, 1997; Junaid et al., 2010c), in which, in-
creased and decreased accumulations of metabolites were
noted at different developmental stages of the seedlings or
fruits that grew, reached a steady state when they became
older. The present results also agreed with the mineral analysis
carried out in various date palm cultivars, whereas, no con-
stant trends were found at the developmental stages of differ-
ent date palm cultivars fruit (Attalla et al., 1988; Al-Shahib
and Marshall, 2002). In the present study results also showed
a great variation in water soluble vitamin contents within the
fruit developing stages and date palm cultivars. Similarly, in
other plant system it has also been established that the meta-
bolic pathway was not only restricted to certain tissues, but
it was also modulated by different developmental and environ-
mental conditions (Pasquali et al.,1992; Yohe, 2002; Adebiyi
et al., 2005; Junaid et al., 2010b). Secondly, it has also been re-
ported that the mineral content of the date fruit may be inu-
enced by the level of environment and the soil characteristic as
well as by the amount of fertilizers used by the trees (Al-Juburi
et al., 1994; Al-Hooti et al., 1997). Recent study on water sol-
uble vitamins analysis indicates that water soluble vitamin pro-
duction was tissue specic and environmental condition
dependent (Junaid et al., 2008).
4. Conclusion
A remarkable variation in water-soluble vitamins yield was no-
ticed in fruits of six date palm cultivars. Matured fruits proved
to be highly economical for the production of vitamin B1, B3,
B5, B6; however, the remaining others were present in the B2,
B9, B12. Based on the present investigation it could be con-
cluded that immature and matured date palm fruits could be
used as water soluble vitamins supplements for human con-
sumption in the diet of vegetarians.
Acknowledgment
The rst author (Dr. Junaid Aslam) is thankful to the Chair-
man, Board of Trustees of Dubai Pharmacy College for pro-
viding all facilities to carry out the present research work.
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