Colorimetric quantification of α-tocopherol (vitamin E) in pure form and different comestible samples by using newly synthesized tetrazolium salts

Article abstract of DOI:10.1007/s13738-020-02038-2

A quick, susceptible, and viable spectrophotometric procedure for the assay of α-tocopherol by newly synthesized tetrazolium reagents 2-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium bromide (PTMPT) and 2-phenyl-3-(2-thiazolyl)-5-(4-nitrophenyl)tet

Full text of DOI:10.1007/s13738-020-02038-2

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-02038-2
ORIGINAL PAPER
Colorimetric quantifcation oꢀ α‑tocopherol (vitamin E) in pure
ꢀ
orm and diꢁerent comestible samples by using newly synthesized
tetrazolium salts
Ashwani Kumar1 · Mamta Kamboj1
Received: 28 February 2020 / Accepted: 12 August 2020
©
Iranian Chemical Society 2020
Abstract
A quick, susceptible, and viable spectrophotometric procedure for the assay of α-tocopherol by newly synthesized tetrazolium
reagents 2-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium bromide (PTMPT) and 2-phenyl-3-(2-thiazolyl)-5-(4-
nitrophenyl)tetrazolium bromide (PTNPT) is reported. The reduction behavior of α-tocopherol is used to quantify it in pure
form and diꢀerent edibles. Colorless tetrazolium salt-reduced in the pink-colored formazan by α-tocopherol gives absorption
maxima at 526 nm. The reduction process occurred in the basic medium, so the pH of the reaction medium was maintained
in the range 8–9. Beer’s law was obeyed in the concentration range of 0.2–15.0 μg/mL with PTMPT and 0.1–0.4 μg/mL with
3
−1
−1
4
−1
−1
PTNPT. The molar absorption coeꢁcient is 4.21×10 L mol cm and 6.4×10 L mol cm with PTMPT and PTNPT,
respectively. Limit of detection is 0.14 μg/mL and 0.03 μg/mL, and limit of quantiꢂcation is 0.42 μg/mL and 0.08 μg/mL
for α-tocopherol with PTMPT and PTNPT, respectively. As the proposed method is easy and user-friendly, distinct edibles
are quantiꢂed by using the proposed method, and the results show good agreement with the other existing methods. The
samples were saponiꢂed ꢂrst to convert α-tocopheryl acetate in its active form, i.e., α-tocopherol. Saponiꢂcation had done
2
at temperature 70 °C for 2 h on heating mental. The values of the correlation coeꢁcient (R ) are 0.955 and 0.998 with the
relative standard deviation of 0.8% and 4.6% with PTMPT and PTNPT, respectively. The proposed method is eꢀectively
used for the assay of α-tocopherol in diꢀerent pharmaceuticals, fruits, cereals, nuts, and milk samples.
Keywords α-tocopherol · PTMPT · PTNPT · Spectrophotometer · Quantiꢂcation
Introduction
system helps in preventing diseases inactive tissues of the
body from the agents [5]. Bioavailability of nutrients changes
with age and the kind of dietary admissions. Mostly, the eat-
ing habits regulate physiological conditions and the number
of vitamins within the body. The human body does not inte-
grate vitamins aside from vitamin D. Therefore intake of
vitamin E is required and obtained through dietary routine
[6]. Vitamin E has a lipophilic character, so it can protect
foods by closely binds with lipids in edible products [7].
Vitamin E in supplements is usually present as α-tocopheryl
acetate, a form of α-tocopherol that protects its ability to
function as an antioxidant [8]. The essential sources from
where one can get vitamin E are oats, natural products, and
quality milk products [9]. Alongside elevated levels of basic
unsaturated fats, vegetable oils [10] additionally contain reg-
ular micro-vitamin; for example, consumable oils contain
noteworthy wholesome constituents [11, 12]. The insuꢁ-
cient amount of α-tocopherol in the body leads to the devel-
opment of a disease called ataxia [13]. Quantiꢂcation of
Naturally, vitamin E crops up in eight particular structures
that fuse four tocopherols (alpha, beta, gamma, and delta)
and four tocotrienols (alpha, beta, gamma, and delta). Out
of all, α-tocopherol is the most powerful, frequent, and the
only form of vitamin E that meets human requirements [1].
Although non-α-tocopherols have similar antioxidant nature,
they are poorly recognized by the hepatic α-tocopherol trans-
fer protein (α-TTP) [2]. Vitamin E has antioxidant behavior
[
3] which helps in the deactivation and inhibition of free
radicals, as a result of which vitamin E plays a major role
in enhancing immune response [4]. The enhanced immune
*
Ashwani Kumar
akumarchem@kuk.ac.in
1
Department of Chemistry, Kurukshetra University,
Kurukshetra 136119, India
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Journal of the Iranian Chemical Society
vitamin E is troublesome in complex samples, for example,
eatable material because the vitamin is available in low ꢂxa-
tions [14]. Thusly, for the productive measurement of vita-
min E one needs to perform seclusion before its assurance,
and this conꢂnement is ꢂnished by saponiꢂcation method-
ology [15]. In the literature, an assortment of investigative
systems has been utilized for the examination of vitamin E
in various examples, for example, biological ꢃuids in dos-
age forms, aquatic organisms, and feeds. These incorporates
high-performance liquid chromatography (HPLC methods)
Materials and methods
Reagents and solutions
α-Tocopherol was snapped up from Sigma-Aldrich
(assay ≥ 95.5%). Anisaldehyde (98%), 2-aminothiazol
(97%), 4-nitrobenzaldehyde (98%), phenylhydrazine
(97%), and N-bromosuccinimide (99%) were purchased
from Sigma-Aldrich, while dry methanol (99.5%), DMSO,
ethyl acetate (99%) were of analytical grade and purchased
from CDH chemicals. Potassium hydroxide, sodium
hydroxide, and glycerol (98%) were purchased from CDH
chemicals. Petroleum ether used was of analytical grade
and purchased from CDH chemicals. All investigated sam-
ples such as pharmaceuticals, milk samples, processed
milk samples (Yakult), fruit samples, oats, nuts, syrup
that were used for the quantiꢂcation of α-tocopherol were
purchased from a well-branded manufacturer from a local
market in Kurukshetra, North West Province, India.
The stock solution of α-tocopherol (1000 μg) was
prepared by dissolving 0.1 g of it in 100 mL of absolute
ethanol. The working solution (50 μg/mL) for the quan-
tiꢂcation of α-tocopherol was prepared by dilution of
stock solution with absolute ethanol. 0.01% solution of
2-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium
bromide (PTMPT) and 0.015% solution of 2-phenyl-3-(2-
thiazolyl)-5-(4-nitrophenyl)tetrazolium bromide (PTNPT)
were prepared by dissolving 0.01 g and 0.015 g, respec-
tively, in 100 mL double distilled water. The solution of
60% KOH was prepared by dissolving 60 g of KOH pellets
in 100 mL of double distilled water. Sodium hydroxide
solution (1.0 M, 0.01 M) was prepared by dissolving the
measured amount in double-distilled water.
[
16–18], gas chromatography [19, 20], Fourier transform
infrared spectroscopy (FTIR) [21, 22], electroanalytical
techniques [23], spectroꢃuorimetry [24]. The evaluation of
α-tocopherol in various food oils has been done by utiliz-
ing a few investigative systems, for example, FT-NIR [25].
All the above systems are very costly, tedious, hard to deal
with, and not eꢀectively accessible. The spectrophotomet-
ric technique [26–39] has been utilized as an option for the
quantiꢂcation of vitamin E because of its minimal eꢀort,
less time utilization, and more eꢀectiveness. In the proposed
methodology, we utilize the spectrophotometric strategy
rather than some other investigative procedure because
of its precision and cost-viability. In the current strategy,
the reducing property of α-tocopherol has been utilized to
evaluate it in distinct edibles. From literature examinations
[
29, 30], it has been discovered that tetrazolium salt can be
easily reduced by various reducing agents. Exploring this
perception, another tetrazolium salt, for example, 2-phenyl-
3
-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium bromide
(
PTMPT) and 2-phenyl-3-(2-thiazolyl)-5-(4-nitrophenyl)
tetrazolium bromide (PTNPT) has been synthesized. This
synthesized tetrazolium has utilized as a potential spectro-
photometric investigative reagent for the measurement of
α-tocopherol in diꢀerent pharmaceuticals and edible. The
current technique includes a prompt decrease of reagents
2
-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium
Equipments
bromide (PTMPT) and 2-phenyl-3-(2-thiazolyl)-5-(4-nitro-
phenyl)tetrazolium bromide (PTNPT) into hued formazan
The measurement of absorbance at selected wavelengths
was done by Systronics UV–visible Spectrophotometer
117, India, equipped with a quartz cell of 1 cm light path.
FTIR spectrum of reagents 2-phenyl-3-(2-thiazolyl)-5-(4-
methoxyphenyl)tetrazolium bromide (PTMPT) and 2-phe-
nyl-3-(2-thiazolyl)-5-(4-nitrophenyl)tetrazolium bromide
(PTNPT) was recorded with Beckman IR-20 Spectropho-
tometer, (Philadelphia). ESI–MS spectra of reagents were
recorded with Xevo G2-S Q-T (Waters, USA). A Hanna-
H198100 pH meter (UK) was used to measure the pH
of solutions. All the required amount of chemicals was
weighed on Afcoset 300 balance (Japan), within certain
(
λmax 526 nm) in the presence of NaOH (pH 8–9), which
forms the reason for the measurement of α-tocopherol. The
existing spectrophotometric techniques have genuine disad-
vantages; for example, some require heat and other require
time for the ꢂnish of the response. In any case, the current
technique is profoundly easy, exact, and precise when con-
trasted with these current strategies, because neither warm-
ing nor sitting tight time is required for the fulꢂllment of
the response. We contrast our strategy and other existing
writing strategies, which show great concurrence with the
consequences of the proposed technique.
−
3
limits of 10 . Voltammogram was recorded using the Ivi-
umStat Electrochemical analyzer (Netherlands).
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Journal of the Iranian Chemical Society
Experimental
washed, and the product of the reaction was dried at room
temperature for 4 h [yield 95%, melting point 180–182 °C
for R=OCH3 and yield 96%, melting point 184–185 °C for
Synthesis of tetrazolium salts
R=NO ].
2
Synthesis of 2-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)
tetrazolium bromide and 2-phenyl-3-(2-thiazolyl)-5-(4-nitro-
phenyl)tetrazolium bromide comprises three diꢀerent steps
Step 2: synthesis
of 2‑phenyl‑3‑(2‑thiazolyl)‑5‑(4‑alkylphenyl)
formazan (II)
[
29]. Firstly, 4-alkylphenylhydrazone was synthesized by the
reaction of 4-alkylbenzaldehyde and phenylhydrazine. The
second step involved the diazotization of 4-alkylphenylhy-
drazone with 2-aminothiazol. The ꢂnal step is the conversion
of 2-phenyl-3-(2-thiazol)-5-(4-alkylphenyl) formazan (II)
into 2-phenyl-3-(2-thiazolyl)-5-(4-alkylphenyl)tetrazolium
bromide. The outlines for the synthesis of 2-phenyl-3-(2-
thiazolyl)-5-(4-alkylphenyl)tetrazolium bromide are illus-
trated in Scheme 1.
The preparation of 2-Phenyl-3-(2-thiazolyl)-5-(4-alkylphe-
nyl)formazan (II) was done using the diazotization proce-
dure. For this, 1.5 g of (I) and three times of anhydrous
sodium acetate in (1:1) methanol/DMSO solution were
added. The reaction mixture was stirred, and the temperature
◦
of contents was maintained at 0 C (solution I). Diazonium
salt solution was prepared by dropwise addition of 0.69 g
of sodium nitrite in water into 1.0 g of 2-aminothiazolyl
◦
in dilute HCl at temperature<5 C (solution II). Then, pre-
Step 1: synthesis of 4‑alkylphenylhydrazone (I)
pared diazonium salt solution (solution II) was added into
◦
the above solution (solution I) with constant stirring at 0 C.
2
3
.0 g of 4-alkylbenzaldehyde (0.01 mol) was dissolved in
0 mL dry methanol taken in a 100-mL round-bottom ꢃask.
The pH of the solution was maintained slightly basic (pH
8.0–9.0) because formazan formation takes place in a basic
medium. The contents of the reaction mixture were stirred
at room temperature overnight. Blue or black precipitates of
formazan that is insoluble in water were separated out. The
precipitates were ꢂltered, they were washed with distilled
Then, 1.6 mL of phenylhydrazine (0.01 mol) was added
dropwise with the addition of 1–2 drops of glacial acetic
acid in the above reaction mixture in a round-bottom ꢃask.
°
The mixture contents were reꢃuxed for 2 h at 70 C. The con-
tents of the ꢃask were cooled, and the yellow-colored pre-
cipitates of 4-alkylphenylhydrazone were ꢂltered. The pre-
cipitates with a small quantity of methanol (2–3 mL) were
water, and they were recrystallized with absolute ethanol
◦
[yield 53.2%, melting point 141-142 C for R = OCH and
3
◦
yield 52.7%, melting point 145-146 C for R=NO ].
2
Reflux
CHO
+
H N.HN
2
R
R
CH
N.NH
o
7
0 C
4-Alkylbenzaldehyde
PhenylhydrazIne
4-Alkylphenylhydrazone (I)
Methanol:DMSO
1:1)
pH = 8 - 9
N
S
(
N Cl
2
R
C
N
Oxidation
NBS
R
CH
N
N.NH
N
N
N
N
N
S
S
N
2
-Phenyl-3-(2-thiazolyl)-5-(4-alkylphenyl)
2
-Phenyl-3-(2-thiazolyl)-5-(4-
Tetrazolium Bromide (III)
alkylphenyl) formazan (II)
Scheme 1 Synthesis of 2-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium bromide (PTMPT) and 2-phenyl-3-(2-thiazolyl)-5-(4-nitrophe-
nyl)tetrazolium bromide (PTNPT) (R=OCH for PTMPT and R=NO for PTNPT)
3
2
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Journal of the Iranian Chemical Society
Step 3: synthesis
of the synthesized reagent is 338.4178 and 353.0871 for
PTMPT and PTNPT, respectively. Mass spectroscopy is
found to be in good agreement with the proposed structure
of tetrazolium salt PTMPT and PTNPT; MS–ESI–TOF
of 2‑phenyl‑3‑(2‑thiazolyl)‑5‑(4‑alkylphenyl)
tetrazolium bromide by the oxidation of formazan
(III)
(
m/z) found in 338.4172 and 353.0871, respectively, shown
Synthesis of 2-phenyl-3-(2-thiazolyl)-5-(4-alkylphenyl)
tetrazolium bromide was done by the oxidation of formazan
synthesized in step 2. For this, the weigh amount (0.29 g)
of formazan was dissolved in 30 mL of ethyl acetate and
N-bromosuccinimide was added in it for its oxidation. As the
color of formazan disappears, the formation of tetrazolium
salt is conꢂrmed. The ethyl acetate evaporated, the dry pre-
cipitates were dissolved in methanol, and this solution was
ꢂltered using carbon black. 1/3 of the original solution evap-
orated, and then ethyl acetate (15–20 mL) was added in it.
The resulting solution was allowed to stand overnight. Faint
yellow or colorless precipitates of tetrazolium salt seperates
in Figs. 3 and 4, respectively.
Sample preparation
Pharmaceutical ꢀormulations
Most of the pharmaceutical samples contain α-tocopherol
in its stable form, i.e., α-tocopherol acetate. To quantify
α-tocopherol in different pharmaceutical formulations,
saponiꢂcation has been done to get α-tocopherol in free
form. To saponify, ꢂve capsules (each capsule contains
400 mg of α-tocopherol) had been taken, which had been
mixed thoroughly to get homogeneity. Into a 100-mL round-
bottom ꢃask, 0.1 g of an oily mass of α-tocopherol and 2 mL
of KOH solution (60%), 10 ml glycerol, and 25 ml abso-
lute ethanol had been taken. The reaction mixture has been
reꢃuxed for 2 h at 70 °C [15]. Then, the reaction mixture
was cooled to room temperature followed by its extraction
in separating funnel by addition of 30 mL petroleum ether
in the reaction mixture for 10 min. The aqueous layer was
removed, and the ether layer evaporated to dryness and then
the dried mass was dissolved in 100 mL of absolute ethanol
to get a stock solution of concentration 1000 μg/mL, which
was further diluted with absolute ethanol to get the working
solution of 50 μg/mL. The solution so prepared has been
used for the quantitative analysis of α-tocopherol.
out [yield 65%, melting point 193–195 °C for R=OCH and
3
yield 58.2%, melting point 189–190 °C for R=NO ].
2
The FTIR spectrum of the reagents PTMPT and PTNPT
−
1
has been recorded in the range between 4000 and 400 cm
at room temperature with Beckman IR-20 Spectrophotom-
eter. The spectrum has been recorded at room tempera-
ture using potassium bromide pellets. The IR absorption
−
1
band appears at: for PTMPT IR data (cm ): 1697(C=N),
1
2
1
3
180(CH O-Ph), 1605(C=C), 3400–3000(N–H stretching),
3
−
1
970(C–H stretching) (Fig. 1) and for PTNPT IR (cm ):
697(C = N), 1528(N–O stretching), 1180(H C-O-Ph),
3
400–3000(N–H stretching) (Fig. 2).
+
The ESI–MS spectra of the reagents PTMPT and
PTNPT recorded in D O with Xevo G2-S Q-T (Waters,
2
USA) give a base peak at 336.4178 and 351.0871 which
is under [M−2] peak. This indicates the molecular weight
Fig. 1 Infrared spectra of 2-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium bromide (PTMPT)
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Journal of the Iranian Chemical Society
Fig. 2 Infrared spectra of 2-phenyl-3-(2-thiazolyl)-5-(4-nitrophenyl)tetrazolium bromide (PTNPT)
Fig. 3 Mass spectrum of 2-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium bromide (PTMPT); MS–ESI-TOF (m/z) for C H N OS
1
7
16
5
calcd: 338.4178
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Journal of the Iranian Chemical Society
+
Fig. 4 Mass spectrum of 2-phenyl-3-(2-thiazolyl)-5-(4-nitrophenyl)tetrazolium bromide (PTNPT); MS–ESI-TOF (m/z) for C H N O S calcd:
1
6
13
6
2
3
53.0815
Milk samples
Miscellaneous sample
For the quantiꢂcation of α-tocopherol weighed milk sam-
ple equivalent to 0.1 g (cow milk, buꢀalo milk, Yakult
processed milk, protein powder, infant milk powder) was
transferred to a 100-mL round-bottom ꢃask, for saponiꢂca-
tion, as described for the pharmaceutical formulations. The
resulting dried mass after the saponiꢂcation procedure has
been then dissolved in 100 mL absolute ethanol. This solu-
tion has been further diluted with 100 mL absolute ethanol
to get the working solution.
Each of the other investigated samples, which have not been
quoted above, i.e., Proteinx, Quaker Oats, and cloves, also
contains a signiꢂcant amount of α-tocopherol. To quantify
α-tocopherol, 0.1 g (solid sample), 2 mL (Mac total Syrup)
of each sample has been taken in the round-bottom ꢃask and
saponiꢂcation has been done using the above procedure. The
resulting viscous mass has been then dissolved in 100 mL of
the absolute ethanol, which has been diluted with absolute
ethanol up to 100 mL to get the working solution and ana-
lyzed by the prescribed procedure.
Nuts sample
Experimental procedure
Nuts that contain a remarkable amount of α-tocopherol.
0
.1 g of dried, well-crushed nut samples (almond, cashew
Quantiꢀcation of α‑tocopherol using
2‑phenyl‑3‑(2‑thiazolyl)‑5‑(4‑methoxyphenyl)
tetrazolium bromide (PTMPT)
nuts, and pistachio) have been transferred to a 100-mL
round-bottom ꢃask for saponiꢂcation following the above
procedure, and after that, the dried mass has been dissolved
in 100 mL of absolute ethanol taken in measuring ꢃask.
The resulting solution has been further diluted with 100 mL
absolute ethanol to get a solution, which has been used for
further analysis.
Aliquots of 50 μg of α-tocopherol were added to a 10-mL
volumetric ꢃask. Then, 2.0 mL of reagent (PTMPT, 0.01%)
solution was added to it followed by the addition of 1.0 mL
of 1 M sodium hydroxide solution. The pink-colored
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Journal of the Iranian Chemical Society
formazan formed because of the reduction of colorless tetra-
zolium reagent by α-tocopherol. The pink-colored formazan
gives absorption maxima at 526 nm.
at 479 nm and 595 nm separately for both the complexes.
Extraction was ꢂnished utilizing n-hexane for α-tocopherol
evaluation in palatable oils and ghee tests. Likewise, ferro-
zine-Fe(II) framework has been utilized for the evaluation
of vitamin A and α-tocopherol on a similar example. It was
presumed that the greater part of these techniques, include
metals in the investigation. By exploring the literature, we
have developed two new strategies [26, 34] for the quantita-
tive investigation of α-tocopherol in diꢀerent pharmaceutical
and consumable samples. In this way, we infer that these
spectrophotometric techniques are roundabout one. Moreo-
ver, these systems require either sitting tight time or warm-
ing for the ꢂnish of the investigation, just as the contribution
of metal particles makes these methods dull and profoundly
helpless for impedance in the examination. Execution of
an immediate technique for the test of α-tocopherol was
ꢂnished utilizing triphenyltetrazolium salt as a systematic
reagent [31]. This method requires warming for 10 min on
a water shower at 90± 2 °C, for the reduction of tetrazolium
salt by α-tocopherol. Therefore, an easy system, which is
without holding up time just as warming, is required for the
quantitative examination of α-tocopherol.
Quantiꢀcation of α‑tocopherol using
2
‑phenyl‑3‑(2‑thiazolyl)‑5‑(4‑nitrophenyl)
tetrazolium bromide (PTNPT)
To construct the calibration graph using tetrazolium salt, the
analytical procedure was as follows: To 10 mL of volumetric
ꢃask aliquots of 50 μg of α-tocopherol were added followed
by the addition of 2 mL of tetrazolium salt solution (0.015%)
and 0.02 mL of 0.01 M sodium hydroxide solution. The vol-
ume of the volumetric ꢃask was raised to 10 mL using a
DMSO solvent containing NH OH (1 drop of ammonia in
4
1
00 mL of DMSO). The complex gives absorption maxima
at 526 nm.
Quantiꢀcation of α‑tocopherol in diꢁerent
pharmaceuticals, milk samples, nuts, cereals,
and fruits samples
In diꢀerent edible samples, quantiꢂcation of α-tocopherol
has been done by following the same procedure as described
above, for the pure α-tocopherol form. The absorbance inten-
sity observed for these samples is equivalent to the amount
of α-tocopherol present in them, calculated by using the cali-
bration graph. A comparison of our results with the amount
of α-tocopherol was made, and the labeled and the observed
values from the newly proposed method show good agree-
ment with other existing methods.
In the present investigation, we have evaluated
α-tocopherol by utilizing its reducing property. The synthe-
sis of new tetrazolium salts was ꢂnished utilizing literature
strategies [29, 30]. In writing, extraordinary tetrazolium salts
have been utilized for the measurement of α-tocopherol. In
every one of those strategies, either warming or waiting time
is required for the reduction of a dismal tetrazolium salt
into relating hued formazan by α-tocopherol [31]. The two
recently incorporated tetrazolium salts get reduced imme-
diately by the α-tocopherol (Fig. 5) within the sight of a
modest quantity of base (pH 8–9) neither warming nor hang-
ing tight time is required for the proposed technique. The
measure of α-tocopherol was determined by estimating the
absorbance of the shaded formazan utilizing a UV–visible
spectrophotometer. We contrast our technique and the cur-
rent writing strategy, which shows great concurrence with
the after-eꢀects of the present method.
Results and discussion
Various spectrophotometric techniques have been utilized
for the measurement of α-tocopherol in pharmaceuticals and
various edibles. In the vast majority of these current strate-
gies, metal–ligand complexation has been utilized for the
measurement, where α-tocopherol reduces metal particles
present in higher oxidation states to lower oxidation states,
which at that point formed a colored complex with some
particular reagents. Fe(III) reduced to Fe(II) with the assis-
tance of α-tocopherol [32], and the reduced metal particle
structure forms complex with the speciꢂc reagent, which
has absorption maxima at 562 nm. Cu (II)-neocuproine
framework was used for the quantiꢂcation of α-tocopherol
Optimization of diꢁerent reaction variables
The reduction of tetrazolium salts, i.e., PTMPT and PTNPT,
requires an appropriate amount of base. For PTMPT as the
amount of base increases, the absorption values increase
up to 1.0 mL of 1 M NaOH solution; after that, further
addition of base will decrease the absorption value. In the
case of PTNPT, as the amount of 0.01 M base increases,
the absorption increases up to 0.02 mL and further addition
of base shows a decrease in absorption value. So, 0.02 mL
of 0.01 M was considered suꢁcient for the quantiꢂcation
of α-tocopherol using PTNPT. The eꢀect of reagent con-
centration has also been studied, and it has been found that
[
36], and the absorption maxima for Cu (II)-neocuproine
was found at 450 nm. The reagents bathocuproine and 2, 4,
6
-Tris-(2-pyridyl)- s-triazine builds complex with Cu (I) and
Fe (II) ions separately [33]. The quantitative investigation
has been done in the presence of buꢀer (pH 4), and the maxi-
mum absorbance for the buildings framed has been found
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Journal of the Iranian Chemical Society
R
C
N
R
C
N.NH
N
α
-Tocopherol
N
N
N
N
Reduction
N
S
S
N
2
-Phenyl-3-(2-thiazolyl)-5-(4-
2
-Phenyl-3-(2-thiazolyl)-5-(4-alkylphenyl)
alkylphenyl) formazan
(Pink Color)
Tetrazolium Bromide
(Colorless)
Fig. 5 Reduction of 2-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium bromide (PTMPT) and 2-phenyl-3-(2-thiazolyl)-5-(4-nitrophenyl)
tetrazolium bromide (PTNPT) (R=OCH for PTMPT and R=NO for PTNPT)
3
2
as the amount of tetrazolium salt solution increases, there
was an increase in the absorption value. By optimizing all
the factors, 2 mL of both the reagents PTMPT and PTNPT
was found to be adequate for maximum color development.
To ensure the maximum reproducibility, the reagent must
be added before the addition of NaOH. Otherwise, low
color intensity was observed due to the incomplete oxida-
tion–reduction reaction. It has been noted that α-tocopherol
has been easily oxidized by air due to its photochemical
activity. To overcome this diꢁculty, the experiments were
conducted in the dim light/in the darkened room/or covering
the ꢃask with aluminum foil that prevents the photochemical
oxidation of α-tocopherol.
the concentration range 0.2 μg/mL to 15 μg/mL for PTMPT
and 0.1 μg/mL to 0.4 μg/mL for PTNPT.
The diꢀerent statistical parameters had been optimized,
and satisfactory results are listed in Table 1.
Calibration graph for the quantiꢀcation
of α‑tocopherol at 526 nm
The UV–visible absorption spectra for α-tocopherol with
both the reagents PTMPT and PTNPT were obtained by
scanning of pink-colored formazan from 400 to 700 nm
using a UV–visible spectrophotometer. The maximum
absorption of formazan for both the reagents was obtained
at 526 nm (Fig. 6). The calibration graph for α-tocopherol
using both the reagents was constructed by the addi-
tion of aliquots of 0.01% PTMPT and 0.015% PTNPT in
10-mL volumetric ꢃask containing aliquots of 50 μg/5 μg
of α-tocopherol for PTMPT and PTNPT, respectively.
The addition of sodium hydroxide to the above contents
gives pink-colored formazan with absorption maxima
at 526 nm. The calibration graph for α-tocopherol with
Analytical points of merit
The optimization of diꢀerent reaction variables has been
done to get the maximum absorption value. The calibra-
tion graph was constructed between the absorbance of
the reduced PTMPT/PTNPT and the concentration of
α-tocopherol, which shows a linear relationship in between
Table 1 Statistical parameters for the quantiꢂcation of α-tocopherol
Sr. no. Parameter
Value
Parameter
Value
PTMPT
PTNPT
PTMPT PTNPT
1.
2.
3.
4.
5.
6.
7.
8.
λmax. (nm)
526
526
0.1–0.4
6.4×10
0.006×10
0.016
Correlation coeꢁcient (r)
0.955
0.998
Beer’s limit (μg/mL)
0.2–15.0
4.21×10
0.11×10
0.030
Molar absorptivity (L mol− cm−1
1
)
3
3
4
Relative standard deviation (%) 0.87
LOD
LOQ
4.6
0.03
0.08
2
−3
Sandell’s sensitivity (μg/cm )
0.14
0.42
Standard deviation
Regression equation
Slope (m)
Y
=0.013x+0.202 Y =0.167x+0.008
0.013
0.202
0.167
0.008
Intercept (c)
Y=mx+c where x is in μg/mL
LOD=3S /b (S =standard deviation of response, b=slope of calibration curve)
a
a
LOQ=10S /b (S =standard deviation of response, b=slope of calibration curve)
a
a
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Journal of the Iranian Chemical Society
Fig. 6 Absorption spectra
of reduced 2-phenyl-3-(2-
thiazolyl)-5-(4-methoxy-
phenyl)tetrazolium bromide
(
PTMPT) (0.01%, 2 mL) and
2
5
-phenyl-3-(2-thiazolyl)-
-(4-nitrophenyl)tetrazolium
bromide (PTNPT) (0.015%,
2
mL) against reagent blank;
α-tocopherol (50 μg/mL,3 mL),
NaOH (1.0 M, 1.0 mL) for
PTMPT and α-tocopherol (5 μg/
mL, 0.8 mL), NaOH (0.01 M,
0
.02 mL), total volume of solu-
tion=10 mL
Fig. 7 Calibration graph:
absorbance versus concentra-
tion of α-tocopherol, PTMPT
(
0.01%, 2 mL), NaOH (1.0 M,
1
.0 mL), total volume of solu-
tion=10 mL
2
-phenyl-3-(2-thiazolyl)-5-(4-methoxyphenyl)tetrazolium
of PTMPT/PTNPT by α-tocopherol. The peaks observed
in the cyclic voltammogram of the reagent in the pres-
ence and absence of α-tocopherol give an idea of electron
change during the reduction process. The voltammogram
of α-tocopherol+PTMPT/PTNPT+NaOH was recorded in
ethanol at room temperature using KCl as supporting electro-
lyte, having a scan rate of 0.1 V/s, within the potential range
- 0.1 V to + 1.0 V. The graph between I/mA and E/V for
bromide and 2-phenyl-3-(2-thiazolyl)-5-(4-nitrophenyl)
tetrazolium bromide is shown in Figs. 7 and 8, respectively.
Cyclic voltammetric characteristics
Cyclic voltammetric studies had been done using the Ivi-
umStat Electrochemical analyzer to measure the reduction
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Journal of the Iranian Chemical Society
Fig. 8 Calibration graph:
absorbance versus concentra-
tion of α-tocopherol, PTNPT
(
(
0.015%, 2 mL), NaOH
0.01 M, 0.02 mL), total volume
of solution=10 mL
of curve (A, B) observed in absence of α-tocopherol had
Epa = -0.023 V and Epc = 0.011 V and in the presence of
α-tocopherol had Epa = -0.036 V and Epc = 0.027 V for
PTNPT (Fig. 10).
Interferences
Some metals also have a reducing character that can inter-
fere in the quantiꢂcation of α-tocopherol. To eliminate these
interferences, the extraction of samples in petroleum ether
must be done after saponiꢂcation. Water-soluble impuri-
ties and vitamins such as vitamin C were removed by this
method because it is the vitamin E, which is soluble in petro-
leum ether, while other vitamins not. The other probable
error was removed by standard addition method, and the
experimental error for the vitamin E samples was minimized
by making a triplicate set of observations because all the
samples were prepared similarly.
Fig. 9 Cyclic voltammogram of 2-phenyl-3-(2-thiazolyl)-5-(4-
A
methoxyphenyl) tetrazolium bromide (PTMPT) in the absence and
B
presence of α-tocopherol (15 μg/mL), using KCL as supporting
Statistical tools for validation of method
electrolyte
The validation of the proposed method has been carried out
by ICH recommendations 18.
reagent in the presence of α-tocopherol and reagent alone is
represented by curves A and B, respectively. From the graph,
it is evident that the reduction potential for α-tocopherol
with the reagent and the reagent alone is of having diꢀerent
values. The potential of curve (A, B) observed in the absence
of α-tocopherol had Epa= -0.0154 V and Epc= 0.0121 V
and in the presence of α-tocopherol had Epa = -0.0584 V
and Epc=0.0160 V for PTMPT (Fig. 9) and the potentials
Linearity and range
The present method describes the quantiꢂcation of vita-
min E using newly synthesized tetrazolium salts. The vari-
ables involved in the quantiꢂcation of α-tocopherol was
validated and optimized to evaluate the linearity of the
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Journal of the Iranian Chemical Society
Fig. 10 Cyclic voltammogram
of 2-phenyl-3-(2-thiazolyl)-
5
-(4-nitrophenyl) tetrazolium
bromide (PTNPT) in the
A
B
absence and presence of
α-tocopherol (0.4 μg/mL), using
KCL as supporting electrolyte
proposed method by analyzing the diꢀerent concentration
of vitamin E. Beer’s law was obeyed in the range of con-
centration 0.2–15.0 μg/mL for PTMPT and 0.1–0.4 μg/
mL for PTNPT. Indication of good linearity was observed
when the value of the correlation coeꢁcient was found
Precision and accuracy
The measurement of precision was done using standard
addition method, and for each concentration, absorbance
measurement was repeated thrice. The proposed analytical
method was found to have high precision and have % RSD
less than 2. Percentage recoveries of the vitamin E formula-
tions were attributed to the accuracy of the proposed method
(Tables 2 and 3). For each solution, the regression equation
was used to calculate the concentration values (n=3). The
high accuracy of the proposed method was characterized
2
2
near unity (R = 0.955 for PTMPT and R = 0.998). The
other parameters like the intercept, slope, molar absorptiv-
ity, Sandell’s sensitivity, and correlation coeꢁcient were
calculated and are presented in Table 1.
Table 2 Quantitative analysis of α-tocopherol in pharmaceutical formulations using PTMPT
Sr. no. Pharmaceutical sample
Amount of vitamin Amount of vita-
Amount
Recovery % Relative
standard
present (C ) (μg)
min added (C )
found (C )
P
A
F
Brand
Manufacturer
(
μg)
(μg)
deviation
(
RSD) %
1.
2.
3.
4.
Enew
Merck Ltd. (CM Care)
Vintek Pharmaceuticals
20
4.0
23.8
29.6
31.7
24.5
31.6
33.3
23.6
29.9
32.7
25.1
31.3
32.8
100.8
101.3
100.9
97.9
94.9
96.1
101.6
100.3
97.8
95.6
95.8
± 0.6
± 2.8
± 0.8
± 1.2
± 0.4
± 0.4
± 1.1
± 0.8
± 1.4
± 0.8
± 0.6
± 0.5
2
2
0
0
10.0
12.0
4.0
10.0
12.0
4.0
10.0
12.0
4.0
10.0
12.0
Evitek-400
20
2
2
0
0
Native Forte Franco Indian Pharmaceutical
Ltd.
20
2
2
0
0
EVOC-400 Indizen Pharmaceutical
20
2
2
0
0
97.5
%
Recovery=C +C /C for three replicate measurements (n=3)
P A F
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Journal of the Iranian Chemical Society
Table 3 Quantitative analysis of α-tocopherol in pharmaceutical formulations using PTNPT
Sr. no. Pharmaceutical sample
Amount of vitamin Amount of vita-
Amount
Recovery % Relative
standard
present (C ) (μg)
min added (C )
found (C )
P
A
F
Brand
Manufacturer
(
μg)
(μg)
deviation
(
RSD) %
1.
2.
3.
4.
Enew
Merck Ltd. (CM Care)
Vintek Pharmaceuticals
20
0.2
0.3
0.4
0.2
0.3
0.4
0.2
0.3
0.4
0.2
0.3
0.4
19.8
20.1
20.0
20.5
19.6
20.3
20.6
20.1
19.7
20.1
19.3
19.8
102.0
100.9
102.0
98.5
103.5
100.5
98.1
100.9
103.5
98.5
105.2
103.1
± 0.5
± 0.9
± 0.8
± 0.3
± 0.7
± 0.8
± 0.8
± 1.0
± 0.8
± 1.2
± 0.8
± 0.8
2
2
0
0
Evitek-400
20
2
2
0
0
Native Forte Franco Indian Pharmaceutical
Ltd.
20
2
2
0
0
EVOC-400 Indizen Pharmaceutical
20
2
2
0
0
%
Recovery=C +C /C for three replicate measurements (n=3)
P A F
2
by a low coeꢁcient of variation (R = 0.985) and % RSD
< 2). The concentrations of different samples contain-
the slope of the calibration curve for each utilizing theories
conditions: LOD=3.3×SD/slope and LOQ=10×SD/slope.
(
ing α-tocopherol had been analyzed using the proposed
method three times intra- and inter-day. Good results had
been found, and an acceptable amount has been observed as
shown in Tables 2 and 3.
Analytical applications
The quantiꢂcation of vitamin E in diꢀerent edibles has
been done using the proposed method and the results
are found to be in close agreement as per the labelled
amount of edibles. The closeness of the results are listed
in Table 4. The analyzed amount of α-tocopherol in edible
material such as milk samples, nuts, and miscellaneous
samples (syrup, oats, and cloves) along with the absorb-
ance value is shown in Figs. 11 and 12.
Limit of detection and quantiꢀcation
Low estimations of LOD and LOQ given in Table 1 demon-
strated high aꢀectability of the developed methods. These
methodologies are dependent on the SD of the reaction and
Table 4 Vitamin E content in various comestible samples
Sr. no.
Sample
Manufacturer
Proposed method
PTMPT (μg/g)
Labeled amount on
product
PTNPT (μg/g)
1
2
3
4
5
6
7
8
9
1
.
.
.
.
.
.
.
.
Pistachio
Almonds
–
–
–
0.4
0.29
0.26
0.28
0.15
0.36
0.30
0.38
0.37
0.29
0.36
0.28
–
–
0.2
0.33
–
–
0.25
0.33
0.46(from
nutrition
data)
0.21
0.32
0.23
0.38
0.32
0.21
0.23
0.21
0.15
Cashew nuts
Fermented milk drink
Infant milk powder
Cow milk
Buꢀalo Milk
Proteinx
Yakult
Nestle India ltd.
–
–
Nutricia International Private Limited
Tirupati lifesciences
Symega food ingredients ltd.
.
0.
Mactotal syrup
Quaker oats
1
1.
Cloves
–
0.30
0.32
0.35
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Journal of the Iranian Chemical Society
Comparison of the proposed method with other
spectrophotometric methods
The result of the proposed method has been compared with
the other existing UV–visible spectrophotometric methods
in Table 5. It was observed that the proposed method is sim-
ple, reliable, and quicker than other many spectrophotomet-
ric methods. The main advantage of the proposed method
is its sensitivity and compatibility. The present procedure is
not complicated and less time-consuming than other existing
spectrophotometric methods.
Conclusion
Here, a simple, highly sensitive, direct, easy to handle, and
facile procedure was proposed for the quantification of
α-tocopherol (vitamin E) in pure form, various pharmaceu-
tical formulations, and comestible food samples. The quanti-
tative analysis is done by spectrophotometric method, which
is cost-eꢀective and easy to handle and less time-consuming
in comparison with the literature techniques such as HPLC,
UPLC and gas chromatography, which are quite expensive
and not available easily. The proposed procedure is magniꢂ-
cent for the evaluation of α-tocopherol (vitamin E) in dif-
ferent eatables and is quite competent for the agile analysis
of vitamin E.
Fig. 11 Quantitation of α-tocopherol in diꢀerent comestible edibles
using PTMPT
Fig. 12 Quantitation of α-tocopherol in diꢀerent comestible edibles
using PTNPT
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Journal of the Iranian Chemical Society
Table 5 Comparison of proposed method with other reported spectrophotometric methods for the quantitation of α-tocopherol
Sr. no. Reagent
Mode
λmax (nm) Time for
Remarks
References
completion of
reaction
1
.
Cu(I)-bathocuproine
Indirect 479
5 min
Proper analytical parameters were
not ꢂxed
Devi et al. [33]
2
3
.
.
Fe(II)-2,2′-bipyridine
Cu(II)-neocuproine
Indirect 520
Indirect 450
4 min
30 min
Less sensitive method
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Tutem et al. [36]
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time required
4
5
.
.
Fe(II)-2,4,6-Tripyridyl-s-triazine Indirect 595
Fe(II)-ferrozine Indirect 562
60 s
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Jadoon et al. [32]
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.
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10 s
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10 min
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PTMPT/PTNPT
Direct 526
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Acknowledgements The authors admiringly conꢂrm the ꢂnancial
assistance provided by UGC and CSIR, New Delhi, India, to carry
out this work (UGC-JRF Award no. 2121510026). The authors are
thankful to MNIT (Materials Research Centre), Jaipur, for providing
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