1. INTRODUCTION:
Vitamins A and E belong to the
group of fat soluble vitamins. Vitamin A is only required in small amounts,
but plays an important role in the organism. For this reason vitamins have to
be consumed regularly via the food or by intake of pharmaceutical vitamin
preparations. Vitamin A is present in animal products, such as milk, butter,
yolk of egg, fats and in particularly large quantities in fish liver oils
(daily intake of retinol 300ug for infants and young children, 500–750 µg for
children of age 9–15 years, 750 µg for adolescents adults and 1200 µg for
lactating women). Vitamin A plays a key role in many essential biologic
processes, and is necessary not only for vision but also for embryonic
development and the regulation of proliferation and differentiation of many
cell types. Of particular importance for public health is the role of vitamin A
in reproduction: deficiency as well as excess is teratogenic in animals (Combs
Jr. 2008).
Vitamin E (tocopherol) naturally
occurs in four forms as α, β, γ and δ tocopherols and four
corresponding unsaturated analogues, tocotrienols, α-Tocopherol, the most
pharmacologically important vitamin E agent, is a peroxyl radical scavenger
that is protective for polyunsaturated fatty acids within membranes and
lipoproteins (Siluk et al., 2007). Vitamin E is essential for such body
functions as growth, reproduction, prevention of various diseases, and
protection of the integrity of tissues. The naturally occurring
α-tocopherols and tocotrienols exist in the unesterified forms and are
distributed widely in vegetable oils, nuts, green leafy vegetables, seeds, fruits
and fortified cereals are common food sources of vitamin E (Hoppe and Krennrich
2000).
It is also an antioxidant and is
believed to play a preventive role in diseases associated with oxidative stress
like cancer, cardiovascular diseases, cataracts, age-related macular
degeneration, central neurode-generative diseases and diabetes mellitus. While
recent epidemiological studies have suggested that antioxidative vitamins play
a protective role in coronary heart disease and some cancers , others have indicated
that increased vitamin E intake may be linked to increased mortality, although
the latter finding have been disputed (Siluk et al 2007). The concentration of
a-tocopherol in human blood serum ranges from 5 to 15 mg/mL (Combs Jr. 2008).
Various methods have been
developed for the determination of vitamin A and E in biological and
pharmaceutical samples such as colorimetric/spectrophotometery (Amin 2001;
Prieto et al., 1999; Tutem et al., 1997; Devi et al., 2004; Jadoon et al.,
2010; Rishi et al. 2011), fluorimetry (Razagui et al., 1992), voltammetry
(Mikheeva et al., 2007), capillary zone electrophoresis (Yinfa et al., 1996),
HPLC (Kienen et al. 2008; Semeraro et al. 2009; Driskell et al. 1982), and flow
injection chemiluminescence (Yaqoob et al. 2009; Waseem et al. 2009; Asgher et
al., 2011).
The chromogenic specific reagent
ferrozine [3-(2- pyridyl-5,6-bis(phenylsulfonic acid)-1,2,4 triazine] proposed
by Stookey (1970)
which reacts with divalent Fe to form a stable magenta complex species. The advantages of ferrozine
(Fz) over other Fe(II) selective reagents are higher molar absorption
coefficient, water solubility, stability and low viscosity. Fz is a very
sensitive and selective reagent for iron(II) giving a 1:3 (Fe:Fz) magenta
chelate with a sharp absorption peak at 562 nm and a molar absorptivity of 27
900 L/mol.cm compares
favorably with 22,600 for 2,4,6-tris(2-pyridyl)-1,3,5-triazin (TPTZ), 22,143
for bathophenanthroline, and 11,100 for 1,10-phenanthroline. Interferences are
at a minimum (Stookey 1970). This is an important advantage with respect to Spectrophotometery
is a well accepted analytical technique owing to its versatility, flexibility
and ease of operation (Khalid et al., 2011; Waseem et al., 2010; 2011). This
paper presents a manual Spectrophotometric method for the determination of
vitamin A (Retinol) and E (α-Tocopherol) with the detection system using
Fe (II)-Fz in pharmaceuticals, infant milk powder and in blood serum samples.
The experimental results show that Fe (III) is reduced to Fe (II) by both
vitamins in buffered solution carrying surfactants.
2. EXPERIMENTAL:
2.1
Materials and Reagents:
All reagents used were of analytical grade,
unless stated otherwise and all solutions were prepared in ultra-high-purity
(UHP) deionised water (Elga, Purelab Option, UK). Iron (III) stock solutions
(1000 mg/L) were prepared by dissolving 0.48 g of FeCl3.6H2O in
0.01M HCl. A working iron (III) solution was prepared daily by diluting the
stock solution with UHP water. Ferrozine (FW 492.47, 97%, Aldrich) stock
solution (1000 mg/L) was prepared by dissolving 0.1 g in 100 mL UHP water.
Working solutions were prepared daily by diluting stock solution with UHP
water. Buffers (acetate, phosphate, phthalate) 0.1M were prepared from their
respective salts in UHP water. Surfactant standard solutions, 0.1%, were
prepared by dissolving 0.1 g of Triton X-100, Brij-35 and CTAB individually in
100 mL of UHP water and stored in dark brown bottles. Working solutions were
prepared from these stock solutions by appropriate dilution with UHP water and
protected from light. Vitamins standard stock solution (100 mg/L) were prepared
by dissolving 10 mg of each reactive (retinol, retinyl acetate, retinyl
palmitate, α- tocopherol, α-tocopheryl acetate) in 100 mL of ethanol
using dark brown volumetric flasks, stored in the dark at -20ºC. Working
solutions were prepared from the stock solutions by appropriate dilution with
ethanol solution (5.0%, v/v) and shielded from light. Standard solutions (1000
mg/L) of sodium, potassium, calcium, magnesium, nitrate, sulphate and chloride
ions from their respective salts, glycerol, maltose, lactose, mannitol, methyl
cellulose, sucrose, starch, albumin, PVA in UHP water, and retinol, retinyl
palmitate and vitamin D3, cholesterol, vitamin K, EPA, DHA fatty acids in
ethanol were prepared respectively. Various working solutions were prepared
from these stock solutions by appropriate dilution with ethanol solution (5.0%,
v/v), protected from light and used for interference studies. Pharmaceutical
formulations and Infant powdered milk containing vitamin E: DL-α-
tocopherol acetate was collected from local market.
2.2.
Sample Preparation:
Pharmaceutical solid samples (five tablets)
were ground with a mortar and pestle and a known quantity of the ground
material was weighed and vitamin A and E were extracted twice with 5.0 mL
methanol. In case of liquid samples, a known volume was taken and diluted in 10
mL of methanol. Samples were taken in glass tubes protected from light and
saponified with addition of 1.0 mL of KOH, 50% and 2.0 mL of ethanol and
incubated in a water bath at 45°C for 2 h with intermittent mixing and purging
of nitrogen gas. After incubation, 1.0 mL of water was added and extracted five
times with 5.0 mL ether. The organic layer was recovered and evaporated to
dryness in a water bath at 37°C under a stream of nitrogen. The residue was
re-dissolved by vortex-mixing in 5.0 mL methanol and diluted appropriately with
carrier solution, methanol 5% solution containing triton X-100.
Commercially available pharmaceutical formulations’
containing vitamin E exists as DL- α-tocopheryl acetate, and is quite
stable to oxidation. Tocopheryl acetate was converted into α-tocopherol by
previously reported procedures (Lubna et al., 2011). From each sample, two
capsules were placed into a bottle containing 200 mL of ethanol and sonicated
for 10 min at room temperature. Standard solutions were prepared from each
sample and stock solutions of α- tocopheryl acetate in a 20 mL brown glass
bottle, 25 μl of concentrated sulfuric acid (as a catalyst) and 10 mL of
methanol were added to each bottle. The bottles were covered with aluminium
foil by leaving a space for evaporation; the bottles were then heated at 70–80
ºC in a water bath for 80–120 min by intermittent purging of nitrogen gas; during
this period the contents of the bottles were evaporated to almost dryness. The
end product of transesterification was dissolved in 10 mL of ethanol and
analyzed accordingly. For extraction of vitamins from commercially available
infant milk powder, previously reported procedure (lubna et al 2011; Jadoon et
al., 2010) was adapted as described. 5.0 g milk powder was weighed and
transferred to a 100 mL Erlemeyer flask, homogenized in hot water (50 mL
containing 0.1 g ascorbic acid) with constant stirring for at room temperature
for 10 min. The mixture was saponified with 60 mL of alcoholic potassium
hydroxide (60%). Vitamins were extracted with hexane three times with 25 mL
portions and then hexane was evaporated to dryness at 45Ċ with purging of
nitrogen gas. The residue was dissolved in methanol (5.0 mL) and used for
analysis.
Blood specimens were taken from several
healthy volunteers, collected in tubes (protected from light), centrifuged at
5000 rpm for 10 min, and 2.7mL of serum fraction was collected. A 500 mL serum
aliquot from each sample in the absence and presence of internal standards (250
and 100 mg/L for retinol and tocopherol, respectively) were treated with 500 mL
methanol and vortexed for 30 s; vitamins were extracted five times with 4.0mL
hexane and the organic layer was removed. The organic solvent was evaporated
with a stream of nitrogen and the residue was re-dissolved in 0.5mL of
methanol. The samples were appropriately diluted with carrier solution and
analyzed for retinol and tocopherol accordingly.
2.3.Instrumentation:
The absorbance of the complex was monitored
at 562 nm using a spectrophotometer (Jenway, 6505, UK) equipped with quartz
(suprasil, Hellma, UK) sample holder, the output of the spectrophotometer is
obtained using by connecting it to a chart recorder (Kipp and Zonen BD40,
Holland). The temperature of solution was maintained using a water bath
(Clifton, Nickel Electro Ltd. England).
2.4.Proposed Procedure:
All the solutions were mixed using sonicator
and transferred in to the 3 mL quartz cuvette for reference (blank) and samples
(carrying vit. A and E). The detector output from spectrophotometer was
recorded using a chart recorder.
3. RESULTS AND DISCUSSION:
3.1. Selection of manual parameters:
In order to establish the best possible
conditions for the determination of Vitamin A and E, various experimental
parameters were investigated using a univariate approach. The key parameters
optimized were buffer type and pH, concentration of solvent/surfactant for
carrier stream and sample preparation, Fe(III) and Ferrozine concentrations,
temperature and reaction time. All of these studies were performed with a 5
mg/L of vitamin A and E standard solution having 5% ethanol and the absorbance
was monitored at 562 nm with a UV/Vis spectrophotometer after two minutes of
reagents mixing. Each selected concentration is then used for the optimization
of subsequent parameter.
Ferrozine-Ferrous complex can be made in
water and is stable at pH between 4-9, (Stookey 1970) however to keep the pH
same at all situations, buffers can be used between this range keeping in view
of Fe(III) reduction by both vitamins and their stability. Phosphate, acetate,
and phthalate buffers were used (0.1M, pH 6), phosphate buffer was found to be
suitable, hence the pH of which was optimized in the range of 4-8. pH 6 was
selected with suitable response compared with other pHs as lower pH may destroy
vitamin A and higher pH can cause precipitation of Fe(III) (Fig 1).
Fig. 1 Variation of peak height absorbance
with respect to pH.
Vitamin A and E are less soluble in water
compared to organic polar or non polar solvents, therefore it is necessary to
use water miscible organic solvent or surfactant to dissolve them. Ethanol (5%)
and several surfactants such as TritonX-100, Brij-35, SDS, and CTAB were used
(Fig. 2).
Fig. 2-Variation of peak height absorbance
with respect to the concentration of surfactants.
Figure 2 shows that the higher response is
observed with Triton X-100 concentration of 0.01% compared to ethanol alone and
other surfactants carrying 5% ethanol and therefore selected to use for sample
carrier stream/sample preparation and used for further experiments (Lubna et
al., 2011).
Fig. 3 Variation of peak height
absorbance with respect to the concentration of Triton X-100.
Fe (III) concentration on the formation of
magenta iron (II)-Ferrozine complex was investigated in the range from 1-30
mg/L as shown in Fig. 4.
Fig. 4. Variation of peak height absorbance
with respect to the concentration of Fe (III).
Maximum peak height absorbance was observed
at 10 mg/L of iron(III) above which peak absorbance does not change
significantly and increases base line (Fe(III) forms a weak complex with
Ferrozine at lower pH). Therefore, Fe (III) concentration of 10 mg/L was
selected and used for further studies with reproducible base line.
The Ferrozine reagent proposed by Stookey (1970) which reacts with divalent Fe to
form a stable magenta complex of 3:1. The maximum absorbance of which is
recorded at 562 nm with a molar absorption coefficient close to 30,000 l mol−1 cm−1, which is
far higher than the bathophenanthroline or 1,10-phenanthroline used previously
(Rishi et al. 2011 JCSP). The concentration of Ferrozine was studied in the range
of 10-50 mg/L using 10 mg/L of Fe (III) concentration. The absorbance increased
with the increase in Ferrozine concentration up to 30 mg/L and further increase
in concentration resulted in decrease in peak height absorbance and increase in
base line (Fig. 6).
Fig. 5 Variation of peak height
absorbance with respect to the concentration of Ferrozine.
Therefore, Ferrozine concentration of 30
mg/L was selected and used subsequently. Reaction rate of Fe (III) reduction
can be increased by increasing temperature; hence temperature was also
investigated in the range of 25 to 85 ºC. Increase in temperature of the
reagents caused a positive change in absorbance up to 70 ºC above which it is
almost constant.
Fig. 6 Variation of peak height absorbance
with respect to the temperature of solution.
The effect of reaction time for reagent
mixing and reduction was also checked, vitamin A shows slow reaction kinetics
as compared to vit. E (Fig. 7).
Fig. 7 Variation of peak height absorbance
with respect to the reaction time for reduction.
Reaction time of two minutes for vit E
reduction was found to be suitable; however vitamin A requires almost double
(four minutes) for significant reduction. Five minutes of reaction time however
was selected for the quantitative determination of both the vitamins.
3.1. Analytical figures of merit
Under the selected conditions describe
above, linear calibration graphs of peak absorbance versus vitamin A and E
concentration were obtained. Vitamin A shows a linear calibration graph in the
concentration range of 0.1–10 μg/mL with a limit of detection (3s) of 0.06
μg/mL (Fig 10).
Fig.
8 Calibration graph for the determination of vitamin A
The regression equation A=0.0896c+0.0089
(where A=absorbance; c=concentration
in μg/mL). The coefficient of determination was r2=0.9974 with
relative standard deviations (n = 4) in the range of 0.8–2.8%. The
proposed method allowed 10 injections per hour. Vitamin E gives a calibration
graph linear in the concentration range of 0.1–20 μg/mL with a limit of
detection (3s) of 0.03 μg/mL (Fig 9).
Fig.
9 Calibration graph for the determination of vitamin E.
The regression equation, A=0.0909c
+ 0.0015 (where A=absorbance; c=concentration
in μg/mL). The coefficient of determination was r2=0.9993
with relative standard deviations (n = 4) in the range of 1.1– 2.6% with
a sample throughput of 30 injections per hour.
3.2. Interferences
In order to assess the selectivity of the
proposed Manual Spectrophotometric method, the influences of common foreign
species/ excipients in drugs including lactose, mannitol, maltose, sucrose,
starch, and cellulose, gum acacia, PVA, albumin, inorganic ions e.g., sodium,
potassium, calcium, magnesium, zinc, copper, manganese, nitrate, chloride and
sulfate ions and organic compound e.g., cholesterol, β-carotene,
vitamin D3, vitamin K on vitamin A and E determination were investigated under
the conditions established. Interferrents were tested under the optimized
conditions containing a standard solution of 1.0 µg/mL of vitamin A and 0.5
µg/mL of vitamin E individually. The tolerance of each foreign species was
taken as the largest concentration yielding less than ±5% of the error of the
adoptive concentrations of vitamins. As shown in Table 1, no clear interference
could be found with most of the compounds for vitamin A and E determinations.
Selective extraction procedure, however, insures the absence of most of the
interference described previously and is demonstrated by the HPLC reference
method analysis.
Table
1.Maximum tolerable concentration for the determination of vitamin A and E (n
= 4).
|
Species
added
|
Tolerance
ratio (%)
|
|
Vit
A
(1
ppm)
|
Vit
E (0.5ppm)
|
|
Sodium,
potassium, calcium, magnesium,
|
300
|
300
|
|
Chloride,
sulfate, nitrate, sucrose, starch, cellulose
|
200
|
300
|
|
Glucose,
gum acacia, magnesium stearate, dextrin
|
100
|
100
|
|
Zinc,
copper, manganese
|
50
|
50
|
|
Mannitol,
glycerol, methyl cellulose, cholesterol, β-carotene, vitamin D3 and K,
EPA, DHA, polyvinyl alcohol, albumin
|
40
|
60
|
|
Uric
acid, ascorbic acid
|
1
|
1
|
3.3. Method Validation
The proposed Spectrophotometric method for
the determination of vitamin A and E in pharmaceuticals, infant milk formula
and human blood serum was validated by comparing with HPLC reference method
(Driskell et al. 1982). Statistical tests F and t were applied
for the results given in Tables 2 and 3 between proposed and reference
methods. The calculated results showed that there is no significant difference
between the two methods at 95% confidence level.
3.4. Analytical applications
The proposed Spectrophotometric method was
applied to determine vitamin A and E in pharmaceuticals, infant milk formula
and blood serum. The pharmaceuticals containing vitamin A (as retinol) alone
or along with vitamin E (as tocopheryl acetate) can be analyzed directly, as
tocopheryl acetate does not cause any interference for retinol determination.
However, vitamin E (tocopherol) determination requires transesterification
(sample containing Tocopherol acetate does not show any reducing property), the
presence of retinol, if any, could be destroyed which does not cause any
interference (Waseem et al. 2009). It could be seen from Table 2 that there
were no significant differences between the labeled contents in pharmaceutical
samples/infant milk formula and those obtained by the proposed Manual-spect
method and reference method.
For blood serum samples, vitamin E
(tocopherol) was observed interfering with vitamin A (retinol) determination.
Normal blood serum concentrations of retinol and tocopherol are in the ranges
0.3–0.6 and 5.0–15 mg/L, respectively. However, difference in reaction rates
can be used for the determination of both vitamins.
Vitamin A interference can also be removed
by destroying it with few drops of H2SO4 as vitamin E is
quite stable at lower pH and analyzing at 1.0 mL/min flow rate. It can be seen
from Table 3 that there were no significant differences between the HPLC
reference method and those obtained by the proposed method for vitamin A and E
determinations in human serum samples. The results given in Table 3 confirm
that the proposed method is not liable to
interferences. They seem to be accurate, and the method was successfully
applied to the determination of vitamins A and E in the presence of common
excipients.
REFERENCES
[1] Amin, A. S. 2001.
Colorimetric determination of tocopheryl acetate (vitamin E) in pure form and
in multivitamin capsules. Eur. J. Pharm. Biopharm. 51(3): 267–272.
[2] Asgher M,
Yaqoob M, Waseem A, Nabi A. Flow injection methods for the determination of
Retinol and -Tocopherol using lucigenin enhanced chemiluminescence.
Luminescence, 26(6) (2011) 416–423.
[3] Combs
G.F. Jr., The Vitamins: Fundamental Aspects in Nutrition and Health, Elsevier
Academic Press, USA, (2008).
[4] Devi I,
Memons. A., Khuhawar M.Y. Spectrophotometric analysis of Vitamin E using Cu
(l)-Bathocuproine,and Fe(ll)-2,4,6 tris-(2-pyridyl)tarizine complexes. Journal
of the Chemical Society of Pakistan, 2004, 26(3), 239-243.
[5] Driskell,
W. J., W. J. Neese, C. C. Bryant, and M. M. Bashor. 1982. Measurement of
vitamin A and vitamin E in human serum by high-performance liquid
chromatography. J. Chromatogram. B 231(2): 439–444.
[6] Fu P, Xia
Q, Boudreau MD, Howard PC, Tolleson W, Wamer W. Physiological role of retinyl
palmitate in the skin. Vitamins and Hormones – Vitamin A, 75:223–56.
[7] Helrich
K, Official Methods of Analysis of the Association of Official Analytical
Chemists, Washington, DC, 15th ed., p. 1070.
[8] Hoppe PP,
Krennrich G. Bioavailability and potency of natural-source and all-recemic 
-tocopherol
in human: a dispute. Euro J. Nutr. 2000, 39, 183-93.
[9] Jadoon S,
Waseem A, Yaqoob M and Nabi A. Flow injection Spectrophotometric determination
of vitamin E in pharmaceuticals, milk powder and blood serum using potassium
ferricyanide-Fe(III) detection system. Chinese Chemical Letters, 21(6)
(2010) 712–715.
[10] Khalid A,
Waseem A, Afzal I, Yaqoob M, Nabi A, Yasinzai MM. Flow Injection
Spectrophotometric and Spectrofluorimetric methods for the Determination of
Candesartan Cilexetil in Pharmaceuticals. Scientific Research and Essays. 6(29)
(2011) 6203–6208.
[11] Kienen,
V., W. F. Costa, J. V. Visentainer, N. E. Souza, and C. C. Oliveira. 2008.
Development of a green chromatographic method for determination of fat-soluble
vitamins in food and pharmaceutical supplement. Talanta 75(1): 141–146.
[12] Mikheeva E.V., Anisimova L.S. Voltammetric
determination of vitamin E (α-Tocopherol acetate) in multicomponent
vitaminized mixtures. J. Anal. Chem. 62 (2007) 373.
[13] Prieto P., Pineda M., Aguilar M.
Spectrophotometric quantitation of antioxidant capacity through the formation
of a phosphomolybdenum complex: specific application to the determination of
vitamin E. Anal. Biochem. 269 (1999) 337.
[14] Razagui, I.
B., P. J. Barlow, and K. D. A. Taylor. 1992. A spectrofluorimetric procedure
for the determination of a-tocopherol in nutritional supplement products. Food
Chem. 44(3): 221–226.
[15] Rishi L,
Jadoon S, Waseem A., Yaqoob M., Nabi A. Flow-injection methods for the
determination of α-tocopherol with spectrophotometric detection. Journal
of The Chemical Society of Pakistan, 33(4) (2011) 508–514.
[16] Semeraro, A.
I., M. Altieri, Patriarca, and A. Menditto. 2009. Evaluation of uncertainty of
measurement from method validation data: An application to the simultaneous
determination of retinol and a-tocopherol in human serum by HPLC. J.
Chromatogr. B 877(11–12): 1209–1215.
[17]
Siluk D, Oliveira RV, Esther-Rodriguez-Rosas M, Ling S, Bos A,
Ferrucci L, Irving W. Wainer. A validated liquid chromatography method for
the simultaneous determination of vitamins A and E in human plasma. J Pharm
Biomed Anal. 2007, 15; 44(4): 1001–1007.
[18] Tutem E.,
Apak R., Gunaydi E., Sözgen K. Spectrophotometric determination of vitamin E
(α-tocopherol) using copper(II)-neocuproine reagent Talanta 44 (1997) 249.
[19] Waseem A,
Rishi L, Yaqoob M and Nabi A. A Flow-Injection determination of Cysteine, N-Acetyl
cysteine and Glutathione in Pharmaceuticals using Potassium Ferricyanide-Fe
(III) Spectrophotometric system. Chemical Research in Chinese Universities,
26(6) (2010) 893–898.
[20] Waseem A,
Rishi L, Yaqoob M and Nabi A. Flow Injection Determination of Retinol and
Tocopherol in Pharmaceuticals with acidic Potassium permanganate
Chemiluminescence. Analytical Sciences, 25(3) (2009) 407-412.
[21] Waseem A,
Yaqoob M and Nabi A, Hussain I. Flow-Injection Spectrophotometric procedure for
the determination of Uric Acid in Urine using Prussian blue reaction. Chemical
Research in Chinese Universities 27(6) (2011) 929–933.
[22] Yaqoob M,
Waseem A and Nabi A. Flow-Injection Determination of Vitamin A in
Pharmaceuticals Formulations using Tris(2,2'-bipyridyl)Ru(II)-Ce(IV)
Chemiluminescence Detection. Luminescence 24(5) (2009) 276-281.
[23] Yinfa, M.
1996. Quantitative retinol assay for serum and dried blood spots. US Patent
5532166