Spectrochimica Acta Part A 72 (2009) 989–993

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Enzymatic determination of vitamin A in pharmaceutical formulations with

spectrophotometric detection
Lubna Rishi, Mohammad Asgher, Mohammad Yaqoob ∗ , Amir Waseem, Abdul Nabi
Department of Chemistry, University of Balochistan, Sariab Road, Quetta, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: An enzymatic method for the determination vitamin A (retinol) is reported using soluble and immobilized
Received 20 September 2008 alcohol dehydrogenase, isolated from rabbit liver. The reaction is based on the oxidation of retinol and
Accepted 12 December 2008 simultaneous reduction of NAD+ to NADH followed by spectrophotometric detection at 340 nm. The cal-
ibration graph was linear over the range of 2.0–10 ␮M with correlation coefficients of 0.9967 and 0.9992
Keywords: (n = 5) for soluble and immobilized alcohol dehydrogenase respectively, with relative standard deviations
Vitamin A
(n = 3) in the range of 0.5–1.2%. The limit of detection was lower than 1.0 ␮M. The proposed method was
Alcohol dehydrogenase
applied to determine vitamin A in pharmaceuticals, and the results obtained were in reasonable agree-
Spectrophotometry
Pharmaceutical formulations
ment with the amount labeled. The results were compared using spectrophotometric reference method,
and no significant difference was found between the results of the both methods.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction samples. These includes; spectrophotometry/colorimetry [7–11],

Vitamin A, or retinol in its various forms functions as a hormone
and it is also an essential component of the visual cycle. Deficiency
of vitamin A leads to a variety of symptoms in humans, includ-
ing dryness of the skin, eyes, and mucus membranes; retarded
development and growth; and night blindness, an early symptoms
commonly used in diagnosing vitamin A deficiency [1,2]. Recent
interest has focused on the potential role of vitamin A status in
modulating the effect of HIV infection, particularly in the vertical
transmission from mother to infant [3]. Vitamin A might have addi-
tional roles, e.g., as protection against some forms of cancers and in
the treatment of skin disorders [4].
Vitamin A is only required in small amounts, but plays an impor-
tant 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 quanti-
ties in fish liver oils (daily intake of retinol 300 ␮g for infants and
young children, 500–750 ␮g for children of age 9–15 years, 750 ␮g
for adolescents adults and 1200 ␮g for lactating women). It has been
shown that carotene is converted into vitamin A in liver; hence good
source of carotene, such as green vegetables are good potential of
vitamin A [5,6].
Various methods have been reported for the determination
of vitamin A (retinol) in biological, food and pharmaceutical
∗ Corresponding author. Tel.: +92 819211266; fax: +92 819211277.
E-mail address: yaqoob2001@hotmail.com (M. Yaqoob).
high-performance liquid chromatography [12–15] voltammetry
[16], fluorimetry [17] and chemiluminescence [18,19].
The determination of vitamin A in pharmaceutical preparations
has always presented considerable analytical problems. The most
widely used methods include an ultraviolet-spectrophotometric
technique, in which vitamin A is assayed by measurement of its
ultraviolet absorption, the accuracy of these methods is usually
impaired by the presence of other substances, which either absorb
in the same region of the spectrum as vitamin A or give a simi-
lar color reaction with other impurities. The separation of vitamin
A from the accompanying vitamins D and E in pharmaceutical
products, prior to its physicochemical determination, is therefore
of prime importance. The separation methods used is generally
chromatography but, in spite of the time-consuming nature of this
procedure, the results obtained are often unreliable.
Craig et al. [7] reported a spectrophotometric method for vita-
min A using glacial acetic acid saturated with iron sulfate. Addition
of sulfuric acid (concentrated) with stirring caused formation of a
blue color which almost instantly changed to bright pink measured
at wavelength 520 nm with a stability of the complex for 12 min.
The calibration graph was linear over the range 20–100 ␮g vitamin
A. Lucy and Lichti [8] have reported the formation of retinol as a
molecular complex with iodine to produce a blue-green molecu-
lar complex measured at 610 nm which rapidly decomposes, giving
iodide and an unknown species at 870 nm. The presence of water
greatly facilitates the production of iodide from retinol and iodine.
Bayfield [9] reported a colorimetric method for vitamin A in com-
mercial and animal tissue samples using trichloroacetic acid as
the chromogenic agent in chloroform at wavelength 620 nm. The

1386-1425/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2008.12.024
990 L. Rishi et al. / Spectrochimica Acta Part A 72 (2009) 989–993
calibration graph was linear over the range 1.0–7.0 ␮g/mL vitamin
A.
Ibrahim et al. [10] reported two spectrophotometric methods for
the assay of retinol via charge transfer complexes in drug formula-
tions. The first method is based on the reaction of retinol with iodine
to give a molecular charge transfer complex measured at 241 nm
after 5 min reaction time. The second method depends on the for-
mation of a highly colored stable radical anion between retinol
and 7,7,8,8,-tetracyanoquinodimethane gives a bluish green chro-
mogen measured at 843 nm after 45 min reaction time. Both the
methods obey Beer’s law in a concentration range of 2.5–25 ␮g/mL.
These experiments were performed in chloroform and acetonitrile,
respectively. Spatny et al. [11] reported a dual channel flow injection
analysis method for the determination of vitamin A in an aqueous
solution. Iodine was reduced by retinol to produce the iodide ion,
the absorbance band of which was measured at 226 nm. A linear
calibration curve was obtained over the range 0.1–100 ␮g/mL with
the limit of detection 0.053 ␮g/mL.
The use of enzymes in flow-injection assays has increased
rapidly and the advent of effective procedures for their immobi-
lization on various carriers has widen the opportunities for their
practical application. Such water-insoluble enzyme derivatives can
be packed into columns and the combination of a flow-injection
system with an immobilized enzyme reactor offers many advan-
tages [20–22].
In the present work, alcohol dehydrogenase (ADH) is isolated
and purified from rabbit liver and used in soluble and immobilized
form for the determination of vitamin A in pharmaceuticals. The
enzymatic reaction is based on the oxidation of retinol to retinal and
simultaneous reduction of NAD+ to NADH followed by spectropho-
tometric detection at 340 nm. The reduction of NAD+ catalyzed by
ADH in the presence of retinol can be given as
alcohol dehydrogenase
Retinol + NAD+  Retinal + NADH + H+
max =340 nm
2. Experimental
2.1. Materials and methods
Alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase;
EC 1.1.1.1) was isolated and purified from rabbit liver; 1.07 U/mg
according to the reported procedures [23,24]. NADH (disodium salt,
98%) and NAD+ (free acid 100%) were obtained from Sigma (St.
Louis, USA). All other chemicals were analytical grade (E. Merck,
Dramstadt, Germany and Fluka, Buchs, Switzerland) and ultra-
high purity (UHP) deionised water for all solutions was purified
using a Milli-Q-water purification system (0.067 ␮S cm−1 , ELGA
Purelab Option, UK). Retinol stock solution (10 mM) was prepared
by dissolving the required quantity of retinol in methanol (HPLC
grade) in brown bottle and stored at −4 ◦ C in the dark. Working
standards were prepared by diluting aliquots of the stock solu-
tion daily in methanol and stored at −4 ◦ C in the dark during the
day.
2.2. Preparation of crude extract
Rabbit livers, weighing 65 g portions were perfused with ice
cold KCl solution (150 mM), removed and washed. The livers were
minced and homogenized with 3.0 V of cold phosphate buffer
(10 mM pH 7.4) containing dithiothreitol (0.1 mM) in a Minohum
Omni mixer for two periods of 10 s, and stirred at 4 ◦ C for 2 h. The
homogenate was centrifuged at 13,000 rpm for 30 min at 4 ◦ C and
the tissue debris was discarded. To the supernatant containing ADH
activity (160 mL, containing dithiothreitol, 0.1 mM) solid ammo-
nium sulfate was added with constant stirring over a period of
30 min to 21% saturation at 4 ◦ C. A few drops of NaOH (100 mM)
was added to the supernatant to maintain a pH of 7.4. After stand-
ing for 20 min, the suspension was centrifuged at 13,000 rpm for
20 min and the precipitate was discarded.
2.3. Enzyme purification
The resulting supernatant (145 mL) was stirred with 20 g of
DEAE-Sephadex resin, equilibrated in Tris–HCl (10 mM, pH 7.9), on
ice for 30 min to remove proteins by absorption on the resin; rabbit
liver ADH is not absorbed on the resin. The suspension was filtered
through a Buchner funnel under vacuum using an aspirator. In the
resulting solution (130 mL), dithiothreitol (DDT, 0.1 mM) was added
as a stabilizer. This solution was applied to a CM-Sephadex C-50 col-
umn (2.5 cm × 15 cm) equilibrated in phosphate buffer (50 mM, pH
7.5 containing dithiothreitol 0.1 mM). The column was washed with
the equilibrating buffer containing NAD+ (0.1 mM) until enzyme
activity and protein were no longer detectable in the effluent. The
elution of the enzyme from the column was carried out with a lin-
ear gradient of NAD+ (0.1–1.0 mM) in the buffer. The fractions with
high activity were pooled and concentrated (6.0 mL). The concen-
trated solution of ADH was applied to a Sephadex G-100 column
(2.5 cm × 60 cm) equilibrated with phosphate buffer (50 mM, pH
7.5) containing dithiothreitol (0.1 mM) and eluted with the same
buffer. Fractions with enzyme activity were pooled (45 mL) and
concentrated (15 mL), stored at −20 ◦ C until use. All the above steps
were carried out at 4 ◦ C. The enzyme activity was measured by the
method given below. The purified enzyme preparation resulted in
the appearance of a dark band on electrophoresis (SDS-PAGE) with
Coomassie staining with a molecular weight of 37 ± 1 kDa. The spe-
cific activity and percentage yield for each fraction are listed in
Table 1.
2.4. Enzyme assay
A 1.3 mL of pyrophosphate buffer, 1.5 mL of NAD+ (1.0 mM),
0.1 mL of ethanol (95%) were taken in test tube, mixed thoroughly
and incubated at 37 ◦ C for 5 min. ADH activity was measured by
adding 0.1 mL of enzyme solution to the reaction mixture. The
progress of the reaction was followed at 340 nm with a UV–vis
spectrophotometer (Jenway, 6505, UK) for approximately 10 min.
In blank the enzyme was replaced by 0.1 mL pyrophosphate buffer
(50 mM, pH 8.8). One unit of ADH is defined as the amount of
enzyme which causes the reduction of 1.0 ␮mol of NAD+ per minute
under the assay conditions.
2.5. Protein assay
Protein concentration was determined by measuring the
absorbance at 280 nm or by the colorimetric method [25] using
bovine serum albumin as a standard.
2.6. Electrophoresis
Polyacrylamide gel electrophoresis in denaturing conditions in
the presence of sodium dodecyl sulfate (SDS-PAGE) was performed
as described [26], using 15% polyacrylaminde gel. The electrophore-
sis was performed on a vertical slab mini gel apparatus (Model
Mini-Protean II cell, BioRad, USA). The protein was stained with
Coomassie Blue R-250.
2.7. Immobilization of ADH
The enzyme used in the present study was immobilized on con-
trolled pore glass (CPG), according to previous procedures [27,28].
ADH (20 U) was immobilized in 0.5 g aliquots of the derivatized
 L. Rishi et al. / Spectrochimica Acta Part A 72 (2009) 989–993
Table 1
Alcohol dehydrogenase purification.
Fraction Total protein (mg) Total units (U)
Crude extract 4125 624
21% (NH4 )2 SO4 2110 410
DEAE-Sephadex 635 340
CM-Sephadex C-50 242 215
Sephadex G-100 158 167
glass beads. The immobilization was carried out incubating the
derivatized glass beads overnight at 4 ◦ C with the enzyme dissolved
in 0.5 mL of phosphate buffer (100 mM, pH 6.0). After comple-
tion of the immobilization reaction, the beads were washed with
the same buffer and packed in a glass column (2.0 mm × 80 mm),
washed with a stream of phosphate buffer (50 mM, pH 11) and uti-
lized as needed. When not in use, the column was stored at 4 ◦ C in
glycine buffer (50 mM, pH 11). Almost 75–80% of the enzyme were
bound to the support by measuring the protein contents [25] of
the residues to evaluate the yield of the immobilization procedure
and no deterioration in enzyme activity was noticed after use for
2 months.
2.8. Manual procedure for ethanol/retinol determination
From each standard solution of ethanol/retinol prepared in
water and methanol respectively, 0.1 mL was mixed with 1.3 mL
of pyrophosphate (50 mM, pH 10.5) and 1.5 mL of NAD+ (10 mM).
The reaction mixture was incubated for selected time periods (e.g.
5 min) in the thermostated water bath at 37 ◦ C. At zero time, 0.1 mL
of alcohol dehydrogenase enzyme was added and the reactants
were mixed gently. The liberated NADH was measured at 340 nm for
approximately 10 min (at 37 ◦ C) using UV–vis spectrophotometer
(Jenway 6505, UK) equipped with 10 mm silica cuvettes. A ther-
mostated water bath was used to maintain the temperature during
incubation
2.9. Flow-injection procedure for ethanol/retinol determination
A flow injection manifold used for retinol determination is
shown in Fig. 1. A peristaltic pump (Ismatec Reglo 100, 4 channels,
Switzerland) was used to propel the buffer solution (glycine/NaOH
buffer 50 mM, pH 11 containing 1.0 mM NAD+ ) at 0.8 mL min−1 . A
rotary injection valve (Rheodyne 5020, Anachem, Luton, UK), was
used to inject 60 ␮L retinol standards into the buffer stream passed
through the enzyme column. The sample zone is then stopped in the
enzyme column (2.0 mm × 80 mm) for enzymatic reaction. After
120 s the pump was reactivated and the absorbance of NADH was
monitored at 340 nm using a spectrophotometer (Jenway, 6505, UK)
with a flow-through cell (100 ␮L) connected to a chart recorder
(Kipp & Zonen BD40, Holland). The immobilized enzyme column
was thermostated using a circulating water bath at 37 ◦ C by flow-
ing water through a water jacked around the enzyme column when
in operation and kept at 4 ◦ C when not in use.
Fig. 1. Flow-injection manifold for the determination of vitamin A.
991
Specific activity (U/mg) Purification (fold) Yield (%)
0.15 1.0 100
0.19 1.27 65.7
0.53 3.53 54.5
0.89 5.93 34.4
1.07 7.1 26.7
3. Results and discussion
Several purified ADHs have been demonstrated to catalyze
retinol oxidation in vitro including human classes I, II and IV ADHs
[29,30], rat classes I and IV ADHs [31], mouse class IV ADH [32]
and chick class VII ADH [33]. These ADHs are able to metabolize
all trans, 9-cis, and 13-cis retinoid isomers. Class III ADH does not
catalyze retinol oxidation and neither class V nor class VI have been
examined for retinol activity [31,29].
Early studies indicated that liver ADH, known as class I ADH,
catalyzes the oxidation of retinol to retinal in the presence of NAD
[34,24]. This enzyme also able to oxidize ethanol to acetaldehyde,
and can in fact utilize many alcohols as a substrate [35]. In mam-
mals, only class I ADH has evolved to efficiently oxidize ethanol,
suggesting that the additional ADH classes have evolved to oxidize
other alcohols. Since alcohol dehydrogenation is a reversible reac-
tion, ADHs can also catalyze the reduction of aldehydes such as
retinal when assayed with NADH in vitro. ADH is a cytosolic dimeric
zinc-dependent enzyme with a subunit molecular weight of 40 kDa
[35].
3.1. Optimization studies
The flow-injection spectrophotometric manifold (Fig. 1) and
manual method for retinol were optimized by investigating the
effect of various parameters including reagent concentrations,
buffer pH and temperature. Results are reported in Table 2. All
studies were carried out with 0.5 mM ethanol solution.
3.2. Effect of buffer pH
The effect of pH optimum on the activity of soluble and immobi-
lized ADH was investigated using pyrophosphate and glycine/NaOH
buffers (50 and 100 mM), respectively in the range of 7.0–11.0. The
maximum absorbance for reduction of NAD+ to NADH was observed
at pH 10.5 and 11, respectively for soluble and immobilized ADH and
were used subsequently.
3.3. Effect of NAD+
The cofactors ␤-nicotinamide adenine dinucleotide (NAD+ ) and
its reduced form (NADH) are used by over 250 dehydrogenases
and as such play major role in many biological redox reactions.
The effect of NAD+ concentration on the bioconversion process
was investigated over the range of 0.1–10 mM using pyrophos-
phate and glycine/NaOH buffers (50 and 100 mM, pH 10.5 and
11), respectively. The NAD+ concentration of 5.0 mM gave maxi-
mum absorbance for soluble ADH and 1.0 mM for immobilized ADH,
which were used subsequently.
3.4. Effect of temperature
The effect of temperature on the activity of soluble ADH was
investigated by incubating the reaction mixture in a thermostated
water bath for 10 min at various temperatures (range studied;
20–60 ◦ C). For immobilized ADH, the column was thermostated
992 L. Rishi et al. / Spectrochimica Acta Part A 72 (2009) 989–993

Table 2
Ranges investigated and optimized conditions for the enzymatic determination of vitamin A (n = 3).

Parameter Range studied Optimized values

Soluble ADH Immobilized ADH

Pyrophosphate and glycine/NaOH buffers; 50 and 100 mM (pH) 6.5–11.0 10.5 11.0
NAD+ (mM) 0.1–10.0 5.0 1.0
Temperature (◦ C) 20–60 37 37
Incubation time (min) 0–30 10 2.0

Table 3
Analytical characteristics of ethanol and retinol.

Figures of merit Ethanol (soluble and immobilized ADH) Retinol (soluble and immobilized ADH)

Linear range (␮M) 2.0–10 2.0–10

Correlation coefficienta (r2 ) 0.9952 and 0.9990 0.9967 and 0.9992
Calibration equation (y, absorbance; x, concentration in ␮M) y = 0.0041x − 0.0004 and y = 0.003x − 0.0004 y = 0.0049x + 0.0006 and y = 0.004x + 0.0007
RSD range, % (n = 4) 0.4–1.0 0.5–1.2
Limit of detection (␮M), S/N = 2 <1.0 <1.0
a
The correlation coefficient was calculated using five different concentration of each analyte.

in a water jacket by circulating water. The results are shown in
Table 2. An increase in absorbance with increase in temperature
was observed up to 50 ◦ C; however, the reaction mixtures were
maintained at 37 ◦ C to protect the enzyme from denaturation and
to increase the life time of the enzyme column.
3.5. Effect of pre-incubation time/stop-flow time
The effect of incubation time on the absorbance was studied
over the range of 0–30 min at 37 ◦ C given in Table 2. The increase
in absorbance observed by increasing the time from 0 to 20 min.
further increase in incubation time had no significant effect on the
absorbance. To have a better sample throughput, 10 min incubation
time was selected for further studies. In case of flow-injection pro-
cedure using immobilized ADH column, the sample was allowed to
stop in column for better conversion of NAD to NADH. The effect of
stop flow time were studied over the range of 0–5 min. Maximum
absorbance was obtained for a stop time of 2 min and was used for
further studies.
3.6. Analytical figures of merit
Standard solutions of ethanol and retinol were treated accord-
ing to the proposed method for soluble and immobilized ADH
under optimum conditions. The analytical characteristics are given
in Table 3. The limit of detection was lower than 1.0 ␮M with relative
standard deviations 0.5–1.2% for both soluble and immobilized ADH
over the range investigated with a sample throughput of 25 h−1
with flow-injection manifold.
3.7. Validation of the proposed method
The proposed enzymatic methods for the determination of
retinol were validated by comparing with spectrophotometric
method [9]. A saturated solution of trichloroacetic acid (TCA)
was prepared in chloroform immediately before use by dissolving
approximately 50 g TCA in 25 mL chloroform. Standard solutions
of vitamin A (retinol) were prepared in chloroform in the range
2.0–10 × 10−6 mol/L. Aliquots up to 1.0 mL from each standard were
taken in a series of test tubes followed by 2.0 mL of TCA reagent
and the absorbance was measured at 620 nm using a UV–vis spec-
trophotometer (Jenway, Model 6505, UK). vitamin A containing
capsules were analyzed by dissolving appropriate quantity in chlo-
roform and treating an aliquot of 1.0 mL with TCA, the concentration
was determined from the calibration graph prepared from the
standards. The method is compared with the proposed methods
(Table 4).
3.8. Application to pharmaceutical formulations
The proposed method using soluble and immobilized ADH was
applied to vitamin A capsules (Accucaps Industries Ltd., Canada),
quoted vitamins A and E 100,000 and 20 IU, respectively. The sam-
ple solution were prepared by dissolving 0.1-mL aliquot in 10 mL of
methanol and diluted appropriately with methanol and was ana-
lyzed with the developed method.
Seven Seas (UK) quoted vitamins A, D and E, 200, 1.68 and
200 ␮g/capsule with unsaturated fatty acids EPA and DHA, 26 and
24 mg/capsule, respectively. A 0.1-mL aliquot from capsule was
taken in a brown glass tube protected from light was dissolved in
10 mL of methanol. From this solution appropriate aliquots were
analyzed with the proposed enzymatic methods.
Another sample of vitamin-A palmitate (Fluka, Buchs, Switzer-
land), 5.0 mg portion of the compound taken in a brown glass
tube protected from light was dissolved and saponified with the
addition of 1.0 mL of KOH (50%) and 2.0 mL of methanol and incu-
bated 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 with n-hexane (2.0 mL × 5.0 mL). The organic
layer was recovered and evaporated to dryness in a water bath at

Table 4
Determination of vitamin A in three pharmaceutical formulations (n = 3).

Sample Vitamin A quoted Vitamin A found

Proposed method Spectrophotometric reference method

Soluble ADH Immobilized ADH

1 100,000 IU 99,524 ± 60 IU 99,645 ± 40 IU 99,605 ± 40 IU

2 200 ␮g 188 ± 10 ␮g 190 ± 15 ␮g 182 ± 15 ␮g
3 5.0 mg 2.68 ± 0.2 mg – 2.65 ± 0.3 mg

Sample 1: Accucaps Industries Ltd., Canada, 2: Seven Seas, UK, and 3: Fluka, Buchs, Switzerland.
 L. Rishi et al. / Spectrochimica Acta Part A 72 (2009) 989–993
37 ◦ C under a stream of nitrogen. The residue was re-dissolved by
vortex-mixing in 5.0 mL methanol and the sample was then ana-
lyzed by the proposed method for the determination of vitamin A.
The results obtained are given in Table 4 which was in reasonable
agreement with the amount labeled. No significant difference was
found between the results of the two methods (soluble ADH and
spectrophotometric).
4. Conclusions
An enzymatic method is established for the determination of
retinol using soluble and immobilized ADH isolated from rabbit
livers. The limit of detection was lower than 1.0 ␮M retinol. The
methods were applied to commercially available capsules and the
results were in reasonable agreement with the amount of retinol
quoted. In addition, the purification procedure for ADH is very sim-
ple and economic especially when these enzymes are not available
commercially. A single band with a molecular weight of 37 ± 1 kDa
was found to be associated with alcohol dehydrogenase. This value
is similar to the method reported previously [23].
Acknowledgements
The authors are grateful to the Higher Education Commission,
Pakistan and the Pakistan Science Foundation for financial support
(Grant Nos. 20-640/R&D/06 and CHEM/B-BU 252, respectively).
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