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PAPER www.rsc.org/methods | Analytical Methods

A quantitative analysis of Zidovudine containing formulation by FT-IR and

UV spectroscopy
Ashok Peepliwal,* Sagar D. Vyawahare and Chandrakant G. Bonde
Received 26th May 2010, Accepted 30th July 2010
DOI: 10.1039/c0ay00341g

In this study IR and UV spectroscopic procedures are describe for the quantitative determination of
Zidovudine (AZT) from solid dosage form. For IR spectroscopic method (KBr disc technique) has
been used and Ursodeoxycholic acid (UDCA) was used as an internal standard. The specific absorption
bands at 2105 and 2931 cm1 were chosen for AZT and UDCA respectively. In this method, beer’s law
Published on 05 October 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00341G

was obeyed in the concentration range 0.8–2.0% w/w in KBr disc. The regression equation was found to
be y ¼ 1.088x + 1.194 with correlation coefficient 0.9819, and the assay was found to be 99.8% with
RSD of 2.157%. The UV spectroscopic method was also used in the quantitative determination of AZT
in solid dosage form. 266 nm was chosen as lmax. The regression equation was found to be y ¼ 0.0434x
+ 0.027, and the correlation coefficient 0.9989. The assay was found to be 99.81% with RSD of 0.273%
by the UV spectroscopic method.

1. Introduction
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Zidovudine (formerly called azidothymidine [AZT]) (Fig. 1),

a pyrimidine nucleoside analogue chemically designated as 30 -
azido-30 -deoxythymidine is active against HIV-1 infection. It is
used in combination with other medications to control HIV-1
infection. It also lowers the risk of getting HIV disease compli-
cations (e.g., new infections, cancer). It belongs to a class of
drugs known as nucleoside reverse transcriptase inhibitors
(NRTI). It is not a cure for HIV infection and it does not prevent
the spread of HIV to others through sexual contact or blood
contamination (e.g., sharing needles). It is also used for the
prevention of maternal-fetal transmission of HIV-1 infection.1,2 Fig. 1 Structure of Zidovudine.
The previously published methods for AZT analysis described
only using high performance liquid chromatography assay with
either UV, tandem mass spectroscopy, LC-MS/MS or HPTLC. 2.2. Chemicals
The earlier methods are expensive, time consuming and high AZT supplied by Matrix Pharma, Nashik, UDCA internal
technical skills are required. IR spectroscopic method for the standard for IR by Sigma Aldrich USA, potassium bromide (IR
determination of AZT in solid dosage form was suggested for the spectroscopy grade), all the reagents and chemicals were of
first time. The objectives of this study were firstly to develop and analytical grade. Retrovir tablets containing 300 mg of AZT
then validate two analytical methods for the quantification of were purchased from local pharmacies in Dhulia-India.
AZT containing solid dosage form.3–6

2.3. Base line technique

2. Experimental The baseline technique was used for the quantitative analysis of
drug substance. The following Fig. 2 showed the application of
2.1. Apparatus
baseline technique. The values of PB and PO were calculated
FT-IR spectrophotometer: Perkin Elmer, Spectrum Rx- using the infra-red spectrum of the samples and, using the
1(Version 5.3.1, Operated on HP computer with Windows 2003). equation of regression, the quantitative analysis was carried out.
UV-Vis spectrophotometer: double-beam Perkin Elmer,
Lambda-25 loaded, WinLab software (Version 5.2.0, operated
on HP computer with Windows 2003). A 1 cm quartz cell over 2.4. IR spectroscopic method
the range of 200–400 nm was employed.
2.4.1. Stock solutions. The stock solution of AZT (2 mg ml1)
and UDCA (1 mg ml1) were prepared in chloroform. These
School of Pharmacy and Technology Management, SVKM’s Narsee
solutions were stable at 2–8  C for 2 weeks.
Monjee Institute of Management Science (NMIMS), Shirpur, Dhule,
Maharashtra, India 425405. E-mail: ashok_peepliwal@yahoo.com; Fax:
+02563-286552; Tel: +02563-286549 2.4.2. Method validation

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Fig. 2 Measurement of PB and PO points of the absorption peaks by
base line technique (Application of base line technique).
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2.4.2.1. Linearity. 1.0, 1.5, 2.0, 2.5, 3 ml of solution of AZT
and 1.5, 1.0, 2.0, 1.5, 2.0 ml of solution UDCA were pipetted and
poured into a 250 mg KBr powder in porcelain dishes separately.
The dishes were kept in a hot air oven for 20 min at 60  C to
evaporate the moisture and solvent from the mixture. Then
a series of synthetic standard mixtures of AZT-UDCA (2–1.5),
(3–1), (4–2), (5–1.5), (5–2) mg were transferred into dishes
separately. The powders were mixed through with an agate pestle
and homogenous fine powder was obtained, after this 125 mg
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discs were prepared for each mixture and employed for quanti-
tative measurement. Each disk of 125 mg contained 2 mg, 3 mg,
4 mg, 5 mg and 6 mg AZT respectively so a linear curve could be
plotted. The calibration curve for AZT was prepared using the
LogPB–LogPO values of AZT-UDCA in synthetic standard
mixtures. The correlation of coefficient for AZT was found to be
0.9819 with equation line of y ¼ 1.0883x + 1.1944.
2.4.2.2. Accuracy and precision. Varying concentrations of
AZT were prepared with internal standard in KBr disk range
from 80%, 100% and 120%. PB and PO points of the absorption
peaks were determined and LogPB–LogPO values of AZT-
UDCA calculated by base line technique as depicted in Fig. 2.
The intra-day and inter-day accuracy precision was studied. The
percentage RSD of the method was calculated at these three
levels.
2.4.2.3. Recovery study. The recovery study was performed
at 80%, 100% and 120%. 4 mg of AZT and concentration 3.2 mg,
4 mg, and 4.8 mg added into the disk containing AZT-UDCA
standard mixture of 4 mg AZT. The recoveries of three levels
were estimated and %RSD was calculated.
2.4.2.4. Ruggedness. Ruggedness of the proposed methods
was determined by analysis of aliquots from homogeneous slot in
by different analysts and at different percentage relative
humidity (%RH  5%) using similar operational and environ-
mental conditions; the % R.S.D. was calculated.
2.4.3. Assay of AZT tablet by IR spectroscopy method. Ten
tablets were weighed powdered and a portion of powder equiv-
alent to 40 mg AZT was accurately weighed and extracted with
chloroform. The solution was filtered in to 20 ml volumetric flask
and the volume was made up to the mark to get the solution of
2 mg ml1. The solution of UDCA, 1 mg ml1 was prepared by
dissolving 20 mg of UDCA in 20 ml of chloroform.
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Then 2 ml of solution of AZT and 2 ml of solution of UDCA
were pipetted and poured into a 250 mg KBr powder in porcelain
dishes separately. The dishes were kept in hot air oven for 20 min
at 60  C to evaporate the moisture and solvent from the mixture.
The synthetic standard mixture of AZT-UDCA (4–2) mg was
transferred into dishes separately. The powders were mixed
through with an agate pestle and homogenous fine powder was
obtained of which 125 mg of disc was prepared by using KBr.
Likewise five homogeneous discs were prepared and employed
for quantitative measurement.
2.5 UV spectroscopic method
2.5.1. Preparation of stock solution. Accurately weighed 20
mg of AZT transferred to 20 ml volumetric flasks. It was dis-
solved in 10 ml methanol shaken manually for 5 min. The volume
was made up to the mark with methanol to obtain final strength
1000 mg ml1.
2.5.2. Determination of l max. From the stock solution, 0.1
ml and 0.3 ml of AZT solution was transferred to 10 ml volu-
metric flasks and the volume was adjusted to the mark with the
same solvent to obtain concentrations of 10 and 30 mg ml1
respectively. The solutions were scanned in the UV range 200–
400 nm. The scanning spectrums of the drug are shown in Fig. 3
and Fig. 4 and the wavelength 266 nm was selected for further
study.
2.5.3 Determination of E (1%, 1 cm). 0.3 ml aliquot of AZT
from stock solution was transferred to six 10 ml volumetric flaks
and volume was adjusted to the mark with methanol to obtain six
individual concentrations of 30 mg ml1. The absorbance of each
solution was measured at 266.0 nm. E (1%, 1 cm) values of drug
were calculated using eqn (1) and results of E (1%, 1 cm) of the
drug are shown in Table 1:
E (1%, 1 cm) ¼ Absorbance/Concentration (g/100ml) (1)
2.5.4. Method validation
2.5.4.1. Linearity. Different concentration of AZT from 10–
50 mg mL1 were prepared using the stock solution to prepare the
calibration curve of AZT. The slope and intercept was calculated
from the regression line equation.
Fig. 3 Scanning spectrum of 10 mg ml1 AZT solution in the UV range
of 200–400 nm.
This journal is ª The Royal Society of Chemistry 2010

Fig. 4 Scanning spectrum of 30 mg ml1 AZT solution in the UV range
of 200–400 nm.
Published on 05 October 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00341G
Table 1 E (1%, 1cm) values of AZT at 266.0 nm
Absorptivity at 266 nm
a
Mean
SDb
%RSDc
a
Absorptivity values are the mean of six determinations. b
Standard Deviation. c RSD is Relative Standard Deviation.
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2.5.4.2. Precision (intra-day and inter-day). Accuracy and
precision study was carried out at three concentration levels from
80%, 100% and 120% of 30 mg mL1. The study was performed
for intra-day and inter-day to assess the variation in analysis.
2.5.4.3. Ruggedness. Ruggedness of the proposed methods
was determined by analysis of aliquots from homogeneous slot in
different laboratories, by different analysts, using similar oper-
ational and environmental conditions; the % R.S.D. was calcu-
lated.
2.5.4.4. Recovery study. To check the accuracy of the
developed methods and to study the interference of formulation
additives, analytical recovery experiments were carried out by
standard addition methods, at 80, 100 and 120% level. From the
total amount of drug found, the percentage recovery was
calculated.
2.5.6. Assay of marketed formulation (Retrovir). Contents of
twenty ‘Retrovir’ tablets containing 300 mg of AZT were
weighed and ground to fine powder. A quantity of sample
equivalent to 20 mg of AZT was transferred into 20 ml volu-
metric flask containing approximately 15 ml methanol, sonicated
for 10 min; the volume was made up to the mark and filtered
through Whatmann filter paper (No. 41). An appropriate volume
0.3 ml of this solution was transferred to 10 ml volumetric flasks
and dissolved, the volume was adjusted to mark with methanol.
The absorbance’s of the solutions were measured at 266.0 nm
against blank.
3.0. Results
3.1. IR spectroscopy method
3.1.1. Linearity. The calibration curve for AZT was prepared
using the LogPB–LogPO values of AZT-UDCA in synthetic
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Fig. 5 Linearity of AZT.
standard mixtures. The correlation of coefficient for AZT was
449.83 found 0.9819 with equation line of y ¼ 1.0883x + 1.1944 (Fig. 5).
0.81 The linearity values of LogPB–LogPO are presented in Table 2.
0.1798
S.D. is 3.1.2. Accuracy and precision. The developed method of AZT
shows high levels of accuracy and precision at 80%, 100% and
120% levels as given in Table 3 and 4. The intra-day and inter-
day percentage %RSD for three levels is found below 1.72.
3.1.3. Recovery study. At the level of three concentrations of
3.2 mg, 4 mg, and 4.8 mg, the recovery was more than 78% and
the %RSD was found to be less than .059. as data presented in
Table 5 show.
3.1.4. Ruggedness. Ruggedness of the proposed method
shows that the different analysts and different percentage relative
humidity (%RH  5%) does not affect the method and the
accuracy of the method is more than 98% and %RSD was found
below 3.728 as shown in Table 6–7.
3.2. Assay of AZT tablet by IR spectroscopy method
The proposed method was used for the tablet formulation of
AZT (Retrovir) and found the % assay is 99.8% with %RSD of
2.15 as given in Table 8.
4. UV spectroscopy method
4.1. Linearity
The method is linear in the range of 10–50 mg mL1. The equation
of regression lie was found to be at y ¼ 0.0427 + 0.038 where the
intercept is 0.038 and slope is 0.0427 for AZT as depicted in
Fig. 6. The equation was used to determine the content of AZT in
tablet formulation under the method.
4.2. Recovery studies by UV-spectroscopy
To check the accuracy of the developed methods and to study the
interference of formulation additives, analytical recovery exper-
iments were carried out by standard addition method, at 80, 100
and 120% level. From the total amount of drug found, the
percentage recovery was calculated. The results are reported in
Table 9.
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Table 3 Intra-day accuracy and precision

Y ¼ TAZT/TUDCA
AZT 2105

Levels Disc Wt/mg Conc./mg PB–PO Log PB–Log PO

2.7576
4.3746

4.9633
3.8574
3.26
80% 124.500 3.200 14.340 0.132
124.300 3.200 14.310 0.131
124.600 3.200 14.370 0.133
Mean 124.467 3.200 14.340 0.132
X ¼ CAZT/CUDCA
SD 0.153 0.000 0.030 0.001
%RSD 0.123 0.000 0.209 0.758
100% 125.900 4.000 15.020 0.166
125.300 4.000 14.990 0.165
125.700 4.000 15.030 0.168

Where, CAZT ¼ AZT conc. in KBr disc, CUDCA¼ UDCA conc. in KBr disc, TAZT¼ LogPB–LogPO values of AZT, TUDCA¼ LogPB–LogPO values of UDCA.
1.33

3.33 Mean 125.633 4.000 15.013 0.166

2.5
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3
2

SD 0.306 0.000 0.021 0.002

%RSD 0.243 0.000 0.139 0.918
120% 1.26.7 4.800 17.810 0.192
Log PB  Log PO

126.200 4.800 17.830 0.198

126.800 4.800 17.780 0.196
Mean 126.500 4.800 17.807 0.195
SD 0.424 0.000 0.025 0.003
%RSD 0.335 0.000 0.141 1.564
0.033
0.028

0.048
0.081
0.05
PB  PO
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Table 4 Inter-day accuracy and precision (3 days)

3.94
3.73
3.96
4.28
3.95

AZT 2105
UDCA 2931 cm1

Levels Disc Wt/mg Conc./mg PB–PO Log PB–Log PO

Conc./mg

80% 124.500 3.200 14.354 0.136

124.300 3.200 14.324 0.134
1.5

1.5

124.600 3.200 14.325 0.138

1
2

Mean 124.467 3.200 14.334 0.136

SD 0.153 0.000 0.017 0.002
%RSD 0.123 0.000 0.119 1.471
Log PB  Log PO

100% 125.900 4.000 15.420 0.165

125.300 4.000 14.980 0.169
Table 2 LogP–LogPO values found for AZT-UDCA in synthetic standard mixturesa

125.700 4.000 15.450 0.164

Mean 125.633 4.000 15.283 0.166
SD 0.306 0.000 0.263 0.003
0.091
0.123
0.163
0.238
0.312

%RSD 0.243 0.000 1.722 1.594

120% 1.26.7 4.800 17.860 0.195
126.200 4.800 17.810 0.192
126.800 4.800 17.700 0.199
PB–PO

Mean 126.500 4.800 17.790 0.195

11.78
13.52
14.98
22.88
23.53

SD 0.424 0.000 0.082 0.004

%RSD 0.335 0.000 0.460 1.798
AZT 2105 cm1

Conc./mg

2
3
4
5
6

4.3. Precision and ruggedness parameters

Precision was determined as repeatability, intra-day and inter-
Disc Wt/mg

day variations. Repeatability was determined by analyzing AZT

solution (30 mg mL1) for six times. Intra-day precision was
125.2

123.8
123.5

determined by analyzing 30 mg ml1 solution of AZT three times

126
124

on the same day. Inter-day precision was determined by

analyzing the same concentration of solutions for three different
days over a period of one week. The results were reported in
Synthetic Mixture

Table 10. Ruggedness of the proposed methods was determined

by analysis of aliquots from homogeneous slot in different
laboratories, by different analysts, using similar operational and
environmental conditions; the %RSD reported in Table 11 was
St1
St2
St3
St4
St5

found to be less than 2%.

a

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Table 5 Recoveries of AZT at three level 80%, 100% and 120%

AZT 2105

Levels Disc Wt/mg Added Conc./mg PB-PO LogPB–LogPO % found

80% 122.500 3.200 14.295 0.139 99.81

128.700 3.200 14.289 0.136 99.79
127.600 3.200 14.332 0.136 99.79
Mean 126.267 3.200 14.305 0.137 99.797
SD 3.308 0.000 0.023 0.002 0.012
%RSD 2.620 0.000 0.163 1.264 0.012
100% 123.980 4.000 15.457 0.169 99.87
127.300 4.000 14.893 0.170 99.89
128.750 4.000 15.278 0.164 99.78
Mean 126.677 4.000 15.209 0.168 99.847
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SD 2.445 0.000 0.288 0.003 0.059

%RSD 1.930 0.000 1.895 1.917 0.059
120% 125.780 4.800 17.895 0.189 99.97
129.250 4.800 17.938 0.199 99.99
125.880 4.800 17.849 0.192 99.92
Mean 126.970 4.800 17.894 0.193 99.960
SD 1.975 0.000 0.045 0.005 0.036
%RSD 1.556 0.000 0.249 2.566 0.036

hand, the internal standard has no absorption band at 2105 cm1

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4.4 Determination of AZT in bulk and tablet using the

developed method
The developed UV spectroscopy method was also used for the
determination of AZT in bulk and tablet. The percentage
amount found in bulk is 99.76% with %RSD of 0.192 and in
tablet 99.81% with %RSD of 0.272 as given in Table 11 and 12.
5.0. Discussion
The IR spectra of discs were taken, for which 2105 cm1 for AZT
(Fig. 7) and 2931 cm1 for UDCA (Fig. 7) absorption bands were
used. Fig. 8 represents the IR Spectrum of commercial tablet
(Retrovir) containing UDCA. The base line technique was used
for the determination of PB and PO values of absorption bands.
The logarithmic differences of these values as absorbance (y)
were plotted against concentration (x) in order to calculate the
regression equation. Internal standard was used in order to
eliminate some unforeseen defaults which originated from the
application of the method. For this purpose UDCA was chosen
as the internal standard with the absorption band at 2931 cm1
where no absorption band is available for AZT. On the other
where AZT has absorption. In this method, the linear concen-
tration range was obtained as 0.8–2.0% w/w. This range is so
narrow because in this study more attention was paid to hold PB
and PO points between 80–20% as the transmittance value which
were used for AZT and UDCA. Especially when the PO point is
under 20% transmittance, any small error for the determination
of this point fairly affect the results.7–13 Quantitative determi-
nation is based on concentration-absorption relationship of
Beer’s law. PB and PO points of the absorption peaks were
assigned with base line technique as shown in Fig. 2. The
regression equation was formed by using the AZT/UDCA
concentration ratio as (x) values and the ratio of Log PB–Log PO
of AZT and Log PB–Log PO of UDCA values as (y). Absorbance
values were measured by logarithmic subtraction of PB and PO
points. The regression equation was calculated by using the ratio
concentration/absorbance of AZT and UDCA (Table 2). At 2105
cm1 the regression equation was found to be y ¼ 1.088x + 1.194,
R2 ¼ 0.9819 and the assay was found to be 99.8% with RSD
2.157%.
UV spectroscopic method was used as another comparison
method for quantitative determination of AZT in commercial

Table 6 Ruggedness of AZT performed by two analysts

Analyst-I Analyst-II

AZT 2105 AZT 2105

LogPB LogPB
Disc Wt Conc./mg PB-PO LogPO Disc Wt Conc./mg PB-PO LogPO

AZT 125.900 4.000 15.756 0.161 AZT 125.900 4.000 15.980 0.175
125.300 4.000 14.834 0.167 125.300 4.000 14.950 0.170
125.700 4.000 15.678 0.169 125.700 4.000 15.890 0.176
Mean 125.633 4.000 15.423 0.166 Mean 125.633 4.000 15.607 0.174
SD 0.306 0.000 0.511 0.004 SD 0.306 0.000 0.570 0.003
%RSD 0.243 0.000 3.315 2.513 %RSD 0.243 0.000 3.655 1.968

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Table 7 Ruggedness of AZT performed at different relative humidity

35% RH 40% RH

AZT 2105 AZT 2105

LogPB LogPB
Disc Wt Conc./mg PB-PO LogPO Disc Wt Conc./mg PB-PO LogPO

AZT 128.400 4.000 14.348 0.179 AZT 128.400 4.000 14.798 0.169
122.380 4.000 15.278 0.174 122.380 4.000 15.898 0.177
128.790 4.000 15.350 0.171 128.790 4.000 15.450 0.178
Mean 126.523 4.000 14.992 0.175 Mean 126.523 4.000 15.382 0.175
SD 3.594 0.000 .559 0.004 SD 3.594 0.000 0.553 0.005
%RSD 2.840 0.000 5.295 3.728 %RSD 2.840 0.000 3.596 2.824
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45% RH

AZT 2105

LogPB
Disc Wt Conc./mg PB–PO LogPO

AZT 128.400 4.000 15.120 0.165

122.380 4.000 14.560 0.167
128.790 4.000 15.140 0.174
Mean 126.523 4.000 14.940 0.169
SD 3.594 0.000 0.329 0.005
RSD 2.840 0.000 2.204 2.802
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Table 8 Assay results of commercial samples (Retrovir tablet 300 mg of Table 10 Summary of repeatability, precision and ruggedness
AZT) by IR spectroscopy.b
Parameters (UV Spectroscopy)
IR-Spectroscopy AZT %RSD
Parameters AZT
Repeatability 0.416
Label Claim 300 Intra-day 0.225
%Drug Contenta 99.8 Inter-day 0.112
SD 2.152 Ruggedness
%RSD 2.157 Analyst I 0.555
Analyst II 0.337
a b
Value for Drug content (%) is the mean of five estimations. S.D. is
standard deviation and R.S.D. is relative standard deviation.
Table 11 Determination of AZT in bulk

Amount Amount % Amount

taken/mg ml1 Absorbance found/mg ml1 found

30 1.353 30.00 100

1.346 29.84 99.48
1.35 29.93 99.78
1.351 29.96 99.85
1.349 29.91 99.7
Mean 1.349 29.93 99.76
SD 0.19
%RSD 0.192

Fig. 6 Calibration curve of AZT over the concentration range of 10–50

ppm. Table 12 Determination of AZT in tablet formulation

Amount Amount % Amount

taken/mg ml1 Absorbance found/mg ml1 found
Table 9 Recovery studies of AZT
30 1.355 30.04 100.14
Excess Drug %Recoverya %RSD 1.349 29.91 99.7
1.345 29.82 99.41
80 100.09 0.208 1.351 29.96 99.85
100 99.85 0.375 1.352 29.98 99.93
120 99.94 0.205 Mean 1.35 29.94 99.81
SD 0.27
a
%Recovery is mean of three estimations. %RSD 0.273

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Fig. 7 IR Spectrum of AZT and UDCA in KBr.

Fig. 8 IR Spectrum of commercial tablet (Retrovir) containing UDCA in KBr.

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tablets. A linear concentration was obtained as 10–50 mg ml1 in
this method. The regression equation was found to be y ¼
0.0434x + 0.027 and the correlation coefficient 0.9984. E (1%, 1
cm) was calculated as 449.83 in methanol. The assay was found
to be 99.81% with %RSD 0.273%.
Although various methods like HPLC-UV, LC-MS/MS,
HPTLC are available, these methods are expensive due to use of
expensive detectors, costly solvents, columns and also more time
needed to develop and validate the method.14–16 The present IR
spectroscopy method is a simple, rapid, accurate and cost
effective analytical tool for AZT determination in tablet
formulations.
Published on 05 October 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00341G
6.0. Conclusion
This paper describes the application of IR spectroscopic method
and UV spectroscopic method to determine AZT in formulation.
The IR spectroscopic method can be use as an alternative for the
determination of AZT in solid dosage forms. The proposed
method is simple, precise, accurate and rapid for the determi-
nation of Zidovudine (AZT) in tablet dosage form. Analysis of
authentic samples containing AZT showed no interference from
the common additives and excipients, demonstrating that the
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recommended procedure is well suited for the assay and evalu-
ation of drugs in pharmaceutical preparations. The developed
method can be easily and conveniently adopted for routine
quality control analysis.
Acknowledgements
We are thankful to Matrix Pharma, Nashik and Sigma Aldrich,
USA for providing us the gift sample of Zidovudine (AZT) and
Ursodeoxycholic acid (UDCA) respectively. We are also
thankful to SPTM, SVKM’s NMIMS (Shirpur campus) for
providing the research facilities to carryout work.
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