PHARMACEUTICAL AND BIOLOGICAL EVALUATIONS

February 2018; Vol. 5 (Issue 1): 1-9.

www.onlinepbe.com ISSN 2394-0859

Research Article DOI: http://dx.doi.org/10.26510/2394-0859.pbe.2018.02

Development and statistically validated UV

spectrophotometric determination of testosterone in gel
formulation
Kartik D. Bhagat, Anvesha V. Ganorkar, Atul T. Hemke, Krishna R. Gupta*

Department of Pharmaceutical Chemistry, Smt Kishoritai Bhoyar College of Pharmacy, Kamptee, Nagpur,
Maharashtra, India

*For correspondence ABSTRACT

Dr. Krishna R. Gupta,
Department of Pharmaceutical
Chemistry, Smt Kishoritai
Bhoyar College of Pharmacy,
Kamptee, Nagpur,
Maharashtra, India.
Email: krg1903@gmail.com
Received: 21 December 2017
Revised: 10 January 2018
Accepted: 11 January 2018
Objective: A simple, precise and accurate UV-spectrophotometric
method is developed and statistically validated for estimation of
Testosterone in gel formulation. The proposed method includes using
regression equation, area under curve (AUC), first order derivative and
second order derivative spectroscopic method.
Methods: based on measurement of absorbance at a selected wavelength
using UV-visible spectrophotometer with 1cm matched quartz cell and
acetonitrile as a solvent. All developed methods obeyed Beer’s-lambert’s
law in the concentration range of 5-25μg/mL, with correlation coefficient
value less than 1.
Results: The percent amount of drug estimated was nearly 100%, found
to be a good agreement with label claim of marketed gel formulation. The
recovery study was carried out at three different levels, the validation
study data was found to be statistically significant as all the statistical
parameters are within the acceptance range (% RSD <2.0 and S.D. <±2.0).
Conclusions: The results of estimation and validation parameters like
accuracy, precision, ruggedness, linearity and range were studied for all
the developed methods and were found to be within limits. The results
obtained were statistically compared using paired t-test and one way
ANOVA analysis. The proposed method can be adopted for routine
quality control for estimation of drug in formulation.
Keywords: Testosterone, UV-Visible spectroscopy, Validation, Derivative,
Area under curve

Introduction levels of testosterone in men may lead to

Testosterone is an anabolic steroid and primary
sex hormone in males. It plays a key role in the
development of male reproductive tissues; it is
involved in the development of primary and
secondary sexual characters. Deficiency in the
abnormalities including frailty and bone loss.1
Testosterone is also used as a medication to
treat male hypogonadism and certain types
of breast cancer. With increase in age of male
humans the testosterone levels gradually falls

©Pharmaceutical and Biological Evaluations 1

Bhagat KD. et al. Pharmaceutical and Biological Evaluations 2018; Vol. 5 (1): 1-9.
down, to treat this deficiency synthetic
testosterone are sometimes prescribed to older
men.2 Testosterone acts through binding to and
activating the androgen receptor.3
Average levels of testosterone are about 7–8
times more in adult males as compared to adult
females. Daily production of testosterone is about
20 times greater in men due to high metabolic
consumption in males. Females are also more
sensitive to the hormone.4
Figure 1: Chemical structure of testosterone.
Testosterone chemically is, Androst-4-en-3-one,
17-hydroxy-, (17β)- or 17β-Hydroxyandrost-4-
en-3-one (Figure 1).5 Literature survey revealed
that methods are reported for estimation of
testosterone on cream sold freely on internet
website, steroid metabolite for doping control in
urine analysis, in fish serum, in oil based
injectables and in equine plasma by LC-MS but
no methods are reported by in gel formulation.6-10
The proposed work represents four new simple,
economical, and rapid UV-spectrophotometric
methods for the quantification of Testosterone in
bulk and its gel formulation. The developed
methods were validated for accuracy, precision,
ruggedness and sensitivity as per ICH guidelines.
Materials and Methods
Chemicals and reagents
Pharmaceutical grade testosterone standard was
obtained as generous gift sample by Alkem
laboratory Pvt. Ltd. Mumbai, Maharashtra, India.
HPLC grade Acetonitrile purchase from MERCK
Life Science Pvt. Ltd. and distilled water was
used. The pharmaceutical dosage form used in
this study is 1%W/W testosterone gel formulation
manufactured by sun pharmaceutical Ind. Ltd.
©Pharmaceutical and Biological Evaluations
Instruments
The instrumentation used for the method
development and validation are UV-
Spectrophotometer (Model JascoV-630 and
Shimadzu-1700) double beam with 1 cm quartz
cell. Sonicator are used is PCi Mumbai, Model
No.3.5L 100H and the Weighing balance use is
Shimadzu AUX220 and analytical balance.
Preparation of working standard solution
The standard stock solution was prepared by
dissolving 10.0 mg of testosterone in 10.0mL
volumetric flask with 10.0 mL of acetonitrile to
get a concentration of 1 mg/mL. From the above
stock solution containing 1.0 mL was pipette and
diluted to 10.0 mL in a volumetric flask upto the
mark with acetonitrile (concentration of 100
μg/mL). Again 1.0 mL of working standard
solution was pipetted in a 10.0 mL volumetric
flask and volume up to the mark with acetonitrile
(concentration 10μg/mL).
Selection of wavelengths for analysis for
testosterone
Method I (regression equation): The working
standard solution of 10μg/mL was prepared and
scanned in the UV range 400–200 nm;
Testosterone showed a maximum absorbance at
237.4 nm.
Method II (area under curve): From the zero
order spectrum of Testosterone, the AUC
between a wavelength range 232.4-242.4 nm was
considered for the analysis.
Method III (first order derivative UV-
spectrophotometry): The zero order absorption
spectrum of Testosterone was transformed to first
order derivative using derivative mode of
spectrophotometer and the two amplitudes
recorded at 250.4 nm and 226.2nm.
Method IV (second order derivative UV-
spectrophotometry): The zero order absorption
spectrum of Testosterone was transformed to
second order derivative and the amplitudes were
recorded at 258.6 nm and 239.6 nm. The
selections of wavelength for the proposed
methods are shown in Figure 2 (a-c).
2
Bhagat KD. et al. Pharmaceutical and Biological Evaluations 2018; Vol. 5 (1): 1-9.

1 correlation coefficient was found to be 0.993 and

0.9901 respectively.
0.8

Preparation of sample solution

0.6

Abs
To determine the content of Testosterone from
0.4 marketed gel formulation; average content per
sachet was calculated. An amount of gel
0.2 equivalent to 100.0 mg of testosterone was
weighed and transferred to a 10.0 mL volumetric
0 flask. To it sufficient amount of acetonitrile was
200 250 300 350 400
Wavelength [nm] added, sonicated for 30 min and the solution was
diluted up to mark with the same solvent. The
Figure 2 (a): Zero order spectrum of content of the flask was filtered through
testosterone showing AUC between selected Whatmann filter paper (No. 41). From the filtrate
wavelength. solution pipette out 1.0 ml and add 10.0 ml
volumetric flask and make up the volume up to
0.07
the mark and get the final concentration of
0.05
10μg/ml. The absorbance was measured at
selected wavelengths as described above and
concentrations in the sample were determined
using regression equation (method 1); Comparing
Abs
0
the AUC of standard and sample at selected
wavelength (method 2) and comparing the
derivative absorbance of standard and sample at
selected wavelength (method 3 and 4)
respectively.
-0.05
200 250 300 350
Wavelength [nm]
Validation of methods
Figure 2 (b): First order derivative spectrum. The proposed method was validated as per ICH
0.002
guidelines.

0.001
Accuracy

0
Abs
-0.001
-0.002
200 250
Wavelength [nm]
Figure 2 (c): Second order spectrums.
Preparation of calibration curve
Appropriate dilutions of standard stock solution
were made using acetonitrile to get final
concentration in the range of 5-25 μg/ml.
Absorbance and area under curve were measured
of each prepared solution at above selected
wavelengths. The calibration curve was plotted
between concentrations vs. absorbance/AUC,
The accuracy of proposed method was
ascertained on the basis of recovery studies
performed by standard addition method. The pre-
analyzed gel equivalent to 100 mg was weighed;
a known amount of standard drug was added at
300 350
different levels covering the range 50-150% of
target. The resultant solutions were then analyzed
by the proposed methods. At each level, triplicate
samples were analyzed to check repeatability and
from the data, the amount of standard drug
recovered was estimated.
Precision
The precision of the analytical method expresses Commented [SM1]:
the closeness of agreement between a series of
measurements obtained from multiple sampling
of the same homogeneous sample under the
prescribed conditions. The precision of the

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Bhagat KD. et al. Pharmaceutical and Biological Evaluations 2018; Vol. 5 (1): 1-9.
methods can be studied as; intra-day variation,
inter-day variation studies. Intra-day study was
carried out by analyzing the 10 μg/ml of sample
for five times within the day while in inter-day
study same solution was analyzed on five
different days.
Ruggedness
Ruggedness of proposed methods was performed
to examine effect of non-procedure related
factors such as instruments, wavelength, and
analysts. For this study Testosterone (10μg/mL)
was analyzed by proposed methods using two
different analyst, two different wavelength, and
two different UV-spectrophotometers (Jasco V-
630 and Shimadzu-1700) restraining similar
operational and environmental conditions.
Linearity
The linearity of an analytical procedure is the
ability to obtain test results that are directly
proportional to the concentration (amount) of an
analyte in the sample within a given range. Five
solutions of sample of different percent of label
claim (80-120%) were prepared, analyzed by
proposed methods and the obtained data were
utilized to plot linearity curve as Percent of label
claim vs absorbance.
Results and Discussion
Testosterone was found to be highly soluble in
acetonitrile and stable in acetonitrile, hence
working standard solutions were prepared of
desired concentrations were prepared throughout
experimentation. All developed methods were
found to obey Beer’s-lambert’s law in the
concentration range of 5-25 μg/mL with
correlation coefficient value less than 1. Further
the proposed methods were applied to the phar-
maceutical formulation for assay of testosterone
in gel formulation. The recovery study was
carried out at three different levels 50-150%. All
developed methods were validated as per ICH
guidelines.
Analysis of marketed formulation
The percentage amount of testosterone estimated
from gel formulation using method I to IV was
found to be M-I= 101.8, M-II=99.6, M-III=101.2
& 99.0 and M-IV=100.2 & 100.8 respectively.
©Pharmaceutical and Biological Evaluations
The % amount estimated from gel formulation
indicates that there was no interference from
excipients present in it (Table 1).
Method validation
Developed methods were validated for linearity,
accuracy, precision, ruggedness and sensitivity as
per the ICH guidelines.
Accuracy
Results of recovery studies are shown in Table 2.
The % RSD value was found to be less than 2
indicating that the methods are accurate.
Linearity
From the linear regression data, it was found that
the linearity curve showed good linear
relationship over the concentration range of 80-
120% of label claim. The linearity curves are
shown in Figure 3 (a-f).
Ruggedness
The results of ruggedness study were found in the
acceptable range with% RSD values less than 2
by all proposed methods as shown in Table 3a and
3b. The results showed no statistical differences
between different operators and instruments
suggesting that the developed methods were
rugged.
Precision
The precision of the method was expressed in
terms of% RSD. The obtained results showed
reproducibility of the assay. The% RSD values
were found within limit indicates that the
methods were found to be precise. Results are
shown in Table 4(a) and 4(b).
Statistical analysis of results
Paired t-test
Paired t-test is the method of validating a new
procedure is to compare the results using sample
of varying compositions with the values obtained
by an accepted method are shown in Table 5
(a).11,12
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Table 1: Result of % estimation from gel formulation.

Sr. Wt. of gel % label claim

no. (mg) M-I M-II M-III M-IV
~100.0 237.4 nm AUC 250.4 nm 226.2 nm 258.6 nm 239.6 nm
1 102.80 99.92 101.91 100.98 99.96 102.80
2 102.02 99.41 102.67 101.58 99.41 102.76
3 102.40 100.40 101.88 98.08 101.99 100.15
4 102.24 100.17 101.60 98.12 102.30 100.20
5 101.74 100.08 101.74 98.63 100.55 100.27
Mean 101.8 99.6 101.2 99.0 100.2 100.8
±SD 0.447 0.548 0.447 1.414 1.304 1.095
%RSD 0.439 0.550 0.442 1.428 1.301 1.086
(M-I: Regression equation method; M-II: Area under curve; M-III: First order derivative; M-IV: second order
derivative.

Table 2: Result of recovery study.

Sr Total amount estimated (mg) Amt. of drug recovered % Recovery
No of drug (mg)
M-I M-II M-III M-IV M-I M-II M-III M-IV M-I M-II M-III M-IV
250.4 239.6 250.4 239.6 250.4 239.6
1. 0.1519 0.1520 0.1518 0.1519 0.05 0.05 0.05 0.05 100.2 100.4 100.0 100.2
2. 0.2019 0.2018 0.2020 0.2019 0.10 0.10 0.10 0.10 100.1 100 100.4 100.1
3. 0.2521 0.2520 0.2521 0.2521 0.15 0.15 0.15 0.15 100.2 100.1 100.2 100.3
Mean 100.1 100.1 100.2 100.2
±SD 0.058 0.21 0.20 0.10
%RSD 0.057 0.207 0.199 0.099
Table 3a: Result for instrument variation.

Sr. Instruments Wt. of % label claim

No. gel (mg) MI M II M III M IV
237.4 AUC 250.4 226.4 258.5 239.6
1. Shimadzu 1600 101.81 100.5 100.20 99.98 99.65 99.49
2. Jasco V-630 ~100.0 99.16 98.45 101.12 99.57 98.57 101.38
Mean 100.48 99 100.5 99.77 99.11 100.43
±SD 1.41 1.414 0.707 0.289 0.707 1.336
% RSD 1.414 1.428 0.703 0.290 0.72 1.330
Table 3b: Result of analyst to analyst variation.

% label claim
Sr. Different Wt. of gel MI M II M III M IV
No. Analyst (mg) 237.4 AUC 250.4 226.4 258.5 239.6
1. Analyst-I 101.74 100.02 101.74 99.33 100.50 100.77
2. Analyst-II ~100.0 102.02 100.67 102.02 101.58 99.96 102.00
Mean 101.5 100.34 101.5 100 99.5 101.38
±SD 0.707 0.45 0.707 1.414 0.707 0.869
% RSD 0.697 0.458 0.697 1.414 0.711 0.857

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Table 4 (a): Results for intraday study.

Time Wt. of % label claim

Sr. interval gel M-I M-II M-III M-IV
no (h) (mg) 237.4 nm AUC 250.4 nm 226.2 nm 258.6 nm 239.6 nm
1 0 100.12 100.22 99.81 98.84 99.44 99.39
2. 1 101.26 99.85 102.63 99.27 102.67 102.00
3. 2 ~100.0 102.22 99.41 102.05 98.88 99.05 102.02
4. 3 102.53 100.35 102.83 98.20 100.93 99.27
5. 4 102.40 99.79 102.29 98.88 99.50 99.61
Mean 101.4 99.4 101.4 98.2 99.8 100.2
±SD 0.894 0.548 1.342 0.447 1.304 1.643
%RSD 0.882 0.551 1.323 0.455 1.307 1.640
Table 4 (b): Results for interday study.

Time Wt. of % label claim

Sr. interval gel M-I M-II M-III M-IV
No. (Day) (mg) 237.4 nm AUC 250.4 nm 226.2 nm 258.6 nm 239.6 nm
1 0 102.38 100.70 102.24 99.35 99.19 112.36
2. 1 99.15 98.00 98.25 89.27 96.25 93.98
3. 2 ~100.0 100.08 98.79 97.00 88.68 98.08 95.07
4. 3 102.26 99.06 98.00 88.03 96.86 94.03
5. 4 101.66 99.26 98.11 87.95 97.51 98.00
6. 5 102.53 100.23 98.62 87.31 96.17 99.73
Mean 101 99.00 98.5 89.667 97.00 98.5
±SD 1.265 0.894 1.761 4.633 1.256 7.007
%RSD 1.252 0.903 1.788 5.167 1.304 7.114

1.2 1
1 y = 0.0074x + 0.1098 0.8 y = 0.0095x - 0.2355
Absorbance
Absorbance

R² = 0.993 R² = 0.9971
0.8
0.6
0.6
0.4
0.4
0.2 0.2

0 0
0 50 100 150 0 50 100 150
A B
Concentration (μg/ml) Concentration (μg/ml)

0
0.05 -0.005 0 50 100 150
0.04
Absorbance

-0.01 y = -0.0002x - 0.0079

Absorbance

0.03 -0.015 R² = 0.9961

0.02 -0.02
y = 0.0003x + 0.0126 -0.025
0.01 R² = 0.9927 -0.03
0
-0.035
0 50 100 150
C Concentration (μg/ml)
D
Concentration (μg/ml)

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Bhagat KD. et al. Pharmaceutical and Biological Evaluations 2018; Vol. 5 (1): 1-9.

0.001 0
0 50 100 150
0.0008 -0.0002
-0.0004 y = -6E-06x - 0.0002
Absorbance

Absorbance
0.0006 R² = 0.9914
-0.0006
0.0004 y = 7E-06x - 1E-05
R² = 0.999 -0.0008
0.0002
-0.001
0
0 50 100 150 -0.0012
E Concentration (μg/ml) F Concentration (μg/ml)

Figure 3 (A-F): Plot of linearity and range for method I-IV. (A) M-I: Plot of linearity and range,
(B) M-II plot of AUC, (C) M-III first order derivative at 250.4 nm, (D) M-III first order
derivative at 226.2 nm, (E) M-IV (258.6), (F) M-IV (239.6).

Table 5 (a): Observation table for paired T-test.

Sr. No. Paired Results

1 M-I vs. M-II 0.4899
2 M-I vs. M-III(a) 1.8910
3 M-I vs. M-III(b) 3.8340
4 M-I vs. M-IV(a) 2.726
5 M-I vs. M-IV(b) 1.704
Table 5 (b): Observation table for one way ANOVA t-test.

Bonferroni's multiple Mean diff. t Significant? Summary 95% CI of diff

comparison test P<0.05?
M 1 vs. M 2 2.244 3.244 No ns -0.05983 to 4.548
M 1 vs. M3a 0.2800 0.4048 No ns -2.024 to 2.584
M 1 vs. M3b 2.762 3.993 Yes * 0.4582 to 5.066
M 1 vs. M4a 1.398 2.021 No ns -0.9058 to 3.702
M 1 vs. M4b 1.004 1.451 No ns -1.300 to 3.308
M 2 vs. M3a -1.964 2.839 No ns -4.268 to 0.3398
M 2 vs. M3b 0.5180 0.7489 No ns -1.786 to 2.822
M 2 vs. M4a -0.8460 1.223 No ns -3.150 to 1.458
M 2 vs. M4b -1.240 1.793 No ns -3.544 to 1.064
M3a vs. M3b 2.482 3.588 Yes * 0.1782 to 4.786
M3a vs. M4a 1.118 1.616 No ns -1.186 to 3.422
M3a vs. M4b 0.7240 1.047 No ns -1.580 to 3.028
M3b vs. M4a -1.364 1.972 No ns -3.668 to 0.9398
M3b vs. M4b -1.758 2.542 No ns -4.062 to 0.5458
M4a vs. M4b -0.3940 0.5696 No ns -2.698 to 1.910

Comparison of results the analytical procedure has been accurate and/or

When new analytical method is developed, it is
usual practice to compare the values obtained
from sets of results with either a true value or
mean or other sets of data to determine whether
precise, or if it is superior to another method.; the
statistical technique is using to the one way
repetitive measures ANOVA analysis in
Bonferroni's multiple comparison test observing
the data for t-value and significance (p<0.05) to

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Bhagat KD. et al. Pharmaceutical and Biological Evaluations 2018; Vol. 5 (1): 1-9.
the comprise of each methods. The significance
is finding on to the two methods is (M1 vs. M3b
& M3a vs. M3b) but the remaining methods there
is not outcome to the significance value (Figure
4). The results are tabulated in the Table 5 (b).
150
100 * McGraw-Hill, 2000.
% Claim
50
0
M1 M2 M3a M3b M4a M4b
Method
Figure 4: One way ANOVA t-test.
Conclusions
The UV-Spectrophotometric methods that were
developed for the determination of Testosterone
are based on Calibration curve, derivative and
area under curve techniques. The method were
validated and found to be simple, sensitive,
accurate, and precise. Hence, it can be used
successfully for routine analysis of testosterone
from its gel formulation.
Acknowledgements
The authors were thankful to Principal of Smt.
Kishoritai Bhoyar College of Pharmacy,
Kamptee, Nagpur (MS) for providing necessary
help for the work.
Funding: No funding sources
Conflict of interest: None declared
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