Research Article

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

Dr. Krishna R. Gupta,

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








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


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 levels of testosterone in men may lead to 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 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.


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).

Figure 2 (a): Zero order spectrum of testosterone showing AUC between selected wavelength.

Figure 2 (b): First order derivative spectrum.

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, correlation coefficient was found to be 0.993 and 0.9901 respectively.

Preparation of sample solution

To determine the content of Testosterone from marketed gel formulation; average content per sachet was calculated. An amount of gel equivalent to 100.0 mg of testosterone was weighed and transferred to a 10.0 mL volumetric flask. To it sufficient amount of acetonitrile was added, sonicated for 30 min and the solution was diluted up to mark with the same solvent. The content of the flask was filtered through Whatmann filter paper (No. 41). From the filtrate solution pipette out 1.0 ml and add 10.0 ml volumetric flask and make up the volume up to the mark and get the final concentration of 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 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.

Validation of methods

The proposed method was validated as per ICH guidelines.


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 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.


The precision of the analytical method expresses 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 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 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.


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 pharmaceutical 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. 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.


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.


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).


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.


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

Table 1: Result of % estimation from gel formulation.

Sr. no. Wt. of gel (mg) % label claim
  ~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 No Total amount estimated (mg) of drug Amt. of drug recovered (mg) % Recovery
M-I M-II M-III 250.4 M-IV 239.6 M-I M-II M-III 250.4 M-IV 239.6 M-I M-II M-III 250.4 M-IV 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. No. Instruments Wt. of gel (mg) % label claim
237.4 AUC 250.4 226.4 258.5 239.6
1. Shimadzu 1600 ~100.0 101.81 100.5 100.20 99.98 99.65 99.49
2. Jasco V-630 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.

Sr. No. Different Analyst Wt. of gel (mg) % label claim
237.4 AUC 250.4 226.4 258.5 239.6
1. Analyst-I ~100.0 101.74 100.02 101.74 99.33 100.50 100.77
2. Analyst-II 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

Table 4 (a): Results for intraday study.

Sr. no Time interval (h) Wt. of gel (mg) % label claim
237.4 nm AUC 250.4 nm 226.2 nm 258.6 nm 239.6 nm
1 0 ~100.0 100.12 100.22 99.81 98.84 99.44 99.39
2. 1 101.26 99.85 102.63 99.27 102.67 102.00
3. 2 102.22 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.

Sr. No. Time interval (Day) Wt. of gel (mg) % label claim
237.4 nm AUC 250.4 nm 226.2 nm 258.6 nm 239.6 nm
1 0 ~100.0 102.38 100.70 102.24 99.35 99.19 112.36
2. 1 99.15 98.00 98.25 89.27 96.25 93.98
3. 2 100.08 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


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.




M-I vs. M-II



M-I vs. M-III(a)



M-I vs. M-III(b)



M-I vs. M-IV(a)



M-I vs. M-IV(b)


Table 5 (b): Observation table for one way ANOVA t-test.

Bonferroni's multiple comparison test

Mean diff.





95% CI of diff

M 1 vs. M 2





-0.05983 to 4.548

M 1 vs. M3a





-2.024 to 2.584

M 1 vs. M3b





0.4582 to 5.066

M 1 vs. M4a





-0.9058 to 3.702

M 1 vs. M4b





-1.300 to 3.308

M 2 vs. M3a





-4.268 to 0.3398

M 2 vs. M3b





-1.786 to 2.822

M 2 vs. M4a





-3.150 to 1.458

M 2 vs. M4b





-3.544 to 1.064

M3a vs. M3b





0.1782 to 4.786

M3a vs. M4a





-1.186 to 3.422

M3a vs. M4b





-1.580 to 3.028

M3b vs. M4a





-3.668 to 0.9398

M3b vs. M4b





-4.062 to 0.5458

M4a vs. M4b





-2.698 to 1.910

Comparison of results

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 the analytical procedure has been accurate and/or 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 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).

Figure 4: One way ANOVA t-test.


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.


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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