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 Table of Contents  
Year : 2012  |  Volume : 4  |  Issue : 4  |  Page : 318-321  

Determination of azathioprine in bulk and pharmaceutical dosage form by HPTLC

Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist. Dhule, India

Date of Submission12-May-2012
Date of Decision18-May-2012
Date of Acceptance28-May-2012
Date of Web Publication07-Nov-2012

Correspondence Address:
Pritam S Jain
Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist. Dhule,
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-7406.103263

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Introduction: An HPTLC method for analysis of Azathioprine in bulk and pharmaceutical formulation has been established and validated. Materials and Methods: The analyte was separated on aluminium plates precoated with silica gel 60 F254. The mobile phase was Ethyl acetate: Methanol: Triethylamine (4:1:0.5v/v). Quantification was done by densitometric scanning at 300nm. Results: Response was a linear function of Azathioprine concentration in the range of 200-1200 ng/band. The limit of detection and quantification for Azathioprine were 18.58 and 59.14 ng/band, respectively. Average recovery was 100.1% which shows that the method was free from interference from excipients present in the formulation. Conclusion: The established method enabled accurate, precise, and rapid analysis of Azathioprine in bulk as well as pharmaceutical formulation.

Keywords: Azathioprine, HPTLC, validation

How to cite this article:
Jain PS, Thakre P, Chaudhari AJ, Chavhan ML, Surana SJ. Determination of azathioprine in bulk and pharmaceutical dosage form by HPTLC. J Pharm Bioall Sci 2012;4:318-21

How to cite this URL:
Jain PS, Thakre P, Chaudhari AJ, Chavhan ML, Surana SJ. Determination of azathioprine in bulk and pharmaceutical dosage form by HPTLC. J Pharm Bioall Sci [serial online] 2012 [cited 2022 Jul 2];4:318-21. Available from:

Azathioprine is chemically 6-[(1-methyl-4-nitro-1H imidazol- 5yl) sulfanyl]-7H-purine. [1] It has a marked effect on T-lymphocytes [2] and immunosuppressive action, and is given orally or by the intravenous (IV) route. [3] It is co-administered with cyclosporine and corticoids to prevent rejection after transplantation. [4] It is also used in systemic anti-inflammatory states, such as rheumatoid arthritis, lupus erythematosus, colitis ulcerosa, autoimmunological hepatitis and Crohn's disease. [5]

Several methods have been described for determination of Azathioprinein pharmaceutical preparations, including spectrophotometry, [6] HPLC [7] and NMR. [8] The proposed method was validated as per the International Conference on Harmonization (ICH) guidelines. [9],[10]

   Materials and Methods Top


The tablet formulation was procured from a local pharmacy. Pure Azathioprine was obtained from Alkem Lab., Mumbai, India. All chemicals were of analytical grade. All dilutions were performed in standard volumetric flasks.


The samples were spotted in the form of bands of 6 mm width with a Camag microliter syringe (100/μL) on pre-coated silica gel aluminum plates 60 F-254 (10 cm × 10 cm with 250 mm thickness, E. Merck, Wilmington, USA) using a Camag Linomat-5 applicator. The plates were pre-washed with methanol and activated at 100°C for 5 min prior to chromatography. The slit dimension was kept at 6.00 mm × 0.45 mm (micro) and 20 mm/s scanning speed was employed. The mobile phase consisted of ethyl acetate: methanol:triethylamine (4:1:0.5 v/v), and 7.5 mL of mobile phase was used. Linear ascending development was carried out in a 10 cm × 10 cm twin trough glass chamber (Camag, Switzerland) saturated with the mobile phase. The optimized chamber saturation time for the mobile phase was 15 min at room temperature (25°C ± 2). The length of the chromatogram run was approximately 8 cm. Subsequent to development, the thin layer chromatography plates were dried in a current of air with the help of an air dryer. Densitometric scanning was performed on a Camag TLC scanner 3 and was operated by winCATS software.

Stock and working standard solution

Azathioprine (25 mg) was accurately weighed into a 25 mL volumetric flask and dissolved in a minimum volume of methanol, and diluted to the required volume with methanol to furnish a solution of concentration 1000 ng/μL. This was used as stock solution. Calibration standards were prepared over the concentration range 200-1200 ng/band for Azathioprine by appropriate dilutions of the above-mentioned standard stock solution in a 10 mL volumetric flask with methanol.

Calibration curve

Separate stock standard solutions of Azathioprine were used for the preparation of calibration standard solutions. All calibration standards were prepared freshly every day and were found to be stable during the analysis time. The plate was developed, dried and scanned as described above. After densitometric scanning, the peak area was recorded for each concentration and a calibration plot was obtained by plotting average peak area against concentration of Azathioprine (ng/spot). The slope and correlation coefficient were also determined.

   Method Validation Top

Validation of the optimized HPTLC method was carried out with respect to the following parameters:


Repeatability of sample application and measurement of peak area were carried out using six replicates of the same band (800 ng/band of Azathioprine). The intra-and inter-day variation for the determination of Azathioprine was carried out at three different concentration levels of 600, 800 and 1000 ng/band.

Limit of detection and limit of quantification

In order to determine the limit of detection (LOD) and limit of quantification (LOQ), Azathioprine concentrations in the lower part of the linear range of the calibration curve were used. Azathioprine solutions of 200, 240, 280, 320 360 and 400 ng/ band were prepared and applied on the plate. The LOD and LOQ were calculated using the equation LOD = 3.3 × N/B and LOQ = 10 × N/B, where, N is the standard deviation of the peak areas of the drugs (n = 3), taken as a measure of noise, and B is the slope of the corresponding calibration curve.


The specificity of the method was ascertained by analyzing standard drug and sample. The spot for Azathioprine in the sample was confirmed by comparing the Rf values and spectra of the spot with that of the standard. The peak purity of Azathioprine was assessed by comparing the spectra at three different levels, i.e., peak start (S), peak apex (M) and peak end (E) positions of the spot.


Ruggedness of the method was performed by spotting 800 ng/band of Azathioprine by two different analysts keeping the same experimental and environmental conditions.


The analyzed samples were spotted with extra 80, 100 and 120% of standard Azathioprine, and the mixture was analyzed by the proposed method. This was done to check the recovery of the drug at different levels in the formulations.


By introducing small changes in the mobile phase composition, the effects on the results were examined. Mobile phases having different compositions of ethyl acetate: methanol:triethylamine (5:2.5 v/v) were tried and chromatograms were run. The amount of mobile phase, temperature and relative humidity was varied in the range of ± 5%. The plates were pre-washed by methanol and activated at 80 ± 10°C for 2, 5 and 7 min prior to chromatography. Time from spotting to chromatography and from chromatography to scanning was varied from 10, 15 and 20 min.

Application of the proposed method to the tablet formulation

To determine the concentration of Azathioprine in tablets (labelled claim: 100 mg per tablet), the contents of 20 tablets were weighed, their mean weight was determined and they were finely powdered. The powder, equivalent to 100 mg of Azathioprine, was weighed. The drug from the powder was extracted with methanol. To ensure complete extraction of the drug, it was sonicated for 30 min and the volume was made up to 10 mL. The resulting solution was filtered using a 0.45 μm filter (Mill filter, Milford, MA, USA). The above solution (800 ng/ band) was applied on a TLC plate, followed by development and scanning, as described above.

   Results and Discussion Top

Development of optimum mobile phase

The TLC procedure was optimized with a view to developing a stability-indicating assay method. Initially, ethyl acetate: methanol (4:1v/v) gave good resolution with Rf value of 0.48 for Azathioprine, but typical peak nature was missing and tailing appeared. Finally, the mobile phase consisting of ethyl acetate: methanol: triethylamine (4:1:0.5 v/v) gave a sharp and well-defined peak at R f value of 0.49 [Figure 1]. Well-defined spots were obtained when the chamber was saturated with the mobile phase for 15 min at room temperature.
Figure 1: Densitogram of Azathioprine

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Calibration curve

The linear regression data for the calibration curves showed a good linear relationship over the concentration range 200-1200 ng/band. Linear regression equation was found to be Y = 7.154 X + 1293.9 and R 2 = 0.998.

Validation of the method


The precision of the developed HPTLC method was expressed in terms of %relative standard deviation (%RSD). The results depicted revealed high precision of the method, which is presented in [Table 1].
Table 1: Precision

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Detection limit and quantification limit was calculated by the method described above. The LOQ and LOD were found to be 18.58 and 59.14 ng/spot, respectively. This indicated adequate sensitivity of the method.


The peak purity of Azathioprine was assessed by comparing the spectra at peak start, peak apex and peak end positions of the spot, i.e., r2 (S, M) = 0.999 and r2 (M, E) = 0.999. Good correlation (r2 = 0.999) was also obtained between standard and sample spectra of Azathioprine [Figure 2].
Figure 2: A typical overlain spectrum of standard drug and drug extracted from the tablet

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When the method was performed by two different analysts under the same experimental and environmental conditions, it was found to be rugged [Table 2].

Recovery study

The proposed method when used for extraction and subsequent estimation of Azathioprine from the pharmaceutical dosage form after overspotting with 80, 100 and 120% of additional drug, affording good recovery of Azathioprine. The amounts of drug added and determined and the % recovery are listed in [Table 3].
Table 2: Summary of validation parameter

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Table 3: Recovery study*

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Robustness of the method

The standard deviation of peak areas was calculated for each parameter, and the %RSD was found to be less than 2% The low values of %RSD [Table 4] indicated that the method was robust.
Table 4: Robustness of the method*

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Analysis of the marketed formulation

A single spot at R f 0.49 was observed in the chromatogram of the drug samples extracted from the tablets. There was no interference from the excipients commonly present in the tablet. The %drug content and %RSD were calculated. The low %RSD value indicated the suitability of this method for the routine analysis of Azathioprine in pharmaceutical dosage forms.

   Conclusion Top

This HPTLC method is simple, precise, specific and accurate. Statistical analysis proved that the method is reproducible and selective for the analysis of Azathioprine as the bulk drug and in tablet formulations.

   Acknowledgment Top

The authors are thankful to the Principal, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur (Maharashtra), India, for providing the required facilities and valuable guidance to carry out this research work.

   References Top

1.O'Neil MJ. The merck index. 14 th ed. NJ: USA: Merck and Co. INC., White House Station; 1996. p. 672.  Back to cited text no. 1
2.2 Bhaskar M, Manohara YN, Gayasuddin M, Balaraju M, Bharath Kumar T. Spectrophotometric determination of azathioprine in bulk and pharmaceutical dosage forms. Int J Chem Tech Res 2010;2:376-8.  Back to cited text no. 2
3.Smita S, Mukesh S. Spectrophotometric and atomic absorption spectrometric determination and validation of azathioprine in API and pharmaceutical dosage form. J Optoele Biomed Mate 2010;2:213-6.  Back to cited text no. 3
4.Thierry D, Roselyne B. Simultaneous determination of 6-thioguanine and methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by HPLC. Clin Chem 1998;44:551-5.  Back to cited text no. 4
5.Jianbo W, Pan Z, Suqin H. Direct determination of azathioprine in human fluids and pharmaceutical formulation using flow injection chemiluminescence analysis. J Chin Chem Soc 2011;58:1-7.  Back to cited text no. 5
6.Lakshmi C, Reddy M. Spectrophotometric determination of azathioprine in pharmaceutical formulations. Talanta 1998;47:1279-86.  Back to cited text no. 6
7.Binscheck T, Meyer H, Wellhoner H. High-performance liquid chromatographic assay for the measurement of azathioprine in human serum samples. J Chromatogr B Biomed Appl 1996;675:287-94.  Back to cited text no. 7
8.Goger N, Parlatan H, Berkkan A, Ozden T. Quantitative determination of azathioprine in tablets by 1H NMR spectroscopy. J Pharm Biomed Anal 1999;21:685-9.  Back to cited text no. 8
9.ICH. Q2A Text on validation of analytical procedures, International Conference on Harmonization. Oct 1994.  Back to cited text no. 9
10.ICH. Q3B Validation of analytical procedures: Methodology, International Conference on Harmonization. Nov 1996.  Back to cited text no. 10


  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4]

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