INTRODUCTION:

Azathioprine (AZT), a purine analogue, primarily serves to prevent organ rejection following transplantation and is additionally beneficial in managing various autoimmune conditions.^1-3 Administered orally or via intravenous (IV) route, azathioprine exerts an immunosuppressive effect.

It is official in the United States Pharmacopoeia, the European Pharmacopoeia, and the British Pharmacopoeia.^4-5 AZT is chemically represented as 6-[(1-methyl-4-nitro-1H-imidazol-5-yl) sulfanyl]-7H-purine, illustrated in Figure 1.⁶ AZT behaves as a precursor for mercaptopurine, inhibiting a crucial enzyme necessary for DNA synthesis. Therefore, it primarily impacts rapidly multiplying cells, especially the T cells and B cells within the immune system⁷. AZT works in different ways to affect cellular immunity. It stops lymphocytes from activating, differentiating, stimulating, and reacting with other lymphocytes in the lab. It also lowers the activity of natural killer lymphocytes.^8-9. Presently, AZT is the established therapy for patients suffering from persistently active inflammatory bowel syndrome (IBS¹⁰. Moreover, it is employed in the management of anti-inflammatory conditions, including rheumatoid arthritis, Crohn's disease, lupus erythematous, ulcerative colitis, and autoimmune hepatitis¹¹.

Fig. 1: Structure of Azathioprine

Following an exhaustive review of the existing literature, we have identified and documented only a limited number of HPLC procedures. However, scholars have reported numerous methodologies that employ different techniques to detect azathioprine. These include spectrophotometry^12-13 atomic absorption spectrometry⁶, chemiluminescence (CL)¹¹, 1H NMR¹⁴, HPTLC¹⁵, and UPLC¹⁶. However, a significant proportion of these methods are either complex or require the use of expensive analytical instruments.

Several scholars have recorded a range of LC-MS/MS and HPLC techniques for identifying azathioprine and its active metabolic derivatives in blood, bloodstains, liquid components of blood, and urine^17-27. Nevertheless, these techniques precisely quantify azathioprine and its active metabolic substrates in the blood of humans and serum precursors simultaneously. Despite the fact that LC-MS/MS assays offer greater speed and sensitivity compared to LC-UV methods, the equipment required is often prohibitively expensive for many laboratories, especially those in developing countries.

Interestingly, a number of authors, including^28-33, have reported straightforward HPLC methods utilizing UV detection to measure AZT levels. However, the currently existing RP-HPLC method presents drawbacks such as peak tailing, prolonged run time, reduced sensitivity and selectivity, and high costs. To address these limitations, we made an effort to develop a new RP-HPLC method that is uncomplicated, precise, accurate, and cost-effective for estimating azathioprine in tablet dosage form. We developed a novel HPLC-UV method in this research to achieve a rapid analysis turnaround time for determining azathioprine while keeping costs relatively low.

MATERIAL AND METHODS:

Chemicals and reagents:

Azathioprine (AZT) was generously provided as a gift sample by Neon Laboratories Limited, Palghar (Maharashtra, India). HPLC grade acetonitrile (ACN), analytical reagent grade Trifluoroacetic acid (TFA), were acquired from Merck Specialties Pvt. Ltd., Mumbai, India. Commercially available 50mg azathioprine tablets (AZORAN) manufactured by RPG Life Sciences Limited, India were purchased from a local pharmacy store. The water used in mobile phase preparation was freshly prepared from LiChrosolv® and filtered using 0.45μm nylon 66 membrane filters, which were bought from Sigma-Aldrich, India. All of the reagents used in the study were freshly prepared, analyzed, and used.

Instrumentation:

A HPLC system (Agilent 1260 Infinity II) equipped with data processing software OpenLab EZChrom (version 4.8), a UV-Vis detector (G4212B DAD), a quaternary gradient pump (G1311B), and an auto injector (G7129A) was used for the analysis. The column used for separation was Agilent Zorbax Bonus-RP with a size of 250 x 4.6mm and an internal particle size of 5μm. An analytical balance (Aczet CY224C), an ultra sonicator (Labman LMUC-6), and a nylon 6.6 membranous filter (0.45microns, 47mm) (Pall Pvt. Ltd) were used during the study.

Preparation of reagents and standards:

Preparation of mobile phase:

To prepare the mobile phase, we mixed 0.1% TFA in H₂O with ACN in a 55:45 ratio. The mobile phase was degassed using the Infinity Standard Degasser (G1322A), which involved vacuum-filtering it through 0.45-micrometer nylon membrane strainers for 10 minutes before utilization.

Preparation of standard stock solution:

The stock solution of the standard was created by dissolving 5mg of AZT standard in a 10ml volumetric flask. Then, 5ml of diluent 0.1% TFA water: ACN (50:50) was added, mixed until the drug was completely dissolved, and the final volume was made up using diluents to get a final amount of 500 µg/ml, which was then filtered over a 0.45μ membrane mesh. A 1ml aliquot of this solution was pipetted, resulting in a final concentration of 50μg/ml for AZT, achieved by diluting it to 10ml with diluents^34-38.

Selection of wavelength for investigation of azathioprine:

The sensitivity of the HPLC method, employing a UV detector, relies on the choice of wavelength.^39-42. Detecting the drug requires an optimal wavelength that maximizes absorption and responsiveness. We scanned the solution of AZT (50 µg/ml) over the range 200–400 nm and recorded the UV spectrum of the drug. The spectrum indicated that AZT displayed peak absorbance at a wavelength of 278nm (depicted in Figure 2).

Fig. 2: Selection of Wavelength.

Method Optimization:

Optimized Chromatographic Condition:

The mobile phase, consisting of a mixture of ACN and 0.1% TFA in water with a ratio of 45:55, was determined to be the most effective composition. This solvent system successfully separated the components in an ideal manner. The Agilent Zorbax Bonus-RP C18 was employed as the stationary phase in the chosen technique, with dimensions of 250 x 4.6mm and an internal particle size of 5µm. The mobile phase was degassed using the Infinity Standard Degasser (G1322A), which involved vacuum-filtering it through 0.45μm nylon membrane filters for 10 minutes before utilization. Prior to injecting the solutions, allow the column to reach equilibrium for a minimum of 30 minutes using the mobile phase of system⁴³. The apparatus circulated the mobile phase through the chromatographic column at a flow speed of 1.0ml/min and conducted UV recognition at the 278nm wavelength for a duration of 9min. The volume of the injection was 10 microliters. During the analysis, we consistently kept the column at a temperature of 30°C.

Analysis of tablet:

A total of 10 tablets of AZT (Azoran) have been weighed, and the mean weight was calculated. The tablets underwent pulverization in a mortar using a pestle to acquire a finely ground powder. We precisely measured and transferred the weight of AZT, which is equivalent to 5mg, into a volumetric flask with a capacity of 10 ml. We introduced 5mL of a solution that includes 0.1% TFA and ACN in a 50:50 v/v ratio to the mixture. Subsequently, we subjected the mixture to sonication for a duration of 15 minutes. The volume was adjusted to reach the desired level by adding the solvent system. The final volume was adjusted to the desired level by adding the same solution, resulting in a sample stock solution of AZT with a concentration of 500 μg/ml. In addition, we employed a 0.45μm filtration membrane to purify the solution. By pipetting 1ml of this solution and adding diluents up to a total volume of 10ml, the ultimate concentration of AZT was diluted to 50μg/ml. We determined the purity by:

Sample area

% Assay = --------------------- × 100

Standard area

Method validation:

In accordance with Q2 (R1) International Conference on Harmonization (ICH) procedures from 2005, we performed the validation of the optimized chromatographic method. The following characteristics, like specificity, precision, linearity, accuracy, range, system suitability, detection limit (LOD), and quantitation limit (LOQ), were considered for validation ⁴⁴.

RESULTS AND DISCUSSION:

Specificity:

To validate the method’s specificity, we collected chromatograms of the blank and sample under optimal analytical parameters and compared them with the standard solution ^45-46. The method's specificity implies that there were no interferences from the mobile phase, placebo, or other excipients co-eluted with the drug. The clear segregation of the AZT peaks in the HPLC chromatogram, even when excipients are present, signifies its purity and showcases the method's specificity, unaffected by contaminants or excipients, as shown in Figure 3.

Fig. 3: HPLC Chromatogram of standard AZT

Precision:

To achieve system precision, we performed five separate determinations at 100% of the test concentration (50 μg/ml)^47-49. For method precision, we used five different sample preparations of AZT from the same homogenous sample. We studied the precision of an analytical method by performing repeatability. We determined the peak area for each measurement and expressed it in terms of the mean, the standard deviation, and the RSD. We calculated the RSD to be 0.06, as mentioned in Table 4. The obtained percentage RSD values (<2) prove that the developed procedure is precise. The precision studies revealed a high degree of reliability and predictability for the method.

Table 1: Precision values of AZT by the proposed method

Sample ID	Peak Area
Rep 1	3913587
Rep 2	3919319
Rep 3	3913832
Rep 4	3915914
Rep 5	3914936
AVG	3915518
STDEV	2319.186
% RSD	0.06

Linearity:

From a standard stock solution, we prepared a set of AZT solutions and injected them into the HPLC system at five separate concentration ranges (80, 90, 100, 110, and 120%), which are 40 to 60μg/ml (AZT), as shown in Table 1. After filtering the solutions using a Millipore syringe filter, we injected 10µl into the HPLC system and recorded their chromatograms for 9min. We documented the area under each peak and formed a calibration curve by plotting the peak extent against the concentration of the analyte (refer to Figure 4). We determined the slope (a), intercept (b), and correlation coefficient (r²) by applying the linear regression equation. We found that the peak area and the concentration had a significant linear relationship. The coefficient of determination (r²) value of 0.9959, as indicated in Table 2, confirmed the proportionality of the calibration curves.

Table 2: Linearity Study

Concentration (µg/ml)	Peak Area (Mean ± %RSD)
40	3134326 ± 0.067
45	3427382 ± 0.058
50	3913587 ± 0.070
55	4306061 ± 0.061
60	4696625 ± 0.066

Fig. 4: Calibration curve of AZT

Accuracy:

The accuracy was measured over 3 concentration levels: 80, 100, and 120%, within Beer's limit for the drug. We then added known amounts of the pure samples, each with a concentration of 50 µg/ml, to the previously analyzed sample solutions. Three of these solutions were were introduced into the HPLC system to find out the percentage recoveries. We evaluated recovery by comparing the measured concentration to the one added and evaluating it in terms of %RSD. The developed HPLC method yielded results (Table 3) within the limits, ranging from 99.92 to 100.06%, demonstrating its accuracy.

Range:

By plotting the curve, we used the range between the upper and lower limits of the calibration curves to calculate the analytical method's range. The range was from 40 to 60 µg/ml of the test concentration.

System Suitability:

For the system suitability test, we injected five replicates of freshly prepared standard solutions of AZT. Retention time (RT), theoretical plates (TP), and asymmetry of the standard chromatogram were tested to ensure the system’s suitability⁴⁹. The system shows reliable and consistent results, as seen in the low percent RSD values (Table 4). The elution time is 2.67 minutes, indicating the system's precision. The mean TP is 11737.20±0.3992, demonstrating the system's ability to separate sample components effectively. The peak asymmetry falls within the acceptable range of 0.9 to 1.2, with a mean value of 1.06±1.0553, showing that the system consistently produces symmetrical chromatographic peaks. In simpler terms, the proposed system is exact, reliable, and suitable for the intended study.

Table 4: System Suitability Study

Sample ID	RT	TP	Asymmetry
Rep 1	2.67	11686	1.04
Rep 2	2.67	11763	1.04
Rep 3	2.67	11715	1.04
Rep 4	2.67	11717	1.02
Rep 5	2.67	11805	1.05
AVG	2.67	11737.2	1.04
STDEV	0	46.8636	0.0109
% RSD	0.00	0.3992	1.0553

Sensitivity:

The determination of LOD and LOQ was conducted to validate the method's sensitivity. This involved calculating the standard deviation of the response and the slope of the corresponding calibration curve. The resulting LOD and LOQ values were 6.17 and 18.69 µg/ml, separately, indicating high sensitivity of the method.

Analysis of tablet:

The results of testing the AZT drug in its commercial form showed a concentration of 49.85μg/ml, which is an impressive 98.89% of the labelled amount, demonstrating high precision with a low percent RSD value of 0.4578. These results clearly reveals that the method is sensitive and capable of providing accurate and reliable estimates of the amount of AZT in available formulations.

ABBREVIATIONS:

% RSD: Percentage Relative Standard Deviation, µg/ml: Microgram per millilitre, μg: Microgram, µm: Micrometer, ACN: Acetonitrile, AVG-Average, AZT: Azathioprine, CL: Chemiluminescence, H NMR: Proton Nuclear Magnetic Resonance, HPLC: High-Performance Liquid Chromatography, HPLC-UV: High Performance Liquid Chromatography-Ultraviolet detection, HPTLC: High-Performance Thin-Layer Chromatography, IBS: Inflammatory bowel syndrome, ICH: International Conference on Harmonization, IV: Intravenous, LC-MS/MS: Liquid Chromatography-Mass Spectrometry/ Mass Spectrometry, LC-UV: Liquid Chromatography with Ultraviolet Detection, LOD: Limit of Detection, LOQ: Limit of Quantification, ml: Millilitre, MS/MS: Tandem mass spectrometry, Rep-Replicate, RP- Reverse Phase, RT: Retention time, STDEV-Standard deviation, TFA: Trifluoroacetic acid, TP: Theoretical plate number, UPLC: Ultra-Performance Liquid Chromatography, UV: Ultraviolet.

ACKNOWLEDGEMENTS:

Principal of Appasaheb Birnale College of Pharmacy in Sangli (Maharashtra), is acknowledged for offering the essential facilities required for conducting the research. Additionally, the authors are grateful to Neon Laboratories Limited, Palghar (Maharashtra, India), for providing the azathioprine sample used in the research study.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 29.12.2023 Revised on 20.08.2024

Accepted on 29.01.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5551-5556.

DOI: 10.52711/0974-360X.2025.00800

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sample	Amount Spiked (µg/ml)	Area	Amount Recovered (µg/ml)	AVG	STDEV	% RSD
80%	39.88	3134326	39.90	100.06	0.0070	0.01
100%	49.85	3913587	49.83	100.00	0.1035	0.10
120%	59.82	4696625	59.79	99.92	0.0477	0.05