Analytical Method Development and Validation of Spectrophotometric Method for Estimation of Tofacitinib citrate in oral and Topical Formulations
Sana Tabassum*, Dr. Makula Ajitha
Centre for Pharmaceutical Sciences, Jawaharlal Nehru Technological University,
Hyderabad, Telangana - 500085 India.
*Corresponding Author E-mail: sanaksm1996@gmail.com
ABSTRACT:
Tofacitinib, administered as Tofacitinib citrate, is a member of a novel class of therapeutic agents known as Janus kinase (JAK) inhibitors. It is approved by the US FDA for the treatment of moderate to severe conditions such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, and polyarticular course juvenile idiopathic arthritis. In the present study, a simple and cost-effective UV spectrophotometric method was developed for its analysis. The method primarily utilizes ethanol as an eco-friendly green solvent, with a small quantity of N,N-dimethylformamide as a co-solvent. Method development included verification of compliance with the Beer-Lambert Law, ensuring linearity within the concentration range of 2–20μg/mL, with a correlation coefficient (r˛) of 1.000. The maximum absorbance (λmax) for the developed method was found to be 249nm. Validation of the method was carried out in accordance with ICH guidelines, confirming that the method is simple, accurate, precise, sensitive, linear, and rugged. The developed method is suitable for both qualitative and quantitative estimation of Tofacitinib citrate in bulk drug as well as pharmaceutical formulations. Percent assay was successfully performed for extended-release tablets and transdermal formulations. The method enables calculation of percent label claim using either a standard solution or a calibration curve. Due to its simplicity, cost-effectiveness, and use of predominantly green solvent, this method is highly suitable for routine analysis of Tofacitinib citrate in pharmaceutical industries and analytical laboratories.
KEYWORDS: Tofacitinib, Rheumatoid arthritis, Beer-Lambert’s law, Validation, ICH.
INTRODUCTION:
Janus kinase inhibitors (JAKi) represent a new class of oral drugs counteracting the activation of JAKs, which are cytosolic enzymes presiding over many biologic functions, including the activation of the inflammatory cascade in immune cells. Rheumatoid arthritis (RA) is a complicated, long-lasting autoimmune condition that causes synovitis and gradually destroys joints.1-3 In both the early and severe stages of the disease, synthetic and biological disease-modifying antirheumatic medications have been the backbone of care for individuals with RA. To date, some oral JAKi have already been licensed for the treatment of rheumatoid arthritis and other immune-mediated diseases such as Tofacitinib, Baricitinib, Upadacitinib, Peficitinib, Filgotinib, Ruxolitinib, etc. Tofacitinib is FDA approved for the treatment of moderate to severe rheumatoid arthritis (RA), psoriatic arthritis (PA), and ankylosing spondylitis, ulcerative colitis (UC), polyarticular course juvenile idiopathic arthritis (pcJIA). It is also employed for the treatment of ulcerative colitis, vitiligo, psoriasis, alopecia areata, atopic dermatitis, lichen planus, and lupus erythematosus. It is a second-generation selective Janus kinase (JAK) inhibitor targeting the JAK1 enzyme. Pubchem directory indicates Molecular formula of Tofacitinib citrate is C22H28N6O8 and molecular weight is 504.5g/mol. Chemical name of Tofacitinib citrate is 2-hydroxypropane-1,2,3-tricarboxylic acid;3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d] pyrimidin-4-yl) amino] piperidin-1-yl]-3-oxopropanenitrile. The category of tofacitinib is Janus kinase inhibitor, antineoplastic and immunomodulating agent and it is used in treatment of rheumatoid arthritis.4-6
Tofacitinib citrate is an oral Janus kinase inhibitor for the treatment of rheumatoid arthritis. Cytokines work within a complex regulatory network in RA, signaling through different intracellular kinase pathways to modulate the recruitment, activation, and function of immune cells and other leukocytes. Several research works elucidated the safety and efficacy of Tofacitinib drug. The chemical structure of Tofacitinib citrate is represented in Figure 1. Tofacitinib, a first oral non-biologic disease-modifying anti-rheumatic drug (DMARD) can be used as monotherapy or in combination with methotrexate or other non-biologic DMARD’s, for treating adults with moderate or severe rheumatoid arthritis. It is contraindicated for use with biologic DMARDs or with immunosuppressive agents, such as Azathioprine and cyclosporine.7-9
Some methods are developed for performance of assay of Tofacitinib citrate by few UV spectrophotometric and costly HPLC methods.9-15
Figure 1 Chemical structure of Tofacitinib citrate7
MATERIAL AND METHODS:
Materials:
Analytical grade ethanol and N, N-dimethylacetamide of Merck grade were used throughout the study. The standard active pharmaceutical ingredient (API) of tofacitinib citrate was obtained as a gifted sample. Commercial tablet formulations, and TOFE ER tablets (11mg) and transdermal patch. Containing 5mg of tofacitinib citrate respectively, were used for analysis. Whatman filter paper (Grade 1), composed of cellulose and of Merck grade, was used for filtration purposes.
Instrumentation:
Instruments used for this method are Double beam Shimadzu UV-Visible Spectrophotometer of UV 1800 model, Cuvettes made up of glass quartz of 1cm. Weighing was done using Analytical Calibrated Electronic Microbalance (Mettler Toledo). Calibrated glass wares such as Volumetric flask, measuring cylinder, pipettes, test tubes and beakers were used made up of borosilicate glass of Class-A grade. Ultrasonic Bath XUBA series sonicator was used for sonication. BSCO Hot air oven was used for drying of glass wares.
Solubility Determination:
A small quantity of tofacitinib citrate standard was placed in a clean, dry test tube, and a small volume of water was added to assess its solubility. The drug was found to be insoluble in water, as the resulting solution was not clear. Subsequently, a small quantity of the standard was taken in another dry test tube and dissolved in a small volume of ethanol. The drug appeared to be initially soluble, producing a clear solution; however, the formation of some insoluble particles was observed upon standing. Further, the solubility was evaluated using a ethanol. The drug was found to be solutble
Preparation of Standard Stock Solutions of Tofacitinib Citrate for Linearity:
Preparation of Diluent:
Ethanol was used as diluent.
Accurately weighed about 5mg of tofacitinib citrate and transferred it into a clean, dry 10mL volumetric flask. A small volume of diluent was added and the solution was sonicated for approximately 2minutes to ensure complete dissolution. The volume was then made up to 10mL with the same diluent.
0.04mL of the stock solution was pipetted into a 10mL volumetric flask, and the volume was made up to 10mL with diluent. The solution was then shaken well after capping.
0.1mL of the stock solution was transferred into a 10mL volumetric flask. The volume was adjusted to 10mL with diluent and mixed thoroughly after capping.
0.2mL of the stock solution was pipetted into a 10mL volumetric flask. The volume was made up to 10mL with diluent and mixed well after capping.
0.3mL of the stock solution was transferred into a 10mL volumetric flask. The volume was adjusted to 10mL with diluent and shaken well after capping.
0.4 mL of the stock solution was pipetted into a 10mL volumetric flask. The volume was made up to 10mL with diluent and mixed thoroughly after capping.
The working standard solution III of tofacitinib citrate was scanned in the UV range of 200–400nm. The absorption spectrum is shown in Figure 2. The maximum absorbance (λmax) was found to be 249nm.
Figure 2: Spectrum of Tofacitinib citrate
The developed method demonstrated linearity over the concentration range of approximately 2 to 20µg/mL. The correlation coefficient (r˛) was found to be 1.00, indicating excellent linear relationship between concentration and absorbance, as shown in Figure 3.
Twenty tablets of tofacitinib citrate (TOFE ER 11mg) were accurately weighed and transferred into a clean, dry mortar. The tablets were crushed into a fine powder using a pestle. Powder equivalent to 11mg of tofacitinib citrate was accurately weighed and transferred into a 10 mL volumetric flask. Approximately 5mL of diluent was added, and the mixture was sonicated for about 30 minutes to ensure complete extraction of the drug. The volume was then made up to 10mL with the same diluent. The resulting solution was mixed well and filtered through Whatman filter paper. From the filtrate, 0.2mL was pipetted into a 10mL volumetric flask and diluted to volume with diluent to obtain Sample Solution I. Further, 5mL of Sample Solution I was diluted to 10 mL with diluent to obtain Sample Solution II. The absorbance of Sample Solution II was measured at 249 nm using a UV spectrophotometer against a blank (diluent). The concentration of the drug was determined using the standard calibration curve method, and the amount of drug and percentage label claim were calculated as shown in Table 1.
Figure 3: Calibration Curve of Tofacitinib citrate at 249 nm.
A known area of the transdermal patch (e.g., 1 cm˛ or the entire patch) containing an amount equivalent to 5mg of tofacitinib citrate was cut and transferred into a 10mL volumetric flask. Approximately 5mL of diluent was added, and the mixture was sonicated for about 15 minutes to facilitate drug extraction. The volume was then made up to 10mL with diluent, and the solution was mixed thoroughly. The resulting solution was filtered through Whatman filter paper. From the filtrate, 0.2mL was pipetted into a 10 mL volumetric flask and diluted to volume with diluent to obtain the sample solution. The absorbance was measured at 249nm against a blank (diluent). The concentration of the drug was determined using the standard calibration curve method, and the amount of drug and percentage label claim were calculated as shown in Table 2.
Table 1: Results of Assay / Determination of Tofacitinib Citrate in TOFE ER Tablets (Precision Evaluation)
|
Sample |
Concentration of drug (μg/mL) |
Amount of the drug (Tofacitinib) found (mg) |
% Label Claim |
% RSD of % Label claim of six samples |
|
Sample-1 |
9.99 |
5.00 |
99.9 % |
1.9 % |
|
Sample-2 |
9.78 |
4.89 |
97.8 % |
|
|
Sample-3 |
9.84 |
4.92 |
98.4 % |
|
|
Sample-4 |
10.23 |
5.12 |
102.3 % |
|
|
Sample-5 |
9.89 |
4.95 |
98.9 % |
|
|
Sample-6 |
10.2 |
5.10 |
102.0 % |
Table 2: Results of Assay/Determination of Tofacitinib Citrate in Transdermal patch (Precision):
|
Sample |
Concentration of drug (μg/mL) |
Amount of the drug (Tofacitinib) found (mg) |
% Label Claim |
% RSD of % Label claim of six samples |
|
Sample-1 |
10.90 |
10.90 |
99.1 % |
1.7 % |
|
Sample-2 |
10.89 |
10.89 |
99.0 % |
|
|
Sample-3 |
11.10 |
11.10 |
100.9 % |
|
|
Sample-4 |
10.88 |
10.88 |
98.9 % |
|
|
Sample-5 |
10.79 |
10.79 |
98.1 % |
|
|
Sample-6 |
10.52 |
10.52 |
95.6 % |
The percentage label claim of tofacitinib citrate was found to be within acceptable limits. The %RSD of the assay values for six replicate samples was not more than 2%, indicating good precision. Therefore, the developed method was found to be accurate. The repeatability study complied with ICH guidelines and was found to be satisfactory.
Validation of Proposed Method and It’s Results:
The developed method was validated as per ICH guidelines.
Accuracy:
Accuracy (recovery) was evaluated at three different levels by spiking known amounts of standard solution to the sample. The study was carried out at 50%, 100%, and 120% of the target concentration (10µg/mL). Recovery experiments were performed in triplicate at each level. The amount of drug added and percentage recovery were calculated for each replicate. The mean percentage recovery at each level was determined and the results were tabulated in Table 3.
Table 3: Results of Accuracy:
|
S.No |
Recovery Level |
Sample |
Amount added, µg/mL |
Amount Found, µg/mL |
% Recovery |
Average % Recovery |
|
1 |
50 % |
Sample-1 |
5 |
4.79 |
95.8 % |
98.5 % |
|
Sample-2 |
5 |
4.89 |
97.8 % |
|||
|
Sample-3 |
5 |
5.10 |
102.0 % |
|||
|
2 |
100 % |
Sample-1 |
10 |
10.20 |
102.0 % |
102.0 % |
|
Sample-2 |
10 |
10.10 |
101.0 % |
|||
|
Sample-3 |
10 |
10.20 |
102.0 % |
|||
|
3 |
120 % |
Sample-1 |
12 |
12.10 |
100.8 % |
100.8 % |
|
Sample-2 |
12 |
12.35 |
102.9 % |
|||
|
Sample-3 |
12 |
12.22 |
101.8 % |
Based on the obtained results, accuracy at three different levels was found to be within acceptable limits and was considered to comply with ICH guidelines for the assay of tablets.
Precision – Intraday Precision:
Intraday precision was evaluated by analyzing a standard solution of 10µg/mL in the morning and afternoon. The analysis was performed in triplicate (n = 3) following the same procedure as used for the assay of tablets. The amount of drug and percentage label claim were calculated, and the %RSD for the replicate samples was determined. The results are presented in Table 4.
Table 4: Results of Intraday Precision:
|
Time |
% Label Claim |
|||||
|
Sample-1 |
Sample-2 |
Sample-3 |
Average |
SD |
% RSD |
|
|
Morning |
98.9 |
102.1 |
100.1 |
100.4 |
1.6 |
1.6 |
|
Afternoon |
102.1 |
100.9 |
99.9 |
101.0 |
1.1 |
1.1 |
|
Average % RSD |
|
1.35 |
||||
Based on the above results, intraday precision was found to comply with ICH limits and was therefore considered satisfactory.
Ruggedness – Different Analyst:
Ruggedness was evaluated by three different analysts using a standard solution of 10µg/mL in triplicate (n = 3), following the same procedure as developed for the assay of tablets. The amount of drug and percentage label claim were calculated for each analysis. The %RSD was determined for the samples analyzed by each analyst as well as across the three different analysts. The results are presented in Table 5.
Table 5: Results of Ruggedness-Different Analyst:
|
Analyst |
% Label Claim |
|||||
|
Sample-1 |
Sample-2 |
Sample-3 |
Average |
SD |
% RSD |
|
|
Analyst-1 |
96.9 |
98.2 |
96.0 |
97.0 |
1.1 |
1.1 |
|
Analyst-2 |
101.1 |
100.2 |
100.3 |
100.5 |
0.5 |
0.5 |
|
Analyst-3 |
98.9 |
100.0 |
99.2 |
99.4 |
0.6 |
0.6 |
|
Average % RSD |
|
0.8 |
||||
Based on the above results, ruggedness with different analysts was found to comply with ICH limits and was therefore considered satisfactory.
Ruggedness – Different Equipment:
Ruggedness was evaluated using two different instruments of the same model by analyzing a standard solution of 10 µg/mL in triplicate (n = 3), following the same procedure as developed for the assay of tablets. The amount of drug and percentage label claim were calculated. The %RSD was determined for the replicate samples. The results are presented in Table 5.
Table 6: Results of Ruggedness-Different Instrument:
|
Instrument |
% Label Claim |
|||||
|
Sample-1 |
Sample-2 |
Sample-3 |
Average |
SD |
% RSD |
|
|
Equipment-1 |
99.9 |
98.9 |
97.6 |
98.8 |
1.2 |
1.2 |
|
Equipment-2 |
100.2 |
101.9 |
100.9 |
101.0 |
0.9 |
0.8 |
|
Average % RSD |
|
1.0 |
||||
Based on above results ruggedness on different instrument was found to be passed per ICH limits.
RESULTS:
|
Parameters |
Observations |
|
Linearity Range |
2 – 20 µg/mL |
|
Correlation Coefficient ® |
1.00 |
|
Slope |
0.151 |
|
Intercept |
0.000 |
|
Regression equation |
Y = 0.151x + 0.000 |
|
LOD value |
0.54 |
|
LOQ value |
1.63 |
|
Precision of Assay Samples |
% RSD was found to be within limits |
|
% Recovery |
% Recovery was found to be within limits |
|
Intraday Precision |
% RSD was found to be within limits |
|
Ruggedness-Different Analyst |
% RSD was found to be within limits |
|
Ruggedness-Different Analyst |
% RSD was found to be within limits |
CONCLUSION:
Tofacitinib citrate is a therapeutic agent used in the treatment of arthritis and ulcerative colitis. Quality control tests such as assay, dissolution, and related substances are routinely performed for various pharmaceutical formulations and stability studies, which require significant use of chemicals, solvents, and analytical instruments. Being a potent drug, tofacitinib citrate is administered at very low doses, making its handling in analytical laboratories challenging due to the need for analysis at low concentration levels. Therefore, a sensitive spectrophotometric method was developed to detect and quantify the drug at lower concentrations. The proposed method for the assay of tofacitinib citrate in tablets and topical formulations is simple, eco-friendly, cost-effective, and easy to perform. The method employs UV spectrophotometry, eliminating the need for expensive analytical instrumentation. The percentage label claim for tablet formulations was found to be within acceptable limits. Linearity was established using standard solutions, confirming that the method meets acceptance criteria. The developed and validated method was found to be specific, linear, precise, accurate, robust, and rugged, making it suitable for routine quality control analysis.
ACKNOWLEDGEMENT:
We are thankful to ANRF - Anusandhan National Research Foundation (Formerly SERB) for sanctioning the project and providing financial assistance through SERB granted project. We are thankful for the management of JNTU University for providing us instruments and laboratory to perform our study. We are thankful to Dr. CH. Shilpa Chakra (Associate Professor & Head of the Department, Centre for Nano Science and Technology, JNTUH UCEST, Hyderabad, Telangana) for supporting us and providing necessary resources.
CONFLICT OF INTEREST:
We declare no conflicts of interest.
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Received on 01.04.2026 Revised on 05.05.2026 Accepted on 06.06.2026 Published on 10.07.2026 Available online from July 25, 2026 Asian Journal of Pharmaceutical Analysis. 2026; 16(3):185-190. DOI: 10.52711/2231-5675.2026.00028 ©Asian Pharma Press All Right Reserved
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