1. INTRODUCTION:

Tofacitinib is the name of the active medicinal component. Tofacitinib is a janus kinase inhibitor, antineoplastic, and immunomodulating drug used to treat rheumatoid arthritis.¹ Chemically, it is (3R,4R).-4-Methyl-3-(methyl-7H-pyrrolo[2,3-d] pyrimidin-4-ylamino)-ß-oxo-1-piperidinepropanenitrile,2-hydroxy-1,2,3-propanetri carboxylate (1:1).

It's being researched as a treatment for psoriasis, inflammatory bowel disease, and other immunological illnesses, as well as for preventing organ transplant rejection² Tofacitinib has also been tested in clinical trials for the prevention of organ transplant rejection, and it is also being investigated for the treatment of psoriasis³ Cytokines participate within a complex regulatory network in RA, signaling through various intracellular kinase pathways to affect immune cell recruitment, activation, and function⁴ A review of the literature finds that few methods have been described for the quantification of tofacitinib using HPLC, and no Stability suggesting analytical approach has been documented for estimating Tofacitinib from Tofacitinib Citrate Extended Release tablet dosage form⁵ Figure 1 depicts the chemical structure of Tofacitinib.

Several studies have been conducted to determine the safety and efficacy of Tofacitinib. So far, only a few techniques for assessing tofacitinib in pharmaceutical dosage forms using RP-HPLC methods have been published. According to a literature review, there is no such simple RP-HPLC method for determining tofacitinib solubility in pharmaceutical dosage forms⁶ Tofacitinib, a first oral non-biologic disease-modifying anti-rheumatic drug (DMARD), is used to treat adults with moderate to severe rheumatoid arthritis as monotherapy or in combination with methotrexate or other non-biologic DMARDs. It should not be combined with biologic DMARDs or immunosuppressive drugs like azathioprine or cyclosporine⁷ Tofacitinib citrate immediate release tablet dosage form will provide valuable information that can be used to assess the drug's inherent stability under various stressed conditions, ultimately improving formulation and manufacturing process⁸ The developed method is suitable for industry and academia for quantifying tofacitinib in nano formulations in an easy and cost-effective manner. It's used to assess the entrapment efficiency, stability, and in vitro release profile of tofacitinib-loaded SLNs. The % recovery of tofacitinib in the skin matrix was calculated, and the method was successfully applied to study the dermatokinetics of tofacitinib when applied topically⁹ However, a thorough investigation revealed that the stability indicating HPLC method for quantitative determination of Tofacitinib Citrate and its related impurities in active pharmaceutical ingredient has yet to be reported. As a result, it was determined that an accurate, rapid, and specific stability indicating method for Tofacitinib Citrate's 'Related substances' was required. Dihydroimpurity is a potential process-related impurity that was also well separated from the main peak. There is no method in the literature for measuring impurities. We developed a new accurate and stability indicating RP-HPLC method for the determination of Tofacitinib¹⁰

Fig. 1: Structure of Tofacitinib

2. MATERIALS AND METHODS:

Instrument used:

The liquid chromatographic system consists of WATERS HPLC 2695 SYSTEM equipped with quaternary pumps, Photo Diode Array detector and Auto sampler integrated with Empower 2 Software. The analytes were monitored at 254 nm. Chromatographic analysis was performed on Kromosil C18 column having 4.8 mm× 150 mm i.e. and 5μm particle size.

Materials used:

API of Tofacitinib was obtained from Hetero laboratories in Hyderabad, India. The Merck Millipore technology was used to distill and purify the water. Rankem in India supplied HPLC quality methanol.

Chromatographic Conditions:

The Kromosil C18 (4.8mm x 150mm, 5m) was employed, equilibrated with mobile phase Methanol and Water in a 40:60 (v/v) ratio, and the flow rate was kept at 1.0mL/min. The detection wavelength using the UV detector was 291 NM, the injection volume was 10L, and the run time was fixed at 6.0 minutes.

Preparation of Mobile Phase:

A mixture of methanol and Trifluoroacetic in the ratio of 70:30% v/v was prepared and used. Before proceeding to analysis mobile phase was sonicated, filtered and degassed by 0.45µ membrane filter.

Preparation of Standard Stock Solution:

Tofacitinib pure medication (10mg) was weighed and transferred to a 10mL volumetric flask, followed by 10mL of mobile phase (1000g/mL concentration). Various aliquots of this stock solution are produced and injected.

Assay Procedure:

Tofacitinib bulk medication was carefully weighed and put into a 10mL volumetric flask before being made up to volume with mobile phase. For 5minutes, the solution was sonicated.

3. RESULTS AND DISCUSSION:

a. System Suitability:

Table 1 summarizes the findings from the technique validation and system appropriateness investigations.

Table 1:

S. No.	Tofacitinib
Inj	RT(min)	USP Plate Count	Tailing
1	2.272	6868	1.39
2	2.273	6618	1.39
3	2.274	6448	1.39
4	2.276	6594	1.40
5	2.280	6717	1.33
6	2.281	6950	1.32

b. Linearity:

Tofacitinib has a linear concentration range of 10-60 g/mL. Table 2 and Figure 2 indicate the outcome.

Table 2:

Sr. No	Conc (μg/mL)	Peak area
1	10	235561
2	20	476879
3	30	714265
4	40	957610
5	50	1198447
6	60	1411469

Figure 2:

c. Accuracy:

The method's accuracy was tested at three distinct concentration levels, namely 50%, 100%, and 150%, and revealed affordable %recoveries in the range of 99 - 102.20% for Tofacitinib. Table 3 displays the findings.

Table 3:

% Level	Amount Spiked (μg/mL)	Amount recovered (μg/mL)	% Recovery	Mean % Recovery
50%	20	19.91	99.57	99.73%
	20	19.82	99.10
	20	19.82	99.08
100%	40	40.04	100.10
	40	40.08	100.19
	40	39.78	99.44
150%	60	60.29	100.49
	60	60.08	100.13
	60	59.68	99.47

d. Precision:

The accuracy research was graded using the % RSD value. The %RSD was discovered to be less than 2%. Table 4 displays the results of the precision research.

Table 4:

S. No	Area of Tofacitinib
1.	954234
2.	951726
3.	955212
4.	959154
5.	954717
6.	957499
Mean	955424
S.D	2602.1
%RSD	0.3

e. Detection and quantification limits:

LOD sample Preparation:

0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flasks and made up with diluents. From the above solutions 0.3ml each of Tofacitinib, solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluents.

LOQ sample Preparation:

0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flask and made up with diluent. From the above solutions 0.9ml each of Tofacitinib, solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluent. LOD & LOQ results were shown in table no 5.

Table 5:

Sr. No.	Parameter	Value µg/mL
1	Limit of Detection	0.05
2	Limit of Quantification	0.14

f. Robustness:

Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min), mobile phase minus (50:50A), mobile phase plus (60B:40A), temperature minus (27°C) and temperature plus (33°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

The robustness of the procedure was investigated by making purposeful modifications to the chromatographic settings and observing the effects on the findings. These adjustments had no negative impact on the drug content, as seen by the low values of % relative standard deviation (less than 1%). Table 6 displays the outcomes.

Table 6:

S. No.	Condition	%RSD of Tofacitinib
1	Flow rate (-) 0.9ml/min	0.6
2	Flow rate (+) 1.1ml/min	0.2
3	Mobile phase (-) 50B:50A	0.3
4	Mobile phase (+) 60B:40A	0.8
5	Temperature (-) 27°C	0.3
6	Temperature (+) 33°C	0.6

4. CONCLUSION:

Tofacitinib in pharmaceutical dosage form is determined using a straightforward, precise, accurate, sensitive, and specific RP-HPLC technique. Tofacitinib retention time was determined to be 2.259 minutes. Tofacitinib’s RSD was discovered to be 0.5%. Tofacitinib’s RSD of Method Precision was determined to be 0.4%. Tofacitinib recovery was 99.73%. Tofacitinib regression equation yielded LOD and LOQ values of 0.05 and 0.14, respectively. Because retention durations and run times were reduced, the technique created was easy and cost-effective, and it may be used in frequent quality control tests in industries.

5. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 17.10.2023 Revised on 12.04.2024

Accepted on 19.07.2024 Published on 10.12.2024

Available online on December 30, 2024

Asian Journal of Pharmaceutical Analysis. 2024; 14(4):207-210.

DOI: 10.52711/2231-5675.2024.00037