Tofacitinib is chemically known as 3-[(3R,4R)-4-methyl-3- [methyl (7H-Pyrrolo [2, pyrimidine-4yl) amino] piperidin-1-yl]-3-oxopropanenitrile. It is an oral Janus kinase inhibitor for the treatment of rheumatoid arthritis . Cytokines work within a complex regulatory network in RA, signalling through different intracellular kinase pathways to modulate the recruitment, activation, and function of immune cells and other leukocytes [2-6]. Several research works elucidated the safety and efficacy of Tofacitinib drug [7-14]. The chemical structure of Tofacitinib was displayed in Figure 1.
The research review demonstrates that not one of the major pharmacopoeias such as USP, EP, JP, and BP reported any LC methods. Only a limited number of techniques have been published too far for the tofacitinib assessment in pharmaceutical dosage forms employing RP-HPLC methods [15–17] and HPTLC .
According to a research review, there is no such straightforward RP-HPLC method for analysing tofacitinib's solubility in pharmaceutical dosage forms. Therefore, we attempted to create a straightforward HPLC method for tofacitinib in the dose form of tofacitinib tablets. According to the ICH guidelines, a straightforward HPLC approach is described in the current work for the dissolution investigation of tofacitinib in tofacitinib tablet dosage form [19, 20].
Materials and Methods
Chemicals and reagents
Working standard for tofacitinib (Clearsynth, Hyderabad, India), the mentioned formulation was acquired on the neighbourhood market. Hydrochloric acid and orthophosphoric acid (Merck, Bombay, India), acetonitrile (J.T. Baker, USA), triethylamine, and ultra-pure water (Milli-Q system, Millipore, Bedford, MA, USA) were utilized in this stduy. In addition, analytical and HPLC-grade chemicals and solvents were used in this investigation.
For analysis, an Agilent 1260 high-performance liquid Chromatographic system with an auto sampler and PDA detector was deployed. OpenLab software was utilized to record the data. We adopted a pH metre (Thermo Orion Model), Bandelin ultrasonic bath, dissolution (Make: Electro lab), and an analytical balance (Mettler Toledo Model).
Waters X-Bridge shield RP-18 (150 × 4.6 mm, 5) was subjected to chromatographic analysis. The mobile phase was composed of acetonitrile and pH 3.5 triethylamine buffer in an 85:15 v/v ratio. The injection volume was 20 µL, the flow rate was 1.0 mL/min, the column oven and sampler cooler temperatures were ambient, and detection was carried out at 215 nm using a photodiode array detector (PDA).
Preparation of buffer solution
1.0 mL of triethylamine was transferred into 1000 mL of water sonicated to dissolve and mixed well, and then pH was adjusted to 3.5 with ortho phosphoric acid solution. After that, it was filtered through 0.45 µm membrane filter.
Preparation of mobile phase
An 85:15 (% volume/volume) mixture of pH 3.5 Triethylamine buffer solution and acetonitrile were produced, which was well mixed and sonicated to remove gas.
The dissolution system (n=6) and dissolution USP apparatus Type - I were used to conduct the dissolution test for the tofacitinib formulation (Basket). 0.1N Hydrochloric acid was the medium. Dissolution media was poured into a 900 mL container, the bath was kept at 37 °C, and the basket's speed was set at 100 RPM. The sample was taken out in aliquots and filtered using a 0.45 m PVDF membrane filter for 30 minutes.
Preparation of dissolution medium (0.1 N Hydrochloric acid)
8.9 mL of concentrated Hydrochloric acid was transferred into 1000 mL volumetric flask, containing 500 mL of water, made up to the volume with water and mixed well.
Preparation of diluent
The dissolution medium (0.1 N Hydrochloric acid) was used as a diluent.
Preparation of standard solution
Tofacitinib working standard was accurately weighed and deposited into a 100 mL volumetric flask, and then 50 mL of diluent was added, the contents were sonicated for two minutes to dissolve them, and the remaining diluent was used to make up the volume. The solution was further diluted by adding 4 mL to a 100 mL volumetric flask, making up the volume with the diluent, and thoroughly mixing it (the volume of the standard solution contains 11.2/g of tofacitinib).
Preparation of test solution
In each of six dissolution tubes, which contain 900 mL of dissolving medium that has been adjusted to 37 °C ± 0.5 °C, one tablet was put and there should be no air bubbles on the tablet's surface before starting the device. At the allotted time, 15 mL of the sample solution was removed from each dissolution vessel, the sample was kept at least 1 cm from the vessel wall and in a region that is halfway between the medium's surface and the top of the revolving paddle. Next, it was filtered through a 0.45 m PVDF syringe filter; the first 2 mL of filtrate were then discarded and the remaining solution was injected straight into an HPLC column. Tofacitinib was present in the sample solution at a concentration of 11.1 g/mL.
Preparation of placebo solution
900 mL of dissolution media that has been equilibrated to 37±0.5 °C has been placed in each of the six dissolving vessels after being precisely weighed and divided into placebo powder (equal to 10 mg of tofacitinib). There should be no air bubbles on the tablet's surface before starting the device. After the allotted time has passed, 15 mL of sample solution from each dissolving vessel should be collected. Samples should be taken out of areas that are at least 1 cm from the vessel wall and halfway between the medium's surface and the top of the revolving paddle, and then it was filtered through a 0.45 m PVDF syringe filter and the first 2 mL of filtrate were discarded. Thereafter, the remaining solution was injected straight into an HPLC column.
The maximum UV absorbance (max) of tofacitinib drug material was demonstrated at 215 nm when its standard solution of 10 g/mL was scanned between 200 and 400 nm.
Mobile phase made up of various solvent compositions were tested to achieve the best separation to produce a suitable and reliable HPLC method for the tofacitinib assessment in the dosage form of tofacitinib tablets. It was attempted using a mobile phase composed of Triethylamine buffer, pH-adjusted to 3.5, Acetonitrile in the ratios of 50:50, 65:35, 75:25, and 85:15 % v/v. When tofacitinib was injected, increased retention times and unsatisfactory peak tailing were seen, and the tofacitinib peak was not properly eluted in the ratios of 50:50 % v/v and 65:35 % v/v. The peak form was good for the following trial, but there was some small tailing. The mobile phase is 75:25 v/v. The mobile phase for the subsequent attempt is composed of 85:15 % v/v. Tofacitinib was eluted to produce a nice peak and meet the requirements for system appropriateness. Figure 2 depicts the chromatogram for the Tofacitinib standard. Table 1 presents the method's results for system appropriateness.
Results and Discussion
Specificity: Blank and placebo interference
The blank, placebo solution, standard, and sample solution were prepared and analysed as per method. Blank and placebo were injected in the above mentioned chromatographic conditions and the blank and placebo chromatograms were recorded. Chromatogram of blank solution in Figure 3 showed no peak at the retention time of Tofacitinib peak. This indicates that the blank solution used in standard and sample preparation do not interfere in estimation of Tofacitinib in Tofacitinib tablets formulation. Similarly, chromatogram of placebo solution in Figure 4 showed no peaks at the retention time of Tofacitinib peak. This indicates that the placebo used in sample preparation do not interfere in estimation of Tofacitinib in Tofacitinib tablets formulation (Figure 5). The observations are listed in Table 2.
Figure 5: Typical chromatogram sample
System precision was demonstrated by preparing standard solution as per method and injected the same into HPLC system in six replicate injections of standard solution. The area of analyte peak was recorded for these standard injections. The system precision was evaluated by computing the % relative SD for the peak area of these standard readings. The observations are provided in Table 3.
The six replicate standard solutions' relative standard deviation was found to be less than the specified limit, or 0.07%.
Method precision was demonstrated by performing dissolution for six units as per method. The % drug dissolved of each unit was quantified for the sample. The method precision was evaluated by calculate the individual, mean % drug dissolved, and % relative SD for each set of samples. The results of the precision study are listed in Table 4.
The method precision was established as mentioned in the developed method and the results are found satisfactory. The % relative SD of 6 units is 0.26%.
Intermediate precision of the method was demonstrated by carrying out method precision study in six preparations of a same sample. Representing a single batch by two different analysts on different days. These samples were prepared as per the method.
The method precision was evaluated by calculate the individual, mean % drug dissolved, % relative SD for each set of samples and overall mean % drug dissolved, % relative SD for both method precision and intermediate precision. The results of the precision study are presented in Tables 5 and 6.
By creating solutions with concentrations ranging from 20% to 150% of the usual concentration level, the linearity of area for tofacitinib was demonstrated. The peak area of these solutions was recorded when they were added to the HPLC apparatus. It was done to plot concentration versus area response. The relationship between concentration and area response co-efficient of determination was assessed, as depicted in Figure 6. The observations are listed in Table 7.
By creating recovery samples of tofacitinib at 20%, 100%, and 150% of the intended stock sample concentration level, the test method's accuracy was put to the test. For each concentration level, the recovery samples were made in three copies. The % recovery of each sample was computed for the amount added after the afore mentioned samples were recorded.
By calculating the relative standard deviation of the findings of the three recovery samples, we evaluated the recovery accuracy at each level. The procedure and data collected, which are shown in Table 8, were judged to be accurate.
The standard and sample solutions for solution stability were created under a variety of circumstances, including bench top at room temperature and in a refrigerator at 2 to 8 °C. By contrasting initially prepared standard and sample solutions with recently prepared standard solutions, the stability of standard and sample solutions was ascertained. Tables 9 and 10 include tabular lists of the observations. Solution stability parameter was established, and the standard and sample solutions are stable up to 48 hours at bench top and in refrigerator (2-8 °C) conditions.
Filter validation for the sample solution was carried out by centrifuging one portion of the solution and filtering the remaining portion through 0.45 m PVDF and 0.45 m Nylon filters. The observations are listed in Table 11.
Filter validation parameter was established. Based on the above results and observations, 0.45 µm PVDF and 0.45 µm Nylon filterers are suitable for filtration.
The robustness of the approach was evaluated by making small adjustments to the chromatographic and dissolving settings, such as the mobile phase ratio, buffer pH, flow rate, temperature, medium volume, and rpm. The following altered conditions each received an injection of standard solution. The observations are listed in Table 12.
Method is robust for changes like flow rate, column oven temperature, pH variation, organic phase of mobile phase, volume of medium, and rpm.
The technique created for the precise measurement of tofacitinib in the sample after dissolution. Tofacitinib could be completely dissolved after 30 minutes when used with a USP Type-I apparatus (Basket) spinning at 100 rpm in 900 mL of 0.1N hydrochloric acid. This procedure was validated for a number of parameters in accordance with the ICH guidelines, including accuracy, precision, linearity, specificity, system suitability, solution stability, filter study, and robustness. The outcomes met the standards for acceptance. The new approach can therefore be successfully used for the routine analysis of tofacitinib in bulk and pharmaceutical dose forms because it is easy to use, precise, affordable, eco-friendly, and safe.
The authors are grateful to the Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur and Andhra Pradesh, India, for providing the facilities to carry out this research work.
No potential conflict of interest was reported by the authors.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.
Narapereddy Krishna Prasad
Alladi Devi Sravanthi
HOW TO CITE THIS ARTICLE
Narapereddy Krishna Prasad, Alladi Devi Sravanthi. Tofacitinib in Pharmaceutical Solid Dosage from Dissolution Study: Development and Validation of RP-HPLC Method. J. Med. Chem. Sci., 2023, 6(9) 2218-2227