Development and Validation of Stability Indicating RP-HPLC Method for Quantitative Estimation of Tofacitinib in Tofacitinib Tablets Dosage Form

Abstract

Aim: The main purpose of the research work is to develop an effective, sensitive, provident and uncomplicated reverse phase HPLC technique for the estimation of Tofacitinib in Tofacitinib tablets dosage form. Study Design: HPLC based grounded quantification studies. Methodology: Estimation of Tofacitinib in Tofacitinib tablet formulation. The separation was set up by using a stationary phase Kromasil C18 (150 x 4.6 mm, 5µ) and the mobile phase comported of pH 4.0 phosphate buffer and acetonitrile in the rate of (80:20 volume/volume). The flow rate was 1.5 mL/min. Tofacitinib was detected using UV sensor at the wavelength of 215 nm. composed of pH 4.0 phosphate buffer and acetonitrile in the rate of (80:20 volume/volume). The flux rate was 1.5 mL/min. Column temperature 25°C and sample temperature 25°C and injection volume 20µL, performance time was 20 moment. Results And Conclusion: The citified technique was validated for numerous parameter as per ICH guidelines like accuracy, precision, linearity, specificity, system suitability, result stability and robustness. The results attained were within the undertaking criteria. So, it can be concluded that the formulated system is easy, specific, cost-efficient, sustainable, and safe and can be successfully employed for the procedure analysis of Tofacitinib in tab and pharmaceutical dosage forms.

Keywords

Tofacitinib; liquid chromatography; forced degradation; validation.

Introduction

Drug Profile

Figure 2. Chemical Structure of Tofacitinib

Molecular Formula :  C16H20N6O

Molecular Weight   : 312.37g/mol

Chemical Name       : 3-((3R, 4R)-4-methyl-3-(methyl (7H-pyrrolo [2, 3-d] pyrimidin-4-yl) amino) piperidin-1-yl)-3-oxopropanenitril

Melting Point           :  198-202° C

Solubility Category: N, N-Dimethylacetamide

Pka: 5.2

Review of Literature for Tofacitinib

A comprehensive review of literature has been performed to determine the availability of analytical reports for determining Tofacitinib in pharmaceutical and biological matrices. The detailed literature survey for cited analytes is as follows

Analysis of Tofacitinib using HPLC includes;

Kaleswararao et. al; A reversed-phase RP-HPLC method for Tofacitinib was developed using a C18 column (150 x 4.6 mm), 3 μm as the stationary phase. The mobile phase comprised 0.4 % perchloric acid and acetonitrile in the ratio of 85:15 % v/v. The mobile phase flow rate was maintained at 1.2 mL/min, with the temperature set at ambient level. The column temperature was maintained at 40ºC to get early elution of analyte and sample cooler temperature at 10 ºC. Texwipe swabs with part number TX761D have been selected as swab sticks for sample collection as the risk of fibre interference is absent. [33]

Sankar as et. al; A novel RP-HPLC method has been developed and validated for the estimation of Tofacitinib in its tablet dosage form. The analysis was performed using an isocratic system on a C18 column with the dimensions of (150 mm X 4.6 mm) and 5 micron particles at ambient temperature. . The mobile phase consisted of methanol: water in a ratio of 90:10% (v/v), with a flow rate maintained at 0.8 ml/min. Detection was conducted at a wavelength of 285.9 nm. Validation of the method was conducted in accordance with ICH guidelines, covering system suitability, linearity, accuracy, and precision. The linear range for TDF was determined to be 50-150 µg/mL, with accuracy and precision well within acceptable limits. [34]

Sivaprasadu et. al; A new method was developed for the estimation and validation of TFC in its pure form and pharmaceutical dosage form using RP-HPLC. mobile phase composed of phosphate buffer (pH 7.0) and acetonitrile (45:55, %v/v) at a flow rate of 0.5 mL/min under isocratic elution. Analytes were monitored via a UV detector at 210 nm and with the column oven temperature at 30°C for a 20-min analysis. . Precision (%RSD) for Impurity-A and Impurity-B and tofacitinib met specifications at 2.4%, 0.8%, and 0.0%, respectively. Accuracy ranged from 86% to 100% for impurities, with LOD at 0.03% and LOQ at 0.05%–0.06%. Correlation coefficients exceeded 0.999 for impurities and tofacitinib citrate. Solution stability was confirmed for 24 h at room temperature . Validation of the method was performed following various parameterssuch as accuracy, precision, linearity, and specificity according to the ICH guidelines.[35]

Ozbay et. al; An analytical method was developed for Tofacitinib and one of its impurities, requiring synthesis and characterization of the impurity. s. The newly developed method exhibits simplicity, specificity, precision, and sensitivity. Experimental procedures utilized a Shimadzu Prominence 20A HPLC system equipped with a Inertsil ODS 3V C18 column (5µm particle size, 4.6 X 250 mm dimensions). The mobile phase, consisting of 0.05M ammonium acetate buffer at pH 5.0 and acetonitrile (65:35 v/v) in isocratic mode with a flow rate of 1.0 mL/min, facilitated accurate detection of the Tofacitinib peak at 230 nm wavelength. Comprehensive validation, including assessments of linearity, accuracy, precision, and robustness, was conducted in accordance with ICH requirements.

 Aim

The aim of this research is to develop and validate a rapid, simple, To Develop Stability indicating Method for determination of Tofacitinib in Tofacitinib tablet dosage Form by using RP HPLC and UV Spectroscopy.

OBJECTIVE

• Explore the accessibility of standard and commercially available preparations of Tofacitinib.
• Review of literature survey
• Preliminary investigation using AT-IR, and UV-Spectroscopy
• Selection of solvent for dissolving the Tofacitnib .
• To Develop Stability indicating Method for determination of Tofacitinib Form its Multiple dosage Form by using RP HPLC / UV PDA Detector.
• Validation of developed methods as per ICH Q2(R1) guideline
• Application of developed methods for determination of Tofacitinib in their respective marketed preparation.

Plan of work

The main intention of the research work is to develop and effective, sensitive, economical and reverse phase HPLC technique for the estimation of Tofacitinib in Tofacitinib tablets dosage form.

Mobile Phase ( Buffer)

Instead of various buffer we can uses acidic acid,formic acid Phosphate buffer, ammonium acetate.

Forced Degradation Study :- A forced degradation study (FDS) in HPLC (High-Performance Liquid Chromatography) is crucial for drug development, intentionally stressing a drug substance (API) with acid, base, heat, light, and oxidation to generate impurities, proving the HPLC method is stability-indicating, meaning it separates API from degradants, identifying potential degradation pathways, and ensuring method specificity for regulatory submission. The primary purpose is to help develop and validate a stability-indicating method using High-Performance Liquid Chromatography (HPLC) that can accurately separate, detect, and quantify the drug substance and its various degradation products.

Method For RP-HPLC

• Chemical and Reagent
• Instrumentation
• preaparation of pH 4.0 phosphate buffer solution
• preparation of mobile phase
• preparation of diluent
• preparation of standard solution
• preparation of test solution (for Tofacitinib tablet 5.0mg)

Development of Method

• Selection of solvent
• Selection of chromatographic layer and detection wavelength
• Preparation of stock standard solution
• Selection of stationary phase
• Selection and optimization of mobile phase
• Optimization of chromatographic conditions
• Testing of system suitability parameters

Validation of Method

• Accuracy (% recovery)
• Precision studies (Intra-day, Inter-day, and Repeatability)
• Sensitivity studies (DL & QL)
• Robustness
• Specificity and selectivity

Methodology

Development and Validation Method

RP- HPLC Method

1. 1. Chemicals and Reagents

Analytical-grade Potassium dihydrogen phosphate, Orthophosphoric acid, Acetonitrile, Hydrochloric acid, Sodium hydroxide, Hydrogen peroxide and water, reagents and chemicals.

1. 2. Instrumentation

Agilent HPLC model:1260 with DAD, Bandelin ultrasonic bath, pH Meter (Thermo Orion Model) and Analytical Balance (Metller Toledo Model) were used in the present assay.

1. 3. Preparation of pH 4.0 phosphate buffer solution

Weighed accurately 1.36 g of potassium dihydrogen orthophosphate into 1000 mL of water sonicated to dissolve and mix well then pH was adjusted to 4.0 with ortho phosphoric acid solution. Filtered through 0.45 µm membrane filter.

1. 4. Preparation of mobile phase

Prepared a mixture of pH 4.0 phosphate buffer solution and acetonitrile in the proportion of 80:20 (%volume/volume) mixed well and sonicated to degas.

1. 5. Preparation of diluent

Prepared a mixture of irrigate and cyanomethane in the proportion of 50:50 (%volume/volume) mixed well and sonicated to degas.

1. 6. Preparation of standard solution

Weighed correctly and transferred 25.7 mg of Tofacitinib working standard into a 50 mL volumetric thermos sonicated for 2 minutes to dissolve the contents and made upto the quantity with diluent. Further diluted this solution 5 mL in to 50 mL volumetric thermos and made up the quantity with diluent and mixed well. (The concentration of the standard solution containing, Tofacitinib 50 ppm)

1. 7. Preparation of test solution (for Tofacitinib tablets 5.0 mg)

Weighed accurate 10 tablets (Tofacitinib tablets 5 mg) and transferred (equivalent to 50 mg of Tofacitinib) into 200 mL volumetric flask, added 120 mL of diluent and sonicated for 30 minutes, with intermediate shaking, cool to room temperature and diluted to quantity with diluent and mixed well. Filtered the solution through 0.45 µm PVDF syringe filter. Further diluted 5.0 mL of the filtrate solution in to 25 mL volumetric thermos, diluted to quantity with diluent, and mixed well. (The concentration of the solution contains 50.0 µg/mL of Tofacitinib).

1. 8. Preparation of test solution (for Tofacitinib tablets 10.0 mg)

Weighed correctly 5 tablets (Tofacitinib tablets 10 mg) and transferred (equivalent to 50 mg of Tofacitinib) into 200 mL volumetric thermos, added 120 mL of diluent and sonicated for 30 minutes, with intermediate shaking, cool to room temperature and diluted to quantity with diluent and mixed well. Filtered the solution through 0.45 µm PVDF needle filter. Further diluted 5.0 mL of the filtrate solution in to 25 mL volumetric thermos, diluted to quantity with diluent, and mixed well. (The concentration of the solution contains 50.0 µg/mL of Tofacitinib).

1. 9. Preparation of placebo solution

Weighed correctly and transferred placebo powder (equivalent to 50 mg of Tofacitinib) into 200 mL volumetric thermos, added 120 mL of diluent and sonicated for 30 minutes, with intermediate shaking, cool to room temperature and diluted to measure with diluent and merge well. Filtered the solution through 0.45 µm PVDF needle filter. Further diluted 5.0 mL of the filtrate solution into 25 mL volumetric thermos, diluted to quantity with diluent and mixed well. (The concentration of the solution contains 50.0 µg/mL of Tofacitinib).

1. 10. Chromatographic conditions

Chromatographic analysis was executed on Kromasil C18 (150 x 4.6 mm, 5µ) mobile phase consisting of pH 4.0 phosphate buffer and acetonitrile in the proportion of (80:20 volume/volume). The run velocity was 1.5 mL/min, the column oven temperature was 25°C and the sampler cooler temperature was 25°C, the inoculation volume was 20 μL, and detection was implemented at 215 nm using a photodiode array detector (PDA).

Method Development

To develop a appropriate and robust HPLC technique for the determination of Tofacitinib in Tofacitinib in tablets dosage form, different mobile phases were employed to achieve a good peak shape. The technique development was started with Hypersil BDS C18 (150x4.6mm, 5µm) with the following different mobile phase compositions like 0.1% orthophosphoric acid buffer and acetonitrile in the proportion of 85:15 volume/volume. It was observed that when Tofacitinib was injected, higher retention time and zenith tailing were not acceptable. The column stationary phase was not suitable for the component. For the subsequent trial change the column from Hypersil BDS to Kromasil C18. The compound Tofacitinib was eluted at blank volume, and the peak shape was inadequate.

For the subsequent trial the mobile segment consisted of pH 4.0 phosphate buffer and acetonitrile in the proportion of 80:20 volume/volume respectively, flow rate 1.5 mL/min, column temperature 25°C and sampler cooler maintained 25°C. UV detection was performed at 215nm. The compound Tofacitinib was eluted at 10.30 minutes and the peak shape was establish to be good. The chromatogram of Tofacitinib standard using the proposed method . System suitability results of the technique are presented.

Validation Method

1. System Precision

The standard solution was organized as per the test technique, infused into the HPLC system six times, and evaluated the % RSD for the vicinity responses.The data were shown in Table 3.The relative standard deviation of six replicates standard solution consequences were establish to be within the specification limit i.e.0.16%.

2. Linearity of Detector Response

The linearity of an analytical technique is its ability to obtain test results which has a definite mathematical relation to the concentration of the analyte. The linearity of response for Tofacitinib was determined in the range of 50% to 150% (24.88-74.64 µg/mL for Tofacitinib). The calibration curve of the analytical technique was assessed by plotting concentration versus peak area and represented graphically. The correlation coefficient [r2] was initiate to be 1.000. Therefore, the HPLC technique was established to be a linear standard curve that was calculated and to demonstrate the linearity of the present technique. From the data obtained which is given in Table 5 and the technique was established to be linear within the proposed range.

3. Accuracy

The accuracy of the test technique was demonstrated by preparing recovery samples of Tofacitinib at 50% to 150% of the target concentration level. The recovery samples were organized in triplicate arrangements on Tofacitinib API spiked to placebo and analyzed as per the recommend technique for apiece attentiveness level except 50% and 150%. The above samples were chromatographed, and the percentage recovery of apiece sample was calculated for the amount added. Evaluated the exactitude of the recovery at apiece level by computing the relative standard deviation of six preparations for 50% and 150% level recovery samples consequences. The data obtained which given in Table 6 and the technique was established to be accurate.

4. Robustness Studies

To authenticate the technique robustness the chromatographic performance at distorted circumstances was assessed evaluated to the ostensible conditions of the technique. The standard solution was infused at each of the following altered circumstances. The technique is robust for altered like flow rate, column oven temperature, pH variation, and the organic phase of the mobile phase.

5. Filter Validation

Executed the filter validation for sample solution, one piece of the solution was centrifuged, and the other piece of the solution was filtered through 0.45 µm PVDF and 0.45 µm Nylon filters. Filter validation parameters were established. Based on the above consequences and observations 0.45 µm PVDF filterers are suitable for filtration.

DISCUSSION

RP-HPLC technique for inference of Tofacitinib in Tofacitinib tablets dosage form was urbanized and authenticated as per ICH instructions. A simple, accurate and reproducible reverse phase HPLC technique was urbanized for the connection of Tofacitinib in Tofacitinib tablets dosage form. The optimized technique consists of a mobile segment consisting of pH 4.0 phosphate buffer and acetonitrile in the proportion of (80:20 volume/volume) with Kromasil C18 (150 x 4.6mm, 5µ) column. The retention time of Tofacitinib was established to be 10.30 minutes. The urbanized technique was authenticated as per ICH Q2A (R1) guideline.

CONCLUSION

The urbanized technique was authenticated for various parameters as per ICH guidelines like accuracy, precision, linearity, specificity, system suitability, solution stability and robustness. The result obtained were within the acceptance criteria. So, it can be concluded that the urbanized technique is simple, precise, cost-effective, eco-friendly, and safe and can be successfully employed for the regular analysis of Tofacitinib in bulk and pharmaceutical dosage forms.

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