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1 Impact factor: 3.958/ICV: 4.10 ISSN: Pharma Science Monitor 8(2), Apr-Jun 2017 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: DEVELOPMENT AND VALIDATION OFUVSPECTROPHOTOMETRIC METHOD FOR THE ESTIMATION OF TOFACITINIB CITRATE N.V. Thakariya*, S.B. Ezhava Department of Quality Assurance, L. M. College of Pharmacy, Navrangpura, Ahmedabad , Gujarat, India. ABSTRACT Tofacitinib is a drug of the Janus kinase (JAK) inhibitor class, It is currently approved for the treatment of rheumatoid arthritis (RA) in the United States and other countries. A simple, sensitive and economical UV spectrophotometric method has been developed for the estimation of Tofacitinib Citrate in bulk drug and laboratory prepared mixture. The proposed method obeyed Beer s law in the concentration range of 5-25 µg/ml with correlation coefficientr= Accuracy was confirmed by recovery studies and mean recovery was found to be ±0.65. The intraday and inter day precision were found to be within limits.the proposed methods have adequate specificity, sensitivity and reproducibility for quality control assay of Tofacitinib Citrate in bulk and laboratory prepared mixture. KEYWORDS: Tofacitinib Citrate, UV Spectrophotometric method, Analytical method validation. INTRODUCTION Chemically, Tofacitinib Citrate is 2-hydroxypropane-1,2,3-tricarboxylic acid; 3-[(3R,4R)-4- methyl-3-[methyl({7h-pyrrolo[2,3-d]pyrimidin-4l})amino]piperidin-1-yl]-3-xopropanenitrile[1]. It is a drug of the janus kinase (JAK) inhibitor class. It is currently approved for the treatment of rheumatoid arthritis (RA) in the United States and other countries. Besides rheumatoid arthritis, Tofacitinib has also been studied in clinical trials for the prevention of organ transplant rejection, and is currently under investigation for the treatment of psoriasis. Known adverse effects include nausea and headache as well as more serious immunologic and hematological adverse effects[1-2].
2 Impact factor: 3.958/ICV: 4.10 ISSN: Figure 1: Tofacitinib Citrate A survey of literature revealed that RP- HPLC[3,5,7] and LC-MS-MS[4,6,8,9], methods have been reported for determination of Tofacitinib in rat plasma, bulk and its dosage forms. However, there was no method reported for Tofacitinib Citrate by UV method till date.so, the aim of work was to develop and validate an analytical method by UV-Visible Spectrophotometer for the estimation of Tofacitinib Citrate. In present study, simple, economical, accurate, reproducible analytical method with better detection range for estimation of Tofacitinib Citrate were developed and validatedd as per the ICH guideline Q2(R1).To apply the above method for estimation of Tofacitinib Citrate in its synthetic mixture. MATERIALS AND METHOD Instruments and Reagents A double beam UV 1700 Pharmaspec (Shimadzu, Japan) UV-Visible spectrophotometer having two matched quartz cells with 1cm light path was employed for spectral measurement. Shimadzu libror AEG-220 balance andfrontline FS-4 sonicator were used for weighing and sonication, respectively. Tofacitinib Citrate API was gifted by Pharm ACE Research Laboratory, Ahmedabad, Gujarat, India. Methanol used was AR grade and purchased from CDH Chemicals, New Delhi, India. Preparation of Standard Stock solution Standard solution: 10 mg of the drug (Tofacitinib Citrate ) was accurately weighed and transferred into 10 ml volumetric flask containing 5 ml methanol. It was sonicated and volume was made upto the mark with methanol (1000μg/mL) (S1) Working solutions: 1 ml was taken from S1 and was transferred to a 10 mlvolumetric flask and the volume made up to the mark with methanol (S2 = 100 μg/ml).
3 Impact factor: 3.958/ICV: 4.10 ISSN: Aliquots of 0.5, 1, 1.5, 2, 2.5 ml from the working solution (S2) were pipetted out in the series and transferred to 10 ml volumetric flasks and made upto the mark with methanol to get 5μg/mL, 10μg/mL, 15μg/mL, 20μg/mL and 25μg/mL concentrations. Preparation of synthetic mixture: 5mg of the drug (Tofacitinib Citrate) was accurately weighed and mixed with the excipients as croscarmellose sodium 10 mg, HPMC (20 mg),lactose monohydrate (150 mg),peg 3350 (25 mg), magnesium stearete (10 mg), microcrystalline cellulose(20 mg) and titanium dioxide (5 mg) and the final synthetic mixture was mixed uniformly. Preparation of solution: The whole synthetic mixture was taken in 50 ml volumetric flask and diluted with methanol. Than it was sonicated for 10 min and volume was made up to mark with methanol. This solution was filtered and the filtrate used for further dilution. From this filtrate 1.5mL solution is pipetted out and transferred into 10mL volumetric flask and diluted with methanol up to the mark. UV Spectrophotometric method The absorption spectra of standard and test solutions of Tofacitinib Citrate were recorded in the range of nm. It showed max at 287 nm (Figure 2). Hence 287 nm wave length was selected to measure absorbance of Tofacitinib Citrate solutions. Figure 2: Spectrum of Tofacitinib CItrate(10 μg/ml) in methanol
4 Impact factor: 3.958/ICV: 4.10 ISSN: METHOD VALIDATION The developed UV Spectrophotometric method were validated for linearity range, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ) parameter as per ICH guideline Q2 (R1). Linearity The linearity of analytical method is its ability to obtain test results which are directly proportional to the concentration (amount) of analyte i n the sample within given range. The linearity of developed method wasdetermined at five concentration levels ranging from 5-25μg/mL. Working standard solution of Tofacitinib Citrate were prepared by suitable dilution of the standard stock solution with methanol to obtain concentration of Tofacitinib Citrate in the range of 5-25μg/mL. The calibration curve was constructed by plotting concentration of Tofacitinib Citrate (µg/ml) versus absorbance. Precision The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. Intraday precision was performed by analyzing three different concentrations of Tofacitinib Citrate (5 µg/ml,15 µg/ml,25 µg/ml) three times in a day and calculate percentage relative standard deviation (% RSD). Inter day precision was performed by analyzing three different concentrations of Tofacitinib Citrate (5 µg/ml, 15 µg/ml, 25 µg/ml) three times in different day and calculate percentage relative standard deviation (% RSD). Repeatability was performed by analyzing Tofacitinib Citrate solution (15 µg/ml) six times and calculate % RSD. Limit of Detection (LOD) and Limit of Quantitation (LOQ): The lowest amount of analyte in a sample that can be detected, but not necessary quantify under the stated experimental conditions is known as LOD while the minimum amount of analyte in a sample that can be quantitated with suitable precision and accuracy is known as LOQ. The LOD and LOQ values were calculated by using calibration curve. The LOD and LOQ are calculated by using formula given in the ICH guideline Q2(R1). LOD = (3.3 σ) /S and LOQ = (10 σ) /S Where σ = Standard deviation of the Y intercept S = Slope of the calibration curve equation
5 Impact factor: 3.958/ICV: 4.10 ISSN: Accuracy The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. To examine the accuracy of the developed method, recovery studies were carried out by standard addition method at three different concentration levels (80%, 100% 120%) in triplicate by spiking standard Tofacitinib Citrate solution in placebo solution. The accuracy of the method was determined by calculating % recovery of Tofacitinib Citrate by spiking in placebo. RESULTS AND DISCUSSIONS The goal of the present study was to develop rapid, precise, accurate and economic UV spectrophotometer method for the analysis of Tofacitinib Citrate in bulk and synthetic mixtureand validate developed method as per ICH guideline Q2(R1). As Tofacitinib Citrate showed wavelength maxima at 287 nm; 287 nm wave length was selected to measure absorbance of Tofacitinib Citrate solutions in method. Linearity Linear relationship in the concentration range of 5-25 µg/ml. The regression equation was found as y = 0.032x with correlation coefficient (figure-3). 1.5 Linearity of Tofacitinib Citrate Absorbance y = 0.032x R² = Concentration (µg/ml) Figure 3: Calibration curve of Tofacitinib Citrate LOD & LOQ The calculated LOD and LOQ values were found to be µg/mL and µg/ml. These method were validated for intraday and inter day precision. Precision
6 Impact factor: 3.958/ICV: 4.10 ISSN: The relative standard deviation (RSD) for intraday and inter day precision were found to be % and % respectively indicating good precision. Repeatability was found to be 0.34% respectively. Table 1: Intraday and Inter day precision Concentration Intraday precision Inter day precision (µg/ml) Mean Abs±S.D. %RSD Mean Abs±S.D. %RSD (n=3) (n=3) ± ± ± ± ± ± Accuracy The accuracy of the developed method, recovery studies were carried out at three different concentration levels in triplicate by spiking standard Tofacitinib Citrate solution in placebo solution which was made up of commonly used tablet excipients and the results obtained are given in Table 2. The mean percent recoveries obtained as ±0.65%, respectively indicate good accuracy of the method. Table 2: Result of Accuracy study Sr. No Level (%) Amount spiked in placebo (μg/ml) Amount of Tofacitinib Citrate recovered (μg/ml) Mean±S.D. % Recovery Mean±S.D ± ± ± ± ± ±0.05
7 Impact factor: 3.958/ICV: 4.10 ISSN: Assay The proposed method was successfully applied for the analysis of Tofacitinib Citrate synthetic mixture (5 mg/mixture) and the result obtained are given in Table 3. The average drug content was found to be ±0.75% respectively. Table 3:Estimation of Tofacitinib Citrate in synthetic mixture by UV method Formulation Amount Amount %Assay(n=3) claimed(mg) obtained(mg) Mean±SD (n=3) Mean±SD Tofacitinib synthetic mixture Citrate 5mg/mixture 5.016± ±0.75 CONCLUSION The results of the study showed that the proposed UV method are simple, rapid, precise, accurate, specific and sensitive for estimation of Tofacitinib Citrate without any interference from excipients. ACKNOWLEDGEMENTS Authors are thankful to the Principal, L.M. College of Pharmacy, Ahmedabad, Gujarat, India for providing the necessary facilities. REFERENCES Sampath Kumar Reddy Govind, Nagaraju Ch. V. S., Rajan S. T., EshwaraiahS.,Kishore M. and Chakravarthy I. E. Stability indicating HPLC method for the quantification of Tofacitinib citrate and its related substances. Der Pharma Chemica, 2014, 6(2), Ali S. Abdelhameed, Mohamed W. Attwa and Adnan A. Kadi An LC-MS/MS method for rapid and sensitive high-throughput simultaneous determination of various protein kinase inhibitors in human plasma. Biomed Chromatogr, 2016 Jul Vijay Kumar S, VinayDhiman, Kalpesh Kumar Giri, KuldeepSharma,MohdZainuddin and Ramesh Mullangi Development and validation of a RP-HPLC method for the quantitation of Tofacitinib in rat plasma and its application to a pharmacokinetic study. Biomed Chromatogr, 2015Sep,29(9),
8 Impact factor: 3.958/ICV: 4.10 ISSN: Kuldeep Sharma, KalpeshkumarGiri, VinayDhiman, AbhishekDixit,MohdZainuddin and Ramesh Mullangi A validated LC-MS/MS assay for simultaneous quantification of methotrexate and Tofacitinib in rat plasma: application to a pharmacokinetic study. Biomed Chromatogr, 2015May,29(5), Na-na Wang, Dao-lin Zhang and Xin-hui Jiang Detection of Xeljanz Enantiomers in Diethyl Amine Active Pharmaceutical Ingredients and Tablets. Chirality2015, 27, Karen J. Klamerus, Christine Alvey, Lei Li, Bo Feng, Rong Wang, Irina Kaplan, Haihong Shi, Martin E. Dowty and SriramKrishnaswami Evaluation of the Potential Interaction Between Tofacitinib and Drugs That Undergo Renal Tubular Secretion Using Metformin, an In Vivo Marker of Renal Organic Cation Transporter Nov;3(6): SujathaMenon, Richard Riese, Ronnie Wang, Christine W Alvey, Haihong Shi, Wendy Petit and SriramKrishnaswami Evaluation of the effect of Tofacitinib on the pharmacokinetics of oral contraceptive steroids in healthy female volunteers. ClinPharmacol Drug Dev, 2016 Sep;5(5): International Council on Harmonization of Technical requirements for the registration of pharmaceuticals for human use (ICH), Validation of analytical proced ures: text and methodology Q2 (R1), 2005.
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