Abstract
Objective: The proposed research work was conducted to develop a single reverse-phase high-performance chromatography (RP-HPLC) method capable of separating two Pharmacopoeial related impurities as well as degradation product of Tolfenamic acid (TA). The drug was subjected to various stress conditions recommended under ICH Q1A (R2) guidelines.
 Methods: The desired separation of two Pharmacopoeial impurities and one degradant generated under oxidative stress was carried out using Sunfire ODS C-18 (250 x 4.6 mm, 5 µm) column maintained at 40 °C. Isocratic elution was carried out using acetonitrile and ammonium dihydrogen orthophosphate buffer (10 mmol, pH 2.5) in the ratio of 80:20 v/v. The detection was carried out at 205 nm using flow rate of 1 ml/min. The developed method was validated as per ICH Q2 (R1) guidelines for specificity, linearity, accuracy, precision, Limit of detection (LOD), Limit of Quantification (LOQ) and robustness.
 Results: Linearity response of TA was found at a concentration range of 10-100µg/ml, with a correlation coefficient of 0.9987. The Pharmacopoeial impurity A and impurity B showed linearity results at concentration of 0.1-1µg/ml, with correlation coefficient of 0.9984 for Impurity A and 0.9989 for Impurity B. The % recovery during accuracy studies for TA and the two impurities were within the acceptance range of 95-105%. LOD and LOQ for TA were found to be 4.561µg/ml and 133.771µg/ml respectively. For impurity A, LOD and LOQ were found to be 0.035 µg/ml and 0.106 µg/ml and for Impurity B, LOD and LOQ were 0.042 µg/ml and 0.128 µg/ml. With slight variation of organic phase in mobile phase and flow rate the method exhibited good robustness. Under forced degradation studies the drug was found stable under hydrolytic, photolytic and thermal stress conditions, but was found susceptible for degradation under oxidative stress with appearance of a degradant peak. From on the RRT values of Pharmacopoeial impurities and the formed degradant it was inferred that the developed method is selective for the drug in the presence of impurities or degradants.
 Conclusion: The developed stability-indicating method is found to be simple, rapid, accurate, precise and robust as compared to other proposed methods while determining TA in presence of its Pharmacopoeial impurities and degradation products. Hence the developed method can be used for analysis of stability samples of TA in presence of its related impurities.
Highlights
Tolfenamic acid (TA) is 2-[(3-chloro-2-methylphenyl) amino] benzoic acid [1]
TA belongs to the class of fenamates of the nonsteroidal anti-inflammatory drug (NSAID) and is used for treatment of inflammation and pain in humans as well as animals
Optimum separation of the drug from its specified impurities was obtained when the pH of the buffer was fixed at 2.5 and the composition of acetonitrile and buffer was in the ratio of 80:20%v/v under isocratic elution
Summary
Tolfenamic acid (TA) is 2-[(3-chloro-2-methylphenyl) amino] benzoic acid (fig. 1) [1]. Tolfenamic acid (TA) is 2-[(3-chloro-2-methylphenyl) amino] benzoic acid British pharmacopoeia lists three impurities in the monograph of TA TA belongs to the class of fenamates of the nonsteroidal anti-inflammatory drug (NSAID) and is used for treatment of inflammation and pain in humans as well as animals. Used for the remedy of acute migraine attacks and for the relief of pain in conditions such as osteoarthritis, dysmenorrhea and rheumatoid arthritis [3,4]. TA has gained high popularity recently for its anticancer activity against different types of cancer [5,6,7,8]. The concentration of all the related substances associated with drug as per ICH guidelines must be less than 0.2% of the concentration of drug except for isopropyl ethers and monoester, whereas the limit should not exceed 0.2% at different storage concentrations [9]
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