Development and Validation of RP-UPLC Method for 2,6-Dimethylaniline, Its Isomers, and Related Compounds Using Design of Experiments

Abstract

2,6-Dimethlyaniline (2,6-DMA) is a key starting material which is used in the synthesis of many classes of drugs, such as anesthetics drugs Lidocaine (Xylocaine®), Bupivacaine, Mepivacaine, Etidocaine, Ropivacaine, Pyrrocaine, Xylazine and anti-anginal drug like Ranolazine and anti-diarrheal drug like Lidamidine. 2,6-DMA together with its five positional isomers and related compounds were separated using a simple isocratic and reverse-phase ultra-performance liquid chromatographic (UPLC) method within a shorter runtime. The developed UPLC method was capable to detect and quantify the impurities at lower levels, i.e., Limit of Detection (LOD) 0.007 µg mL−1 and Limit of Quantification (LOQ) 0.02 µg mL−1. The chromatographic separation of the impurities was successfully accomplished on Acquity UPLC CSH Phenyl hexyl (100 mm × 2.1 mm × 1.7 µm) column at a flow rate of 0.3 mL min−1 and signal detection of 210 nm at a sampling rate of 10 points second−1. The column oven temperature was maintained at 40 °C. A mixture of sodium phosphate buffer (10 mM, pH 3.5)—acetonitrile (86:14, v/v) was used as mobile phase with an isocratic mode of elution. Optimization of the chromatographic conditions was carried out by conducting column scouting experiments, studying effect of PH, and organic ratio on individual peak separation. The current method was developed with a systematic approach. Robustness of the developed method was assessed by varying critical factors, such as PH of the buffer, organic ratio, and column temperature, at two levels [low (−), high (+)]. Final method conditions were selected using global desirability with numerical optimization. The resolution (Rs) between each isomer was > 1.5, and column efficiency (N) > 1900 in all the varied conditions. The developed method was fully validated according to ICH Q2 guidelines and USP general chapter requirements.