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 < 1226 > general chapter requirements.