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A green, accurate and specific high-performance thin-layer chromatographic (HPTLC) method was developed and validated for simultaneous quantitative determination of pyridostigmine bromide (PR), impurity B (IMP B);3-hydroxy-N-methylpyridinium bromide and impurity A (IMP A); pyridin-3-yl-dimethylcarbamate. The two pharmacopeial impurities are also its main inactive metabolites. Furthermore, IMP B is known to be its alkaline-induced degradation product. Achievable separation of the studied components required silica gel HPTLC F 254  plates as a stationary phase and acetone: acetic acid (80:20, v/v) as a developing system. Scanning of the separated bands was done at 260 nm. According to green solvent selection guidelines, acetone and acetic acid are eco-friendly solvents. Validation of the developed method was insured by its acquiesce to international conference on harmonization (ICH) guidelines. The introduced method was successfully achieved for the quantitative determination of PR, IMP B and IMP A in the range of 0.4–10, 2–11 and 0.4–3.5 μg/band, respectively. Successful application of the developed method was done for determination of PR in human plasma in the range of 0.6–10 μg/band, so the proposed HPTLC can be applied in the pharmacokinetic studies. The studied drug was also analyzed in Mestinon® tablets using the developed method.

Green Simultaneous Chromatographic Separation of Pyridostigmine Bromide and Its Related Substances in Pure Form, Tablets and Spiked Human Plasma