Determination of related substances in diazepam rectal gel by high-performance liquid chromatography

Objective To establish a high-performance liquid chromatography method for the determination of related substances in diazepam rectal gel.

Methods The Agilent ZORBAX Eclipse Plus C18 (4.6 mm×150.0 mm, 3.5 μm) column was used with a mobile phase consisting of 10 mmol/L potassium dihydrogen phosphate buffer (adjusted to pH value to 6.0 with 0.5 mmol/L sodium hydroxide solution) and methanol. Gradient elution was performed at a flow rate of 1.0 mL/min, with a column temperature of 30 ℃ and a detection wave length of 234 nm.

Results The chromatographic peaks of diazepam and known impurities Ⅰ to Ⅶ showed good resolution. Diazepam and known impurities Ⅰ, Ⅱ, Ⅲ, and Ⅳ exhibited good linearity in the concentrations ranging of 0.01 to 10.05 μg/mL, 0.01 to 9.92 μg/mL, 0.01 to 10.24 μg/mL, and 0.01 to 10.18 μg/mL, respectively (r²≥0.999), with average recoveries ranging from 99% to 101%. The excipients in the formulation did not interfere with the determination of the principal component and related substances.

Conclusion This high-performance liquid chromatography method is highly specific and reproducible, making it suitable for the detection of related substances in diazepam rectal gel.