1220 infinity 2 lc system

An Agilent Technologies 1220 Infinity II LC system was used in pilocarpine HCl quantification based on a method developed by Fan et al. (1996 (link)) and outlined by El Deeb et al. (2006 (link)). The mobile phase was a mixture of solution A, containing 13.5 mL phosphoric acid, 3 mL trimethylamine and 983.5 mL deionized water, and solution B as methanol in a ratio of 98:2. A standard Gemini^® 5 μm C18 110A LC column 150 × 4.6 mm was used under ambient experimental conditions (18–21 °C). Flow rate was set at 1.5 mL/min and injection volume was 20μL. The UV absorbance wavelength of pilocarpine hydrochloride was determined to be 215 nm and as such, UV detection was done at this wavelength. Validation was carried out according to ICH guidelines Q2R1 (2005 ) over a linearity range of 7.8125–500 µg/mL with coefficient of variation r² = 0.9999. The method was precise with repeatability giving 1.1% RSD; mean % recovery ranging from 78.38 to 103.93%; and limits of detection and quantification at 0.158 μg/mL and 0.528 μg/mL respectively. All but one of the mean recovery RSD% values were under the acceptable 15% upper limit for pharmaceutical analysis (Iyire et al. 2018 (link)). Recovery at the lowest concentration in the range gave the lowest recovery, informing the use of the indirect method for quantifying the amount of pilocarpine hydrochloride entrapped in the niosomes.

ME was analyzed by reverse-phase HPLC (High Performance Liquid Chromatography) using a Zorbax Eclipse XDB-C18 column 4.6 × 150 mm (Agilent) on an Agilent 1220 Infinity II LC System. The mobile phase was delivered at a rate of 1 mL/min with a gradient from A (0.1% HCOOH in H₂O) to B (0.1% HCOOH in CH₃CN) (10% B for 4 min, 10% to 60% B in 10 min, 60% to 100% B in 2 min). The column effluent was monitored at 250 nm.
The quantity of ANDRO contained in the ME was determined by comparison with a range of pure ANDRO (standard).