Syringe membrane filter

High-performance liquid chromatography (HPLC) was used for phenolic profiling. The internal standards were quercetin, gallic acid, caffeic acid, benzoic acid, vanillic acid, cinnamic acid, syringic acid, p-coumaric acid, m-coumaric acid, ferulic acid, sinapic acid, and chlorogenic acid. Phenolic compounds were extracted following the method described by Stalikas (2007) (link) with minor modification. Powdered leaves were extracted in 100% methanol (1:10) and were filtered with a 0.45 um cellulose acetate filter (EMD Millipore, Billerica, MA, United States). An aqueous suspension of the extract was then prepared with double distilled water, the pH was adjusted to 2 with 6 M HCl and the mixture was incubated at 100°C for 3 h. All of the standards and extracts were filtered through a 0.45 μm syringe membrane filter (Millipore) and sonicated for 15 min in a Micro clean 109 bath prior to HPLC. Phenolic compounds were analyzed using gradient HPLC (LC-10AT, SCTL, Shimadzu, Japan). Elution was done for 60 min with a flow rate of 1 mL/min in a gradient system of two mobile phases: A (H₂O₂:Acetic acid 94:6, pH 2.27) and B (100% acetonitrile).

Human nail clippings from healthy volunteers were used for in vitro permeation studies. Nails were hydrated by soaking in normal saline for 1 h before application and seated on a tailor-made adapter meant for nails. The entire assembly was placed in the middle of donor and receiver chambers of a regular Franz diffusion cell (Logan Instruments Ltd., Somerset, NJ, USA). The available area for the drug diffusion was 0.2 cm². Prepared formulations (T1–T6; 500 μL) were individually kept in the donor compartment and allowed the drug to permeate for 6 h. The receiver chamber contains 5 mL of acidified water, whose pH was fixed to pH 3 (using 0.01 N HCl), the temperature was set at 37 °C and rotated at 100 rpm. Samples (1 mL) were withdrawn at regular intervals (2, 4, and 6 h) and replaced with fresh acidified water. The samples withdrawn were filtered using a syringe membrane filter (pore size of 0.2 µm; Millipore, Bedford, MA, USA) and readily estimated by the HPLC method developed. For each formulation, the permeation study was carried out six times and the cumulative amount of drug permeated are represented as means ± SD.