Acquity uplc h class chromatography system

An in vitro release test was performed with flow-through cells (Sotax CE7 smart with pump). Cellulose membrane (Sigma Aldrich (St. Louis, USA)) was mounted in adapters for semi-solid formulations. The donor phase (1 ± 0.1 g) was placed into the adapter. The diffusion area was 1.33 cm². The aqueous receptor medium was 50 mL of purified water. Selected conditions fulfilled the criteria of sink conditions. Samples of the solution were collected at 1, 2, 3, 4, 5, and 6 h and replaced with the same volume of fresh water.
All samples were analyzed by liquid chromatography using the Acquity UPLC H-Class chromatography system (Waters, Milford, MA, USA) equipped with DAD (Waters, Milford, MA, USA), performing detection at 303 nm. Validated UPLC method conditions were C18 column (130 Å, 1.7 µm, 2.1 mm × 50 mm, Waters, Milford, MA, USA), solvent A (acetonitrile) and solvent B 0.5% (v/v) of trifluoracetic acid in ultrapure water) ratio 40–60%; the injection volume was 5 μL; the flow rate was 0.7 mL/min; the column temperature was 25 °C. A standard calibration curve was built up by using standard solutions (4–324 µg/mL). All samples were filtered using a polyvinylidene difluoride filter (pore size 0.2 µm).
The cumulative amounts of released ciclopirox olamine expressed per unit area (cm²) were calculated and discussed in the results section.

Epidermis was separated from dermis by applying the dry heat separation method. DHQ was extracted from separated skin layers with methanol and sonication for 30 minutes in sonication bath (USC1200 THD, VWR). DHQ content in skin extracts was quantified at 289 nm using Acquity UPLC H-Class chromatography system (Waters, Milford, MA, USA). Separation of DHQ from endogenous skin matrix compounds was performed on Acquity UPLC BEH C18 (130 Å, 1.7 µm, 2.1 mm × 50 mm, Waters, Milford, MA, USA) column. The mobile phase was delivered in a linear elution gradient from 20 to 58% of solvent A (acetonitrile) in B (0.1% (v/v) trifluoracetic acid in ultrapure water) for 3 min; the injection volume was 1 µL, flow rate was 0.5 mL/min, and the column temperature was 40 °C. A standard calibration curve was built up by using standard solutions (0.35–28.35 µg/mL). Calibration graphs were plotted according to the linear regression analysis, which gave a correlation coefficient (R²) of 0.9999. The method was tested and validated for DHQ analysis in methanolic extracts of human skin layers.

The analysis was performed by a targeted hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method which has been previously developed from our group [52 (link)] for such studies [53 (link),54 (link),55 (link)] targeting more than 100 hydrophilic endogenous metabolites. The analysis was performed on an ACQUITY UPLC H-Class chromatography system combined with a Xevo TQD mass spectrometer (Waters Corporation, Millford, MA, USA) operating in both positive and negative mode, with an Acquity BEH Amide Column (Waters Ltd., Elstree, UK). The mobile phase consisted of MeCN:H₂O, 95:5 (v/v) and MeCN:H₂O, 30:70 (v/v), both containing 10 mM ammonium formate, pH 6.
The samples were analyzed in a randomized order in two separate batches for each specimen and 5 μL was injected into the system. For the quality assessment of the data, a quality control (QC) sample was prepared for urine or fecal extracts according to published protocols [56 (link)] and analyzed every 10 samples within the analytical batch.