Gis c18 column

The chromatographic separation was carried out on a Shim-pack GIS C18 column (10 mm × 2.1 mm, 3 μm) fitted on a Jasper HPLC system. The mobile phases were 0.1% formic acid (A) and acetonitrile (B). The flow rate was set to 0.3 mL/min. The column temperature was maintained at 40°C. The following gradient program was used: the initial mixture of 90% A and 10% B was held for 0.5 min; the linear gradient was increased to 70% B in 5 min and held for 2 min; then the linear gradient was increased to 90% B in 0.1 min and held for 1 min; then return to the initial conditions in 0.1 min, followed by 3.3 min of equilibration. The total run time was 12 min, and the injection volume was 2 μL. Mass spectra were obtained using an API 3200 equipped with a TurboIon electrospray (ESI) interface set in the positive mode (needle voltage +5500 V). The mass spectrometry conditions were as follows: CUR, 20 psi; Gas 1 and Gas 2, 55 psi; IS: 5500 V; gas temperature: 550°C. The acquisition dwell time for each transition monitored was 50 ms.

The LID content was quantified using high-performance liquid chromatography (HPLC; LC20A) with UV detection (SPD-20A; Shimadzu, Kyoto, Japan). Needles were removed from the baseplate using a razor and collected per patch. They were dissolved in 10 mL phosphate-buffered saline (pH 7.4). HPLC was performed using a Shim-pack GIS C18 column (4.0 × 150 mm, 5 µm) at an injection volume of 50 µL. The mobile phase consisted of 0.1% (v/v) trifluoroacetic acid in acetonitrile water (20:80, v/v). The column temperature and flow rate were 40 °C and 1.0 mL/min, respectively. The detection wavelength was 262 nm. LID was eluted at 5 min. The peak area of LID was used for quantification of unknown samples. The standard curve was linear between 1.25 and 20 µg/mL, with a correlation coefficient of 1.000.