Hypersil bds c18

The Perkin Elmer, Series 200 HPLC Systems supplied with a micro-pump, an auto sampler, and a UV-detector operating at 255 nm was employed to conduct the HPLC analysis. A stainless-steel C-18 reverse-phase column (15 × 0.46 cm) packed with 5 μm particles (Hypersil BDS C18 Thermo Fisher Scientific S.p.A., Milan, Italy) was used. The mobile phase adopted was acetonitrile/water 40:60 v/v, pH 3, eluted at 1 mL/min flow rate. The sample injection volume was 5 μL, while the retention time was 9 min.

We used processed WYJ for in vitro and in vivo experiments. The compounds of processed WYJ were reduced or changed compared with the herbal plant's library. Therefore, we used LC/MS analysis to clarify the compounds of WYJ. The coupling of the UHPLC System (Thermo Fisher Scientific) equipped with a binary pump, an auto sampler, a column thermostat, and DAD detector, and LTQ-Orbitrap XL (Thermo Fisher Scientific) equipped with an electrostatic ionization source (ESI) was used For LC/MS experiments. Data was controlled and processed by Xcalibur software (Thermo Fisher Scientific). The column used in the LC analysis was Thermo Scientific Hypersil BDS C18 (2.1 mm 150 mm, 2.4 μm). Mobile phase A was water with 0.1% formic acid while mobile phase B was acetonitrile. The column temperature was set to 35 and the flow rate was 0.3 mL/min. Elution conditions were summarized as follows: 0–3 min (5%-5%B), 3–45 min (5%∼75%B, 45–45.1 min (75%-5%B), and 45.1–50 min (5%-5%B).
The MS analysis was conducted on both the negative and positive ion modes. Flow rates of sheath gas and auxiliary gas were set at 40 Arb and 20 Arb, respectively. The capillary voltage was set to 35.0 V. The source temperature was set to 350 and the tube lens was set to 110 V. The source voltage was set to 4 kV and 3 kV in the positive and negative ion modes, respectively.

Starting from the analytical conditions reported in literature,^{19 (link)} the column length was decreased from 250 mm to 150 mm (Hypersil BDS C18, 5 μm, 150×4.6 mm, Thermo Scientific, MA, USA) in order to improve tailing factors of the two active ingredients. A Thermo Fisher HPLC system equipped with a P4000 pump and an AS3500 autosampler were employed (Thermo Fisher Scientific Inc., Waltham, MA, USA). A Thermo Scientific UV6000 PDA was set at 250 nm for SX and 238 nm for FP, while the retention time was 3.0 and 6.7 min for SX and FP, respectively. LOQ was about 0.043 μg/mL for salmeterol and 0.035 μg/mL for fluticasone propionate. The linearity of response of salmeterol and fluticasone propionate was evaluated on standard solutions in the concentration range 0.04–13 μg/mL and 0.04–90 μg/mL, respectively, with a correlation coefficient R²: ≥0.99 in both of the cases.

Shimadzu LC-20ad prominence equipped with quaternary pump, and dual wavelength UV detector, column oven and auto sampler (Shimadzu Corporation, Kyoto, Japan) and analytical column Hypersil BDS-C18 (3 µm, 100 mm × 4 mm) (Thermo Fisher Scientific, Phenomenex, USA) were used for chromatographic separation; data acquisition and processing were accomplished with LC solution software. Syringe membrane filters 0.45 μm Millex-HN (Millipore, Bed-ford, MA, USA) for filtration of standards and samples; centrifuge, AX-320 (Tomy Seiko Co., Tokyo, Japan); vortex mixer, Vortex-Genie 2 (Scientific Industries Inc., Bohemia, New York, USA) and ultrasonic machine, B5510J-DTH (Branson, Danbury, CT) were also used. The pH values of the mobile phases were measured using a Hanna instruments pH meter (Hanna Instruments Inc., Cluj-Napoca Jud, Cluj, Romania); vacuum filtration assembly (Millipore filter cellulose nitrate gridded with 0.22 μ size and 47 mm diameter) attached with vacuum pump and glass support, NS 40/35 joints from Sigma-Aldrich (USA) were used for HPLC solvent purification. Flask (Pyrex), volumetric, class A, w/Pyrex standard taper stopper, 1 mL, Corning 5640-1 (Beijing, China) and R-100 rotary evaporator from Buchi Labortechhnik AG (Switzerland) were used for evaporation of solvents.

Concentrations of test molecules were measured using liquid chromatography-tandem mass spectrometry. Samples (10 µl) were injected directly into a Hypersil BDS C18 high-pressure liquid chromatography (HPLC) column (30 × 2.1 mm inner diameter, 3-µm particle size, with guard column; Thermo Fisher Scientific, Grand Island, NY) and a tandem mass spectrometer (PE SCIEX API2000 or API3000; AB SCIEX LLC, Framingham, MA). The mobile phase was 25 mM ammonium formate buffer, pH 3.5. Compounds were eluted with a linear gradient at a flow rate of 300 µl/min. Eluted compounds were ionized using an electrospray interface.
Apparent permeability was measured in both absorptive (apical to basolateral; A to B) and secretory (basolateral to apical; B to A) directions. The efflux ratio for each molecule was calculated as P_app B to A/P_app A to B. The P_app and percent recovery were calculated as P_app = (dC_r/dt) × V_r/(A × C_A) and percent recovery = 100 × [(V_r × C_r^final) + (V_d × C_d^final)]/(V_d × C_N), respectively, where