Accela pda detector

The degradation of M3G at different
concentrations (3.33 × 10^–5, 2 × 10^–4, and 1 × 10^–3 M) was followed
over time at pH 8.1 by HPLC coupled with DAD (HPLC-DAD). Stock solutions
of M3G (1 × 10^–4, 6 × 10^–4, and 3 × 10^–3 M) were prepared in 0.1 M HCl.
Then, 1 mL of NaOH 0.1 M, 1 mL of universal buffer at pH 8.1, and
1 mL of each stock M3G solution were added to a glass vial, and the
solution was analyzed over time by HPLC-DAD on a Thermo Ultimate 3000
liquid chromatograph. The decrease in the concentration of M3G was
followed over time during 7 days in a 250 × 4.6 mm i.d., 2.7 μm
Poroshell 120 reversed-phase C18 column (Agilent) at 25 °C. The
eluents used were (A) 7.5% (v/v) formic acid in water and (B) 7.5%
(v/v) formic acid in acetonitrile at a flow rate of 0.4 mL/min. The
gradient consisted of 3% to 15% B in 11 min, then 25% B in 23 min,
30% B in 27 min, and then isocratic for 4 min. Detection was carried
out from 200 to 800 nm in a Thermo Accela PDA detector. Due to the
low concentration of M3G (3.33 × 10^–5 M) needed
to avoid self-association and the low sensitivity of HPLC analysis
when compared to UV–visible spectroscopy, several aliquots
(1 mL) of the degradation kinetic (pH 8.1) were taken, lyophilized,
and then resuspended in 0.1 mL of 0.1 M HCl to be analyzed by HPLC
to tentatively identify the degradation products.

To study the relationship between pharmacokinetic characteristics and treatment efficacy in vivo, compounds were administered by oral (50 mg/kg) and intramuscular (12.5 mg/kg) methods to healthy BALB/c mice (22~22 g). Subgroups of 3 mice each were bled at 0.25, 0.5, 1, 2, 4, 8, 16, 24, 32, and 48 h post-administration. Then, 300 μl samples of plasma were collected and centrifuged at 2,500 g for 15 min. The compounds in the plasma were extracted on Oasis HLB cartridges (Waters, Massachusetts, USA) with praziquantel as the internal standard. The HPLC-HRMS system used for separation and analysis consisted of an Accela 1250 pump, a PAL HTC autosampler, an Accela PDA detector and an Xcalibur data system (Thermo Fisher Scientific, Waltham, MA, USA). Compounds were separated on a 3-μm Waters Atlantis 2.1 mm × 100 mm column (Massachusetts, USA) with a mobile phase containing methanol and formic acid at a flow rate of 0.35 ml/min. The data were analyzed by the non-compartmental model present in DAS 2.0 (Drug Analysis System, Shanghai University of T.C.M, China). Detailed pharmacokinetic experimental methods, including a representative HPLC-MS chromatogram, a standard curve, and a plot used to confirm precision, are shown in the Supplementary Data.

All experiments were performed on a Thermo Scientific Accela UHPLC system with an Accela 600 Pump, Accela Autosampler and Accela PDA Detector coupled to a Thermo Fisher Scientific LTQ Orbitrap Velos Pro mass spectrometer (Thermo Scientific, Waltham, MA, USA). All samples were centrifuged using a 5424R Eppendorf centrifuge (Eppendorf, Hamburg, Germany). All ACQUITY UPLC columns (BEH C18 (2.1 mm × 100 mm, 1.7 μm, 130 Å), BEH Phenyl (2.1 mm × 100 mm, 1.7 μm, 130 Å) and CSH Phenyl-Hexyl (2.1 mm × 100 mm, 1.7 μm, 130 Å)) and all ACQUITY UPLC VanGuard pre-columns (BEH C18 (2.1 mm × 5 mm, 1.7 μm, 130 Å), BEH Phenyl (2.1 mm × 5 mm, 1.7 μm, 130 Å) and CSH Phenyl-Hexyl (2.1 mm × 5 mm, 1.7 μm, 130 Å)) were purchased from Waters (Milford, MA, USA).