Lc 20ad series hplc system

HPLC was performed on a Shimadzu LC-20AD series HPLC system (Kyoto, Japan) consisting of two LC-20AD pumps, a CTO-20A column oven (set at 35°C) and a SIL-20A autosampler. Analyte separation was achieved on a Phenomenex Luna C₁₈ column (150 × 2.00 mm, 5 μm) preceded by a Phenomenex C₁₈ security guard column, with an isocratic mobile phase consisting of 0.1% formic acid and methanol (20:80, v/v) at a flow rate of 0.3 mL/min.
MS detection was performed on a Thermo Finnigan TSQ quantum mass spectrometer (San Jose, CA, USA). Ultra-high pure nitrogen was used as the sheath and auxiliary gas. The ESI source was operated in negative ion mode under the following conditions: spray voltage, 3.5 kV; capillary temperature, 400°C; sheath gas, 35 arbitrary units; auxiliary gas, 15 arbitrary units. For quantification in multiple-reaction monitoring (MRM) cone voltage, collision energy and precursor to production transition m/z for emodin and IS were as shown in Table 1. For metabolism study of emodin, three phase II metabolites of emodin were monitored simultaneously using MRM mode (Teng et al., 2007 (link)).

The reconstituted venoms were fractionated using a Shimadzu LC-20AD series HPLC system (Kyoto, Japan) using a previously described protocol with modifications [28 (link)]. 200 μg of each venom was loaded onto a 4.6 × 250 mm, C18 (5 μm, 300 Å) reversed-phase column (Shimadzu, Japan), and equilibrated with solution A [0.1% trifluoroacetic acid (TFA) in water (v/v)]. The fractions were eluted at a flow rate of 1 ml/min using the following concentration gradients of solution B [0.1% TFA in 100% acetonitrile (v/v)]: 5–15%, 15–45% and 45–70% for 10, 60 and 10 min, respectively, and the absorbance was monitored at 215 nm.

Frozen leaves were dried in an oven at 60°C until the weight was constant (Zhou et al., 1999 ). Afterward, these leaves were ground into powder smaller than 40 mesh and extracted in a Soxhlet extractor based on Chinese pharmacopeia (2015). 1.000 g dried powder was homogenized in 100 mL chloroform followed by two h reflux at 80°C. The residue was resolved in 50 mL methanol followed by two-hour reflux at 90°C. Then, 20 mL hydrochloride was added to the supernatant, refluxing for 30 min at 90°C and evaporating to dryness. The residue was dissolved in methanol and filtrated through a 0.22 μm membrane for HPLC (Daigle and Conkerton, 1983 ).
Chromatographic separation was implemented on a Shimadzu LC-20AD Series HPLC system (Shimadzu, Duisburg, Germany) equipped with SIL-20A autosampler, SPD-20A UV-VIS detector. ACQUITY UPLC™ BEH C18 column (217 mm × 2.1 mm, 1.7 μm, Waters, Milford, United States) was connected to the whole detection at 25°C. The mobile phase consisted of 10% 0.05 M sodium acetate (A) and 90% acetonitrile (B). A flow rate of 1.0 mL/min was used. Standard curves were established by a series concentration of the corresponding standard (ChemFaces, Wuhan, China) at 360 nm (Mattila et al., 2000 (link)). And the content of total flavonoids was calculated by the formula (Hasler et al., 1992 ):
The data were provided in the (Supplementary Table 1).

The quality of conventional Indian antivenoms and the three batches of second-generation antivenoms was assessed using SE-HPLC, with minor modifications to a previously reported method [28 (link)]. The SE-HPLC system was kept at 25 °C and consisted of a Shimadzu LC-20AD series HPLC system (Kyoto, Japan), a photodiode array detector (PDA), and a Bio-diol column (4.6 × 300 mm, 5 m particle size; Shimadzu, Kyoto, Japan). Proteins were eluted at a flow rate of 1 mL/min for 50 min using a 0.05 M sodium phosphate-0.15 M NaCl buffer (pH 7.0). PDA detector responses were collected at 280 nm with a sample injection volume of 20 μL.

In the Edman degradation analysis of the N-terminal amino acid sequence of the m/z 2332 peptide, purification of the peptide by reverse-phase HPLC was performed using a Shimadzu LC-20AD series HPLC system equipped with a SPD-M20A diode array detector (Shimadzu Corporation, Kyoto, Japan). A Luna C-18 column (5 µm particle size; 4.6 mm ID×250 mm; Phenomenex, Torrance, CA, USA) was utilized for the purification process. The HPLC conditions used were as follows: the column oven temperature was 40°C, the flow rate was 1.0 mL/min, water with 0.1% FA and ACN with 0.1% FA were used as solvents A and B, respectively, the gradient condition was maintained at 10%, as solvent B, for 2 min after a 20-µL sample injection and was linear from 10% to 80% for 15 min. Eluted peptides were monitored at 276 nm. The peptide eluted between 6.6 min and 6.9 min was pooled into a microtube.
The automated Edman degradation of the N-terminal amino acid sequence was performed on RP-HPLC purified peptides using a Procise Model 491 sequencer (Applied Biosystems, Inc., Foster City, CA, USA).

Liquid chromatography was performed on a LC-20 AD series HPLC system equipped with two binary pumps, a thermostatically controlled column compartment set at 40 °C and an auto sample manager maintained at 4 °C (Shimadzu Corporation, Kyoto, Japan). The separation was performed on a Waters XBridge BEH C18 column (2.5 µm, 2.1 × 50 mm) in isocratic elution with 0.1% formic acid in water and methanol (50:50, v:v). The flow rate and the injection volume were set at 0.35 mL/min and 2 µL, respectively.
Tandem mass spectrometry was carried out on a Qtrap 5500 mass spectrometer (AB SCIEX, Ontrario, Canada) equipped with electrospray ionization in the positive ion mode. Linear ion trap (MS³) mode and Multiple Reaction Monitoring (MRM) mode were employed for the data acquisition of OXC, MHD and OXC-d₄ (internal standard, IS). The optimized MS parameters were as follows: ionspray needle voltage 5500 V; heater gas temperature 450 °C; curtain gas 30 psi; nebulizer gas 50 psi; and heater gas 50 psi. Other operation conditions of MS³ transitions and MRM transitions optimized parameters for quantitative analysis of OXC, MHD and OXC-d₄ (IS) are presented in Table 4.

LC–MS/MS was carried out on a LC–20AD series HPLC system (Shimadzu Corporation, Kyoto, Japan) coupled to a Q-TRAP 5500 mass spectrometer (AB Sciex, Foster City, CA, USA). Briefly, HPLC separation was performed on Agilent Poroshell 120 SB–C₁₈ (4.6 mm × 50 mm, 2.7 μm) at 40 °C with a gradient program using eluent A (0.1% formic acid in water) and eluent B (ACN). The gradient program was as follows: 0.0–1.0 min: 4% B; 1.0–2.5 min: 4–95% B; 2.5–4.5 min: 95% B; 4.5–4.6 min: 95–4% B; 4.6–7.0 min: 4% B. The flow rate of mobile phase was set at 0.65 mL/min and the injection volume was 2.0 µL.
An electrospray ionization (ESI) source interface operating in positive mode was used for multiple reaction monitoring (MRM) mode. The operation conditions are following parameters: ion spray voltage, 5500 V; source temperature, 450 °C; curtain gas, 20 psi; ion source gas 1, 60 psi and ion source gas 2, 55 psi; collision gas pressure, medium; entrance potential, 10 V; cell exit potential, 12 V; declustering potential, 80 V; collision energy 18 eV. The monitored MRM transitions were m/z 384.0→141.0 for MER and m/z 390.0→147.0 for MER-d₆.