Ultra c18 column

The extracts (10 mg) were dissolved in ethanol and filtrated through a 0.45 µm syringe filter into a vial bottle. The analysis was performed according to the already reported methods with some modifications [47 (link),75 (link)] and analyzed using an Agilent 1260 Infinity II series, coupled with an electrospray ion (ESI) quadrupole mass spectrometry 6130 (Agilent Tech., Santa Clara, CA, USA). Reverse-phase column chromatography was performed using the Restek Ultra C18 column (250 × 4.6 mm, 4.6 mm, 5 µm) (Restek, Bellefonte, PA, USA). The column was maintained at 30 °C. The gradient elution was carried out using the 5% formic acid as a solvent A and acetonitrile: H₂O: formic acid (85: 10: 5) as a solvent B with a linear gradian elution as 0–8 min, 80% A; 8–24 min, decreased A to 25%; 24–28 min, 25% A; 28–34 min, increased A to 70%; 34–36 min, increased A to 80%; 36–45 min, 80% A. The injection volume for all samples was 5 µL which was monitored at the flow rate of 0.5 mL ml/min. The MS was operated in the negative selected ion monitoring (SIM) as the following condition: dying gas (N2) flow, 12 L/min; dying gas temperature, 350 °C; nebulizer pressure, 60 psi; capillary voltage, 3000 V; fragmentor voltage, 70 V; and the full scan spectra from 100 to 1200 m/z with 250 ms/spectrum. The spectra were processed using Open Lab software (Agilent Tech., Santa Clara, CA, USA).

Lyophilized venoms (10 mg) from D. polylepis, from Kenya and
from South Africa, were dissolved in 0.05 M ammonium acetate pH 4.2 in a final
volume of 5.0 mL, and immediately filtered through a Merck Millipore Amicon
Ultracel 10 K centrifugal filter device with a molecular mass cut-off of 10,000
Da (Tullagreen, Carrigtwohill, IRL), in order to prevent proteolytic cleavage of
peptides by venoms. Filtrated solutions containing low molecular mass fractions
were injected (500 μL) in a reverse-phase HPLC (Prominence, Shimadzu, Japan),
using 0.1% trifluoroacetic acid (TFA) in water, as solvent A, and acetonitrile
and solvent A (9:1) as solvent B. The separations were performed at a flow rate
of 1 mL/min using a Restek Ultra C-18 column (4.6 × 150 mm) and a 20-60%
gradient of solvent B over 20 min. In all cases, elution was followed by the
measurement of ultraviolet absorption (214 nm). The peaks were manually
collected, dried and subjected to enzymatic assays.

Sequential extracts of peeled (bark removed) Tripterygium wilfordii Hook F roots were analyzed for triptolide content using a Shimadzu Prominence LC-2030C system equipped with an Ultra C18 column (250 mm × 4.6 mm, 5 μm dp, Restek, Bellefonte, PA, USA) and a Restek Ultra C18 guard column (10 mm × 2.1 mm, 5 μm dp). The fractions were centrifuged at 3000 rpm for 5 min and evaporated to dryness using Rotovapor R210 (Büchi, New Castle, DE, USA). The residues were dissolved in 1 mL of methanol, filtered through a syringe with a 0.45 μm PFTE membrane, and 20 μL were subjected to the HPLC analysis of triptolide content at 219 nm.

The identification of main phytotoxins was done with a LC-MS system. The chromatographic separation of the compounds was carried out on a Restek Ultra C18 column (250 × 4.5 mm, 5 μm) with a Restek guard-column (10 × 4.0 mm). Two solvent mixtures were used as mobile phases. Solvent A was composed of water/methanol (99.2:0.8, v/v and acidified with formic acid 20 μl L⁻¹) while 100% methanol was used as solvent B. Samples were dissolved in minimum quantity of methanol. The sample injection volume was set at 20 μl. A solvent gradient was adopted for a total run time of 60 min at a flow rate of 1 ml min⁻¹, with all the toxin standards eluting over 20–60 min. The solvent gradient was as follows: equilibration at 5% B for 2 min, from 5 to 35% B in 43 min., from 35 to 100% B in 5 min., 100% B for 10 min., return to initial condition in 2 min.