Mightysil rp 18 column

All peptides were manually synthesized by the 9-fluorenylmethoxycarbonyl strategy with a C-terminal amide as previously described (Katagiri et al., 2010 (link)). The respective amino acids (Kokusan Chemical, Tokyo, Japan) and biotin (Tokyo Chemical Industry, Tokyo, Japan) were condensed using diisopropylcarbodiimide-N-hydroxybenzotriazole on a 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Rink amide resin; Novabiochem, San Diego, CA). The peptides were purified by reverse-phase HPLC on a Mightsil RP-18 GP 250-10 column (Kanto Chemical, Tokyo, Japan) using gradient elution with water/acetonitrile containing 0.1% trifluoroacetic acid. Resulting protected peptide resins were deprotected and cleaved from the resin using trifluoroacetic acid‒thioanisole‒m-cresol‒ethanedithiol‒water (80:5:5:5:5, v/v) at room temperature for 3 hours. Crude peptides were precipitated and washed with diethyl ether and then purified by HPLC using a Mightysil RP-18 column (Kanto Chemical) with a gradient of water/acetonitrile containing 0.1% trifluoroacetic acid. The purity and identity of the synthetic peptides were confirmed by HPLC and by electrospray ionization mass spectroscopy. Mass spectroscopy was performed at the Central Analysis Center, Tokyo University of Pharmacy and Life Sciences (Hachioji, Japan).

The ability of bmGSTT to metabolize each insecticide was determined by high performance liquid chromatography (HPLC) [14] (link). Reaction mixtures (500 µL) contained 120 µM PM, bmGSTT (12 µg), and 5 mM GSH in 50 mM Tris-HCl buffer at pH 8.0. Dehydrochlorinase activity for 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) was assayed by incubating the purified bmGSTT with 0.1 mM DDT and 5 mM GSH in 20 mM Tris buffer (pH 8.0) at 30°C for 2 h. DDT and its metabolites were analyzed by HPLC, as described below, according to a previous report [14] (link), [30] (link).
Reaction mixtures were extracted with three 500 µL portions of ethyl acetate for analysis by HPLC. After removing ethyl acetate, the amounts of each insecticide were determined by HPLC. An HPLC instrument (Prominence, Shimadzu Corp., Kyoto, Japan) was fitted with a 250×4.6 mm Mightysil RP-18 column (Kanto Chemical Co., Inc., Tokyo, Japan) with a flow rate of 1.0 mL/min at 40°C. The mobile phases employed were methanol (MeOH)/acetonitrile/H₂O (70/15/15, v/v/v), MeOH/0.1% acetic acid (70/30, v/v), and MeOH/0.1% acetic acid (85/15, v/v) for detection of DDT, chlorfenapyr (CP), and permethrin (PM), respectively. The concentrations of each insecticide were determined from the corresponding peak areas identified by its authentic sample.

High-performance liquid chromatography (HPLC) was employed to determine the isoflavone content in the FPE using daidzin, daidzein, genistin, and genistein (Sigma-Aldrich) as the standards for quantification. The liquid sample of FPE for analysis was prepared as stated in Section 2.5. The HPLC system was equipped with a UV–VIS detector (Hitachi Chromaster 5420 UV–VIS detector, Hitachi, Ltd., Tokyo, Japan) at 260 nm and a Mightysil RP-18 column (5 μm, 250 mm × 4.6 mm, Kanto Chemical Co., Tokyo, Japan). The HPLC conditions followed the method of Yu and Yang (2019) with a slight modification [16 (link)]. The mobile phase of 0.1% (v/v) trifluoroacetic acid (solvent A) and acetonitrile (solvent B) (Merck, Darmstadt, Germany) was set at a flow rate of 0.8 mL/min with the gradient of solvent A as follows: 90% at 0–10 min, 90–45% at 10–35 min, 45–90% at 35–45 min, and 90% at 45–55 min.

HPLC-FLD for TTX analysis was conducted as described previously [55 (link)]. An Alliance 2690 Separation Module (Waters) connected with the Waters 2487 fluorescence detector. A Mightysil RP-18 column (4.6 mm i.d × 250 mm, particle size 5 µm, Kanto Chemical Co., Inc) was used. The mobile phase for the TTX and TTX analogs was 2 mM heptanesulfonic acid in 10 mM ammonium phosphate buffer (pH 7.0) at a flow rate of 1 mL/min. The eluate was continuously mixed with 4 M NaOH and heated at 110 °C. The intensity of the fluorescence was measured at 505 nm with 384 nm excitation. The same TTX standard was used to compare the retention time of TTX and TTX analogs. The LOD of TTXs was 0.02 μg/g tissue (S/N = 3) and the LOQ of TTXs was 0.06 μg/g tissue; (S/N = 10).

The KSHPH was fractionated using a LC-20AD HPLC system (SHIMADZU Corporation, Kyoto, Japan) of first-stage RP-HPLC on a TSKgel ODS-80TM column (250 × 4.6 mm, Tosoh Corp., Tokyo, Japan). The sample was injected at a volume of 20 µL. The column was pre-equilibrated with eluent A (water containing 0.1% TFA) for 10 min, then peptides were eluted with a linear gradient of eluent B (ACN containing 0.1% TFA) at a flow rate of 1.0 mL/min. On-line UV absorbance scans were performed at 220 nm. Major fractions were collected, lyophilized, and then their antibacterial and ACE inhibitory activities were determined. The fractions which showed strongest antibacterial and/or ACE inhibitory activities, were further separated by second-stage RP-HPLC on a Mightysil RP-18 column (150-4.6, 5 µm, Kanto Chemical Co., Inc., Tokyo, Japan). The elution was conducted at a flow rate of 0.5 mL/min using a linear gradient of ACN containing 0.1% TFA. The elution peaks were detected at a wavelength of 220 nm. After activity tests, the fractions with highest antibacterial and/or ACE inhibitory activities were collected, pooled and lyophilized, respectively. The purity of the active fractions was analyzed by using the same analytical column in the second step of RP-HPLC.

The analysis was performed by a Primaide 1110 pump, a Primaide 1410 UV detector, and a Primaide 1210 auto-sampler (Hitachi, Tokyo, Japan). A Mightysil RP-18 column, 4.6 × 250 mm, 5 μm (Kanto Chemical Co. Inc., Tokyo, Japan) was used. The mobile phase was methanol-Milli Q water (60:40, v/v, pH 3 adjusted by orthophosphoric acid), filtered through a 0.22 μm Millipore filter. The flow rate was 0.6 mL/min, and the sample injection volume was 20 μL. Detection was performed at a wavelength of 288 nm for Sal. B and chloramphenicol (internal standard) at room temperature and detected at a retention time of 6.0 ± 0.1 min and 8.3 ± 0.1 min in Sal. B and chloramphenicol, respectively. The Sal. B standard curve showed power regression at a concentration of 0.1 μg/mL to 10 μg/mL. The limit of detection (LOD) and limit of quantification (LOQ) of Sal. B were 0.1 μg/mL. The inter- and intra-day assay accuracy (% error) and precision (% coefficient of variation (CV)) were −2.6% to −13.2% and 0.8% to 5.1%, respectively, for Sal. B at a concentration of 0.1 μg /mL (Figure S1).