Capcell pak c18

One hundred grapes were detached, counted and weighed to obtain their average berry mass (BM). The TSC and TA were determined by the OIV (2017) method OIV (International Organisation of Vine and Wine) (2017) . The TSS was determined with a hand-held digital Atago PAL-1 m (Atago Co. Ltd., Japan) and indicated by the Brix°. The pH was measured with a Mettler Toledo FE20 Desktop pH Meter (Mettler Toledo Instruments Co., Ltd., Shanghai, China).
The determination of the organic acids in grape fruits by HPLC was modified based on Gao et al. (2004) . HPLC detection (high-performance liquid chromatography, Waters 2699, USA) was conducted using a C18 chromatographic column (4.6 × 250 mm, 5 μm; CAPCELL PAK C18; Shiseido, Japan). We utilized mobile-phase (A) 0.02 M dipotassium phosphate (pH adjusted to 2.3 using phosphoric acid) and mobile-phase (B) methyl alcohol. Mobile-phase A:B = 99:1 was used for isometric elution at a flow rate of 0.5 mL/min. The retention time was used to identify the components of organic acids, and the concentration of organic acids in samples was calculated by external standard method (Destandau et al., 2005 (link)). All 11 standard materials (oxalic acid, tartaric acid, quinic acid, malic acid, shikimic acid, lactic acid, acetic acid, citric acid, fumaric acid, succinic acid and propionic acid) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The solution was then transferred to HPLC vials for injection onto an HPLC system. The HPLC system consisted of an LC-20AD solvent delivery unit (Shimadzu Corporation, Kyoto, Japan) equipped with an SIL-20AC autosampler (Shimadzu Corporation) and an SPD-20AV UV detector (Shimadzu Corporation) set at a wavelength of 206 nm. Samples were injected onto a SHISEIDO CAPCELL PAK C18 (particle size 5 µm, 250 mm × 4.6 mm; SHISEIDO, Tokyo, Japan) and eluted at 1 mL/min with a mobile phase comprising buffer:acetonitrile at a ratio of 98:2. The buffer was prepared from 10 mM KH₂PO₄ and H₃PO₄ at pH 2.5.

Mixture of HDE (90%, 16.7 µL, 120 µmol), tBuOOH (70%, 2 µL, 15 µmol), acetonitrile (300 µL), and 100 mM phosphate buffer (pH 7.4, 900 µL) in a capped Eppendorf tube was incubated at 50 °C with occasional mixing to make an emulsion. After 36 h, 100 µL of the mixture was injected into the HPLC column. Conditions were basically the same as above, except for the elution condition, a linear gradient of ethanol concentration in water, 0–60 min, 15–50%; 60–80 min, 50% (Method 2).
The measurement conditions for mass spectrometry were the same as those for the ipG-adducts, except for the conditions below. Mobile phase A was water, and mobile phase B was methanol. Data were acquired in parallel reaction monitoring (PRM) mode.
For assay of ε-ipG formation, 100 µL of each fraction was mixed with ipG solution (250 μg ipG, 25 μL acetonitrile, 75 μL 100 mM phosphate buffer, pH 7.4), either at 50 °C or 37 °C with tBuOOH (70%, 2μL). After 20 h, 50 μL of each mixture was injected into the HPLC column (Column, Shiseido Capcell Pak C-18, 4.6 × 150 mm, 3 µm), Elution, linear gradient of ethanol concentration in 10 mM ammonium formate, 0–30 min, 15–50% (Method 3).

2,4-heptadienal (90%), 2,4-decadienal (90%), t-butylhydroperoxide (tBuOOH) (70%), hydrogen peroxide (30%), chloroacetaldehyde, and MnO₂ were purchased from Tokyo Chemical Industry Co., Ltd. Japan. Hemin, 2’,3’-O-isopropylideneguanosine (ipG), and calf thymus DNA were purchased from Sigma-Aldrich Chemical Co., USA. Cooking oil, composed of soybean and rapeseed oil, was purchased from a grocery store. The commercial ipG was purified by semi-preparative high-performance liquid chromatography (HPLC, Shiseido Capcell Pak, C-18,10 × 250 mm), using 30% aqueous ethanol for elution. Authentic samples of 8-OH-ipG and ε − ipG were prepared using the methods described in the literature [12 (link)] and [15 (link)], respectively. 1,N²-ethenoguanin (εGua) was obtained by acid hydrolysis (0.1N HCl, 37 °C, 16 h) [16 (link)] of 1,N²-etheno-2’-deoxyguanosine as described [15 (link)].

The HPLC analysis was performed using the Shimadzu HPLC system (Nexera XR; Shimadzu Corp., Kyoto, Japan). The parameters for the HPLC analysis were as follows: column: CAPCELL PAK C18 (Shiseido, Japan); size: 4.6 mm (φ) × 250 mm, detector: ultraviolet (UV) detection at wavelength 280–285 nm; mobile phase: 40% (v/v) acetonitrile/0.1% aqueous phosphoric acid solution; flow rate: 0.4 mL/min; column temperature: 70 °C. The autosampler was used and the injection volume (5–20 μL) differed depending on the assay. Standard hesperidin and HEPT7G were used to validate the HPLC method. The analysis was performed in duplicate, as reported elsewhere (Moriwaki, Kumoi, and Ozeki, 2019).

The alkaloid fraction, the water fraction, matrine, and oxymatrine were analyzed using a Thermo Scientific LC/MS system (Thermo Fisher Scientific, Waltham, Massachusetts, USA), which consisted of an Accella-600 HPLC system with photodiode array detection and an LTQ Orbitrap XL Mass Spectrometer. The LC/MS analysis conditions were as follows. The HPLC conditions: solvent A, 0.1% formic acid in H₂O; solvent B, 0.1% formic acid in MeCN; column, Capcell Pak C-18 (1.5 mm i.d. × 150 mm; Shiseido, Tokyo, Japan); flow, 0.20 ml/min; gradient program, a linear gradient of 2–35% of solvent B in solvent A for 15 min followed by a linear gradient of 35–90% of solvent B for 5 min, and then maintenance of 90% solvent B in solvent A for 5 min; column temp., 25°C; and photodiode array detection, 200–600 nm. The MS conditions: electrospray polarity, positive mode; sheath gas flow, 50 arbitrary units; aux gas flow, 30 arbitrary units; ion spray voltage, 4.6 kV; capillary temp., 350°C; capillary voltage 19.0 V; tube lens, 35 V; and detector, an Orbitrap detector with a high resolution of 60,000.

Female bees of Xylocopa appendiculata circumvolans were collected at Kami-ichi, Toyama in Japan. The venom sacs from five individuals were dissected immediately after collection and extracted with 1:1 acetonitrile-water containing 0.1% TFA (CH₃CN/H₂O/0.1% TFA), and lyophilized.
The lyophilized extracts were subjected to reversed-phase HPLC (Shimadzu Corp., Japan) using CAPCELL PAK C₁₈, 6 × 150 mm (Shiseido Co., Ltd., Japan) with a linear gradient from 5% to 65% CH₃CN/H₂O/0.1% TFA at a flow rate of 1 mL/min over 30 min (Fig. 1). This process released xylopin and xylopinin eluted at 25.1 min and 26.0 min, respectively.Fig. 1

LC-ESI-MS profile of crude venom extracts of Xylocopa appendiculata circumvolans. About 10% of crude venom extract of a single specimen was subjected to reverse-phase HPLC using CAPCELL PAK C₁₈ (1.5 × 150 mm) with linear gradient of 5–65% CH₃CN/H₂O/0.1% formic acid over 20 min at flow rate of 200 μL/min. a UV absorption by PDA. b Total ion current (TIC). Numbers in B show “virtual” fraction number as in Tables 2 to 6