Capcell pak c18 mg column

Samples analysis was carried out on an Agilent 1260 HPLC series liquid chromatography system (Agilent Technologies, Santa Clara, USA), which is equipped with a binary pump, a diode-array detector, an autosampler, and a column temperature controller. Chromatographic separation was performed on a Shiseido Capcell PAK MG-C₁₈ column (250 × 4.6 mm ID, 5 μm; Shiseido, Japan) with the column temperature maintained at 30°C. The mobile phase consisted of acetonitrile (A) and 0.2% formic acid (B) with a linear gradient elution program (0–5 min, 1%-1% A; 5–15 min, 1%–10% A; 15–55 min, 10%–30% A; 55–65 min, 30%–35% A) at a flow rate of 1 mL/min, and the mobile phase was degassed automatically using an electronic degasser system. The injection volume was 10 μL. The detector wavelength was set at 270 nm.

The method described by XU (29 (link)) was used with appropriate modifications to detect isoflavones in soybeans. Homogenates of the samples (1.00 g each) were put into conical flasks with 30 ml of 70% methanol. After ultrasonic treatment for 30 min, the samples were shaken for 2 h in the thermostatic oscillator (60°C). When the conical flasks were cooled to room temperature, the volume was adjusted with 70% methanol to 50.0 ml and centrifuged at 2,795 × g for 10 min. The supernatant was filtered through a 0.45-μm filtration membrane before being subjected to HPLC.
Isoflavones were separated using the Capcell PAK MG C18 column (250 × 4.6 mm i.d., 5 μm, Shiseido, Japan) with a mobile phase of 0.1% aqueous phosphoric acid (A) and acetonitrile (B). The gradient elution conditions were as follows: 18% phase B from 0 to 5 min, 18–32% phase B from 5 to 6 min, and then a holding time of 6–13 min; 32%−30% B from 13 to 14 min, and then a holding time of 2 min; 30–80% phase B from 16 to 17 min, and then a holding time of 3 min; 80–18% phase B from 20 to 21 min followed by a holding time of 21–25 min. The flow rate was set at 1.0 ml/min. The injection volume was 10 μl. The temperature of the column was set at 34°C. The UV detection wavelength was 260 nm.

Samples were de-proteinized with 0.4 mol l⁻¹ perchloric acid and centrifuged at 15,000 g for 20 min at 4 °C. Then, the supernatant was removed and filtered through a 0.22-μm polyvinylidene difluoride membrane. Analysis of histamine in the supernatant was performed by HPLC (ESA, Chelmsford, MA, USA). The system consists of model 582 pump and four channel CoulArray electrochemical detector. After reacting with the derivate o-phthalaldehyde, analytes were separated on a 3-μm, 3 × 50 mm² Capcell Pak MG C18 column (Shiseido, Japan). A two-component gradient elution system was used, with component A of the mobile phase being 100 mmol l⁻¹ Na₂HPO₄, 13% acetonitrile and 22% methanol, pH 6.8, and component B being similar to A except with 5.6% acetonitrile and 9.4% methanol. A gradient elution profile was used as follows: 0–3.5 min, isocratic 100% B; 3.5–20 min, linear ramp to 0% B; 20–22 min, isocratic 0% B; 22–23 min, linear ramp to 100% B; 23–30 min, isocratic 100% B. The flow rate was set to 0.75 ml min⁻¹. The temperature of the column was maintained at 38 °C. The data were acquired and analysed using CoulArray software.