Capcell pak c18 ug120 column

A series of standard solutions of differing known concentrations of VCM in PBS(-) were prepared and injected into a CAPCELL-PAK C18 UG120 column (5 μm, φ 4.6 mm × h 250 mm; Shiseido, Tokyo, Japan) of an Elite LaChrom HPLC system (Hitachi High-Technologies, Tokyo, Japan) equipped with a L-2455 Diode Array Detector (Hitachi High-Technologies). The HPLC conditions were as follows: column temperature, 30°C; mobile phase A, triethylamine buffer (pH 3.2)/acetonitrile/tetrahydrofuran = 92/7/1 (v/v); isocratic elution, phase A (20 min); flow rate, 1 mL/min; wavelength, 280 nm; and injection volume, 20 μL. The peak area of VCM in each standard solution was measured and plotted against the VCM concentration to generate a calibration curve. The concentration of VCM in each eluate sample was then determined by HPLC using the same conditions as those used to generate the standard calibration curve.

The dried rhizomes of KP (Maechu Co., Ltd., Nara, Japan) were extracted twice with 80% ethanol. After filtration, the solution was evaporated under vacuum to yield dry powder.
The contents of 5,7-DMF and 3,5,7,3’,4’-PMF in the extract were analyzed by high-performance liquid chromatography (HPLC) equipped with a CAPCELL PAK C18 UG120 column (4.6 mm × 250 mm, Shiseido, Tokyo, Japan) with a UV detector at 260 nm. The mobile phase was 1% formic acid: acetonitrile (70:30), and the flow rate was 1.0 ml/min. The HPLC chromatogram of the KP extract is shown in Fig. 1. The total amount of polymethoxyflavones (3,5,7-trimethoxyflavone, 3,5,7,4’-tetramethoxyflavone, 5,7,4’-trimethoxyflavone, 5,7-DMF, and 3,5,7,3’,4’-PMF) in the extracted dry powder was 27.5%, in which 5,7-DMF and 3,5,7,3’,4’-PMF were present at 7.45% and 7.2%, respectively.

The right cerebrum, hypothalamus, and its peripherals were homogenized with 0.1 M perchloric acid and 0.02 mM EDTA 2Na. The extract was centrifuged (12,000 rpm, 4°C, 20 min) and the supernatant was collected as the sample. Brain serotonin and dopamine levels were measured in samples using a Prominence HPLC System (Shimadzu, Kyoto, Japan) equipped with a Coulochem II electrochemical detector (ESA Biosciences, Inc., Chelmsford, MA, USA) and Capcell Pak C18 UG120 column (Φ4.6 × 250 mm, 5-μm particle size, Shiseido Co., Ltd., Tokyo, Japan). The mobile phase consisted of buffer (50 mM Na₂HPO₄, 50 mM citric acid, 4.4 mM 1-heptanesulfonic acid sodium salt, and 0.1 mM EDTA 2Na) / acetonitrile / methanol; 1000/35.2/85.8 (v/v/v) and was delivered at a flow rate of 1.0 mL/min. The setting voltage was fixed C.C. = 0.5V, Det.1 = +0.05V, Det.2 = +0.45V.

We seeded 1–2 × 10⁶ primary cells into a 75 cm² tissue culture flask (Corning Inc.) and incubated the cells overnight. The different primary cells were then incubated with 1 mM 5-ALA for 4 h. The cells were washed twice with PBS and detached from the culture flask by 0.25% trypsin and 1 mM EDTA tetrasodium salt solution and phenol red. The cells were centrifuged, resuspended in PBS to a concentration of 1 × 10⁶/mL, then lysed by sonication for 30 s. For the determination of PpIX, 0.2 mL of homogenate was vigorously agitated for 60 s with 0.02 mL of 50% v/v acetic acid and 0.9 mL of N,N-dimethylformamide-2-propanol solution (100:1 by vol.) and centrifuged at 13,150× g for 5 min at 4 °C to collect the supernatant. The supernatants were analyzed with a high performance liquid chromatography system using a Capcell Pak C18 UG120 column (5 μm, 4.6 × 150 mm, Shiseido, Tokyo, Japan), mobile phase of acetonitrile-10 mM tetrabutylammonium hydroxide solution (pH 7.5) (70:30 by vol., flow rate, 1.0 mL/min; elution temperature, 40 °C), and fluorescence detector (Ex. 400 nm, Em. 630 nm).

We analyzed the MDA levels in plasma, erythrocyte, and urine using an HPLC system (Shiseido, Tokyo, Japan) equipped with a fluorescence detector (emission = 527 nm, excitation = 551 nm). A Capcell Pak C18 UG120 column (4.6 mm × 250 mm, 5 μm particle size; Shiseido) was used for separation under isocratic elution with 50 mM phosphate buffer [19 (link),20 (link),21 (link),22 (link)].