Tskgel ods 100v

To observe changes in the appearance and pH and for high-performance liquid
chromatography (HPLC), we first slowly added 0.4 mL of each sample dropwise to
8.4 mL of PPN solution, and we observed the visual changes (color tone/turbidity)
immediately after the dropwise addition. After recording changes in appearance, we stirred the
mixture for 30 sec using a vortex mixer and measured the pH and midazolam concentration
of each sample. The pH was measured using a pH meter (F-72 pH/ION Meter, HORIBA, Ltd.). The
midazolam content was analyzed through HPLC with reference to Kobo’s method,⁸ under the following conditions: column, TS Kgel ODS
- 100 V (5 μm, 4.6 mm×15 cm, Tosoh Corp.); column temperature, room
temperature, flow rate, 0.8 mL/min; detector, UV - 2075 Plus intelligent UV/Vis detector
(JASCO Corp.); detection wavelength, 254 nm, mobile phase, 1 mM citrate-phosphate
buffer (pH 5.0): methanol: acetonitrile=1:1:1 (V/V). The measurement was performed using
an absolute calibration curve method using a midazolam injection solution as a standard
solution. The calibration curve showed linearity in the range 3.1–50.0 μg/mL. We
determined the relationship between the midazolam content as measured through HPLC and the
midazolam concentration of the mixture by fitting the approximate curve. We performed a
Pearson’s regression analysis to determine the correlation.

Metabolite analyses were performed as described by Sugimoto et al. (2014) (link). Briefly, leaf metabolites were extracted by adding 3 × volume of MeOH containing 1 μg mL^–1 formononetin to ca. 100 mg of ground frozen leaf powder. The extract was injected into the LC-MS (3200 QTRAP, SCIEX, Framingham, MA, United States) or LC-high resolution-mass spectrometry (LC-HRMS; Finnigan LTQ-FT, Thermo Fisher Scientific, Waltham, MA, United States). Chromatographic separation was performed using 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient program was started with 20% B for the initial 10 min, 20 to 50% for the next 10 min, followed by an increase from 50 to 95% in 20 min, and a constant 95% B for the final 5 min, with a constant flow rate of 0.2 mL min^–1 and with an ODS column (Mightysil RP-18, 5 μm, 2 mm × 150 mm, Kanto Chemical, Tokyo, Japan) for LC-MS analysis. The gradient program for LC-HRMS analysis was started with 10% B, followed by an increase to 50% in 50 min, then 90% in 10 min followed by a constant 90% B for the final 5 min with a constant flow rate at 0.5 mL min^–1, and the separation was achieved with an ODS column (TSK-gel ODS-100V, 5 μm, 4.6 mm × 250 mm, TOSOH, Japan).

The NTBC contents in the collected effluents and eluents were determined using a high performance liquid chromatograph (HPLC; Merck Hitachi, Tokyo, Japan) equipped with an L-7100 pump, a D-7000 interface, a diode array detector (DAD) L-4500A, an autosampler L-7200, and a column-thermostat Jetstream 2 Plus column. TSKgel ODS-100V (octadecyl, 150 mm × 4.6 mm, 5 µm particle size, TOSOH Bioscience, Tokyo, Japan) was used as the chromatographic column at 24 °C. The mobile phase consisted of ACN (A) and 0.05% TFA in water (B) in isocratic mode (80% A), with a flow rate of 0.5 mL/min and an analytical time of 10 min. The injection volume was 20 μL, and quantitative analysis was performed at the analytical wavelength λ_MAX = 271 nm. The chromatogram of NTBC (c = 20 μg/mL) was presented in Figure 8.
The chromatographic analyses of effluents and eluates were performed in three replicates (n = 3).

Tramadol, ODT, NDT, NODT, and IS in human plasma were separated using a validated LC system (UFLC_XR, Shimadzu Corporation, Kyoto, Japan). The LC system consisted of a CBM-20A system controller, DGU-20A_5R degasser, LC-20AD_XR pump, SIL-20AC_XR autoinjector, and CTO-20AC column oven. Separation was performed using a 3-μm particle ODS column (TSKgel ODS-100 V, 150 × 2.0 mm I.D., Tosoh, Tokyo, Japan) with a guard column (TSKguardgel ODS-100 V, 3 μm particle size, 10 × 2.0 mm I.D., Tosoh). The mobile phase consisted of methanol and 0.15 % formic acid in water (35:65, v/v). The flow rate was 0.2 mL/min and the column temperature was set at 40 °C, and the autoinjector was set at 4 °C. The injection volume was 10 μL.

Supernatants of slr0688i cells were collected by centrifugation at 2500 × g for 10 min at room temperature. Ten milliliter of the supernatant was filtered through a 0.22 µm pore size polyvinylidene difluoride membrane, then frozen by liquid N₂, and lyophilized. The lyophilized material was dissolved in 200 µL of distilled water. Ten microliter of the dissolved sample was mixed with 10 µL of acetonitrile and 180 µL of 10 mM borate-NaOH buffer at pH 8.7, and analyzed by LCMS-IT-TOF (Shimadzu, Kyoto, Japan). The standard NADPH-Na was purchased from Nacalai Tesque, Inc (Kyoto, Japan), and 10 µL of 25 µM NADPH solution was mixed with acetonitrile and borate-NaOH buffer as described above. The LC settings were as follows: Column, TSKgel ODS-100V (Tosoh, Tokyo, Japan); flow rate, 0.3 mL/min; eluent A, 25 mM ammonium formate at pH 6.0; eluent B, 60% acetonitrile. The timetable of the eluent A/B gradient was as follows: 0 min, 3%; 3.70 min, 5%; 3.71 min, 12%; 6.95 min, 30%; 7.95 min, 60%; 7.96 min, 95%. Mass-spectrometry measurements were performed with electrospray ionization in positive ion mode, under dissociation line temperature at 200 °C. Mass spectra were acquired in the mass rage of 100–1200 m/z. Data collection was performed with LCMSsolution Version 3.80; data analysis was performed with Microsoft Excel for Microsoft 365 MSO Version 2202.