Dgu 20a3r

Berberine, 3c and 6k of 1 mg were dissolved in three centrifugal tubes (5 ml) containing phosphate buffer (pH 7.4)-saturated 1-octanol solution (2 mL), followed by the addition of 1-octanol saturated PBS (2 mL). Each tube was vigorously mixed for 1 min and then shaken for 6 h at a speed of 1000 rpm at 37 °C. The mixtures were then centrifuged. 1 mL of 1-octanol phase and 1 mL of buffer were separately collected. Each phase was diluted to a suitable concentration and analysed by HPLC (Shimadzu DGU-20A3R) on a Shimadzu-GL WondaSil C18-WR column (λ = 254 nm) with an eluent of methanol/water (65/35, v/v, 1.0 ml/min). The log D value for each compound was calculated from the ratio of the peak areas using the following equation: Log D = log (Area_oct × x/Area_buf × y). x, y is the dilution factor.

Several Shimadzu LC units (Tokyo, Japan), including an online vacuum degasser (DGU-20A3R), three pumps (LC-20AD_XR), an auto-sampler (SIL-20AC_XR), a column oven (0–85 °C, CTO-20 A), two electronic 2-channel/6-port valves (FCV-12AH), and a controller (CBM-20 A), were used. An ABSciex 5500 Qtrap mass spectrometer (Foster City, CA, USA) equipped with a Turbo V^TM electronic spray ionization (ESI) interface enabled quantitative measurements, while a Shimadzu IT-TOF-MS mounted ESI source was used for high-resolution mass spectrometry.

An ICPMS-2030 mass spectrometer (Shimadzu, Tokyo, Japan), directly coupled with a Prominence LC 20Ai inert system, was used for creating the new speciation method. The LC inert system allowed us to eliminate the possibility of metal background leaching from the mobile phase and enabled us to obtain the lowest possible detection limit, which is required for speciation analysis. The ICP–MS operated at 1.2 kW with 8.0 L·min⁻¹ Ar plasma gas flow, 0.7 L·min⁻¹ nebulizer Ar gas flow, and 1.1 L·min⁻¹ auxiliary Ar gas flow. The concentric (MicroMist) nebulizer with 0.6 mL·min⁻¹ (carrier) argon gas flow was used for nebulizing the HPLC eluate. The sampling depth was set to 5.0 mm. The spray chamber temperature was set to 3 °C. Optimized conditions of the collision cell were −90 V of cell gas voltage, 7.0 V of energy filter voltage, and a 9.0 mL·min⁻¹ cell gas (He) flow rate. The HPLC was equipped with binary pumps LC 20Ai, an autosampler (SIL 20AC), a vacuum degasser (DGU20A3R), a heated column compartment (CTO 20AC), and a controller (CBM 20A) (Shimadzu, Japan). Polypropylene autosampler vials were used, because of the possibility of contamination of the samples with arsenate if using glass vials. The vials were cleaned with dilute nitric acid and thoroughly rinsed with ultrapure deionized water (UPW) (Merck, Kenilworth, NJ, USA) [55 (link)].

The HPLC–DAD analysis of the crude extract and all collected fractions was performed on a Shimadzu HPLC (Tokyo, Japan) equipped with a degasser (DGU-20 A 3R), binary pump (LC-20AD), autosampler (SIL-20 AHT) and DAD detector (SPD-M20 A). The separations were carried out on a Zorbax Eclipse XDB C18 stainless-steel (250 × 4.6 mm, 5 μm) column, following the conditions described by Luca et al. [34 (link)]. Rutamarin content in the crude extract was determined by using the same method with the help of a calibration curve plotted with the corresponding standard (linearity range: 25–250 μg/mL, regression equation: y = 19373x − 31740,regression coefficient R²= 0.9998).

The HPLC analysis was conducted with a Shimadzu LC-20A Prominence Series system (Shimadzu Corporation, Kyoto, Japan) equipped with a quaternary pump (LC-20AD), vacuum degasser (DGU-20A3R), autosampler (SIL-20A), column oven (CTO-20A), and photodiode-array detector (SPD-M20A). The chromatographic data were interpreted using LabSolutions Multi-PDA software. Chromatographic separation was performed on a YMC Triart C18 column (4.6 × 250 mm i.d., 5 μm). The column oven was maintained at 40°C, the detection was conducted at λ = 207 nm, and online UV absorption spectra were recorded in the range of 190 to 400 nm. The gradient elution was performed under the following conditions: initial mobile phase, acetonitrile/0.1% trifluoroacetic acid in water = 15 : 75 (v/v), 0∼15 min; 15 : 75 to 35 : 65 (v/v), 0∼10 min; 35 : 75 to 50 : 50 (v/v), 10∼20 min; 50 : 50 to 100 : 0 (v/v), 20∼25 min; and 100 : 0 (v/v), 25∼30 min. The flow rate was 1.0 mL/min, and the injection volume was 5 μL.