Symmetry c8

Ten milligram of dry LTA was resuspended in 0.5 ml of chromatography start buffer (15% 1-propanol (Sigma Aldrich) in 0.1 M ammonium acetate, pH = 4.7) and centrifuged at 27,000 g for 60 min. The supernatant was subjected to reverse phase high-performance liquid chromatography (RP-HPLC) (JASCO) on a C8 analytic column (Symmetry C8, 5 μm, 3.9 × 150 mm, Waters) using a linear gradient of 15–60% of 1-propanol in 0.1 M ammonium acetate (pH = 4.7). Two milliliter fractions were collected lyophilized and resuspended again for Western blot analysis. The protocol was slightly modified from (Lehner et al., 2001 (link); Grundling and Schneewind, 2007 (link))

Tissues were ground into powder in liquid nitrogen and extracted with acetone. The samples were centrifuged at 10,000× g for 10 min, and the supernatants (80 µl) were mixed with 20 µl of water. Pigments were analyzed by high-performance liquid chromatography (HPLC) using a reversed-phase column (Symmetry C8, 150×4.6 mm; Waters, Milford, MA, USA) according to Zapata et al. [16] . The analysis was performed in triplicate.

Tissues were ground into powder in liquid nitrogen and extracted with acetone. The samples (80 μl) were mixed with 20 μl of water. Pigments were analyzed by high-performance liquid chromatography (HPLC; X-LC, Jasco) using a reversed-phase column (Symmetry C8, 150 × 4.6 mm; Waters) according to Zapata et al.56 .

Basic procedures for the measurement of content of three chlorophyll precursors, protoporphyrin IX, Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethylester were followed by an HPLC-based procedure as described previously [38 (link)]. Extraction of chlorophyll precursors from seedlings was carried out using the same procedure as for chlorophyll extraction described above. The acetone extracts were applied to a HPLC system (LC10A VP system, Shimadzu) equipped with a reverse phase column (Symmetry C8, 150 mm × 4.6 mm, 3.5μm particle size, Waters, http://www.waters.com/) with a guard column (C8, 4.0 mm × 3.0 mm, Phenomenex, http://www.phenomenex.com/). Chlorophyll precursors were eluted by an anomalous gradient from solvent A (methanol:acetonitrile: 0.25 M pyridine = 50:25:25) to solvent B (methanol:acetonitrile:acetone = 20:60:20). Eluents were monitored with a fluorescence detector (RF-550, Shimadzu). For protoporphyrin IX detection, the excitation wavelength was set at 400 nm and fluorescence emission was detected at 634 nm, while for detections of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethylester, the excitation wavelength was set at 417 nm and fluorescence emission was detected at 600 nm. Content of these precursors was estimated through calibration curves determined from standard substances (Frontier Scientific, http://www.frontiersci.com/).

The HPLC-UV method was used to determine the blood plasma concentrations of sorafenib and its metabolite SR_NO. Analyzes were carried out using the HPLC Waters 2695 Separations Module with an autosampler and the Waters 2487 Dual Absorbance Detector [43 (link),44 (link)]. The supernatant volume for each time point used to prepare the HPLC sample was 20 μL.
An ammonium acetate 0.1 M, pH = 3.4 (eluent A) and acetonitrile (eluent B) (1:1, v/v) solution were used as a mobile phase. The flow rate was set at 1.0 mL/min. The linear gradient ran from 60% eluent A and 40% eluent B to 29% eluent A and 71% eluent B. Other HPLC parameters were as follows: the temperature of the column (a reversed-phase Symmetry^® C8, 250 × 4.6 mm, 5.0 μm particle size, Waters Corporation^®, Milford, MA, USA) was 25 °C, the UV detection wavelength was 265 nm, the injection volume was 20 μL. Lapatinib was used as the internal standard (IS). The calibration curves were linear within the range of 0.025−3.0 µg/mL (R² = 0.999), and 0.025−0.40 µg/mL (R² = 0.999), for sorafenib and SR_NO respectively. The high intra- and inter-day precision (coefficients of variation, CV < 13%) and accuracy (%bias < 14%) for sorafenib, and SR_NO were obtained. The lower limit of quantification (LLOQ) was 0.025 µg/mL for both sorafenib and SR_NO.

The levels of SN-38 eluted from the SMPs were characterized by an in vitro elution method. Fifteen milligrams of SMPs were placed in a test tube (N = 3) consisting of 1 mL of phosphate-buffered saline (PBS). The volume of PBS used here was to simulate that of the approximate brain fluid in the rats. The follow-up was placed in an incubator shaker (Deng-Yng Incubator E600, Taipei, Taiwan) with a speed of 100 rpm at 37 °C for 24 h, the eluent was collected and replaced by 1 mL of fresh PBS, for the following 24 h period. The procedure was carried out daily for eight weeks. The concentration of SN-38 in the eluent was examined utilizing a Hitachi L-2200 HPLC analysis (Multisolvent Delivery System, Tokyo, Japan). A Symmetry C8 (5 μm, 4.6 × 250 mm, Waters) column was used to separate the SN-38. The mobile phase contained NaH₂PO₄ (25 mM, pH = 3.1): acetonitrile (Sigma-Aldrich, Saint Louis, MO, USA) (50/50, v/v). The absorbency was monitored at a wavelength of 265 nm and a flow rate of 1.0 mL/min. The retention time was 3.62 mins. All experiments were performed in triplicates and the samples were diluted to bring the unknown concentrations into the range of the assay standard curve. A calibration curve was obtained for each set of measurements (correlation coefficient > 0.99). The eluted concentration was characterized employing the highly sensitive HPLC system.