Agilent 1200 series lc

We referred to the previous method in order to measure paeoniflorin in samples (Yuan et al., 2013 (link)). The dried samples (0.50 g) were weighed and extracted with 50 mL of 50% aqueous methanol with ultrasonication for 30 min. The extracted samples were diluted with 50 mL 50% aqueous methanol and filtered with a 0.45-μm Millipore filter membrane (Millipore, MA, USA) at 25 °C. We used the Agilent 1200 LC Series (Agilent Technologies, Palo Alto, CA, USA) High Performance Liquid Chromatography (HPLC) system to measure the paeoniflorin abundance. The wavelength was set at 230 nm with a flow rate of 1.0 mL/min at a temperature of 25 °C. Standard compounds were purchased from the National Institutes for Food and Drug Control and the linearity of the standard compounds was checked at seven concentration solutions.

The mycelia and filtrate extracts prepared were filtered through a 0.45 µm Millex-HV filter membrane (Millipore, Billerica, MA, USA) before determination of SAL and TYR contents by RP-HPLC analyses based on the method described in Chinese Pharmacopoeia [33 ]. The analytical HPLC system (Agilent 1200 LC Series, Santa Clara, CA, USA) was equipped with a quaternary pump with vacuum degasser, an Eclipse XDB-C₁₈ (4.6 × 250 mm, 5 µm, Agilent), a 10 µL manual injector, a thermostatted column compartment, and a UV detector. The mobile phase consisted of a methanol/water mixture (30:70) at a flow rate of 1.0 mL/min at 30 °C and monitored using a UV detector at 276 nm. HPLC-grade methanol was purchased from Fisher Scientific (Pittsburgh, PA, USA). Ultrapure water was prepared using an ultrapure water system from Arium Comfort I (Sartorius, Göttingen, Germany). The SAL and TYR standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Detections of SAL and TYR in fungal extracts were based on their retention times. Quantifications of SAL and TYR were done using a linear regression equation fitted in a calibration graph as y = 0.88325x + 0.44806 (r = 0.99951) and y = 2.59700x + 2.18000 (r = 0.99960), respectively. The concentration ranges of SAL and TYR were 2 µg/mL to 200 µg/mL and 1.6 µg/mL to 160 µg/mL, respectively.

Samples were prepared by a desalting of proteins into 100 mm ammonium acetate, pH 7.0. A 1200 series Agilent LC was used to inject 5 μl of sample into 5% acetonitrile (0.1% formic acid) and desalted inline to release the cofactor from the enzyme complex. This was eluted over 1 min by 95% acetonitrile. The resulting ions were analyzed by an Agilent QTOF 6510 run in positive mode and deconvoluted using Agilent Masshunter software.