Helpful As well as Stunning ISRIB Strategies

Twenty-five batches of Radix Isatidis from different geographical regions around China were obtained from three pharmaceutical companies producing the same TCM preparations using Radix Isatidis as raw material. All samples were morphologically authenticated by the authors and voucher specimens were deposited with the Department of Chemistry, Tsinghua University, Beijing, China. The root of Isatis indigotica Fort. were pulverized and approximately 0.5?g of ground sample accurately weighed and extracted by ultrasonication (44?kHz, 250?W) with 5?mL water for 20?min at room temperature. The extracts were then filtered through 0.2?��m PTFE syringe filters and 10?��L of each filtrate subjected to HPLC�CDAD�CESI/MS analysis. Stock solutions (ca. 1.0?mg/mL) of the six reference compounds viz uridine, adenine, adenosine, cytidine, guanosine and R,S-goitrin were prepared in 50% methanol and stored at 4?��C until required. Equal volumes of these six stock CYTH4 solutions were mixed and diluted with 50% methanol to obtain a series of seven mixed standard solutions. The mixed standards were filtered through 0.2?��m PTFE syringe filters and 10?��L injected into the HPLC system. HPLC was performed on an Agilent 1200 series system (Agilent Series 1200, Palo Alto, CA, USA) consisting of a binary pump, autosampler and diode array detector. After scanning in the range 200�C400?nm, a wavelength of 254?nm was selected for qualitative and quantitative analysis. Chromatography was performed on a Phenomenex Luna C18 analytical column (250?mm��4.6?mm, 5?��m) at 25?��C using gradient elution at 0.8?mL/min of (A) water and (B) methanol according to the following find more program: 0�C10?min 5% B; 10�C25?min 5�C15% B; 25�C34?min 15�C35% B; 34�C40?min 35�C95% B. The column was re-equilibrated for 10?min between individual runs. Accurate mass determination to generate empirical formulae was performed on an Agilent 1100 Series LC/MSD TOF (Agilent Technologies Corp., Santa Clara, CA, USA) using electrospray ionization (ESI) in both positive (ESI+) and negative ISRIB concentration (ESI?) ion modes. HPLC effluent was introduced into the mass spectrometer with a post-column split of 3:1. Optimized ESI-MS parameters were: capillary voltage 3500?V (ESI?) or 4000?V (ESI+); drying gas 8.0?L/min; nebulizer pressure 40?psi; gas temperature 325?��C; fragmentor voltage 175?V (ESI+) and 190?V (ESI?); skimmer voltage 60?V; octopole dcl 37.5?V (ESI+) or 38.0?V (ESI?); octopole RF 250?V. Full-scan mass spectra were recorded in the range m/z 50�C1500. Continuous calibration was maintained using reference masses of m/z 121.0509 and 922.0098. Analyst QS software (Applied Biosystems, Framingham, MA) was used to process mass data and to generate exact masses corresponding to particular elemental compositions. Instrument tuning was carried out daily using the tuning solution (G1969-85000, Agilent Corp, USA) to ensure a mass error