Varian prostar 335

(⁶⁸Ga)Ga-DOTA-TATE was synthesized by a fully automated, good manufacturing practice (GMP)–compliant procedure using a cassette-based synthesizer (SCINTOMICS GmbH, Fürstenfeldbruck, Germany) connected to a ⁶⁸Ge/⁶⁸Ga-generator (1.2 GBq Ge-68/Ga-68 Generator, itG Isotope Technologies Garching, Garching, Germany) and equipped with a disposable single-use cassette kit (ABX GmbH, Radeberg, Germany). A standardized labeling sequence with 25 μg (17.4 nmol) of unlabeled DOTA-TATE acetate (GMP) (ABX GmbH, Radeberg, Germany) was used. For quality control, (⁶⁸Ga)Ga-DOTA-TATE was analyzed by analytic high-performance liquid chromatography according to the monographs 2462 (Gallium Chloride) and 2482 (Gallium Edotreotide) of the European Pharmacopoeia. Radioanalytic high-performance liquid chromatography was performed on a Varian ProStar high-pressure gradient system equipped with an ultraviolet-visible detector (Varian ProStar 335) and a radiodetector (Berthold LB 3800-20 with LB 6657 probe) using a RP-18 column (Gemini C18 5µ 110A, 250 × 4.6 mm; Phenomenex, Torrance, CA, USA). The eluent had a linear gradient from 100% solvent A (phosphate buffer (pH 2.5)/acetonitrile, 85:15) to 100% solvent B (phosphate buffer (pH 2.5)/acetonitrile, 65:35) over 25 min at a flow of 0.6 mL/min.

Quantification of the major E. alba callus phytochemicals was performed according to Khurshid et al. [26 ]. Briefly, RP-HPLC was used for separation and quantification, equipped with chromatographic Varian liquid system, autosampler Varian Prostar 410, Metachem Degasit, Photodiode Array Detector Varian Prostar 335, Varian Prostar 230 pump. Galaxie software 1.9.3.2 version was used to control the system. At 35 °C separation temperature, Purospher (Merck) RP-18 column was used to perform separation. Mobile phase consisted of two solvents i.e. solvent A (acetic acid (0.2%) in water) and solvent B (acetic acid (0.2%) in methanol). At 0.8 ml/min flow rate during non-linear gradient run, mobile phase composition varied from 0 to 40 min of A–B: 90:10 to 30:70 (v/v), from 41 to 50 min of A–B: 30:70 to 0:100 (v/v), and A–B: 0:100 (v/v) from 51 to 60 min. Compounds of E. alba were detected at 204 nm. Standards (β-amyrin, stigmasterol, coumarin and wedelolactone) were purchased from Sigma-Aldrich. Quantification of eclalbatin and desmethylwedelolacetone were done using the calibration curves of β-amyrin and wedelolactone respectively and were expressed as β-amyrin and wedelolactone equivalent concentration respectively.