To flash chromatography with dichloromethane and methanol (9:1) solvent method to elute the purified products. All of the lipid prodrugs had been recrystallized with cold diethyl ether precipitation. Characterization HPLC–A high efficiency liquid chromatography (HPLC) system comprising Shimadzu LC 20AT pump (Shimadzu, Lenexa, KS), equipped having a fluorescence detector (Perkin Elmer, Series 200 fluorescence detector, Shelton, CT) and a reversed-phase C12 column (4 m, 250 mm 4.six mm, Synergy-max, Phenomenex, Torrance, CA) was utilized. Mobile phase comprised of 15 mm phosphate buffer (pH two.five) and 35 acetonitrile pumped at a flow rate of 1 mlmin-1. All samples had been analyzed at an excitation wavelength of 265 nm and at an emission wavelength of 380 nm. Mass spectrometry–Mass of GCV and its lipid prodrugs (mono- and di-(O-acyl) conjugated) was determined with MDS Sciex API 3200 Triple Quadruple linear QTrap mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA) by infusing the answer having a good ion source for detection.Syntide 2 manufacturer The compounds have been dissolved in HPLC grade methanol and injected at a flow rate of 15 l min-1 into the electrospray supply using a Hamilton 22 syringe pump.X-GAL web The mass spectra have been acquired at 5 s per scan.PMID:24211511 The capillary temperature of 250 and spray voltage of 3.5 kV have been applied. Dry nitrogen was made use of as sheath and auxiliary gas for analysis.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAdv Ophthalmol Vis Syst. Author manuscript; available in PMC 2014 October 30.Cholkar et al.PageMelting point and partition coefficient–Melting point for prodrugs was determined with open capillaries employing melting point instrument (MEL-TEMPII laboratory devices, USA). Briefly, a small quantity of GCV or purified lipid prodrug was transferred to glass capillary tubes and was permitted to settle in the bottom of capillary tube, by tapping. The temperature variety exactly where the strong GCV or prodrug started to melt and entirely turned to liquid was recorded as melting point. Octanol/water partition coefficient was determined with ACD labs computer software. NMR spectroscopy–All the synthesized prodrugs spectra have been recorded on Varian 400 MHz proton (1H) and carbon (13C) nuclear magnetic resonance (NMR) spectrometer (Varian, USA) in deuterated dimethyl sulfoxide (DMSO d6). Tetramethylsilane (TMS) served as internal normal along with the chemical shifts are expressed in parts per million (ppm) relative to TMS. A selection of 5000 6000 scans was accumulated for every single spectrum. Cell culture–In vitro cytotoxicity of novel lipid prodrugs was evaluated with Cell Titer 96Aqueous Non-Radioactive cell proliferation assay kit (Promega, Madison, WI) on human retinal pigment epithelial cell line (ARPE-19 was bought from American Variety Culture Collection (Manassas, VA) at passage number 21). ARPE-19 was cultured and maintained as reported previously [22]. Briefly, ARPE-19 cells were grown inside a culture medium containing DMEM supplemented with 10 (v/v) FBS (heat inactivated), 29 mM NaHCO3, 20 mM HEPES, 100 mg of penicillin and streptomycin each, and 1 nonessential amino acids at pH 7.4. Cells had been grown at 37 , in a humidified atmosphere of 5 CO2 and 90 relative humidity. The growth medium was replaced with fresh medium each other day. Cells with passage number 25 have been cultured in flasks and harvested at 80-90 confluency with TrypLETM Express (a superior replacement for trypsin) (Invitrogen, Carlsbad, CA, USA). Cells have been then plated in 9.