Ultimate 3000 hplc apparatus

NLC and UA-NLC nanocarriers were prepared by high-pressure homogenization technique. CP (solid lipid—2%) with the mygliol-812 (liquid lipid—3%) and an aqueous solution of polysorbate 80 were heated to 70 °C, separately. Then, UA (0.1%) was dissolved in the lipid phase and the aqueous phase was added. The emulsion was submitted to a homogenization process in Ultra-Turrax T25, with S25N—18G dispersing element (IKA^®-Labortechnik, Staufen, Germany) at a stirring speed of 10,400 rpm for 5 min. For the NLC formation, the prepared emulsion was quickly transferred to the high-pressure hot homogenizing equipment (High-Pressure Homogenizer SPCH-10, Stansted Fluid Power), being homogenized during five cycles at 600 bar, and then cooled to 25 °C in an ice bath. The samples were stored in glass bottles at 4 °C. Empty NLC was prepared similarly, without adding UA.
In all experiments, UA was quantified using the UltiMate 3000 HPLC apparatus (Dionex Corporation, Sunnyvale, USA), with a UV-VIS detector, and automatic injector. A reverse-phase C18 column (BDS-Hypesil-C18^®, Thermo Scientific, Waltham, MA, USA) was used with acetonitrile: water (88:12, v:v) mixture as the mobile phase, with a flow rate of 1.0 mL·min⁻¹; UA was detected at 210 nm wavelength [18 (link)].

The purity of the synthesized CoA-thioesters and enzyme reaction products was determined with reverse phase high performance liquid chromatography-mass spectrometry (RP-HPLC-MS) by employing an UltiMate® 3000 HPLC apparatus (Dionex GmbH, Idstein, Germany) connected directly to an LXQ™ Finnigan™ (ThermoScientific, Dreieich, Germany) mass spectrometer. An Acclaim 120 C18 Reversed-Phase LC Column (4.6 × 250 mm, 5 μm, 120 Å pores; Dionex GmbH) was used at 30°C based on a method described earlier with slight modifications (Lindenkamp et al. [2013 (link)]; Schürmann et al. [2011 (link)]). A gradient system was used, with 50 mM ammonium acetate, pH 5.0 adjusted with acetic acid (A), and 100 % (v/v) methanol (B) as eluents. Elution occurred at a flow rate of 0.5 ml/min. Ramping was performed as follows: equilibration with 90 % A for 2 min before injection and afterwards a change from 90% to 10% eluent A in 25 min, followed by holding for 10 min and then returning to 90% eluent A within 10 min. CoA-thioesters were detected at 259 nm by a photodiode array detector. Tuning of the instrument was done by direct infusion of a solution of 0.4 mM CoA as shown before (Schürmann et al. [2011 (link)]).