Hplc fld

The HA-Cis and HA-mPEG-Cis solutions were placed in a dialysis bag, and four different release media with pH values of 5.0, 5.5, 6.8, and 7.4 were used for incubation in the dark at 37°C. The samples were collected at different time points, filtered, and analyzed by HPLC-FLD (Agilent Technologies, USA). The acetonitrile/0.1% formic acid in the water (30/70) was used as mobile phase, and the flow rate was set as 1.0 mL per minute.

The system consisted of an Agilent 1100 Series high-performance liquid chromatograph coupled to a micro vacuum degasser and a fluorescence detector (HPLC-FLD) (Agilent Technologies, Barcelona, Spain) with excitation and emission wavelength of 365 and 435 nm, respectively. Separation was carried out on a column Ace 5 C18, 250 × 4.6 mm, 5 µm particle size (Análisis Vínicos, Ciudad-Real, Spain). A manual injector system equipped with a 100 µL injector loop and a 250 µL syringe was used. The isocratic mobile phase for aflatoxins was methanol/acetonitrile/water (40:10:50, v/v/v), pumped with a flow rate of 1.0 mL/min. The retention times (min) for the analyzed aflatoxins were 9.95 for AFG2, 11.00 for AFG1, 13.51 for AFB2, and 15.09 for AFB1 (Supplementary Material Figure S1). The fluorescence intensity of AFB1 and AFG1 was improved with postcolumn photochemical derivatization using a photochemical reactor for enhanced detection (PHRED) (LCTech UVE, Dorfen, Germany) set at 254 nm. The identification and quantification of aflatoxins in the culture broth samples were performed using the software package OpenLAB CDC 2013 (Agilent Technologies, Barcelona, Spain).

Analysis of tocopherols was performed by HPLC according to Brazaityte et al.’s [25 (link)] methodology with some modifications. About 1 mg of oil was weighed in an Eppendorf tube, and then 1 mL of n-hexane with 1% of BHT was added to the tube. Afterward, the samples were filtrated through a 0.45 µm polytetrafluoroethylene (PTFE) membrane syringe filter (VWR International, Radnor, PA, USA) and were analyzed by HPLC/FLD (fluorescence detector) (Agilent Technologies, Santa Clara, CA, USA). The HPLC measurements were performed using a normal phase column (Phenomenex Luna Silica, 5 μm, 250 mm × 4.6 mm). The HPLC 10A system, equipped with an RF-10A fluorescence detector (Shimadzu, Japan), was used for analysis. Peaks were detected at an excitation wavelength of 295 nm and an emission wavelength of 330 nm. The mobile phase (0.5% isopropanol in hexane) was used at a flow rate of 1 mL min⁻¹. The α-tocopherol, γ-tocopherol, and δ-tocopherol were identified according to the analytical standard. The α-tocopherol, γ-tocopherol, and δ-tocopherol content were expressed per 100 g of oil.