Luna 3u silica column

Lipids were extracted from wandering 3^rd instar larval fat body as previously described [60 (link)]. The lipidomic analyses were carried out on an analytical system comprising an Agilent HPLC 1260 coupled with a SCIEX 5500 QTRAP. Separation of individual classes of polar lipids by normal phase HPLC was carried out using a Phenomenex Luna 3u silica column (i.d. 150x2.0 mm). Multiple reaction monitoring (MRM) transitions were set up for quantitative analysis of various polar lipids. Individual lipid species were quantified by referencing to spiked internal standards. PC-14:0/14:0, LPC-C20, PE-14:0/14:0, PS-14:0/14:0, PA-17:0/17:0, PG-14:0/14:0 were obtained from Avanti Polar Lipids and dioctanoyl phosphatidylinositol (PI, 16:0-PI) was obtained from Echelon Biosciences, Inc. Separation of glycerol lipids (DAG and TAG) by reverse phase HPLC/ESI/MS/MS was carried out on a Phenomenex Kinetex 2.6μ-C18 column (i.d. 4.6x100mm). Using neutral loss-based MS/MS techniques, the levels of TAG were calculated as relative contents to the spiked d5-TAG 48:0 internal standard (CDN Isotopes), while DAG species were quantified using 4ME 16:0 Diether DG as an internal standard (Avanti Polar Lipids). Free cholesterols and ergosterols were analyzed using HPLC/APCI/MS/MS with the corresponding d6-Cho (CDN Isotopes) as the internal standard.

We formulated five experimental diets without or with astaxanthin at different concentrations [0 mg/kg (control), 50 mg/kg, 100 mg/kg, 150 mg/kg, and 200 mg/kg] (Table 1). We used natural astaxanthin derived from Haematococcus pluvialis (purity 10%), which was purchased from Shangcheng Biotechnology Co., Ltd. (Xi'an, China). Fish meal, soybean meal, and rapeseed meal were used as protein sources. Soybean oil was the lipid source. Wheat flour and corn flour were used as carbohydrate sources. All the diets were energetically equal. The dry materials were mixed to homogeneity in a Hobart mixer at the FFRC, and then the wet materials were added, and the mixture was shaped into cold-extruded pellets (2.5 mm diameter). After drying, the feeds were sealed in vacuum-packed bags and kept at −20°C until the start of the experiment. The astaxanthin content was determined by liquid chromatography [17 (link)]. A Luna 3u silica column (150 mm × 4.60 mm; Phenomenex, Torrance, CA, USA) was used for these analyses. The mobile phase was n-hexane and acetone (83 : 17, v/v; flow rate 1.0 mL/min). The detection wavelength was 478 nm, and the injection volume was 20 μL.