Agilent 6890n

The extraction and derivatization of total lipids in plasma and erythrocyte samples were carried out using a modified method of Folch et al. [13 (link)]. The internal standard solution containing methyl undecanoate (C11:0) was added to the samples, and mixed with boron trifluoride and methanol. This mixture was heated at 115 °C for 20 min. After cooling to room temperature, the mixture was extracted with n-hexane. The n-hexane containing methyl esters of total lipids were analyzed by an Agilent 6890N gas chromatography (Agilent Technologies, Palo Alto, CA, USA) equipped with a flame ionization detector at 280 °C and a capillary column (CP-Sil 88, 50 m, 0.25 mm ID, 0.20 μm film thickness). The injector was set as a split mode at 250 °C, with the split ratio of 1:5. The oven temperature was programmed as follows: ramping from 120 °C to 166 °C at 2 °C/min, and holding at 166 °C for 10 min; then ramping to 200 °C at 2 °C/min and holding at 200 °C for 10 min. Individual fatty acids were identified against the reference standards. The data were collected and processed using Agilent OpenLAB software (Agilent Technologies, Santa Clara, CA, USA). Both absolute concentration (μg/mL) and the relative concentration (weight percent of total fatty acids, wt. %) of DHA were calculated.

Soil microbial composition was determined by analyzing the ester-linked phospholipid fatty acid (PLFA) composition of the soil. About 8.0 g of frozen soil was weighed, and lipids were extracted overnight by the modified Bossio and Scow (1998) (link) method, using 23 mL of chloroform: methanol: phosphate buffer (1:2:0.8 v/v/v) solution. The extracts were separated on silica acid columns by sequential elution using organic solvents with increasing polarity, followed by evaporation under N₂. Phospholipids were sequentially saponified and methylated to form fatty acid methyl esters. Individual fatty acid methyl esters were identified and quantified using a gas chromatograph (Agilent 6890N) equipped with the MIDI software package Sherlock MIS version 4.5 (MIDI Inc., Newark, Delaware, USA), and PLFA was analyzed. The MIDI package automatically controlled all gas chromatography operations, including calibration, subsequent sample sequencing, peak integration, and nomenclature. The calibration standards contained a mixture of linear saturated methyl hydroxy fatty acid esters with a length of 10–20 carbons (MIDI Part No. 1208) (He et al., 2019 (link)).

Total fat of milk samples was extracted with Folch reagent as previously described [29 (link)]. Bound fatty acids were methylated using 0.5M sodium methoxide similar to methods described by Christie (1982) using select modifications described by Politz et al., (2013) [30 (link),31 (link)]. Briefly, toluene was added to dried chloroform extract (2:1 v/w). Next, 0.5M sodium methoxide was added in excess to lipid extracts (100:1 v/w) and samples were heated at 60°C for 10 minutes in a water bath. The methylation reaction was quenched with 0.35M glacial acetic acid (1.5:1 v/v) followed by hexane extraction of methyl esters to yield a final FAME concentration of 10mg/ml. Relative abundance of fatty acid methyl esters (FAME) was analyzed using gas chromatography (Agilent 6890N) coupled with flame ionization detection as previously described [32 (link)]. A 100m biscyanopropyl polysiloxane capillary column (Rt-2560, Restek Corp, Bellefonte, PA) was used for separation of FAMEs.

Fatty acid composition of the n-hexane extracts from pepper samples were determined by GC- flame ionization detector (FID, Hewlett Packard, Palo Alto, CA, USA). Firstly, the fatty acids were converted to methyl esters by using BF₃ (14%, methanolic) [16 ]. The chromatographic analysis was performed by using a HP Agilent 6890 N (Palo Alto, CA, USA), HP-88 capillary column (100 m, 0.25 mm i.d. and 0.2 μm), and a flame ionization detector (FID) was used to separate and identify these fatty acids [17 (link)]. Chromatographic conditions were given in our earlier paper [18 (link)].

The standard fatty acid methyl ester (99% purity) was purchased from Sangon Biotech [26 ]. The rapeseed seeds (0.5 g) were ground. Lipids were extracted with 5 mL solvent composed of diethyl ether / hexane petroleum ether (1:1, v/v), and fatty acid components of the total glycerolipids were converted into 3 mL methyl esters by transesterification with 0.5 mol L^-1 NaOH for 30 min. One μL lipid extracted solvent was analyzed by gas chromatograph using Agilent 6890 N. The initial column temperature was 150°C hold 3 min, increasing at 5°C min^-1 to 220°C, and then hold for 10 min. The peaks corresponding to each fatty acid species were recorded and identified by their characteristic retention times.