Thermo ultimate 3000 hplc

Samples were analyzed using a Thermo Ultimate 3000 HPLC (Thermo Fisher Scientific, Bremen, Germany) in tandem with an AB Sciex Qtrap 4500 mass spectrometer (AB Sciex Pte. Ltd., Singapore) combined with an electrospray ionization source. The parameters of both the LC and MS systems were the same as those described in our previous study [8 (link)]. A SeQuant^® ZIC-HILIC column (150 mm × 2.1 mm, 5 µm) was used to separate BMAA using a binary mobile phase. Solvent A was water containing 0.1% formic acid, and solvent B was acetonitrile containing 0.1% formic acid. The injection volume was 5 µL. The gradient was run at 350 μL·min⁻¹ at 30 °C from 95% to 60% B over 19 min, decreased to 40% B at 25 min, increased to 95% B at 27.01 min, and held for 2.99 min before re-equilibration for the next run. Mass spectrometry parameters were set as follows: curtain gas pressure of 40 psi, spray voltage of 5500 V, source temperature of 350 °C, source gas 1 and 2 at a pressure of 55 psi and 50 psi, respectively, and collision cell entrance potential of 10 V. The selective reaction monitoring (SRM) mode with five transitions (m/z 119 ->102, 101, 88, 56, and 44) was used to quantify BMAA and its isomers (Table S1). The limits of detection of BMAA and DAB were 3.4 and 0.65 ng·mL⁻¹, respectively.

Injections (100 µL) were made of each solvent extracted sample on a Thermo Ultimate 3000 HPLC (Thermo-Fisher, Sunnyvale, CA, USA) using an updated method, as previously reported [27 (link)]. The column was a 100 × 3 mm LiChrosorb 5 DIOL (Chrompack, Raritan, NJ, USA), using a 0.5 mL/min flow rate and the following binary gradient: A: 1000:1 hexane:acetic acid; B: 100:1 hexane:isopropyl alcohol with the following ramp: 0 min, 100/0 A/B; 8 min, 100/0; 10 min, 75/25; 40 min, 75/25; 41 min, 100/0 and 60 min, 100/0. Detection was accomplished by diode array detectors (DAD) at 205 nm and 320 nm, and also with the Thermo Charged Aerosol Detector (CAD). Oleic acid, linoleic acid, linolenic acid and the unknown potential free fatty acids (FFA), using linoleic calibration, were appraised at 205 nm and oryzanol at 320 nm. The 254 nm and 280 nm responses were also recorded by the DAD for monitoring, but were not used for any peak analyses. Detection was accomplished by CAD for the steryl esters (StE), triacylglycerols (TAG), stearic/palmitic acid, 1,3-diacylglycerols and 1,2-diacylglycerols (DAG), both using sterol calibration. There were multiple iterations of the various possible DAG compounds, and peaks were therefore broad sets that separated out as 1,2 versus 1,3. The instrument numbers generated were reliable for showing how the classes delivered trends through processing.

Samples were injected in a constrained randomized order. A QC injection was done every 8th sample followed by a blank injection. Prior to the injection of samples, five repeated injections of the QC were done to precondition the column, followed by two blank injections. Following, a 2-fold serial dilution series ranging from 0.5 to 32.0 µL QC was injected. In addition, MS/MS analysis was performed on subpools of samples. The injection volume was 10 µL and a Thermo Accucore aQ RP C18 column (100 × 2.1 mm, 2.6 µm particle size, Thermo Fisher Scientific, Waltham, MA, USA) was used for the chromatography. The analysis was performed on a Thermo Ultimate 3000 HPLC and Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Details of the LC-HRMS analysis have been previously described [26 (link)].