Qtrap 4500 lc ms ms system

Sperm lysates were extracted in 50 μL of lysis buffer (8 M Urea, 0.2 M Ammonium Bicarbonate) with sonication. Each sperm lysate was trypsin-digested and labeled with iTRAQ 4-plex reagent (AB SCIEX) according to the manufacture’s instruction. The labeled peptides were applied to QTRAP^®4500 LC/MS/MS System (AB SCIEX). These experiments were performed in duplicate. Identification and quantification of proteins from sperm lysates were performed using the ProteinPilot software, Version 4.0 (AB SCIEX).

Mogroside MIIE, MIII, MIV, MIVA, Si, MV, IMV, OMV, and MVI were detected by LC-MS/MS following previously described methods [38 ]. The HPLC system consisted of an Agilent Technologies 1260 Series LC system (Agilent, USA) equipped with an automatic degasser, a quaternary pump, and an autosampler. Chromatographic separations were performed on an Agilent Poroshell 120 SB C18 column (100 × 2.1 mm, 2.7 μm) by gradient elution using a mobile phase consisting of (A) water (containing 0.1% formic acid) and (B) acetonitrile (containing 0.1% formic acid) with the following gradient procedure: 0 min, 26% B; 5 min, 32% B; 5.01–5.50 min, 80% B; and 5.51–10.0 min, 26% B. The flow rate was 0.25 mL·min⁻¹, and the injection volume was 10 μL. The column effluent was monitored using a 4500 QTRAP^® LC-MS/MS system (AB Sciex, Toronto, Canada). The compound-dependent instrumental parameters were optimized and listed in AppendixTable A2. Both the standards and samples of nine mogrosides in the LC-MS/MS chromatograms are displayed in AppendixFigure A3.

Perform toxin analysis according to the method described by Nishimura et al. (44 (link)). A crude toxin extract (1 mL) was taken and mixed with 60 µL of 2.5 M NaOH, followed by incubation in a water bath at 70°C for 40 min. After cooling to room temperature, 60 µL of 2.5 M HCl was added to the mixture, which was then filtered through a 0.22-µm spin filter (Pall Corporation, USA) and stored at −20°C for subsequent analysis by liquid chromatography. The reference standards for the three DSTs (OA, DTX1, and DTX2) were procured from the National Research Council of Canada’s Institute for Marine Biosciences, and absolute quantification was performed. Regression curves were constructed based on the known toxin concentrations and spectral areas, enabling the quantification of toxin levels in the samples. Analytical software was utilized to visualize the toxin spectra and facilitate quantification. The determination and quantification of LC/MS were carried out using an HPLC system (Shimadzu Prominence LC-20ADXR) coupled with a tandem mass spectrometer (4500 QTRAP LC-MS/MS system, AB Sciex Instruments, Foster City, CA). Phenomenex Kinetex XB-C18 (150 × 2.1 mm, 2.6 µm) column was utilized for toxin separation, with the mobile phase consisting of acetonitrile (solvent A) and 0.15% formic acid in water (solvent B).