4.4. LC-MS/MS-MRM Method

The five sulfonates, cysteic acid, isethionic acid, 2,3-dihydroxypropane-1-sulfonate, sulfoquinovose and taurine, were analyzed using multiple reaction monitoring with LC-MS/MS. Sample aliquots (15 µL) were injected into the Ultimate 3000 HPLC (Dionex, Sunnyvale, CA, USA) and separated on a BEH amide column (2.1 × 100 mm, 1.7 µm; Waters, Milford, NZ, USA). The column temperature was kept at 60 °C, and the flow rate of the elution solvents was 0.5 mL/min. The elution solvents used were A: 50% acetonitrile and 0.1% formic acid in water, and B: 0.1% formic acid in water. The LC gradient was run for 2 min at 80% A followed by a linear gradient of 3 min to 50% A, which was held for 2 min. This was followed by a rapid increase in A to 80%, which was held for a further 3 min to equilibrate the column.

The sulfonates were identified and quantified using specific MRM traces for the five analytes measured on a QTRAP^® 5500 (AB Sciex, Framingham, MA, USA) in negative mode. The ionization source settings were as follows: ion spray voltage of −4.5 kV, temperature of 500 °C, curtain gas flow of 30, collision gas/medium and ion source gases of 40 and 60. The transition masses, corresponding declustering potentials and collision energies were determined by direct infusion prior to flow injection analysis (Table 1). Data acquisition and analysis were performed using the Analyst^® Software (Version 1.6.2, AB Sciex).