2.6. Quantification of Artificial Sweeteners in Human Plasma
SEPARATION
Standard solutions: Stock solutions of the analytes (roughly 10 mM) were individually prepared by dissolving the exactly weighed solids of acesulfame-K, sodium saccharin and sodium cyclamate in methanol. The internal standard p-toluolsulfonic acid hydrate was prepared in acetonitrile at a concentration of ~10 mM, and the exact concentration was determined by qNMR versus a caffeine standard [Matrix-matched calibration: For matrix-matched calibration, we prepared spiked plasma samples, which were processed and analyzed in triplicates to establish calibration curves, as described in detail in the Quantitative analysis: An aliquot of the plasma sample (100 µL) was mixed with the internal standard working solution (1 mL) in an Eppendorf cup. The mixture was vortexed and centrifuged (12,500 rpm, 4 °C, and 15 min). The clear supernatant was decanted into a new reaction tube and was evaporated in a stream of nitrogen. The residue was taken up in a mixture of water and acetonitrile (95 + 5, Chromatographic separation: The samples were injected onto a Luna phenylhexyl column (150 × 2 mm, 3 µ, Phenomenex Darmstadt, Germany) protected by a guard column of the same material (2 × 2 mm, 3 µ, Phenomenex, Darmstadt, Germany). Eluents were 1% formic acid in acetonitrile (eluent A) and 1% formic acid in water (eluent B). The solvent was delivered at a flow of 300 µL/min. After 2 min of isocratic elution (A/B 5/95), the composition was changed to A/B 95/5 within 3 min following a non-linear gradient. After 1 min of isocratic elution, the starting conditions were re-established within 0.2 min and kept constant (3.8 min) for equilibration.Instrumentation: MS/MS data were acquired on a 3200 triple quadrupole mass spectrometer (ABSciex, Darmstadt, Germany). Potentials applied for the introduction of the compounds into the ion source (Q1 mass, declustering potential) and the fragmentation-related parameters (cell entrance potential, collision energy, cell exit potential) were optimized by using the auto-tune function of the software (Analyst 1.5.1, ABSciex, Darmstadt, Germany). The Q1/Q3 pairs are given in 