Tamara van Donge

Chapter 6 112 Supporting information Supporting Information 1 - Method development Detailed Description of Measuring Ibuprofen Enantiomers using HPLC-MS/MS Materials Racemic (R/S) ibuprofen with a certified concentration (1.000 mg/mL in methanol) and ethyl acetate was bought from Sigma-Aldrich (St. Louis, MO, USA) and racemic ibuprofen-d3 from C/D/N isotopes Inc. (Pointe-Claire, Quebec, Canada). LC-MS grade methanol and water was purchased from Honeywell (Charlotte, NC, USA) and glacial acetic acid from Caledon Laboratories (Georgetown, Canada). Hydrochloric acid (0.15N) was diluted from a stock solution 1.0N. All ratios of dilutions are given as volume ratios. HPLC-MS/MS instrument An Agilent 1200 HPLC system, equipped with a binary pump, autosampler and thermostatted column compartment was attached to a Sciex 4000 QTrap with electrospray ionisation source. Data were processed using the Analyst 1.6.2 software (AB Sciex, Framingham, MA, USA). HPLC setting For isocratic elution, a mixture of methanol and water (80/20) with 0.1% acetic acid was used as mobile phase at a flow rate of 250 μL/min. The ibuprofen enantiomers were separated on a chiral column (Phenomenex Lux Celulose-3, 2 x 150 mm, 3 μm particle size) that was cooled down to 15°C. The injection volume was 5 μL and the total run time was 10 minutes. To reduce potential contamination of the mass spec, the solvent flow was diverted to the waste for the first 4.0 min and after 9.0 minutes. Mass spectrometer setting The electrospray ionisation source was run in negative ionization mode with a capillary voltage of 4.5 kV and 600°C source temperature. In MRM mode, a mass-to-charge ratio ( m/z ) of 205.2 and161.1 for (R/S)-ibuprofen and 208.2 and 164.1/188 for the internal standard (R/S)-ibuprofen-d3 was detected. The declustering potential and collision energy was set to -31 V and -11 V, respectively. Calibration and Quality controls To prepare the calibration curve, one hundred microliter of the certified ibuprofen solution (1.000 mg/mL) was dried under nitrogen and reconstituted in 1000 µL of methanol/water (80/20) resulting in a concentration of 100 µg/mL for (R/S)-ibuprofen. Serial dilution of this solution with methanol/water (80/20) lead to concentrations of 50, 25, 10, 5, 1, 0.5, 0.1, and 0.05 µg/mL. Repeated measurements (n=10) of a concentration of 10 µg/mL have shown that the ratio of the integrated signals of S- and R- is 1.00 and therefore the final concentration