Caplc system

Positive-ion electrospray ionization mass spectrometry analysis was performed on a Micromass Q-TOF ultima global mass spectrometer (Waters) coupled to a Waters CapLC system. Mobile phases were A: 95% H 2 O containing 4.9% acetonitrile and 0.1% formic acid (FA), and B: 95% acetonitrile containing 4.9% H 2 O and 0.1% FA. The injector, loop, column and solvents were cooled to 2 °C to minimize back-exchange using a home-made low temperature box (50 × 50 × 50 cm) maintained at 2 (±0.2) °C by a refrigeration compressor. For mass spectrometry analysis, the desolvation temperature was 413 K and the source temperature was 353 K. Nitrogen was used as both cone gas and desolvation gas with a flow rate of 40 L h -1 and 400 L h -1 , respectively. The collision energy was set up to 10 eV. Global deuterium uptake spectra were acquired in the range of m/z 500-2500. Local deuterium uptake spectra were obtained in the range of m/z 400-1800. MassLynx (ver. 4.0) software was used for analysis and post data processing.

The concentrations of PFOS were measured using combined liquid chromatography-mass spectrometry using a CapLC system (Waters, USA) connected to a Quadrupole-LIT quadrupole mass spectrometer (Applied Biosystems, UK) as it was described in Dorneles et al. (2008) . Aliquots of 5 l were loaded on an Optiguard C18 pre-column (10 mm x 1 mm i.d., Alltech, USA). The analysis was performed on a Fluophase PFP column (50 mm x 1 mm i.d., Thermo, USA) at a flow rate of 40 l/min. The mobile phase was 2 mM NH4OAc (A) / Acetonitrile (B). A gradient elution was used starting at 35 % B and going to 90 % B in 5 min. At 5 min and 6 seconds the initial conditions were resumed. PFOS was measured under (-) electrospray ionisation using the transitions from mother to daughter ion (499 → 80/99) to identify them. The dwell time was 0.1 s. The ES-capillary voltage was set at -4.5 kV and the cone voltage -100 kV. The PFOS concentration was calculated using an un-extracted calibration curve. The limit of detection (LOD) was 0.9 ng/mL and 0.15 ng/g ww for blood and eggs respectively. This was established on a signal to noise ratio (S/N) > 3.

All reactions were carried out in oven dried 10 mL microwave vials. Other glassware (roundbottomed flasks) can be used but must be sealed tightly as gaseous products arise. All ketones and amines were purchased from commercial suppliers (Sigma-Aldrich, Acros Organics, Alfa-Aesar, Fluorochem, TCI and J&K). Calcium carbide was obtained from Sigma-Aldrich in granulated form (pieces) and technical quality (≥ 75% gasvolumetric). Calcium carbide is further ground in a mortar with a pestle until a fine powder is obtained and used immediately. 1 H ( 13 C) NMR spectra were recorded at 400 (100) MHz on a Bruker Avance III HD spectrometer using CDCl3 as solvent and TMS as the internal standard. Assignments were determined using 2D (HSQC, HMBC and DEPT or APT) spectra. Chemical shifts are given in parts per million (ppm), Jvalues are given in hertz (Hz), and number of protons for each signal are also indicated. For high resolution mass spectrometric analysis (HRMS), samples were dissolved in methanol/water (1/1), containing 0.1% formic acid and diluted to a concentration of approximately 10-6 mol/L. 10 µl of this solution was injected using a CapLC system (Waters) and electrosprayed using a standard electrospray source, positive ion mode accurate mass spectra were acquired using a Q-TOF II instrument (Waters). The parent ions [M+H]+ are quoted.