Milli q synthesis water purification system

Tetrodotoxin standard (99% HPLC) was purchased from Wako Chemicals (Tokyo, Japan). The stock solution of TTX was prepared in MeOH with a concentration of 1000 mg/L and stored in a brown glass bottle at −18 °C. Methanol (MeOH, HPLC grade) and acetonitrile (ACN, HPLC grade) were purchased from Fisher Chemicals (Bridgewater, NJ, USA). Acetic acid, ammonium acetate and formic acid (FA) were bought from Sigma Aldrich (St. Louis, MO, USA). Distilled water was prepared by a Milli-Q Synthesis water-purification system (Millipore, Bedford, MA, USA).

Spray-dried WPI from cheese whey, Provon 190, was purchased from Glanbia Nutritionals Inc. (Richfield, ID, USA) and contained 90.1 wt% protein, with 3.6 wt% moisture, 2.9 wt% ash and, by difference, 3.4 wt% lactose and fat (manufacturer’s analysis). The protein composition as measured with gel electrophoresis (SDS-PAGE, 5 replicates) and corrected with an α-LA/β-LG calibration curve was: 67.8 wt% β-LG, 25.0 wt% α-LA and 72 wt.% caseins and minor whey proteins (immunoglobulins (Ig), bovine serum albumin (BSA), lactoferrin (Lf) and casein fragments). The measurement and calculation of GMP contents were performed using an HPLC/mass-spectrometry/total nitrogen combination method (described below), and the composition of the WPI was corrected accordingly, to approximately 18% GMP, 56% β-LG, 20.5% α-LA and 5.5% other whey proteins.
WPI solutions ranging from 20 to 100 g·L⁻¹ were prepared using de-ionized water produced with a Milli-Q Synthesis water purification system (Millipore, Billerica, MA, USA). Liquid carbon dioxide (CO₂) tanks with an eductor tube were purchased from GTS-Welco (Allentown, PA, USA).