Milli q gradient a10 purification system

Certified reference materials for C1/2, GTX1/4, GTX2/3, dcGTX2/3, GTX5, GTX6, STX, NEO, dcSTX, dcNEO, an in-house reference material of C3/4 were obtained from the National Research Council Canada (Halifax, Nova Scotia, Canada). Formic acid and ammonium formate were obtained from Sigma-Aldrich (Oakville, ON, Canada). Acetic acid (AcOH) and hydrochloric acid were purchased from Caledon (Georgetown, ON, Canada). Acetonitrile and methanol (Optima LC-MS grade) were purchased from Fisher Scientific (Mississauga, ON, Canada). Deionized water (18.2 MΩ cm) was obtained from a Milli-Q gradient A10 purification system (Millipore Corp., Billerica, MA, USA).

Sweat was collected using sterilized medium gauze pads (Johnson & Johnson, 7.62 cm $\times$ 30.48 cm) and absolute waterproof tape (Nexcare). Standard urinary cortisol ELISA (0.47–200 ng/mL range) and saliva cortisol (0.5–100 ng/mL) ELISA kits, for sweat and saliva cortisol analysis respectively, were purchased from Eagle Biosciences (Amherst, NH). Methanol (99% ACS grade) was purchased from VWR (Atlanta, GA). Artificial sweat was purchased from Pickering Test Solutions (Mountain View, CA). Cortisol solution in methanol at 1 mg/mL was purchased from MillporeSigma (Burlington, MA). Deionized water is purified (18.2 MΩ cm; total organic content < 6 ppb) using a Millipore Milli-Q Gradient A-10 purification system (Bedford, MA). Spectroscopic measurements were taken using the BioTek Cytation Hybrid Multi-Mode Reader (Agilent Technologies).

Ammonium bicarbonate (NH₄HCO₃), dithiotheritol (DTT), iodoacetamide (IAA), formic acid (FA) and acetonitrile (ACN) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All the reagents used were analytical or HPLC grade. Lactoferrin isolated from bovine milk (purity ≥85%) were purchased from Sigma-Aldrich. Trypsin (sequencing grade modified, V511 20 μg lyophilized enzyme) was purchased from Promega (Madison, WI, USA). Camel milk powder used as the control matrix was obtained from Al Ain Dairy (UAE, Dubai). All the peptide standards were chemically synthesized by SynPeptide Co., Ltd. (Shanghai, China) with purity above 95%. The internal standard (IS) peptide was the synthetic peptide containing a stable isotopically labelled amino acid ([¹³C₆, ¹⁵N₂] lysine). The stock solutions of the synthetic peptides were prepared using 20% acetonitrile/water solution. Ultrapure water was prepared using a Milli-Q Gradient A-10 purification system (Millipore, Bedford, MA, USA) during all the experiments. All chemical agents were prepared with 100 mM NH₄HCO₃ without further purification. Infant formulas were obtained at local retailers.