6. AOACSPIFANMethods-2018Awards

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g) pH meter h) Constant temperature oven capable of maintaining 50 (+/- 1)°C i) Thermometer (-20 to 110°C) j) Vortex mixer k) Sonic water bath l) Stir bars m) Stir plates n) ACRODISC disposable filter assembly, 0.45 micron pore size From Gelman Scientific, cat. # 4217, or equivalent o) 3 cc or 5 cc disposable syringe (s) p) Weighing paper q) Weigh boats r) Funnel(s) s) Water bottle t) Membrane filter, 0.45 micron u) Autosampler vials and caps (see autosampler requirements). v) Analytical Column: Preferred column: YMC PACK C8, 3 um particle, 3.0 50 mm – YMC America, INC, Product No. OC12S03-0503WT, or alternate w) HPLC system with autosampler, column oven to maintain 30°C, Fluorescence Detector, binary or quaternary HPLC Pump with degasser, HPLC peak area integrator or chromatography data processor.

C. Reagents

a) Methanol, HPLC grade b) Sodium Phosphate Monobasic, HPLC grade. c) Phosphoric Acid, (85%) d) Trizma Base e) Hydrochloric Acid, 6N.

f) Laboratory Water – minimum of 18 mega ohm conductivity g) 5-Methyl-DL-tryptophan Sigma Cat # M-0534, or equivalent. h) Protease from Streptomyces griseus: Type XIV (enzyme): Sigma Cat # P-5147, or equivalent.

D. Preparation of Standards and Solutions

a) Hydrochloric Acid, 1.0N Solution. Aliquot 167 mL of 6N HCL (standardized) to a 1000 mL volumetric flask containing approximately 700 mL of laboratory water. Bring to volume with Laboratory Water. Storage: Room temperature. Expiration date: 2 years.

b) Trizma Buffer, 0.1M, pH 8.5. Weigh 6.055 g Trizma base, and transfer to a 400 mL beaker. Bring volume to 300 mL with