AOAC SPIFAN ERP Meeting Book-March 16, 2016

2011.06 (Fol-22) w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

13.) α -Amylase Solution (2 mg/mL): Dissolve 1.0 g α -Amylase in 50 mL of water. Treat with 20mg charcoal per mL of solution. Vortex and PDVF syringe filter. Store at 4-8 °C. until use. Prepare fresh on day of use. Each sample requires 1mL.

Store at -80 °C Use as is from supplier.

14.) Rate Plasma Conjugase:

15.) TCEP Solution (approx. 1M): 1g of TCEP reagent and 3.5mL H 2 O 16.) Extraction Buffer (10mM phosphate buffer, 1% ascorbic acid, pH 6.0) : a.) Weigh and transfer about 1.4 g of sodium phosphate, dibasic, anhydrous (Na 2 HPO 4 ) into a 1000mL beaker. Add 10g of ascorbic acid (sodium ascorbate) and approximately 700mL of DI water. Stir on a stir plate until completely dissolved. b.) Adjust pH with phosphoric acid and/or 10mM sodium hydroxide (10M NaOH) to pH 6.0 c.) Transfer into a 1L volumetric flask and dilute to volume with DI water. Prepare fresh on day of use. d.) Add 0.20mL of Internal Standard Intermediate Standard Solution to the buffer. 16.) HPLC Mobile Phase : a.) 500mLWater, Optima LC/MS, and 5 mL acetic acid b.) Methanol, Optima LC/MS. Sample Preparation: 1.) Accurately weigh 0.1 – 2.0 g of sample, depending on estimated total folates, into 50mL centrifuge tube using analytical balance. Use one additional tube with no sample as the method blank. Add 10mL of Extraction buffer (10mM phosphate buffer, 1% ascorbic acid, pH 6.0) containing mixed internal std each (labeled folic acid and methyl –THF) at 4 ng/mL total of 40 ng each. Add 50uL TCEP Solution. Vortex to mix well 2.)To check the efficacy of the rat plasma conjugase, use 150uL of Rat Plasma Test Solution (20ug/mL Pteroyltri-y-glutamate) in place of homogenized sample in the 50mL centrifuge tube. Add 10mL of Extraction buffer (10mM phosphate buffer, 1% ascorbic acid, pH 6.0). Add 50uL TCEP Solution. Vortex to mix well. 3.) Add 1mL protease solution to each centrifuge tube, fill the head space with nitrogen, cover, vortex to mix and incubate for 3hrs in a water bath at 37 °C. 4.) Inactivate the protease enzyme by placing the samples in a boiling water bath (100 °C) for approximately 5min with shaking. After inactivating the enzyme, cool the samples to room temperature. 5.) Add 1mL α -amylase solution and 300uL of rat plasma conjugase, fill the head space with nitrogen, cover and incubate for a minimum of 16hr in a water bath at 37 °C. 6.) After the 16hr incubation, place the samples in a boiling water bath (100 °C) for approximately 5min. Cool to room temperature. 7.) Centrifuge at room temperature for a minimum of 5 min.

11