AOAC SPIFAN Nutrients ERP (December 7, 2022)

Amino-06 (SLV) 10-2022 FOR ERP USE ONLY DO NOT DISTRIBUTE

F. Mobile phase preparation

a. Eluent A - Ammonium formate (2 mM) Weigh 31,5±0,1 grams of Ammonium formate into a beaker glass. Add exactly 250 mL UHQ water and dissolve. Transfer 1 mL into a volumetric flask of 1 liter. Fill to the mark and homogenize. Prepare the eluent on the day of use fresh. b. Eluent B – Acetonitrile (100%)

G. Preparation of standards

a. Working standard solutions Pipette, according table 2, 500 µL of each prepared taurine standard stock solution (E(a)) in their corresponding volumetric flask (T1-T7) of 50 mL. Add 100 µL of Internal standard stock solution (E(b)) to all volumetric flasks. Fill to the mark and homogenize the prepared taurine working standard solutions. Working standard solutions should be prepared fresh daily. b. Calibration standards Pipette, according table 2, from each taurine working standards (G(a)) 400 µL into a corresponding 15 mL centrifuge tube (T1-T7). Add 1600 µL of acetonitrile by pipetting to every centrifuge tube. Homogenize the freshly prepared calibration standard and filtrate over 0,2 µm syringe filter into an injection vial.

Table 2 Pipetting scheme for working standard and calibration standard solutions

Working standard solutions

Calibration standards

Taurine Stock solution (µl)

IS solution (µl)

working standard (µl)

Acetonitrile (µl)

Taurine calibration Concentrations (mg/L)

T1 T2 T3 T4 T5 T6 T7

500 500 500 500 500 500 500

100 100 100 100 100 100 100

400 400 400 400 400 400 400

1600 1600 1600 1600 1600 1600 1600

0,03 0,25 0,50 1,00 2,49 4,98 9,95

H. Sample analysis a. Sample preparation – Reconstitute powdered samples or liquid concentrates to a Ready to-feed basis according to instructions. For example, weigh 25 grams of powdered sample into a sample container and add 200 grams of 40°C UHQ water to obtain a total weight of 225 grams. Mix well with a magnetic stirrer to form a homogeneous suspension. b. Weigh 2,5 grams of reconstituted (H(a)) or RTF sample in a volumetric flask of 50 mL. Subsequently pipette 100 µL of IS solution (E(b)) and add 20 mL of UHQ water to the volumetric flask. Mix the solution well. Add 2 mL of 50% TCA solution (E(c)) and fill to the mark and homogenize. Let the samples stand for 10 minutes. Filter the clarified samples through filter paper, discard the first 3-5 mL. c. Dilution - Pipet 400 µL filtrate (H(b)) in a 15 mL centrifuge tube and dilute by addition of 1600 µL acetonitrile. Homogenize the diluted sample and subsequently centrifuge the