AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

EXPER IMENTAL

A. Principle

This single laboratory validation (SLV) report describes the method performance of the determination of total taurine in infant formula and adult/pediatric nutritionals by ultra high performance liquid chromatography (UHPLC) with multiple reaction monitoring (MRM) mass spectrometry (MS). Powdered samples are reconstituted according to SPIFAN guidelines in water prior to further analysis. Liquid and reconstituted samples are diluted and extracted in ultra high quality (UHQ) water including 2-Aminoethane-d 4 -sulfonic acid as an internal standard (IS) and trichloroacetic acid (TCA). Deproteinized samples, due to the use of TCA, are filtrated. Subsequently the filtrate is diluted and injected on a hydrophilic interaction liquid chromatography (HILIC) column. Taurine and the IS are separated from the matrix by the means of an aqueous ammonium formate gradient. The components of interest are selectively detected by MRM-MS.

B. Safety precautions

The method employs corrosive, toxic and flammable chemicals such as trichloroacetic acid and acetonitrile. Wear personal protective equipment, for example gloves, laboratory clothing and safety glasses. Refer to material safety data sheets and take appropriate safety precautions. Waste disposal should be in accordance to local requirements.

C. Apparatus

a. Liquid Chromatography instrument. – Consisting of a pump, degassing module, autosampler, column oven, capable of high pressure – For example Shimadzu Nexera X2 or Waters Acquity UPLC b. Triple Quadrupole Mass spectrometer with Electrospray ionization probe.- For example AB SCIEX 4500, 5500+ or Waters XEVO TQ-S c. UHPLC column - Waters Acquity UPLC BEH Amide Column, 130Å, 1,7µm, 2,1x150 mm or equivalent, for example Phenomenex Kinetex 100Å, 2,6µm d. UHPLC Guard column - Waters Acquity UPLC BEH Amide VanGuard Pre-column, 130Å, 1,7µm, 2,1x5 mm or equivalent, for example Phenomenex SecurityGuard ULTRA cartridge, HILIC e. Centrifuge f. Analytical balance – Weighing to ± 0,1 mg g. Magnetic stirrer h. Sonicator bath i. Multipipet j. Adjustable pipettes with volumes of: 20 – 200 µl, 200 – 1000 µl, 2 – 10 ml k. Volumetric glassware - 50, 100, 500 and 1000 mL l. General laboratory glassware