AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

sample at 3000 rpm for 30 minutes. Filter the supernatant through a 0,2 µm syringe filters into an injection vial. d. System blank – Pipet 400 µL UHQ water in a 15 mL centrifuge tube and dilute by addition of 1600 µL acetonitrile. Homogenize the system blank and filter through a 0,2 µm syringe filter into an injection vial. e. Method blank – Weigh 2,5 grams of UHQ water in a 50 mL volumetric flask. Pipette 100 µL of IS solution (E(b)) and 2 mL of 50% TCA solution (E(c)) to the volumetric flask. Fill to the mark and homogenize. Let the samples deproteinize for 10 minutes. Filter the deproteinized samples over filter paper, discard the first 5 mL Pipet 400 µL filtrate in a 15 mL centrifuge tube and dilute by addition of 1600 µL acetonitrile. Homogenize the diluted sample and subsequently centrifuge the sample at 3000 rpm for 30 minutes. Filter the supernatant through a 0,2 µm syringe filter into an injection vial. f. Calibration Standard blank (T0) – Pipet 100 µL of IS stock solution (E(b)) into a 50 mL volumetric flask. Fill to the mark with UHQ water and homogenize. Pipet 400 µL from the flask in a 15 mL centrifuge tube and dilute by addition of 1600 µL acetonitrile. Homogenize the standard blank and filtrate through a 0,2 µm syringe filter into an injection vial.

I.

Chromatographic Conditions

The separation of taurine is performed by a UHPLC system equipped with a HILIC column. The column temperature is kept at 45±1° Celsius. The injection volume is 3,5 µL and the autosampler temperature is 15±1°C. Taurine is separated according to the gradient presented in table 3. Detection of the component of interest is performed by a mass spectrometer.

Table 3. Gradient program

Eluent

Time (min)

Ammonium formate (A (%))

Acetonitrile (B (%))

Flow (mL/min)

0,0 4,5 4,5 5,5 5,5 8,0

4

96 51 14 14 96 96

0,4 0,4 0,4 0,4 0,4 0,4

49 86 86

4 4

J.

MS conditions

The detection of taurine and the IS is performed on a triple quadrupole MS in MRM mode. The settings describes for the setup of AB SCIEX MS are listed in table 4. Ionization is performed in a turbo spray ion source in positive mode and the data acquisition rate is 0,465 s with 1032 collection cycles. In table 4 the ion source parameters are described. The MRM transition parameters of taurine and the IS are defined in table 5. The MS settings in other laboraties should be optimized locally.