AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

Table 2012.12B. Detector waveform

Table 2012.12C. Mobile phase gradients Time, min a Flow, mL/min A, % B, % Time, min b

Potential,

Potential,

Flow, mL/min A, % B, %

Time, s a

V Integration Time, s b

V Integration

0.00 0.20 0.40 0.41 0.60 0.61 1.00

+0.05

0.00 +0.10

0.00 0.4 100 0 0.00 0.4 100 0 10.00 0.4 100 0 14.00 0.4 100 0 10.10 0.4 0 100 14.10 0.6 0 100 20.00 0.4 0 100 30.00 0.6 0 100 20.10 0.4 100 0 30.10 0.4 100 0 30.00 0.4 100 0 40.00 0.4 100 0

+0.05 Start

0.20 +0.10 Start

+0.05 +0.75 +0.75 –0.15 –0.15

End

0.40 +0.10 0.41 –2.00 0.42 –2.00 0.43 +0.60 0.44 –0.10 0.50 –0.10

End

Samples with low levels of sugars.

a

b Default gradient and for samples with high levels of sugars to help elute them from the high-capacity MA1 column.

Carbohydrate triple.

a

Standard quad.

b

and incubated for 2 h with stirring at ambient temperature on a stir plate, then placed in a boiling water bath for 5 min to quench the enzyme. The samples were allowed to cool before performing necessary dilutions with purified water. Finally, the samples were filtered through a 0.45 μm PTFE syringe filter into injection vials. I. Chromatographic Parameters ADionex ICS-3000 system was equipped with a CarboPac MA1, 250×4.0 mm id, column in conjunction with a CarboPac MA1, 50×4.0 mm guard column. Flow rates of 0.4 and 0.6 mL/min were used, with an injection volume of 25 μL. A column temperature of 30°C was maintained. The calibration curve was established by injection of a minimum of two sets of reference standards that bracketed the samples; they were also interspersed throughout the run. The detection settings for the two different waveforms used are shown in Table 2012.12B , and the two different gradient conditions are shown in Table 2012.12C . The default waveform used was the carbohydrate triple potential. The standard quad waveform was also tested and gave similar results.

J. Quantification and Confirmation A calibration curve was obtained using least-squares regression analysis for all peak areas and concentrations of the working standards. Using the calibration curve, the concentration of myo- inositol in the sample solutions was determined. Quantification and identification were accomplished using Empower chromatography software, based on retention time windows established during method development. The concentration of each analyte in a sample was calculated by the following equation: where R = results expressed in percent; C = concentration of the analyte in the injected solution in μg/mL; V = volume of the initial extract in mL; S = sample amount in g; and D = dilution factor, the inverse of any dilution made from the initial extract. References: J. AOAC Int . 95 , 1469(2012)

J. AOAC Int . 96 , 1068(2013) DOI: 10.5740/jaoacint.13-128 AOAC SMPR 2011.007 J. AOAC Int . 95 , 295(2012) DOI: 10.5740/jaoac.int.11-0443

© 2012 AOAC INTERNATIONAL