AOAC OMB Final Action Recommendation (December 2019)-2016.14

2016.14 (Jan. 2019) FOS-03 MLT Report FOR ERP USE ONLY DO NOT DISTRIBUTE

Appendix B: Comments of the participants

Lab code

Comments

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- We did not use a reaction coil, as it is not stated in the protocol for this method. - Finally we settled on using the PA1 as we get better chromatography in our lab using this – not sure exactly why we could not replicate your lab’s chromatography on the PA20. - We did not have to introduce any deviations besides applying slight positive pressure for the SPE cartridges. - We noticed that the chromatograms for the Carbopac-PA1 are not ideal as at the retention time of fructose the baseline increases due to the gradient. The PA20 column is ordered for further investigation and to run the next samples of the MLT. - The issue with the variation in batch quality of sucrase should be solved; quality and performance should be guaranteed. - There was no long chain inulin added (type inulin HP) which surprises us as with the SLV this type was used for the spiking. During the sample preparation with SPE there could be a chance that longer chains are lost. - Sample preparation on 1 day was not feasible due to the SPE step. - The blank was negative in our case so the correction has to be performed as prescribed in the protocol. This gives a plus in the final result, which is not logical. - Double blind samples were grouped in one sequence/day, which would not include the difference between series. - During the SPE step, a positive pressure was necessary to accelerate the elution. - We decided to calibrate by height because the peak is tailing and the integration is difficult. - Can you confirm that this is in fact N,N-diacetylchitobiose? Elicityl use the name chitinbiose but this is not strictly correct. I cannot see chitobiose (which describes the N-deactylated version) for sale on their website. - The preparation method is quite simple, with the only difficulty I encountered is the fact that the SPE columns took a very long time to run. - We do not have a secondary pump available, so none of the conditions described in the study protocol are feasible for us at present. What we have done is just applied our internal chromatographic conditions for sugars in food. Therefore, we had to include slight modifications for the chromatography. Since we have an internal method already developed, we feel more comfortable using our PA10 method rather than PA1 conditions, with sodium azide as eluent reagent. If it is possible, we would like to avoid that reagent. No comments - We noticed that during the SPE purification the sample solution did not pass through the stationary phase of SPE column. In order to load the sample we applied a small pressure on the top of the SPE column. - We used a 4 mm ID column and applying the chromatography conditions reported in the method with the flow adjusted to 1,0 ml/min instead of 0,25 ml/min the peak of chitobiose and fructose were not resolved. No comments.

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