AOAC Gluten Qualitative Validation Guidance-FINAL (July 2023)

Gluten Working Group Comments/Responses (Round 1)

ANNEX B • Main concern is that most of the reference methods based on R5 antibody overestimate with barley and rye flours and are not appropriate for the quantification of gluten in barley and rye. An alternative method, "the wet chemical method", Wehling P./Scherf K. (2) is proposed in Annex B to arrive at the glutent content for wheat, rye and barley flours. However, there are no laboratories which perform this wet chemical method. I think this issue should be resolved or clarified before the Guidance approval. o The guidance does not rely on a reference method, nor on the wet chemical method. The wide allowance for recoveries in gluten and allergen methods (typically 50-150%) is given precisely because these are living, variable commodities. Use of the provided conversion factors should not, in itself, result in out-of-range recovery values. • Says 5.7 further down in document. • The factors for nitrogen to protein calculation differ (5.83 and 5.7). This is probably due to different factors recommended for the different gluten sources (wheat vs rye and barley), but this needs to be further explained • In one case a conversion factor of 5.83 is used, in another case 5.7 is used • It would be 5.83 now o Changed to 5.83 in both instances ANNEX B second paragraph • Replace selectivity by interference (twice, line 1 and 2) o Selectivity is the term used to encompass both cross-reactivity and interference ANNEX B third paragraph “”and we the higher conversion factor…” • … we selected the higher…? o This sentence was rewritten ANNEX B barley gluten conversion page 1 • Double check math - 8.55*0.75 is 6.4 not 4.7 • Above states nitrogen conversion factor of 5.83 • I think this should be 47 mg of gluten per gram of flour: 47,000 mg/kg * 1 kg/1000g = 47 mg/kg • 'This percent value is then multiplied by 10,000 to estimate the mg/kg (ppm) value at 47,000. This is equivalent to 47 μg of gluten per gram of flour' - Should be 'This is equivalent to 47 mg of gluten per gram of flour' • There is an example with barley flour in Annex B where the Dumas nitrogen level of 1.5% is multiplied by 5.7 to convert it to percent crude protein. I think the % nitrogen level should have ANNEX B second paragraph “5.83”