AOAC-RI ERP Book - Gluten.pdf

Ltipo t,.l'At-.: JorJrìNÄr, orr AOAC INl't..RNÄr'roNr\t- Vot-. 96. No. l. 2013 125

Table 4. lnternal LOD, ppm Replicåte Krce

Sausage Cracker

Oats

Sorghum

Corn

Buckwheal

0.70 0.40 0.30 0.60 0.80 o.20 0.20 0.50

0.00 0.20 0.00 0.'í0 0.50 0.40 0.1 0 0.30 0.10 0.50 3.12

0.30 0.20 0.40 0.1 0 0.50 0.40 0.20 0.30

0.00 0.00 0.00 0.û0 0.10 0.00

1.50 0.20 0.00 û.50 0.90 1,00 0.60 1.00 o.20 j.71, 0.49 )a) 1.20

0.40 0.80 0.50 0.40 0.60 0.40 0.50 0.40 0.70 0_80

1.00 0.20 0.1 0 o.20 1.00 0.70 0.40 0.20 0.10 0.60

0.00 0.00 0.30

1

2

3

0_10

5

0.50 0.30 0.20 0.00 0.30 0.00

6

7 I I '10

0_00

0.1 0

0.1 0 0.00

0.00 0.00 c. c2 0,04

0.'10

0.20 0.27

r

c.3 8

1...,1.:

0.27 t/ó

SD N4ean + 3.3 SD Overall LOD = 1.10 ppm

0.35

0.1 9 086

0,16

0.18 o.75

0.1 3

0.71

0.1 6

1.62

1.09

" All samples prev¡ously tested gluten-free

bean I'lour) into 5-100 g batches. One u,as spiked rvith l0 rn[, 2%CMC solulion onl)'f'or a 0 ppm gliadin level. l'he rL.nìaining portions \\'ere spiked using above preparatiort rvith ad.justed concentrations to ensure each respectiYe level (5. 10. 20. and 40 pptn) \vere crealed b¡'spiking l0 mL based on drv *'eight. \\¡ater rias then added. and each batch uas ntixed according 10 tlre recipe and baked al 220"C fìrr 22 r¡in. Samples rvere then dried in a household f'ood deh¡'drator. frozen, and ground to a tne particle size belore lhe¡,uere portioned into 0.25 g sample sizt' in 50 cc pol)propvlene ccnlrilupe tubes and blind-coded. Saurples ucre held fË/at 4'C arraitine arral¡sis. All sanrples u,ere blind-coded and stored at 2-8'C until analyzed. (a) Prepare 80% ethanol exlraction solution by cornbining eight parls ethanol u'ith t\\'o parts (r,/v) distilled s'ater. (b) Prepare sanrple extract dilution solution (PIIS) by dissolr ing thc'contents of'the packet in I I- distilled uater. (c) \\tighout 0.25 g sarnple into a 50 cc scre\\,cap centriligc tube. (d) Add 2.5 rnl renatuling cocktail solution. (e) lf'samples contain lrucku'hcat. chestnut 1'lour. ta¡tnins, or phenolic cornpounds. like chocolate. cofTèe or cocoa. add one level scoop ol'extraction acfdilive . Irol all other commodil\, l)'pes. do not add extraction additive. (f) Cap and vortcx 30 s 1o honrogenize cocktail and sarnple. (g) Incubate 40 rnin at 50'C (n'ater lratlr or oven). (h) Remove sarnples aud let cool 5-10 min. (i) Add 7.5 mL 80% etlranol ancl r.ortex again for l0-20 s, (j) Sltake (150-200 rpm) l'or I h at rooln ternperature olì a rotator (tr¡be on its side). (k) Centrihrge sarnple I0 niin at >2500 rpnr. (l) Dilute the sarnple 2.3 nrl- PBS) (nt) Saniples are re¿rcl-r (if' material does rlot readil¡- settle) lor l:12.5 rvith PBS (200 FL sample + lìrr anall'sis. Sample Preparation and Extract¡on

Procedural Notes

(a) llash buffer.-Prepale the wash bulìèr solution bv diluting the rvash bufïer concentrate in 960 mL distilled or deionized water iu au entpty I L confainer. Ensure transfcr ofall concentrate b1'rinsing the bottle sevelal titnes rvith rr,ater. and su irl to cnsure tllorough ¡uixi¡r_r.1. (b) Sttbsf rale.-K-Blue substl ale is read¡' l'or use. The substrate should be clear to light blue: discard il it has tunted clark blue. Only. pour the needed volurne of substrate into a reagent boat. Do not retum r.urusecl substrate to tlle boltle. Cover the reagent boat to keep the subslrate protected fiorn light until needed. (c) ,4nÍibody, uel/s.-Keep qells sealed in the f-oil pouch until needed. Iìemove u'ells liorn the loil pouch onlv afier samples alr: extl'acted and the te st procedure is set to bcgin.

Precautions

(a) Ilthanol solution is highly fìanlnlable. Kcep containcr'

Table 5. Temperature robustness

Samples, pÞm

Temperature, oC

Run No.1

0.20

4.70

18

9.70

0.00

4.90

9. s0 97ô

24

0.00

4.60

30

Run No. 2

0.60

4.50

18

9.90

0.00 010

4,50

24

9.40

30

4.90

9.50