5. AOACSPDSMethods-2018AwardsV3

65

Table 2017.11C. Positive selectivity study of pea, rice, and soy protein Sample Pea Rice Soy

Sample

Pea

Rice

Soy ND CP ND ND ND ND ND ND CP CP CP CP CP CP

Negative control a

ND b CP d

ND CP ND ND ND ND ND ND ND ND ND ND ND ND

ND CP ND ND ND ND ND ND ND ND ND ND ND ND

Negative control

ND CP ND ND ND ND ND ND ND ND ND ND ND ND

ND CP CP CP CP CP CP CP ND ND ND ND ND ND

LCS c

LCS

Mixed matrices_1 e Mixed matrices_2 Mixed matrices_3 Mixed matrices_4 Mixed matrices_5 Mixed matrices_6

ND ND ND ND ND ND CP CP CP CP CP CP

RICE_10_1 f RICE_10_2 RICE_10_3 RICE_10_4 RICE_10_5 RICE_10_6 SOY_10_1 h SOY_10_2 SOY_10_3 SOY_10_4 SOY_10_5 SOY_10_6

PEA_10_1 g PEA_10_2 PEA_10_3 PEA_10_4 PEA_10_5 PEA_10_6

a  Negative control is BCAA provided by Genysis Brand Solution. b  ND = Not detected. c  LCS = Laboratory control sample. d  CP = Confirmed presence. e  Mixed matrices were made by mixing nonprotein ingredients/adulterants, matrix A and matrix B. Detailed information of nonprotein ingredients/adulterants is listed in Table 2017.11B .

f  Rice sample was prepared by adding 10,000 ppm rice protein in the mixed matrices. g  Pea sample was prepared by adding 10,000 ppm pea protein in the mixed matrices. h  Soy sample was prepared by adding 10,000 ppm soy protein in the mixed matrices.

( e ) Trypsin stock solution (~50 mg/mL) .—Transfer 525 mg trypsin to a 15 mL conical tube and add 10.5 mL digestion buffer. Mix by gentle inverting tube repeatedly until all trypsin is dissolved. ( Do not vortex trypsin solutions. ) Aliquot in 1200 µL portions to 1.5 mL centrifuge tubes. Label “TRYP 50” and store at –20 ° C. Two freeze/thaw cycles are allowed prior to discarding. ( f ) Trypsin working solution (~500 µg/mL) .—Remove one vial of the 50 mg/mL trypsin stock solution from freezer and allow to thaw completely prior to use. Transfer 400 µL to a 50 mL conical tube and dilute to 40 mL using digestion buffer. Mix by gently inverting tube repeatedly. ( Do not vortex trypsin solutions. ) Aliquot in 1500 µL portions to 1.5 mL centrifuge tubes. Label “TRYP 500” and store at –20 ° C. Two freeze/thaw cycles are allowed prior to discarding the solution. ( g ) Alkylating/protection solution (0.5 M iodoacetamide) .— Iodoacetamide is toxic. Use proper personal protective equipment including laboratory coat, gloves, and safety glasses. For each run, a fresh batch of alkylation solution must be prepared immediately before use. Discard unused portion. Per sample, dissolve 185 mg iodoacetamide (MW = 184.96) in 2 mL ultrapure water. Vortex until completely dissolved. ( h ) IS (β-casomorphin 1-4, YPFP) spike solutions .—Prepare IS stocking solution by weighing out ~40 mg β-casomorphin 1-4 peptide into a 25 mL volumetric flask, fill to volume with PBS, and mix well. Prepare a ~100 µg/mL IS intermediate solution in a 25 mL volumetric with PBS. Prepare IS working solution at ~2000 ng/mL in a 50 mL volumetric with PBS. Label “BCAS 2000” and store at –20 ° C.

( k ) Dimethyl sulfoxide (DMSO) .—CAS No. 67-68-5, HPLC grade. ( l ) Reference peptide .—β-Casomorphin 1-4 (bovine) (YPFP). ( m ) Laboratory control sample (LCS) .—For peptide signal tracking. D. Preparation of Reagents, LCS, and MRL Samples ( a ) 1 M Tris buffer .—Transfer 60.57 g Trizma base (MW = 121.14) to a 500 mL graduated cylinder and add ~350 mL ultrapure water. After Trizma base solid dissolves, fill to volume with ultrapure water and transfer to a bottle. ( b ) Basic extraction buffer (5 M urea in 50 mM Tris buffer) .— Transfer 300.3 g urea (MW = 60.06) to a 1 L graduated cylinder and add ~600 mL ultrapure water. Cover cylinder with parafilm and stir until urea is dissolved. (Solution will get cold. Ensure solution comes back to room temperature before continuing to next step.) Add 50 mL of 1 M Tris buffer and fill to volume with ultrapure water. Mix well and then transfer to a bottle. ( c ) Reducing solution (1 M DTT) .—Transfer 1.697 g DTT (MW = 154.2) to a 15 mL conical tube and add 11 mL ultrapure water. Mix by vortexing until solution is clear. Aliquot the solution in 1.1 mL portions to 1.5 mL centrifuge tubes. Label “DTT” and store at –20 ° C. Two freeze/thaw cycles are allowed prior to discarding. ( d ) Digestion buffer (100 mM ammonium bicarbonate) .— Transfer 3.953 g ammonium bicarbonate (MW = 79.06) to a 500 mL graduated cylinder and add 400 mL ultrapure water. Fill to volume with ultrapure water and transfer to a bottle.

© 2018 AOAC INTERNATIONAL