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( k )  Dimethyl sulfoxide (DMSO).— HPLC grade (CAS No. 67- 68-5). ( 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 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

Table 2017.12C. Positive selectivity study of milk protein Sample Casein Whey Negative control a ND b ND LCS c CP d CP Mixed matrices_1 e ND ND Mixed matrices_2 ND ND Mixed matrices_3 ND ND Mixed matrices_4 ND ND Mixed matrices_5 ND ND Mixed matrices_6 ND ND MILK_10_1 f CP CP MILK_10_2 CP CP MILK_10_3 CP CP MILK_10_4 CP CP MILK_10_5 CP CP MILK_10_6 CP CP a  Negative control is BCAA provided by CRO Manufactory. 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 2017.12B . f  Milk sample was prepared by adding 10,000 ppm milk protein in the mixed matrices.

Table 2017.12D. Probability of identification (POI) study of milk protein at low concentration a Sample Casein b Whey Sample

Casein

Whey

Negative control

ND c CP e CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP 100

ND CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP 100

Negative control

ND CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP 100

ND CP CP CP CP CP ND CP CP CP CP CP CP CP CP CP CP CP CP CP 100

LCS d

LCS

MAT A_1000_01 f MAT A_1000_02 MAT A_1000_03 MAT A_1000_04 MAT A_1000_05 MAT A_1000_06 MAT A_1000_07 MAT A_1000_08 MAT A_1000_09 MAT A_1000_10 MAT A_1000_11 MAT A_1000_12 MAT A_1000_13 MAT A_1000_14 MAT A_1000_15 MAT A_1000_16 MAT A_1000_17 MAT A_1000_18

MAT B_1000_01 g MAT B_1000_02 MAT B_1000_03 MAT B_1000_04 MAT B_1000_05 MAT B_1000_06 MAT B_1000_07 MAT B_1000_08 MAT B_1000_09 MAT B_1000_10 MAT B_1000_11 MAT B_1000_12 MAT B_1000_13 MAT B_1000_14 MAT B_1000_15 MAT B_1000_16 MAT B_1000_17 MAT B_1000_18

POI, %

POI

a  Low concentration is at 1000 ppm (0.1%) for milk. b  In this experiment, instead of 1000 ppm as the MRL, milk used 100 ppm as MRL. c  ND = Not detected. d  LCS = Laboratory control sample. e  CP = Confirmed presence. f  Matrix A is preworkout finished goods provided by CRO Manufactory. g  Matrix B is premixed vitamin provided by CRO Manufactory.

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