5. AOACSPDSMethods-2018AwardsV3

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E. Preparation of Test Sample ( a ) Thoroughly mix sample prior to weighing. Transfer 500 mg to a 15 mL centrifuge tube. ( b ) Prepare each sample separately to avoid clumping of material in tube. ( c ) Add 10 mL of extraction buffer followed by vortexing for 10–60 s until the sample material is a homogenous slurry. ( d ) Sonicate for 60 min. (While sonicating, preheat the water bath.) ( e ) Spin the tubes down for 10 min at 4000 g to pellet remaining solids. ( f ) Transfer 4 mL of the supernatant by pipet to a new 15 mL conical centrifuge tube and add 150 µL of the thawed reducing solution. ( g ) Vortex samples to mix. Heat for 30 min in a 55°C water bath. ( h ) Cool to room temperature. (While cooling prepare fresh alkylating solution.) ( i ) Alkylate each sample separately. Add 1.2 mL of the freshly prepared alkylating solution, mix by vortexing, and store the tube protected from light for 30 min at room temperature. ( j ) Following alkylation, add 4 mL of digestion buffer to each sample. Table 2017.11F. Weights required and final % composition Sample 1 2 3 4 Total Weight, g 24.8 25.5 28.4 22.1 100.80 Kjeldahl, % 19.79 19.79 19.79 19.79 79.16 Table 2017.11E. Probability of identification (POI) protein of pea, rice, and soy protein at high concentration a Sample Pea Rice Soy Negative control ND b ND ND LCS c CP d CP CP MAT_A_10000_1 e CP CP CP MAT_A_10000_2 CP CP CP MAT_A_10000_3 CP CP CP MAT_A_10000_4 CP CP CP MAT_A_10000_5 CP CP CP MAT_A_10000_6 CP CP CP MAT_B_10000_1 f CP CP CP MAT_B_10000_2 CP CP CP MAT_B_10000_3 CP CP CP MAT_B_10000_4 CP CP CP MAT_B_10000_5 CP CP CP MAT_B_10000_6 CP CP CP POI, % 100 100 100 a  High concentration is at 10000 ppm (1%). b  ND = Not detected. c  LCS = Laboratory control sample. d  CP = Confirmed presence. e  Matrix A was provided by CRO Manufactory. f  Matrix B is premixed vitamin provided by CRO Manufactory.

Table 2017.11G. Column parameters HPLC column

Luna 3 µm, 150 × 2 mm C18, 200 Å

Column temp.

30°C 5 µL

Recommended injection vol.

( k ) Mix by carefully inverting the tube multiple times. ( l ) Add 250 µL of the thawed trypsin working solution. ( m ) Mix by carefully inverting tube multiple times. ( n ) Incubate samples overnight in an incubator at 37°C. (Record times at start and finish of incubation.) ( o ) Add 400 µL formic acid to quench the samples. ( p ) Mix carefully as addition of formic acid can cause foaming leading to sample loss. Mix by inverting the tube multiple times. ( q ) Centrifuge samples for 10 min at 4000 g to pellet any precipitate. ( r ) Filter a portion of resulting supernatant using a disposable syringe and 0.22/0.45 µm nylon syringe filter into a 1.5 mL centrifuge tube. ( s ) Thaw an aliquot of the 2000 ng/mL internal standard spike solution. ( t ) Prepare final sample at 1:10 dilution with 0.1% formic acid and 200 ng/mL internal standard. ( Example : Add 100 µL sample, 100 µL IS spike solution, and 800 µL of 0.1% formic acid to an autosampler vial.) ( u ) Inject 5 µL sample for analysis. F. Determination ( 2 ) Valco valve 2 position schedule .—Position A: Flow from column diverted to waste; position B: flow from column sent to MS for analysis. See Table 2017.11I for schedule. ( c ) Mass spectrometer conditions.— AB Sciex 4000 Q TRAP. See Tables 2017.11J for instrument settings, 2017.11K for analytes, and 2017.11L for transition parameters. ( d ) Sequence format .—( 1 ) DMSO blank, prep blank, solution blank. ( 4 ) Unknown sample(s) .—Inject unknown samples followed by a DMSO blank. Intersperse a second set of MRL samples after the unknowns. G. Calculations ( a ) Calculation of 1000 ppm MRL .—Calculate the average peak area ratio of each peptide from both sets of MRL: ( 2 ) MRL samples. ( 3 ) DMSO blank. ( a ) Column parameters .— See Table 2017.11G . ( b )( 1 ) Pump gradient .— See Table 2017.11H .

Table 2017.11H. Pump gradient Time, min

A, % B, % Flow rate, mL/min Curve

0.0 0.8 5.1 6.4 7.3 7.5

85 85 50

15 15 50 98 98 15 15

0.3 0.3 0.3 0.3 0.3 0.3 0.3

0 0 0 0 0 0 0

2 2

85 85

10

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