5. AOACSPDSMethods-2018AwardsV3

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Table 2017.11D. Probability of identification (POI) protein study of pea, rice, and soy protein at low concentration a Sample Pea b Rice c Soy d Sample Pea Rice Soy Negative control ND e ND ND Negative control ND ND ND LCS f CP g CP CP LCS CP CP CP MAT A_1000_01 h CP CP CP MAT B_1000_01 i CP CP CP MAT A_1000_02 CP CP CP MAT B_1000_02 CP CP CP MAT A_1000_03 CP CP CP MAT B_1000_03 CP CP CP MAT A_1000_04 CP CP CP MAT B_1000_04 CP CP CP MAT A_1000_05 CP CP CP MAT B_1000_05 CP ND CP MAT A_1000_06 CP CP CP MAT B_1000_06 CP CP CP MAT A_1000_07 CP CP CP MAT B_1000_07 CP CP CP MAT A_1000_08 CP CP ND MAT B_1000_08 CP CP CP MAT A_1000_09 CP CP CP MAT B_1000_09 CP CP CP MAT A_1000_10 CP CP CP MAT B_1000_10 CP CP CP MAT A_1000_11 CP CP CP MAT B_1000_11 CP CP CP MAT A_1000_12 CP CP CP MAT B_1000_12 CP CP CP MAT A_1000_13 CP CP CP MAT B_1000_13 CP CP CP MAT A_1000_14 CP CP CP MAT B_1000_14 CP CP CP MAT A_1000_15 CP CP CP MAT B_1000_15 CP CP CP MAT A_1000_16 CP CP CP MAT B_1000_16 CP CP CP MAT A_1000_17 CP CP CP MAT B_1000_17 CP CP CP MAT A_1000_18 CP CP CP MAT B_1000_18 CP CP CP POI, % 100 100 94.4 POI 100 94.4 100 a  Low concentration is at 1000 ppm (0.1%) for pea, rice, and soy.

( i ) LCS .—A composite of raw material protein samples is prepared ensuring that each protein source is at the same level in the final composite. For each raw material protein samples used in the composite, a Kjeldahl result is used to calculate the weights to be used in the composite. For ~100 g composite from the raw materials, use the following equation to calculate the weight of each: b  In this experiment, instead of 1000 ppm as the MRL, pea used 100 ppm as MRL. c  In this experiment, instead of 1000 ppm as the MRL, rice used 500 ppm as MRL. d  In this experiment, instead of 1000 ppm as the MRL, soy used 100 ppm as MRL. e  ND = Not detected. f  LCS = Laboratory control sample. g  CP = Confirmed presence to be positive. h  Matrix A is preworkout finished goods provided by CRO Manufactory. i  Matrix B is premixed vitamin provided by CRO Manufactory.

Amount of LCS = (10 g mix × 2%)/19.79% = 1.01 g

The LCS spike is mixed with the remaining weight of negative control matrix. ( Example : Above mixed with 8.989 g negative control matrices). ( k ) Mobile phase A (0.1% formic acid in water) .—Pipet 500 µL formic acid into a 500 mL graduated cylinder. Fill to volume with ultrapure water, transfer to a glass bottle, mix well, and degas with sonicator prior to use. ( l ) Mobile phase B (0.1% formic acid in acetonitrile) .—Pipet 500 µL formic acid into a 500 mL graduated cylinder. Fill to volume with acetonitrile, transfer to a glass bottle, mix well, and degas with sonicator prior to use. ( m ) Needle rinse (95% acetonitrile 5% water) .—Pipet 25 mL ultrapure water into a 500 mL graduated cylinder. Fill to volume with acetonitrile, transfer to a glass bottle, mix well, and degas with sonicator prior to use.

Amount of sample X = (100 g composite/No. samples) × (avg. Kjeldahl result/Kjeldahl result for sample X)

See Table 2017.11F for required weights and final % composition. ( j ) MRL samples .—Produce MRL samples at 1000 ppm (0.1%) mixing the LCS and negative control matrices. The average percentage of each raw component is used to calculate amount of LCS needed to spike into a negative control matrix to produce a proscribed level. For example, the calculation for amount of LCS to produce a 10 g mix at initial 20000 ppm (2%) is as below:

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