5. AOACSPDSMethods-2018AwardsV3

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500 mL graduated cylinder and add 400 mL ultrapure water. Fill to volume with ultrapure water and transfer to a bottle. ( 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 gently 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 Table 2017.12E. Probability of identification (POI) of milk protein at high concentration a Sample Casein Whey Negative control ND b ND LCS c CP d CP MAT_A_10000_1 e CP CP MAT_A_10000_2 CP CP MAT_A_10000_3 CP CP MAT_A_10000_4 CP CP MAT_A_10000_5 CP CP MAT_A_10000_6 CP CP MAT_B_10000_1 f CP CP MAT_B_10000_2 CP CP MAT_B_10000_3 CP CP MAT_B_10000_4 CP CP MAT_B_10000_5 CP CP MAT_B_10000_6 CP CP POI, % 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 2017.12F. 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 2017.12G. Column parameters HPLC column

Luna 3 µm, 150 × 2 mm C18, 200 Å

Column temp.

30°C 5 µL

Recommended injection vol.

iodoacetamide (MW = 184.96) in 2 mL ultrapure water. Vortex until completely dissolved. ( h )  Internal standard (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. ( 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 of composite from the raw materials, use the following equation to calculate the weight of each: Amount of sample X = (100 g of composite/No. of samples) × (avg. of Kjeldahl result/Kjeldahl result for sample X) See Table 2017.12F for weights required 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: Amount of LCS = (10 g of 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.

Table 2017.12H. 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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