5. AOACSPDSMethods-2018AwardsV3

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AOAC Official Method 2017.12 Identification of Milk Proteins in Raw Materials and Finished Goods

Table 2017.12A. Negative selectivity study of milk protein Sample Casein Whey Negative control a ND b ND LCS c CP d CP Matrix A-01 e ND ND Matrix A-02 ND ND Matrix A-03 ND ND Matrix A-04 ND ND Matrix A-05 ND ND Matrix A-06 ND ND Matrix B-01 f ND ND Matrix B-02 ND ND Matrix B-03 ND ND Matrix B-04 ND ND Matrix B-05 ND ND Matrix B-06 ND ND a  Negative control is BCAA provided by CRO Manufactory. b  ND = Not detected. c  LCS = Laboratory control sample. d  CP = Confirmed presence. e  Matrix A is preworkout finished goods provided by CRO Manufactory. f  Matrix B is premixed vitamin provided by CRO Manufactory. Nonprotein ingredients and adulterants were added in both matrix A and B. Detailed information is listed in Table 2017.12B .

ESI HPLC-MS/MS First Action 2017 (Applicable for identification of milk protein in raw material and finished good samples.) See Tables 2017.12A – E for results of the single-laboratory study supporting acceptance of the method. A. Principle Solid sample is extracted by 5.0 M urea in 50 mM Tris buffer followed by reducing with 1 M dithiothreitol (DTT) for 30 min and alkylation in 0.5 M iodoacetamide for 30 min. The extract is further digested by trypsin solution at 37°C overnight, and then quenched by formic acid. After filtering by a disposable syringe and 0.22 µm nylon syringe filter, the extract is diluted (1:10) with 0.1% formic acid and 200 ng/mL internal standard and subjected to LC-MS/MS analysis. The method uses β-casomorphin 1-4 peptide as the internal standard (IS). Milk protein identifications are based on comparison with method reporting limit (MRL) samples, which are made from raw materials identified using PCR. B. Apparatus ( a ) Ultra-performance liquid chromatography (UPLC) system .—Shimadzu Nexera. ( b ) Triple quadrupole mass spectrometer detector .—AB Sciex 4000 Q TRAP. ( c ) HPLC column.— C18, 150 × 2 mm, Synergi 4 µ Hydro 80 Å (P/N 00F-4375-B0; Phenomenex.) ( d ) HPLC guard column .—C18 (P/N AJ0-8768; Phenomenex). ( e ) Centrifuge.— Capable of 4000 g . ( f ) Analytical balances .—0.0001 g accuracy. ( g ) Volumetric pipets. ( h ) Pipet tips. ( i ) Disposable syringes. ( j ) Nylon syringe filters .—0.22 µm. ( k ) Disposable centrifuge tubes .—1.5, 15, and 50 mL. ( l ) Autosampler vials . ( m ) Incubator .—Capable of maintaining 37°C. ( n ) Refrigerator.— Capable of maintaining 2–8°C. ( o ) Freezer.— Capable of maintaining –20°C. ( p ) Heated water bath.— Capable of maintaining 55°C. ( q ) Sonication bath. ( r ) Vortex mixer.

C. Reagents ( a ) Ultrapure water .—≥16 MΩ/cm.

( b ) Acetonitrile.— HPLC grade (CAS No. 75-05-8). ( c ) Formic acid.— ≥98.5% purity (CAS No. 64-18-6).

( d ) Iodoacetamide.— Proteomics grade (CAS No. 144-48-9). ( e ) DTT.— Molecular biology grade (CAS No. 3483-12-3). ( f ) Urea.— Molecular biology grade (CAS No. 57-13-6). ( g ) Ammonium bicarbonate.— ≥99.5% pure (CAS No. 1066-33-7). ( h ) Trypsin .—Proteomics grade (CAS No. 9002-07-7). ( i ) Trizma base.— Molecular biology grade (CAS No. 77-86-1). ( j ) Phosphate buffered saline (PBS) concentrate .—Molecular biology grade.

Table 2017.12B. Components of nonprotein ingredients/adulterants in selectivity study Nonprotein ingredient/adulterant

Concn, % Nonprotein ingredient/adulterant Concn, % Nonprotein ingredient/adulterant Concn, %

Creatine Taurine Caffeine Metaline

7.85 5.23 1.75 0.42 1.12 0.50 0.49 0.50

Beta alanine

0.50 0.51 0.51 0.50 0.50 0.50 0.50 0.50

Histidine Citrulline

0.51 0.50 0.50 0.51 0.50 0.52

Serine

Phenylalanine

Tryptophan Aspartic acid

Glutamine Methionine

Urea

Isoleucine

Arginine Leucine

Glycine

Lysine

Valine

Threamine

Tyrosine

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