AOAC Final Action Methods in 2019

provides for a selective and specific determination of tryptophan in nutritional products. The enzymes in the pronase self-digest to produce background tryptophan in the absence of sample. Consequently, the enzyme system is nonspecific for the sample tryptophan, and a blank subtraction is mandatory. Using this approach, recoveries of free tryptophan spikes as well as tryptophan from bovine serum albumin (BSA) spikes are found to be essentially equivalent, indicating near comparable self-digestion rates with and without sample. Sample preparation consists of adding a weighed sample, the enzyme solution, internal standard (5-methyl-DL-tryptophan), and Trizma ® buffer into a tube. A small amount of methanol is added as a bactericidal agent. The preparation is mixed and incubated at 50°C for 16 h (overnight) to ensure complete hydrolysis of all sample types (while many samples are fully hydrolyzed within 6 h, some have been found to require longer; thus, the 16 h minimum for full applicability). After hydrolysis, the sample–enzyme mixture is diluted to 50 mL with methanol–water and filtered. The sample is injected onto a C8 column with reference standards and enzyme blank preparations and the analytes of interest detected and

AOAC Official Method 2017.03 Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula HPLC Following Enzymatic Hydrolysis

First Action 2017 Final Action 2019

[Applicable to the determination of total tryptophan content in infant, pediatric, and adult nutritional products as defined in SMPR 2014.013.] See Table 2017.03A for multilaboratory testing data supporting acceptance of the method. A. Principle Tryptophan is released (hydrolyzed) from intact protein using a combination of proteolytic enzymes found in pronase, isolated from Streptomyces griseus, for this purpose. The pronase enzyme powder contains at least 10 proteolytic enzymes (depending on the source, supplier, etc.) which hydrolyze peptide bonds internally (endoproteases) and externally (exopeptidases), either at the N-terminal end (amino peptidases) or at the carboxy terminus (carboxypeptidases; 1, 2). The protein is thus “attacked” on different sites simultaneously, releasing tryptophan in a relatively short period of time. Following proteolysis, tryptophan is quantitated by reversed-phase isocratic HPLC and fluorescence detection, which Table 2017.03A. Statistical analysis of MLT blind duplicates Sample ID K16NTAV

quantified fluorometrically. B. Apparatus and Materials

( a ) HPLC system with autosampler .—Column oven to maintain 30°C, fluorescence detector, binary or quaternary HPLC pump with

E10NWZC

410057652Z

410457651Z

00729RF00

Total number of laboratories Total number of replicates

10 20

10 20

9

10 20

10 20

18

Overall mean of all data (grand mean), mg/100 g

19.56

18.09

25.41

17.92

90.52

Repeatability SD Reproducibility SD Repeatability RSD, % Reproducibility RSD, %

0.50 0.79

0.33 0.73

0.23 1.08

0.65 1.78

1.22 3.72

2.5 4.1

1.8 4.0

0.9 4.2

3.6 9.9

1.3 4.1

HorRat value

0.56

0.55

0.61

1.35

0.72

Sample ID

00730RF00

4052755861

4044755861

EV4H2R

CLC10-B

Total number of laboratories Total number of replicates

8

10 20

10 20

10 20

10 20

16

Overall mean of all data (grand mean), mg/100 g

70.42

24.79

23.96

21.99

18.14

Repeatability SD Reproducibility SD Repeatability RSD, % Reproducibility RSD, %

1.45 2.97

0.44 1.00

0.51 0.79

0.22 0.88

0.29 0.55

2.1 4.2

1.8 4.0

2.1 3.3

1.0 4.0

1.6 3.0

HorRat value

0.71

0.58

0.47

0.56

0.41

Sample ID

00866RF00

00859RF00

00795RF00

50350017W1

Total number of laboratories Total number of replicates

10 20

9

10 20

10 20

18

Overall mean of all data (grand mean), mg/100 g

23.38

21.35

32.80

22.59

Repeatability SD Reproducibility SD Repeatability RSD, % Reproducibility RSD, %

0.60 1.02

0.32 0.67

0.58 1.09

0.50 0.80

2.5 4.4

1.5 3.1

1.8 3.3

2.2 3.5

HorRat value

0.62

0.44

0.50

0.50

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