AOAC SPIFAN Nutrients ERP-Final Review (Jan. 2019)

D RAHER & W HITE : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 3, 2018 825

Table 2017.03A. Tryptophan SLV data: precision

SPIFAN code a

No. of replicates b

Mean, g/100 g RTF c

Sample type

RSD r

RSD iR

SRM 1849a

CLC10-B

12 12 12 12 12 12 12 12 12 12 12 12 12 12

20.1 25.3 18.3 25.7 24.1 21.4 23.7 32.2 23.2 19.5 18.3 22.4 70.6 91.3

0.37 0.56 4.95 0.82

1.51 0.97 6.92 2.02 1.82 1.33 1.56 2.42 1.69 2.27 1.79 3.68 1.91 1.7

Infant formula powder, partially hydrolyzed milk-based Infant formula powder, partially hydrolyzed soy-based

410057652Z 410457651Z 4052755861 4044755861 00859RF00

Toddler formula powder, milk-based Infant formula powder, milk-based

0.2 0.9

Adult nutritional powder, low-fat

Child formula powder

00866RF00

0.94 0.24

Infant elemental powder

00795RF

Infant formula powder, FOS/GOS-based Infant formula powder, milk-based Infant formula powder, soy-based

50350017W1

2

K16NTAV

0.31

E10NWZC

0.2

Infant formula, RTF milk-based

EV4H2R

0.45 1.86

Adult nutritional, RTF high-protein

00730RF00 00729RF00

Adult nutritional, RTF high-fat

0.3

a Materials from the SPIFAN Sample Kit. b Duplicates on each of 6 days performed by one laboratory. c RTF = Ready-to-feed.

hydrolysis of all sample types. (Although many samples are fully hydrolyzed within 6 h, some have been found to require longer; thus, a 16 h minimum is recommended for full applicability.) After hydrolysis, the sample – enzyme mixture is diluted to 50 mL with methanol – water and filtered. The sample is injected into a C8 column with reference standards and enzyme blank preparations and the analytes of interest detected and quantified fluorometrically. (a) HPLC system. — Equipped with an autosampler, column oven to maintain 30°C, fluorescence detector, binary or quaternary HPLC pump with degasser, and HPLC peak area integrator or chromatography data processor. (b) Analytical column. — YMC PACK C8; 3 µm particle, 3 × 50 mm (YMC America, Inc.; Product No. OC12S03-0503WT). (c) Analytical balance. — Readable to 0.001 mg. (d) Oven. — Constant-temperature oven capable of maintaining 50 ± 1°C. B. Apparatus and Materials

either at the N-terminal end (amino peptidases) or at the C terminus (carboxypeptidases; 5, 6). The protein is thus attacked on different sites simultaneously, releasing Trp in a relatively short period of time. Following proteolysis, Trp is quantitated by reversed-phase isocratic HPLC and fluorescence detection, which provides for a selective and specific determination of Trp in nutritional products. The enzymes in the pronase self-digest to produce background Trp in the absence of a sample. Consequently, the enzyme system is nonspecific for the Trp sample, and a blank subtraction is mandatory. Using this approach, recoveries of free Trp spikes as well as Trp 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

Table 2017.03B. Tryptophan SLV data: accuracy

50% overspike

100% overspike

SPIFAN code

No. of replicates a

Spiking material

Native Trp level, g/100 g

Avg. recovery, %

RSD, %

Avg. recovery, %

RSD, %

Sample type

SRM 1849a

CLC10-B

12 12

BSA BSA

20.06 25.28

99.9

1.83

100.9 104.1

2.4

Infant formula powder, partially hydrolyzed milk-based Infant formula powder, partially hydrolyzed soy-based

410057652Z

104.3

3.3

2.71

410457651Z

11

BSA

18.33

103.7

8.6

101.5

4.8

Adult nutritional powder, low-fat

00859RF00

12 10 12 12 12

BSA BSA BSA BSA BSA

21.39 32.22 19.51 18.27

96.2

5.2 2.5

99.5

3.9 2.7

Infant elemental powder

00795RF

100.7 100.5

102.4 101.7

Infant formula powder, milk-based Infant formula powder, soy-based

K16NTAV

2.99 5.09

1.99 3.53

E10NWZC

93.8

99.5

Adult nutritional, RTF high-fat

00729RF00

91.3

104.8

1.6

104.9

1.3

a Samples spiked in duplicate with two levels of spikes over 3 days. Samples with <12 replicates contained data points that were confirmed outliers.