6. AOACSPIFANMethods-2018Awards

62

8 B runt et al .: J ournal of aoaC I nternatIonal V ol . 100, n o . 3, 2017

content (using the same three different fructan ingredients; Table 2016.14I ). The samples were also analyzed in duplicate on 3 different days, and the recoveries were calculated by comparing the theoretical spike amount with the measured spike amount.

fructan content and adding 3 times the SD to estimate the LOD and adding 10 times the SD to estimate the LOQ. (c) Repeatability and intermediate reproducibility .— Repeatability (r) and intermediate reproducibility (iR) were assessed by analyzing samples (containing fructans) in duplicate on at least 6 different days. Excel, or the in-house statistical package Q-Stat, were used to calculate the SD(r) and SD(iR) using the following equations:

Results

Method Development

n

n

)

(

2

∑ ∑ 2 i

SD n

− x x n i 2

The method essentially consists of three stages: ( 1 ) removal of sucrose and free sugars, ( 2 ) hydrolysis of fructan to release glucose and fructose, and ( 3 ) analysis of the released glucose and fructose by HPAEC–PAD. To optimize all parameters, the final HPAEC–PAD method was first developed. In this case, the two laboratories developed different approaches: NRC used a CarboPac PA20 column, and CCC used a CarboPac PA1 column (representative chromatograms are shown in Figure 1). Each system has a dedicated elution gradient, as described in J . In both cases, the glucose and fructose are well separated from other sugars, including galactose, which may be released from lactose if the inulinase used for fructan hydrolysis is insufficiently specific. The appearance of galactose in the chromatogram can thus be used as an indicator for this side activity. Both laboratories added sodium hydroxide solution postcolumn, before PAD. The postcolumn addition of sodium hydroxide results in improved baseline stability and higher detector sensitivity. The amperometric detector has a thin-layer flow cell. Due to the impedance in the amperometric flow cell and the resulting ohmic drop in the potential of the working electrode, calibration curves of amperometric detectors deviate from linearity, especially at higher analyte concentrations (9); therefore, both laboratories used quadratic calibration models.

1 2 i

)

(

i

i

=

=

1

1

SD r

=

=

+ × 1 2

)

)

)

(

(

(

= SD iR SD b 2

2 SD r

where n = the number of (single or duplicate) determinations; x i = the individual result within the set of single determinations, with i going from 1 to n ; x i1 and x i2 = the two results within the set of a duplicate determination, with i going from 1 to n ; and SD(b) = the SD between the means of duplicates. (d) Recovery .—Recovery was assessed slightly differently in the two different laboratories. At NRC, several different infant formulas (containing no fructans) were spiked with three different levels of three different fructan ingredients (Table 2016.14H ). The fructan content of the ingredients was separately determined following Method 997.08 (3). The spiked samples were then analyzed in duplicate on 3 different days, and the recovery was calculated by comparing the measured amount with the theoretical (expected) amount. At CCC, six samples (containing fructans) were spiked with an additional 50 or 150% of the native fructan

Table 2016.14H. Design of spike-recovery experiment at NRC

Pure fructan ingredient

Table 2016.14I. Design of spike-recovery experiment at CCC

Sample No.

Sample description

Level 0 a

Level 1 b

Level 2 c

Level 3 d

Day 1: Orafti P95 spike, g/100 g

Day 2: NutraFlora P-95 spike, g/100 g

15

Infant Formula Powder, Milk-Based Infant Formula Powder, Soy-Based

None Orafti P95 Orafti HP NutraFlora P-95

Day 3: Orafti HP spike, g/100 g

Sample No.

Sample description

Spike level

16

None Orafti HP NutraFlora P-95

Orafti P95

1

Child Formula Powder Toddler Formula Powder, Milk-Based Infant Formula Powder, Milk-Based Child Formula Powder Infant Formula Powder, FOS/ GOS-Based

Low 0.17 High 0.49 Low 0.17 High 0.49 Low 0.17 High 0.49 Low 0.17 High 0.49 Low 0.017 High 0.049 Low 0.17 High 0.49

0.17 0.50 0.17 0.50 0.17 0.50 0.17 0.50

0.19 0.53 0.19 0.53 0.19 0.53 0.19 0.53

18

Adult Nutritional RTF, High-Protein Adult Nutritional Powder, Low-Fat Infant Formula Powder, Partially

None NutraFlora P-95

Orafti P95 Orafti HP

9

11

None Orafti P95 Orafti HP NutraFlora P-95

10

7

None Orafti HP NutraFlora P-95

Orafti P95

Hydrolyzed Milk-Based

12

13

Infant Elemental Powder

None NutraFlora P-95

Orafti P95 Orafti HP

14

0.017 0.050

0.019 0.053

a Level 0 = 0 g/100 g. b Level 1 = 0.03 g/100 g. c Level 2 = 2 g/100 g. d Level 3 = 5.0 g/100 g.

19 Adult Nutritional RTF, High-Fat

0.18 0.50

0.19 0.53