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B runt et al .: J ournal of aoaC I nternatIonal V ol . 100, n o . 3, 2017 13

do not contain a terminal glucose (Fm type), there will be a small underestimation of fructan depending on the chain length [i.e., the degree of polymerization (DP)]. Thus, the theoretically achievable recovery (due to calculation alone) is less than 100% for many fructan ingredients, depending on the average DP and the GFn-to-Fm ratio (Table 6). The worst case is a fructan ingredient containing 100% Fm-type chains and having an average DP of 3, for which only a 96% recovery is achievable; however, in practice, no such ingredient exists. The most impacted ingredient that we are aware of would be a fructan ingredient with an average DP of around 4 and an Fm-to-GFn ratio of 5. Such a product has a theoretically possible recovery of 97.7%. We believe that this small theoretical underestimation should not be a major issue in most cases and has not had a major impact in this study. However, when the laboratory knows the average DP of the fructan ingredient being used, the calculation can be adapted to avoid the underestimation as follows: ) ( = × × × C C D V m 0.0001 G GB ) ( = × × × C C D V m 0.0001 F FB ) ( ) ( ) ) ( ( = + × × + TF C C DP-1 0.9 1 DP F G = the concentration (μg/mL) of glucose in Solution B; D = the dilution factor between Solution A and Solution B (from Table 2016.14C ); V = the total volume (mL) of Solution A; m = the amount (g) of sample weighed to prepare SolutionA; 0.0001 = the factor to convert analyte concentration (μg/mL) in solution to analyte concentration (g/100 g) in sample; C F = the concentration (g/100 g) of fructose released from fructan; C FB = the concentration (μg/mL) of fructose in Solution B; TF = the total fructan concentration (g/100 g) in the sample; 0.9 = the factor to correct for uptake of water during fructan hydrolysis; and DP = the average DP of the fructan ingredient. where C G fructan; C GB = the concentration (g/100 g) of glucose released from

For the full spike-recovery experiments, three different pure fructan ingredients were used: Orafti HP, Orafti P95, and NutraFlora P-95. The ingredients were separately analyzed using Method 997.08 (3) to confirm their purity. At NRC, six different blank matrixes were spiked at three levels with the above-mentioned three pure fructan ingredients on 3 different days in duplicate. All samples were initially analyzed without using the blank subtraction (Table 3). At the two higher spiking levels, recoveries were, in general, very good (92–104%), with one exception—the Adult Nutritional RTF, High-Protein sample—for which the average recovery was only 86% at the highest spike level (0.03 g/100 g), which is equivalent to the LOQ specified in the SMPRs (5), the recoveries were less good, varying from 101 to 151%, with three matrixes achieving the SMPRs (recoveries of 101–105%) and three matrixes being outside the requirements (recoveries of 114–151%). Because the spike level is very low, a small amount of interference can have a significant impact on the recovery. To correct for this interference, the method using the blank subtraction was applied. Using the blank subtraction, recoveries on the samples with low spike levels are significantly improved to 95–119% (Table 4) but still do not meet the SMPRs in all cases [recoveries for two matrixes exceeded 110% (i.e., sample No. 7 at 117% and sample No. 18 at 119%)]. This improvement demonstrates the need for the blank subtraction for some samples, especially those containing low levels of fructans. At CCC, the six fructan-containing samples were overspiked at about 50 and 150% levels of the original fructan content determined in the precision study. All samples, both nonspiked and spiked, were analyzed without using the blank subtraction. The average recoveries (Table 5) were all within the target range of 90–110% defined in SMPR 2014.002 (5), with the exception of one sample (sample No. 9), which had an average recovery of 89% at the low spike level. Most of the spike-recovery data give acceptable results despite the fact that the method contains an inherent issue that can lead to underestimation of fructan content for some ingredient types. The issue lies in the calculation in which all the fructose is multiplied by a factor of 0.9 to correct for water uptake during hydrolysis. For fructan chains containing a terminal glucose (GFn type), this is not a problem because the glucose is not corrected and 100% recovery can always theoretically be achieved. However, for fructan chains that

Method Specificity

There are potentially two different mechanisms that may cause interference in the method: ( 1 ) an interfering substance could coelute with the glucose or fructose, and ( 2 ) the presence

Table 3. Spike-recovery results at NRC

Level 1

Level 2

Level 3

Spike, g/100 g Recovery, % RSD, %

Spike, g/100 g Recovery, % RSD, %

Spike, g/100 g Recovery, % RSD, %

Sample No.

Sample description

7

Infant Formula Powder, Partially Hydrolyzed Milk-Based

0.031

122

7.3

2.00

103

2.5

5.01

92.0

2.2

11

Adult Nutritional Powder, Low-Fat Infant Elemental Powder Infant Formula Powder, Milk-Based Infant Formula Powder, Soy-Based Adult Nutritional RTF, High-Protein

0.031

102

5.1

1.99

102

2.0

5.02

102

1.6

13 15

0.030 0.031

105 101

5.2 5.0

2.02 2.00

95.7 99.7

1.8 2.2

5.00 5.02

95.5 98.2

6.1 2.4

16

0.030

114

3.0

2.02

104

4.2

5.02

93.6

2.8

18

0.030

151

11

1.99

95.5

2.2

4.95

86.0

3.8