AOACSPIFANMethods-2017Awards

runt

et al

ournal of

aoaC I

nternatIonal

100, n

3, 2017

glucose, sucrose, maltodextrin, and/or starch. The subtraction

of two large values in order to calculate much lower inulin/

FOS values generally results in less precise data with large

SDs. Nevertheless, the method performs well when applied

to products containing relatively low levels of interfering

components.

The principle of Method

999.03

(4) differs from Method

997.08

(3), in that all monosaccharides present after combined

α-glucanases and sucrase treatment are removed by converting

them into alditols (via borohydride reduction). After enzymatic

hydrolysis of the fructans, a colorimetric reducing sugar assay

is then used to make the quantitative analysis. Fructans are thus

accurately determined even in samples with high contents of

monosaccharides, sucrose, maltodextrin, and/or starch, using

relatively simple and inexpensive equipment. However, there

is a drawback to this method: The reducing end groups of the

fructan chains that do not terminate with a glucose (often referred

to as Fm-type chains) are reduced into alditol end groups,

which escape the analysis, resulting in low recoveries (e.g., the

theoretical recoveries of fructobiose (F2) is only 50%, for F3,

it is 67%; for F4, it is 75%, etc.). Fructan chains containing a

terminal glucose (GFn-type chains) do not have a reducing end

group, so these are recovered completely. FOS material prepared

by depolymerization of inulin generally contains high amounts

of F3 and F4, so total recovery can be below 80%. Although the

method is not well suited to the analysis of samples containing

FOS generated by partial hydrolysis of inulin, it is well suited to

the analysis of long-chain fructans in a wide range of products

and is quick and simple to apply.

Neither of the methods is optimal for the determination of

fructans in infant formula or adult nutritionals. Therefore, the

Stakeholder Panel on Infant Formula and Adult Nutritionals

(SPIFAN) issued a call for new methods to meet the AOAC

Standard Method Performance Requirements

(SMPRs

)

defined in SMPR 2014.002 (5). After consideration by the

Expert Review Panel, two methods, including the one described

here and Method

2016.06

, were considered to have acceptable

performance.

Method

2016.06

(6, 7) is based on Method

999.03

(4) and

on a method published by Cuany et al. (8), with a number of

improvements. In Method

2016.06

(6, 7), a simplified sample

preparation was introduced that reduces method turnaround

time and, concomitantly, improves performance. The Cuany

et al. method (8) required knowledge of the fructan type in

the product to select appropriate correction factors (to correct

for the “loss” of the terminal monomer of the fructan chains).

Those correction factors are still required in Method

2016.06

(6, 7); however, a preanalysis step has been introduced to

identify the fructan type and, thus, the appropriate correction

factor, without the analyst needing (potentially confidential)

recipe information.

The method described here has been designed to determine

the fructan content without the need for ingredient-specific

correction factors, thus avoiding the need for preanalysis (or

knowledge of the fructan type in the product), and without

interference from other components such as sucrose and

free sugars. As in Method

999.03

(4), an enzyme mixture is

used to hydrolyze sucrose and α-glucans to their constituent

monosaccharides. Next, following the strategy of Cuany et al.

(8), a graphitized carbon SPE column is used to eliminate the

released glucose and fructose before the enzymatic hydrolysis

of the fructans. The key difference is to avoid any borohydride

treatment, thus avoiding the significant underestimation of

Fm-type fructans. The method was codeveloped in two

laboratories: Nestlé Research Centre (NRC) in Lausanne,

Switzerland, and Carbohydrate Competence Centre of Eurofins

(CCC) in Heerenveen, The Netherlands. Single-laboratory

validations (SLVs) were performed independently in both centers.

AOAC Official Method 2016.14

Fructans in Infant Formula and Adult Nutritionals

HPAEC-PAD

First Action 2016

A. Principle

Samples are reconstituted in water (if required) and further

diluted until the concentration of fructan in solution is such that

after hydrolysis, the fructose and glucose concentration is within

the range covered by the standard curve. The diluted sample is

treated with a mixture of sucrase and α-glucanases to hydrolyze

sucrose and α-glucans, respectively, releasing their constituent

monosaccharides. The sample is passed through an SPE cartridge

packed with graphitized carbon. Salts and monosaccharides pass

through and are washed away, while the fructans are retained.

Fructans are released from the column using an acetonitrile

solution. The released fructans are hydrolyzed with an inulinase

mixture, and the released glucose and fructose are analyzed by

high-performance anion-exchange chromatography (HPAEC)

with pulsed amperometric detection (PAD). The fructan content

is calculated by summing the glucose and 0.9× the fructose

content measured. In some matrixes containing low amounts

of fructans, a blank correction may be necessary and can be

applied. In this study, blank corrections were required only

at the lowest fructan concentration (0.03 g/100 g) and only

for a few products. For unknown matrixes containing fructan

concentrations below 0.1 g/100 g, it is advisable to check

whether the blank correction is required.

B. Materials

Samples from the SPIFAN II SLV Kit were provided by

Covance (Madison, WI) and are listed in Table

2016.14A

. In

addition, two infant formulas from Nestlé (Vevey, Switzerland)

were included in the SLV performed at NRC. Fructan

ingredients used for spiking experiments were Orafti

P95 and

Orafti HP (both from Beneo, Tienen, Belgium) and NutraFlora

P-95 (Ingredion, Inc., Westchester, IL).

The samples were stored in the original package in a dry place

and protected from light until the moment of use. According

to the instructions for this SLV Kit and the AOAC

Standard

Method Performance Requirements

(SMPRs

) defined in

SMPR 2014.002 (5), all powder products except SRM 1849a

were reconstituted by dissolving 25 g powder in 200 g water.

The SRM 1849a was weighed directly or reconstituted by

dissolving 10 g in 90 g water.

In this validation study, three different standards of pure

fructan ingredients were used:

(1)

Orafti HP, a long-chain inulin ingredient.

(2)

Orafti P95, a hydrolyzed inulin ingredient consisting of

both GFn and Fm constituents.