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Dual Lab Validation of a Method for the Determination of Fructans in Infant
Formula & Adult Nutritionals /
25 Jul 2016
2
S U M M A R Y
A method has been co‐developed at Nestlé Research Centre (NRC), Lausanne Switzerland and
Eurofins Carbohydrate Competence Centre (CCC), Heerenveen, The Netherlands. For analysis,
samples are reconstituted in water and further diluted to a suitable concentration. The diluted
sample is treated with a mixture of sucrase and α‐glucanases to hydrolyse sucrose and alpha‐
glucans to their constituent monosaccharides. The sample is passed through a solid phase
extraction 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 hydrolysed with an inulinase mixture, and the
released glucose and fructose are analysed by high performance anion‐exchange chromatography
with pulsed amperometric detection (HPAEC‐PAD). The fructan content is calculated by summing
the glucose and 0.9 × fructose content. The main advantages of the method over existing methods
are (1) sucrose and monosaccharides are eliminated, avoiding their interference in the analysis, (2)
all components of the fructan are measured, thus there is no need for ingredient‐specific correction
factors, (3) the method is sufficiently sensitive to detect low amounts of fructan (0.03 g/100g in
reconstituted products).
Some minor differences in the application of the method exist in the two labs:‐
(1)
Just prior to SPE, the CCC applied a Carrez clean‐up step. This was not performed at NRC
(2)
During SPE, CCC used 1 × 400 µL then 2 × 800 µL of NaCl (1M) while NRC used 2 × 1000 µL of
NaCl (1M)
(3)
For SPE elution CCC used 5 × 400 µL elute solution, NRC used 3 × 400 µL elute solution
(4)
Chromatographic conditions are different since CCC employ a Carbopac PA1 column (2 × 250
mm) and NRC employ a Carbopac PA20 ( 3 × 150 mm).
The method has been validated independently in both laboratories using the SPIFAN material test
kit. Of the 19 matrices in the SPIFAN kit, 6 were found to contain fructans.
At NRC the 6 fructan‐containing SPIFAN samples and 2 Nestlé materials were analysed on 6 different
days in duplicate to determine the precision of the method. Of the 13 fructan‐free samples, 6 were
selected for use in spike/recovery experiments. The 6 matrices were spiked with 3 different fructan
ingredients at three different levels. CCC followed the same SLV procedure using the 6 fructan‐
containing SPIFAN samples.
The results of the two SLVs are summarized in the table below. In most cases the data are better
than required by the SMPR. However, out of the 18 experiments carried out at NRC for the spike‐
recovery experiment, 3 resulted in recoveries slightly outside the SMPR guidelines. The results
outside the SMPR targets were at the extremes of the working range (i.e. at 0.03 g/100g and at 5
g/100g) and the results on the other matrices tested at the same levels were within the target range
of the SMPR. Likewise at Eurofins, out of the 12 spike‐recovery experiments, only one result was
outside of the SMPR at 89%. The method precision was well within the limits for all samples at NRC,
VALIDATION REPORT
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