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Dual Lab Validation of a Method for the Determination of Fructans in Infant
Formula & Adult Nutritionals /
25 Jul 2016
51
A‐7‐b SPE clean‐up
Pipet 400 μl of the centrifuged sample at the top of the GC‐SPE column and flush carefully over the
activated GC‐SPE column.
Flush (at gravity conditions) with successively
‐ 1 x 400 μl 1M sodium chloride solution
‐ 2 x 800 μl 1M sodium chloride solution
‐ 5 x 800 μl demineralized water till just dry
Collect the following 5 respective flushing’s with 400 μl of 25% acetonitrile + 0.05 TFA solution. Suck
the GC‐SPE columns just dry. And homogenize the collected eluates.
A‐8 Hydrolysis of the fructans
Pipet 1.0 ml of the collected GC‐SPE eluate in a 2 ml micro centrifuge tube.
Add 350 μl 0.1M sodium acetate buffer ph = 4.5.
Pipet 0.1 ml enzyme mix B (fructanase) and homogenize.
Incubate the solution during 40 min. in a waterbath adjusted to 40 ± 2 °C
Filter the hydrolysate with a 0.2 μm syringe filter in a chromatographic vial and close the vial with a
screw cap.
The sample is now ready for the chromatographic analysis
A‐9 Preparation of the calibration standard solutions
Primary calibration standard:
dissolve 100mg glucose and 400 mg fructose, accurately weighted at 0.1
mg, in a 500 ml volumetric flask in 100 ml demineralized water. Add 25 ml acetonitrile, make up with
demineralized water to the mark and homogenize. The concentration of the stcock standard solution
is calculated as:
In which
C
stock
= concentration standard in mg/ml
w = weight of the dissolved standard in mg
z = purity of the standard
V = volume of the volumetric flask in ml
In total 5 calibration standards are applied, S1, S2, S3, S4, and S5
The calibration standards S1, S2 and S3 are prepared by two successive dilutions as specified in table
3. Pipet the specified volume of the primary standard in a 15 ml polypropylene tube. Pipet also the
specified amount of demineralized water in the polypropylene tube and homogenize.
Vzw C
stock
/
VALIDATION REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE