Page 102 - Analytical method for food addtives

60 Analytical methods for food additives

Preparation of calibration graphs
Inject 20 µL of each of the standard solutions. Plot the peak area obtained for each
analyte in each standard solution on the vertical axis versus the corresponding
analyte concentration in mg/L, along the horizontal axis, to give the five calibra-
tion graphs.

Sample preparation
Homogenise the sample. The portion of prepared sample not immediately required
for analysis should be placed in an air-tight container and stored in such a way that
deterioration and change in composition are prevented.

Liquid samples not containing insoluble matter
Weigh, to the nearest 0.001 g, about 10 g of prepared sample and dilute with
methanol to 100 mL in a volumetric flask and mix. Pass this solution through a
0.45 µm filter to eliminate any particulate matter.
Confirm that the HPLC system is operating correctly by injecting the combined
20 mg/L standard solution, then inject 20 µL of the sample filtrate onto the HPLC
column. After the analyte peak or peaks have been eluted and a steady base-line is
re-attained repeat the injection. Inject 20 µL of a combined standard solution after
every fourth injection. If the amount of analyte(s) in the extract is high an aliquot
of the extract should be diluted with mobile phase A such that the concentration in
the diluted extract is within the range used in the calibration graphs and an
appropriate dilution factor used in the calculation.

Other samples
Weigh, to the nearest 0.001 g, about 10 g of prepared sample into a centrifuge tube.
Add methanol (20 mL) and close the tube. Vortex mix the sample and methanol to
ensure a uniform suspension and then extract the sample by shaking vigorously for
2 min. Centrifuge at a relative centrifugal force (RCF) of approximately 2630 for
5 min and decant off the methanol layer into a 100 ml volumetric flask. (Note:
Since the centrifuge is to be used with methanolic extracts it should be emphasised
that tubes with screw caps or other suitable closures are required.)
Repeat steps twice with further portions of methanol (20 mL each). It is
particularly important to vortex mix during re-extraction as the solid matter can be
difficult to disperse. Care is also needed in decanting the methanol layer from a
sample containing a high oil content to ensure that none of the oil layer is decanted
with the methanol. Combine the extracts in the 100 ml volumetric flask and make
up to the calibration mark by the addition of methanol. Shake to obtain a
homogeneous solution. (Note: For high fat percentage foodstuffs it is advisable to
include a freezing out stage for the combined extracts at the end of the extraction
procedure. This can be performed by placing the sample in dry ice for approxi-
mately 20 min until the fat has solidified, decanting the methanolic solution and
then proceeding by making to volume with methanol.)
Filter the solution through a filter paper, rejecting the first few mL and collect
about 15 mL. Filter this through a 0.45 µm filter. Carry out the chromatographic