Page 234 - Analytical method for food addtives

156 Analytical methods for food additives

form, hold separatory funnel under hot tap water 5–10 s. Collect extracts in
250 mL separatory funnel and let combined extracts drain slowly into 250 or
500 mL round-bottom flask to aid removal of hexane-oil droplets. (Note: at
this point, 150 mL acetonitrile extract may be stored overnight, refrigerated.)
Evaporate to 3–4 mL, using flash evaporator with ≤40 °C water bath,
within 10 min. (Note: (1) Prolonged evaporation time may cause TBHQ losses.
To decrease evaporation time, use efficient vacuum source and water-ice con-
denser cooling. (2) Use 500 mL flask to reduce ‘bumping’ losses. Take care to
ensure quantitative transfer of extract after evaporation.) Using disposable
pipette, transfer acetonitrile-oil droplet mixture to 10 mL glass-stoppered
graduated cylinder. Rinse flask with small portions non-saturated acetonitrile.
As rinse pools in flask bottom, pipette rinse to cylinder until 5 mL is collected.
Rinse pipette through top and continue to rinse flask with small portions 2-
propanol, transferring rinses to cylinder until 10 mL is collected. Mix cylinder
contents. (Note: Delay in analysing extracted test portion may cause TBHQ loss.)
(b) Chromatography – Using sample loop injection valve, inject 10 µL sample
extract and elute with solvent gradient program for test extracts. Before and
after every 3–4 test injections, or more frequently if differences between
standard peak heights are found to be >5 %, inject 10 µL antioxidant working
standard solution (10 µL/mL) and elute with solvent gradient program for
standards. For analyte peaks off scale or >3x standard, quantitatively dilute
test extract with 2-propanol-acetonitrile (1 + 1) and reinject. Identify peaks by
comparison with retention times of standard.
For reagent blank determination, take 25 mL saturated hexane and follow
extraction (a), from ‘. . . extract with three 50 mL portions of saturated
acetonitrile’. Inject 10 µL reagent blank extract and elute with solvent gradient
program for samples. The reagent blank should have no peaks interfering with
antioxidant determination.
Use electronically determined peak height, or measure peak height to
0.1 mm, using blank gradient chromatogram as guide to follow baseline.
Determine antioxidant peak heights and average antioxidant standard peak
heights (from duplicate injections before and after test injection, corrected for
gradient blank).

Calculation
Calculate concentration of antioxidant as follows:

Antioxidant, µg/g = (R /R ) × (C /W ) × D [12.1]
x s s x
where:
R and R are peak heights from test portion and standard, respectively
x s
C is concentration standard, µg/mL
s
W is test portion weight, g/mL, in undiluted 10 mL test extract
x
D is dilution factor, if injected solution is diluted
(For further information see AOAC official method 983.15.)