Methods of analysis analytical methods for food additives
Extraction a General method – Accurately weigh 5.0 g homogenised test portion into
30 mL centrifuge tube with Teflon-lined screw cap. Add 3.00 mL internal standard solution, 1.5 mL H
2
SO
4
1 + 5, 5 g sand, and 15 mL ether. Screw cap on tightly to avoid leakage. Mechanically shake 5 min and centrifuge 10 min
at 1500 g. Transfer ether layer with disposable pipette to 250 mL separator. Repeat extraction twice with 15 mL ether each time.
Extract combined ether phases twice with 15 mL 0.5 M NaOH and 10 mL saturated NaC1 solution each time. Collect aqueous layers in 250 mL separa-
tor, add 2 drops of methyl orange, and acidify to pH 1 with HC1 1 + 1. Extract with CH
2
C1
2
, using successive portions of 75, 50, and 50 mL. If emulsion forms, add 10 mL saturated NaC1 solution. Drain CH
2
C1
2
extracts through filter containing 15 g anhydrous Na
2
SO
4
into 250 mL round-bottom flask. Evaporate CH
2
C1
2
solution in rotary evaporator at 40 ºC just to dryness. b Cheese and food products with paste-like consistency – Accurately weigh
5.0 g homogenised test portion into 200 mL centrifuge flask. Add 15 mL H
2
O and stir with glass rod until test portion is suspended into aqueous phase. Add
3.00 mL internal standard solution, 1.5 mL H
2
SO
4
1 + 5, and 25 mL ether. Stopper flask carefully and check for leakage. Mechanically shake 5 min and
centrifuge 10 min at 2000 g. Transfer ether layer with disposable pipette to 250 mL separator. Repeat extraction twice with 25 mL ether each time.
Continue as in a, beginning ‘Extract combined ether phases. . .’
Derivatisation and gas chromatography Add 10.0 mL CHC1
3
to residue in 250 mL round-bottom flask. Stopper and shake manually 2 min. Transfer 1.00 mL CHC1
3
solution to 8 mL test tube with Teflon- lined screw cap and add 0.20 mL silylating agent. Cap and let stand 15 min in oven
or H
2
O bath at 60 ºC. Inject duplicate 1 µL portions of residue solution into gas
chromatograph. Start temperature program when solvent peak emerges. Measure peak heights and calculate peak height ratios of sorbic acidcaproic acid. Use
average of duplicate ratios. Peak height ratios for duplicate injections should differ 5 .
Preparation of standard curves Transfer 1.00 mL standard solutions to five 8 mL test tubes with Teflon-lined
screw caps. Add 0.20 mL silylating agent to each tube, cap, and let stand 15 min in oven or H
2
O bath at 60 ºC. Inject duplicate 1 µL portions of standard solutions into
gas chromatograph. Use same conditions as for test portion solution. Measure peak heights and calculate peak height ratios of sorbic acidcaproic acid. Peak height
ratios for duplicate injections should differ 5 . Plot weight ratios x vs. average peak height ratios y for each preservative. Calculate slope and intercept of
standard curve by method of least squares.
Calculation y – a W
′ Preservative, mgkg = ––––– × –– × 1000
[6.1] b W
where b
= slope of standard curve a
= intercept y
= average peak height ratio of preservativeinternal standard W = weight of test portion in g
W ′ = weight of internal standard in mg.
HPLC method for sorbic acid applicable for foodstuffs containing sorbic acid in the range 50–2000 mgkg
11
The following conditions have been shown to be satisfactory: Guard column
Kromasil C18, 5 µm, 10 × 3.2 mm with cartridge holder
Column Kromasil 100–5C18, 250 × 4.6 mm
Detector UV detector
Wavelength 223 nm for benzoic acid and 258 nm for sorbic acid,
methyl 4-, ethyl 4- and propyl 4-hydroxybenzoate Mobile phase
80 citric acidsodium citrate buffer 20 acetonitrile A 60 citric acidsodium citrate buffer 40 acetonitrile B
Gradient system 0–26 min
100 A 26–31 min
go to 100 B 31–45 min
100 B 45–50 min
go to 100 A 50–55 min
100 A Flow rate
1.0 mLmin Injection volume
20 µL
Column temperature Ambient Under these conditions the analytes elute in the order:
1 benzoic acid
2 sorbic acid
3 methyl 4-hydroxybenzoate
4 ethyl 4-hydroxybenzoate
5 propyl 4-hydroxybenzoate
The approximate retention times are 13.9, 17.0, 24.2, 35.8 and 42.9 min respectively. Centrifuge with appropriate centrifuge tubes approximately 50 mL capacity
with screw caps or other suitable closures. Preparation of calibration graphs
Inject 20 µL of each of the standard solutions. Plot the peak area obtained for each