Page 71 - Analytical method for food addtives

E200–3: Sorbic acid and its salts 39

Calculation
y – a W′
Preservative, mg/kg = ––––– × –– × 1000 [6.1]
b W

where
b = slope of standard curve
a = intercept
y = average peak height ratio of preservative/internal 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 mg/kg 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 acid/sodium citrate buffer 20 % acetonitrile (A)
60 % citric acid/sodium 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 mL/min
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