Page 101 - Analytical method for food addtives

E210–13: Benzoic acid 59

based on difference between amount determined in control and amount obtained
after C18 cartridge clean-up.

LC determination of benzoic acid
Make duplicate 10 µL injections of eluted test sample to LC, after injecting
standard. Calculate concentration of benzoic acid in sample as follows:
Benzoic acid, µg/mL = (A/A′) × C × 3 × (1/R) [7.2]
where
A, A′ = peak area of test sample and standard, respectively
C = concentration of standard, µg/mL
3 = dilution factor
R = recovery rate.

HPLC method for benzoic acid applicable for foodstuffs containing benzoic
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.