Page 352 - Analytical method for food addtives

E954: Saccharin 239

Liquid chromatographic method for saccharin in beverages and sweets 17, 18

Principle
Saccharin is determined in soft drinks and juices by reverse phase LC with UV
detection at 254 nm; sweets must first be extracted with ethanol.

Apparatus
a) Liquid chromatograph – isocratic instrument with possibility of using 2
mobile phases, step-gradient, or gradient system. Operating conditions: col-
umn temperature ambient; flow rate 2.0 mL/min; wavelength 254, 280, 207 or
214 nm; injection volume 10–30 µL: sensitivity 0.005–0.02 AUFS; chart
speed 1 cm/min
b) Reverse phase LC column – particle size 10 µm, 30 cm × 3.9 mm i.d., e.g.
µBondapak C18
c) Guard column – particle size 37–50 µm C18
d) Membrane filters – For aqueous solutions, pore size 0.45 µm
e) Centrifuge – minimum 2000 g.

Reagents
a) Mobile phase – prepare 1 % (v/v) acetic acid solution. Mobile phase I: Mix
950 mL 1 % acetic acid solution and 50 mL methanol; de-gas. Mobile phase
II: mix 300 mL 1 % acetic acid solution and 700 mL methanol; de-gas.
b) Ethanol – 99 %
c) Sodium saccharin standard solution – 100 mg/L. Transfer exactly 25 mg
sodium saccharin dihydrate, C H NNaO S·2H O, to 250 mL volumetric flask
7 4 3 2
and dilute to volume with water.
Sample preparation

a) Soft drinks and juices – Decarbonate, filter if particulate matter is present, and
inject
b) Sweets – Weigh 10.00 g samples containing ≥0.5 mg saccharin and add
100.0 g water. Dissolve sample with mixing. Weigh 10.00 g homogenate into
centrifuge tube and add 50 mL ethanol. Mix and let stand overnight. Centri-
fuge and decant aqueous ethanol phase. Wash precipitate twice with 30 mL
ethanol and combine washings with aqueous ethanol phase. Evaporate to
dryness and dissolve and dilute residue to 5.00 mL with water. Filter and inject
directly.

Determination
Inject 10 µL standard solution to determine peak height of saccharin. Repeat
injections until results agree 2 %. Inject sample solution containing c. 1 µg
saccharin twice: chromatograph samples from sample preparation (a) with mobile
phase I and samples from sample preparation (b) with mobile phases I and II.