Determination of Saccharin, Sodium Benzoate and Caffeine in Carbonated Beverages by HPLC (1)

This content was archived on June 24, 2013.

Health protection branch laboratories
Ontario Regional Laboratory
Scarborough

Laboratory Procedure
LPFC-102
April 1980

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Definition: This method is applicable to the determination of saccharin, sodium benzoate and caffeine in carbonated beverages by HPLC.

Scope: This method has been evaluated for the recovery of saccharin, caffeine and sodium benzoate from spiked samples of ginger ale in the 40-800 ppm range for saccharin and caffeine and in the 500-1500 ppm range for sodium benzoate. See Table 1.

Apparatus: 1. Liquid chromatograph. (Note 1)

(a) pump, Perkin - Elmer Series 2 or equivalent;
(b) UV detector, Perkin - Elmer Model LC55B with wavelength set at 254 nm, or equivalent;
(c) injection system, Rheodyne Model 715 or equivalent;
(d) column, stainless steel, 300 X 4 mm i.d. packed with Bondapak/C₁₈, 10 µm;
(e) mobile phase, 15% acetic acid in deionised, membrane filtered water; (Note 2)
(f) flow rate, 1.0 mL/min;
(g) pressure, 1000 psi;
(h) range, 0.02 to 0.16 AUFS.

2. Recorder, 1.0 mV.
3. Magnetic stirrer.

Reagents: 1. Sodium saccharin, USP grade.

(a) obtainable from Merck and Co. Inc., Rahway, N.J. 07065

2. Sodium saccharin, standard solutions.

I. Stock solution, 1.0 mg/mL. (Solution A).
(a) accurately weigh 100 mg of Reagent 1 into a 100 mL volumetric flask;
(b) dissolve in distilled H₂O;
(c) make up to volume with same;
II. Working solution, 0.1 mg/mL. (Solution B).
(a) pipet 10 mL of Solution A into 100 mL volumetric flask;
(b) make up to volume with distilled H₂O;

3. Caffeine, USP grade.

(a) obtainable from Eastman Kodak Company, Eastman Organic Chemicals, Rochester N.Y. 14650

4. Caffeine standard solutions.

I. Stock solution, 1.0 mg/mL. (Solution C).
(a) accurately weigh 100 mg of Reagent 3 into a 100 mL volumetric flask;
(b) dissolve in distilled H₂O;
(c) make up to volume with same;
II. Working solution, 0.1 mg/mL. (Solution D).
(a) pipet 10 mL of Solution C into a 100 mL volumetric flask;
(b) make up to volume with distilled H₂O;

5. Sodium benzoate, USP grade.

(a) obtainable from Mallinckrodt Chemical Works, St. Louis, MO. 63160

6. Sodium benzoate standard solution, 1.0 mg/mL. (Solution E).

(a) accurately weigh 100 mg of Reagent 5 into a 100 mL volumetric flask;
(b) dissolve in distilled H₂O;
(c) make up to volume with same;

7. Mixed external standard solution, 0.5 mg/mL. (Solution F).

(a) accurately weigh 100 mg of Reagents 1, 3 and 5 into a 200 mL volumetric flask;
(b) dissolve in distilled H₂O;
(c) make up to volume with same.

Procedure: A. Preparation of Sample

1. Pour ca 25 mL of sample of carbonated beverage into a 50 mL beaker.
2. Degas by magnetically stirring until effervescence ceases. (Note 3).
3. Filter 2-5 mL aliquot through 0.45 µm pore diameter membrane filter to remove particulate matter.

B. Liquid Chromatography

1. Inject 10 µL of Solutions B, D and E separately into liquid chromatograph to establish retention times of saccharin, caffeine and benzoic acid.
2. Inject 6 µL of Solution F into liquid chromatograph.
3. Measure peak heights for saccharin, benzoic acid and caffeine.
4. Inject 10 µL of prepared sample from Step A (1) into liquid chromatograph. (Note 4).
5. Measure heights of peaks corresponding to saccharin and/or benzoic acid and/or caffeine.

C. Calculations

1. Calculate ppm of caffeine in the sample using the following equation.

C x (PH_sa/PH_st) x (V_st/V_sa) x 1000

2. Calculate ppm of saccharin in the sample using the following equation.

Equation 1 X 0.8929

3. Calculate ppm of benzoic acid in the sample using the following equation.

Equation 1 X 0.8474

where C = concentration in mg/mL of relevant compound in standard solution.
PH_sa = peak height of same compound in sample.
PH_st = peak height of same compound in standard.
V_st = volume of standard injected.
V_sa = volume of sample injected.
1000 = factor to convert from mg to µg.
0.8929 = ratio of molecular weight of sodium saccharin to saccharin.
0.8474 = ratio of molecular weight of sodium benzoate to benzoic acid.

Note 1: Under the chromatographic conditions given sodium saccharin and sodium benzoate elute from the column as saccharin and benzoic acid respectively.

Note 2: Mobile phase, 1 (d), should be degassed each day by magnetically stirring under vacuum for ca 15-30 min.

Note 3: (a) This step avoids bubbles in the syringe.
(b) Period of degassing can vary from ca 15-30 min.

Note 4: In some samples injection volume may have to be reduced to keep saccharin peak on scale.

Reference 1. Private Communication, E.J. Tarter, Ontario Regional Laboratory, Scarborough, Ontario

Background Information

To ensure that import and domestic foods comply with the Canadian Food and Drugs Act and Regulations, a rapid screening procedure is required for the analysis of saccharin and other food additives in carbonated beverages.

AOAC procedures for saccharin, benzoic acid and caffeine are time-consuming, non-specific and of relatively low sensitivity. HPB method FT-17 while specific and sensitive for saccharin, is a multi-step procedure involving extraction, cleanup and derivatization steps prior to GLC quantitation.

D.S. Smyly et al (1) reported using a reverse phase high pressure liquid chromatographic method for the simultaneous separation and determination of saccharin, sodium benzoate and caffeine in soft drinks and other commodities. Since the method requires little or no sample work-up prior to HPLC quantitation, it could have applicability as a rapid screening procedure in HPB regional laboratories.

An Operational Methodology Development Project (OMD) was conducted for the purpose of evaluating the published HPLC procedure (1) in order to simultaneously quantitate saccharin, sodium benzoate and caffeine in carbonated beverages.

(1) D.S. Smyly et al. Journal of the AOAC, 59, 14-19, 1976.

Results and Discussion

Two participating laboratories completed a recovery and check sample study on the procedure as written. Recoveries at levels examined (0 to 1500 ppm) fell in the 82 to 105% range with a coefficient of variation generally under 5%. Recoveries under 90% were attributed to HPLC measurement techniques, which were modified for greater accuracy. Analyses of check samples were completed satisfactorily with most recoveries exceeding 90%. Blanks were reported correctly as "not detected".

Interfering peaks were not observed in Ginger Ale by either participant. The Toronto laboratory also examined a variety of other beverages such as carbonated colas and oranges, as well as mixed fruit punches, without detecting any interfering peaks. In some instances unidentified peaks eluted prior to the saccharin peak, but were well resolved from it.

Recoveries and precision of data are considered sufficiently reliable to warrant the use of this method as a rapid screening procedure for these additives in carbonated beverages.

TABLE 1
RECOVERY STUDY

	LAB #	ADDED ppm	RECOV*	% RECOV	S.D. (ppm)	C.V. (±%)
Na SACCHARIN	1 2	40 40.5	37.9 37.1	94.8 91.6	0.90 0.75	2.4 2.0
	1 2	400 405	405 369	101.4 91.3	11.7 16.9	2.9 4.6
	1 2	800 810	812 723	101.5 89.3	14.6 26.4	1.8 3.7
Na BENZOATE	1 2	500 500	501 524	100.2 104.8	7.4 40.4	1.5 7.7
	1 2	1000 1000	1023 909	102.3 90.9	23.8 43.4	2.3 4.8
	1 2	1500 1500	1529 1331	101.9 88.7	17.4 16.6	1.1 1.3
CAFFEINE	1 2	40 40	40.4 34.8	101.1 87.1	0.88 4.6	2.2 13.1
	1 2	400 400	410 343	102.5 85.8	7.1 15.4	1.7 4.5
	1 2	800 800	798 655	99.7 81.9	7.0 35.1	0.9 5.4

*AVERAGE OF 6 REPLICATES