Determination of Parabens* in Beverages by GLC

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Laboratory Procedure
LPFC-103
April 1979

HEALTH PROTECTION BRANCH LABORATORIES
Ontario Regional Laboratory
SCARBOROUGH

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Definition:

This method is applicable to the determination of methyl, propyl and heptyl parabens in fruit based and non-carbonated beverages.

Scope:

This method has been evaluated for the determination of 0 -1000 ppm levels of methyl and propyl parabens and 0-20 ppm levels of heptyl paraben in fruit drinks. See Table II for recovery data.

Principle:

Sample is acidified, internal standards added, extracted and the acidified extract silylated and analysed by GLC. Quantitation is achieved by calculating area ratio of paraben/internal standard and referring to appropriate standard curve.

Apparatus:

1. Gas chromatograph, Varian Model 2700 or equivalent, equipped with:
   
   
   1. Flame ionization detector;
   2. 6' x ¼" glass column packed with 3% OV-3 on 80-100 mesh Chromosorb W, HP;

   3. Operating parameters	temperatures (°C)
      injector	220
      detector	315
      carrier gas, helium	fr 30 ml/min
      amplifier	10-11*8

   4. Program sequence
      
      
      1. pre-program, 190°C isothermal, 5 min
      2. program, 190-300°C, 8°C/min
      3. post-program, 300°C, isothermal, 6 min
2. Integrator, Hewlett-Packard Model 3380A or equivalent.
3. Reacti-vials, 2-5 ml capacity, Pierce Chem. Co., or equivalent.
4. Reacti-Therm Heating Module, Pierce Chem. Co.
5. Glass syringe, 0.5 ml capacity.

Reagents:

1. Internal standard solutions.
   
   
   1. butyl 4-hydroxybenzoate, 10 mg/ml in chloroform;
   2. n-hexyl 4-hydroxybenzoate, 0.2 mg/ml in chloroform.
2. Chloroform, Caledon "dist-in-glass" grade or equivalent.
3. Sodium sulphate, coarse, granular, fired at 650°C overnight.
4. MSTFA, Chromatographic Specialties, Brockville, Ontario (#48911).

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*Esters of p-hydroxy benzoic acid

Procedure:

A. Preparation of Standard Curve

1. Rinse five 100 ml volumetric flasks with chloroform and label 1/1, .8/.8, .6/.6, .4/.4, .2/.2 .
2. The numerator denotes the weight ratios (methyl and propyl vs butyl) that give area ratios which correspond to 1000, 800, 600, 400 and 200 ppm of methyl and propyl paraben in a sample. The denominator denotes the weight ratios (heptyl vs hexyl) that give area ratios which correspond to 20, 16, 12, 8 and 4 ppm of heptyl paraben in a sample.
3. Add the required weight (mg) of paraben to each flask as specified in Table I and dilute to volume with chloroform. ( Note 1) ( Note 2).
4. Pipet 1 ml of each resulting solution into a 5 ml Reacti-Vial.
5. Add 0.2 ml MSTFA with glass syringe and cap immediately.
6. Heat at 60-70°C for 20 min.
7. Inject 1-3 ml of each solution in triplicate into the gas chromatograph.
8. Compute average area ratios of methyl vs butyl, propyl vs butyl, and heptyl vs hexyl paraben for each weight ratio.
9. Plot the area ratios of methyl, propyl and heptyl paraben against the ppm values for the same paraben as stated in Step 2.

TABLE I

Weight (mg) of Paraben to be added to standard solution flasks

	1/1	.8/.8	.6/.6	.4/.4	.2/.2
Methyl	500	400	300	200	100
Propyl	500	400	300	200	100
Butyl	500	400	300	200	100
Hexyl	10	10	10	10	10
Heptyl	10	8	6	4	2

Note 1: Transfer quantitively methyl, propyl and butyl paraben into flasks from a weighing boat with chloroform.

Note 2: Pipet hexyl and heptyl paraben into flasks using a 2 mg/ml solution of each in chloroform.

B. Determination of Parabens

1. Pipette 10.0 ml non-carbonated drink into a 125 ml separatory funnel.
2. Add 3 ml 25% v/v H₂SO₄.
3. Add 1 ml butyl internal std solution and 1 ml hexyl internal std solution.
4. Extract with 15 ml CHCl₃ by shaking vigorously 20-30 seconds.
5. Filter bottom layer through 15-20 g Na₂SO₄ into a 250 ml flat-bottom boiling flask.
6. Repeat extraction with a second 15 ml portion of CHCl₃ and filter.
7. Evaporate combined filtrate just to dryness on a flash evaporator.
8. Allow flask to cool, add 2 ml CHCl₃, and wash walls of flask by simultaneously rotating and swirling several times.
9. Transfer approximately 1 ml CHCl₃ with a Pasteur pipette to a 5 ml Reacti-Vial.
10. Add 0.2 ml MSTFA and cap immediately.
11. Heat at 60-70°C in heating block for 20 min.
12. Inject 2-3 µl into gas chromatograph. (Note 3)
13. Determine ppm of paraben in sample by calculating area ratio of paraben/internal std and referring to appropriate standard curve. (Note 4)

Note 3: Remove deposits from collector electrode of flame ionization detector daily.

Note 4: For positive samples run a sample blank omitting the addition of internal standard, to determine if interfering peaks are present.

Table II

Recovery of Parabens from Fruit Drink*

	Lab#	Added (Equivalent) ppm	Avg.** Recov. (Equivalent) ppm	Avg. % Rec.	Sth.Dev. ( ± ppm)	C.V. ( ± %)
Methyl Paraben	1	1000	965	96.5	10.5	1.1
	2	1000	968	96.8	19.0	2.0
	1	600	575	95.8	6.3	1.1
	2	600	582	97.0	4.0	0.7
	1	200	178	98.0	9.8	5.5
	2	200	190	95.0	0	0
Propyl Paraben	1	1000	975	97.5	10.5	1.1
	2	1000	978	97.8	11.7	1.2
	1	600	588	98.0	8.2	1.4
	2	600	580	97.0	0	0
	1	200	196	98.0	2.0	1.0
	2	200	200	100.0	0	0
Heptyl Paraben	1	20	18.9	94.5	0.3	1.5
	2	20	20.0	100.0	1.0	5.1
	1	12	11.5	95.8	0.1	0.9
	2	12	11.6	96.7	0.5	4.0
	1	4	3.6	90.8	0.1	2.9
	2	4	3.1	78.0	0.1	3.5

* Hawaiian Punch with 7 fruit juices

** Average of 6 replicates