INSA de Rouen Laboratoire de chimie analytique EC TP ANALYSE

DETERMINATION by HPLC of parabens in cosmetics

The use of preservatives in cosmetic products is widespread but there is a growing controversy about their use. The most used are parabens. The regulatory content of its product must be less than 0.4% for any ester and total not more than 0.8% for the whole of parabens (Cosmetic directive 76/768/EEC). The determination of parabens is widespread and obeys a European regulation (see annex).

List of parabens studied in this TP: Methyl paraben Ethyl paraben Propyl paraben Butyl paraben

Objectives of the work : Implement the European directive for the determination of parabens in - two shampoos for adult - a baby shampoo.

I) Chromatographic separation and calibration

Equipment available: A HPLC Agilent 1100 including: (1) Quaternary pump for the mixture of four solvents under low pressure (2) Vacuum degasser. (3) Injection valve (loop 10 L)
CAUTION DO NOT LEAVE THE VALVE IN AN INTERMEDIATE POSITION! (4) Pursuit 5 C18 column (5m) L = 150 mm d = 4.6 mm ( 5 ) Guard column Pursuit C18 (5 m)

(6) Detector diode array (190 to 950 nm)

Getting started: Actuate the HPLC as explained in the manual. Create an analysis method. Choose a flow of 1 mL/min. An analysis time of 10 minutes. The eluent will contain 50% ACN and 50% water in isocratic mode. The maximum pressure will be 250 bar. The column temperature is 25 C. The detector shall be set preferentially at 254 nm (Bw 8nm) and reference at 350 nm (Bw 50 nm). Set the report parameters as indicated in the manual.

INSA de Rouen Laboratoire de chimie analytique EC TP ANALYSE

Solutions provided: You have: -20 mL of a solution of each paraben at 250 ppm -100 mL of a mother solution containing a mixture of the four parabens at 250 ppm in a 50: 50 (EtOH: water) mixture (unlike the point 4.10 we will not use the 9: 1 EtOH mixture: water) Calibration must be performed according to the standard method. Then, each paraben will be analyzed individually for identification. The first standard solution will be measured three times to check the good repeatability of retention times. Make sure that the criteria for a good separation are checked (selectivity and resolution).

II. Extraction and paraben determination.

You have: -a wash bottle of ethanol: water 50: 50 v/v mixture -50 mL of sulphuric acid approximately 2 mol/L -two adult shampoos -a child shampoo.

Apply the standard method to each sample and calculate the weight percentage of each paraben in in the three products supplied.

A simple report with justification of the operating mode is expected for this TP.

INSA de Rouen Laboratoire de chimie analytique EC TP ANALYSE

Community legislation in force Document 396 L 0045 Seventh Directive 96/45/EC of the Commission of 2 July 1996 relative to analysis methods necessary for the control of the cosmetic products composition (text with EEA relevance) Journal officiel L 213 of the 22/08/1996 DETERMINATION 1. Scope and field of application This method specifies a procedure for the determination of methyl 4-hydroxybenzoate, ethyl 4hydroxybenzoate, propyl 4-hydroxybenzoate, butyl 4-hydroxybnenzoate in cosmetic products. 2. Definition The amounts of the preservatives determined by this method are expressed as percentage by mass. 3. Principle The sample is acidified by adding sulfuric acid and then suspended in a mixture of ethanol and water. After gently heating the mixture to melt the lipid phase to promote quantitative extraction, the mixture is filtered. The preservatives in the filtrate are determined by reversed phase HPLC using isopropyl 4hydroxybenzoate as the internal standard. 4. Reagents 4.1. General All reagents must be of analytical purity and suitable for HPLC where appropriate. Water shall be distilled water, or water of at least equal purity. 4.2. Ethanol, absolute 4.3. Methyl 4-hydroxybenzoate (methylparaben) 4.4. Ethyl 4-hydroxybenzoate (ethylparaben) 4.5. n-Propyl 4-hydroxybenzoate (propylparaben) 4.6. n-Butyl 4-hydroxybenzoate (butylparaben) 4.7. Methanol 4.8. Acetonitrile 4.9. Sulfuric acid solution c(H2SO4)=2mol/l 4.10. Ethanol/water mixture Mix nine volumes of ethanol (4.2) and one volume of water. 4.11. Mobile phase: water /acetonitrile mixture Mix 1 volume of acetonitrile and 1 volume of water. 4.12. Preservative stock solution Accurately weigh approximately 0,025 g methylparaben, 0,025 g ethylparaben, 0,025 g propylparaben, 0,025 g butylparaben in a 100-ml volumetric flask, dissolve and make up to volume with ethanol/water mixture. Kept in a refrigerator the solution is stable for one week. 4.13. Standard preservative solutions From the stock solution (4.12) transfer respectively 20,00 ml, 15,00 ml, 10,00 ml, 5,00 ml and 1,00 ml into 25-ml volumetric flasks. To each flask, add 1,0 ml sulfuric acid solution (4.9) and make up to volume with ethanol/water mixture (4.10). These solutions should be freshly prepared.

INSA de Rouen Laboratoire de chimie analytique EC TP ANALYSE

5. Apparatus Normal laboratory equipment, and: 5.1. Water bath, able to maintain a temperature of 60 C 1 C. 5.2. High performance liquid chromatograph with a UV-detector, wavelength 254 nm 5.3. Analytical column: Stainless steel, 25 cm 4,6 mm i.d. (or 12,5 cm 4,6 mm i.d.) packed with Nucleosil 5C18, or equivalent (see 8.1) 5.4. 100-ml glass tubes with screw cap 6. Procedure 6.1. Sample preparation Weigh approximately 5 g of sample in a 500-ml glass tube with screw cap. Pipette 5,0 ml sulfuric acid solution (4.9) and 50,0 ml ethanol/water mixture (4.10) into the tubes. Close the tube and shake vigorously until a homogeneous suspension is obtained. Shake for at least one minute. Place the tube for five minutes in a waterbath (5.1) kept at 60 C 1 C to facilitate the extraction of the preservatives into the ethanol phase. Immediately cool the tube in a stream of cold water and store the extract in the refrigerator for one hour. (the part in italics won't be performed during this lab's work as it is not necessary) Filter the extract using a filter paper (porosity 0.45 m), transfer quantitatively in a 100 mL volumetric flask, rinsing the extraction tube with ethanol/water mixture (4.10).Transfer around 20 mL of solution in a sample bottle. Store the extracts in the refrigerator and perform the HPLC determination within 24 hours. 6.2. High-performance liquid chromatography (HPLC) 6.2.1. Chromatographic conditions - Mobile phase : water/acetonitrile mixture (4.11) - Flow rate: 1,5 ml/minute (here 1mL/min) - Detection wavelength: 254 nm 6.2.2. Calibration Inject 20l of each of the standard preservative solutions (4.13). From the chromatograms obtained determine the peak area of the standard preservative solutions. Plot a curve for each preservative relating these areas to the concentrations of the standard solutions. Make sure that the calibration curve is a straight line. 6.2.3. Determination Inject 20 l of the sample solution (6.1) into the chromatograph and record the chromatogram. Calculate the peak areas of the investigated preservatives Ascertain that for the standard solutions used in the calibration procedure a linear response is obtained. Ascertain whether the chromatograms obtained for a standard solution and the sample solution meet the following requirements: - the resolution of the worst separated pair shall be at least 1.5. 4

INSA de Rouen Laboratoire de chimie analytique EC TP ANALYSE

If the required separation is not achieved, either a more efficient column should be used, or the mobile phase composition should be adjusted until the requirement is met. - the asymmetry factor As of all peaks obtained shall range between 0,9 to 1,5.

- A steady baseline shall be obtained. 7. Calculation Use the calibration curve (6.2.2) and the peak areas of the investigated preservatives to calculate the concentration of the preservatives in the sample solution. Calculate the methyl 4hydroxybenzoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate contents, as percentage by weight (% m/m) 8. Comments 8.1. Stationary Phase The retention of the solutes in HPLC-determination depends strongly on the type, brand and the history of the stationary phase. It is possible to know whether a column can be used for the separation of the studied conservatives (point 6.2.3) based on the results obtained from reference solutions. In addition to filling materials presented in the method, Hypersil ODS and Zorbax ODS can also be used. You can also optimize the composition of the recommended mobile phase to obtain the desired separation. 8.2. Interferences Under the conditions described in this method, many other components, such as preservatives and cosmetic additives, are eluted. The retention time of a large number of preservatives mentioned in annex VI to the directive of the Council on cosmetic products are cited in: De Kruijf, M.A.H. Rijk, L.A. Pranato-Soetardhi and a. Schouten, (1989). Determination of preservatives in cosmetic products II. High performance liquid chromatographic identification (J.. Chromatography 469, 317-398). 8.3. To protect the analytical column, a column of adequate protection should be used.

Document delivered the: 21/03/1999