Search Legislation

Fourth Commission Directive of 11 October 1985 on the approximation of the laws of the Member States relating to methods of analysis necessary for checking the composition of cosmetic products (85/490/EEC)

 Help about what version

What Version

 Help about advanced features

Advanced Features

 Help about opening options

Opening OptionsExpand opening options

 Help about UK-EU Regulation

Legislation originating from the EU

When the UK left the EU, legislation.gov.uk published EU legislation that had been published by the EU up to IP completion day (31 December 2020 11.00 p.m.). On legislation.gov.uk, these items of legislation are kept up-to-date with any amendments made by the UK since then.

Changes over time for: IDENTIFICATION AND DETERMINATION OF QUININE

 Help about opening options

Status:

EU Directives are published on this site to aid cross referencing from UK legislation. Since IP completion day (31 December 2020 11.00 p.m.) no amendments have been applied to this version.

IDENTIFICATION AND DETERMINATION OF QUININE U.K.

A.IDENTIFICATIONU.K.

1.SCOPE AND FIELD OF APPLICATIONU.K.

This method is intended to detect the presence of quinine in shampoo and hair lotions.

2.PRINCIPLEU.K.

Identification is done by thin layer chromatography on silica gel. Detection of quinine is by the blue fluorescence of quinine in acidic conditions at 360 nm.

For further confirmation, the fluorescence can be eliminated by bromine vapours, and ammonia vapours will cause a yellowish fluorescence to appear.

3.REAGENTSU.K.

All reagents should be of analytical purity.

3.1.Silica gel plates, without fluorescence indicators, 0,25 mm thick, 200 mm × 200 mmU.K.
3.2.Developing solvent: toluene /diethyl ether /dichloromethane /diethylamine /20/20/20/8 (v/v/v/v).U.K.
3.3.Methanol.U.K.
3.4.Sulphuric acid (96 %; ).U.K.
3.5.Diethyl ether.U.K.
3.6.Developing agent: carefully add 5 ml of sulphuric acid (3.4) to 95 ml of diethyl ether (3.5) in a cooled container.U.K.
3.7.Bromine.U.K.
3.8.Ammonium hydroxide solution (28 %; ).U.K.
3.9.Quinine, anhydrous.U.K.
3.10.Standard solution: weigh accurately about 100,0 mg of anhydrous quinine (3.9) into a standard flask and dissolve in 100 ml of methanol (3.3).U.K.
4.APPARATUSU.K.
4.1.Normal equipment for thin layer chromatography.U.K.
4.2.Ultrasonic bath.U.K.
4.3.Millipore filter, FH 0,5 ƒm or equivalent with suitable filtration equipment.U.K.
5.PROCEDUREU.K.
5.1. Preparation of the sample U.K.

Weigh accurately a quantity of the sample which may contain approximately 100 mg of quinine into a 100 ml standard flask, dissolve and make up to the mark with methanol (3.3).

Stopper the flask and leave for one hour at room temperature in an ultrasonic vibrator (4.2). Filter (4.3) and use the filtrate for the chromatography.

5.2. Thin layer chromatography U.K.

Deposit 1,0 μl of standard solution (3.10) and 1,0 μl of sample solution (5.1) on the silica gel plate (3.1). Develop the chromatogram over a distance of 150 mm using solvent 3.2. in a tank previously saturated with solvent (3.2).

5.3. Development U.K.
5.3.1.Dry the plate at room temperature.U.K.
5.3.21.Spray with reagent 3.6.U.K.
5.3.3.Leave the plate to dry for one hour at room temperature.U.K.
5.3.4.Observe under the light from a UV lamp adjusted to a wavelength of 360 nm. Quinine appears as a fluorescent intense blue spot.U.K.

By way of example the table below gives the values of the RF of the main alkaloids related to quinine when developed with solvent 3.2.

AlkaloidRF
Quinine0,20
Quinidine0,29
Cinchonine0,33
Cinchonidine0,27
Hydroquinidine0,17
5.3.5.For further confirmation that quinine is present, the plate is exposed for approximately one hour to bromine vapour (3.7). The fluorescence disappears. When the same plate is exposed to ammonia vapour (3.8), the spots reappear with a brown colour, and when the plate is again examined under UV light at 360 nm a yellowish fluorescence can be observed.U.K.

Detection limit: 0,1 μg of quinine.

B.DETERMINATIONU.K.

1.SCOPE AND FIELD OF APPLICATIONU.K.

This method describes the determination of quinine. It may be used to determine the maximum permitted concentration of 0,5 % (m/m) in shampoos and 0,2 % in hair lotions.

2.DEFINITIONU.K.

The quinine content determined by this method is expressed as a percentage by mass (% m/m) of the product.

3.PRINCIPLEU.K.

After appropriate treatment of the product to be analyzed the determination is done by high-performance liquid chromatography (HPLC).

4.REAGENTSU.K.

All reagents should be of analytical purity and suitable for HPLC.

4.1.Acetonitrile.U.K.
4.2.Potassium dihydrogenorthophosphate (KH2PO4).U.K.
4.3.Orthophosphoric acid (85 %; ).U.K.
4.4.Tetramethylaminium bromide.U.K.
4.5.Quinine, anhydrous.U.K.
4.6.Methanol.U.K.
4.7.Orthophosphoric acid solution (0,1 M): weigh 11,53 g of orthophosphoric acid (4.3) and dissolve in 1 000 nl of water in a graduated flask.U.K.
4.8.Potassium dihyrogenorthophosphate solution (0,1 M): weigh 13,6 g of potassium dihydrogenorthophosphate (4.2) and dissolve in 1 000 ml of water in a graduated flask.U.K.
4.9.Tetramethylammonium bromide solution: dissolve 15,40 g of tetramethylammonium bromide (4.4) in 1 000 ml of water in a graduated flask.U.K.
4.10Eluant: orthophosphoric acid (4.7) /potassium dihydrogenorthophosphate (4.8) /tetramethylammonium bromide (4.9)/water/acetonitrile (4.1) 10/50/100/340/90 (v/v/v/v/v).U.K.

The composition of this mobile phase may be changed in order to achieve a resolution factor R ≥ 1,5.

where

R1 and R2

=

retention times, in minutes, of the peaks,

W1 and W2

=

peak widths at half height, in millimetres,

d'

=

the chart speed, in millimetres per minute.

4.11.Silica treated with octadecylsilane, 10 μm.U.K.
4.12.Standard solutions: weigh accurately approximately 5,0, 10,0, 15,0 and 20,0 mg respectively of quinine anhydrous (4.5) into a set of 100 ml standard flasks. Make up to the mark with methanol (4.6) and shake the contents of the flasks until the quinine dissolves. Filter each sample through a 0,5 μm filter.U.K.
5.APPARATUSU.K.
5.1.Usual laboratory equipment.U.K.
5.2.Ultrasonic bath.U.K.
5.3.High-performance liquid chromatography equipment with a variable wavelength detector.U.K.
5.4.Column: length: 250 mm; internal diameter: 4,6 mm; filling: silica (4.11).U.K.
5.5.Millipore filter FH 0,5 μm, or equivalent, with suitable filtration apparatus.U.K.
6.PROCEDUREU.K.
6.1. Sample preparation U.K.

Weigh accurately into a 100 ml standard flask a quantity of the product sufficient to contain 10,0 mg of anhydrous quinine, add 20 ml of methanol (4.6) and place the flask in an ultrasonic bath (5.2) for 20 minutes. Make up to the mark with methanol (4.6). Mix the solution and then filter an aliquot (5.5).

6.2. Chromatography U.K.
  • Flowrate: 1,0 ml/min.

  • Detector wavelength (5.3): 332 nm.

  • Injection volume: 10 μl of filtered solution (6.1).

  • Measurement: peak area.

6.3. Calibration curve U.K.

Inject at least three times 10,0 μl of each reference solution (4.12), measure the area of the peaks, and calculate the average area at each concentration.

Produce the calibration curve and verify that it is rectilinear.

7.CALCULATIONU.K.
7.1.From the calibration curve (6.3) determine the quantity in μg of anhydrous quinine present in the volume injected (6.2).U.K.
7.2.The concentration of anhydrous quinine in the sample, as a percentage by mass (% m/m), is obtained by the following formula:U.K.

where

B

is the quantity, in micrograms, of anhydrous quinine determined in the 10 microlitres of the filtered solution (6.1).

A

is the mass of the sample in grams (6.1).

8.REPEATABILITY(1) U.K.

For an anhydrous quinine content of 0,5 % (m/m), the difference between the results of two determinations performed in parallel on the same sample must not exceed 0,02 %.

For an anhydrous quinine content of 0,2 % (m/m), the difference between the results of two determinations performed in parallel on the same sample must not exceed 0,01 %.

(1)

ISO 5725.

Back to top

Options/Help

You have chosen to open the Whole Directive

The Whole Directive you have selected contains over 200 provisions and might take some time to download. You may also experience some issues with your browser, such as an alert box that a script is taking a long time to run.

Would you like to continue?

You have chosen to open Schedules only

The Schedules you have selected contains over 200 provisions and might take some time to download. You may also experience some issues with your browser, such as an alert box that a script is taking a long time to run.

Would you like to continue?