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This method will detect alpha-monoglyceryl 4-aminobenzoate (glycerol 1-(4-aminobenzoate). It will also detect ethyl 4-aminobenzoate (benzocaine INN) which may be present as an impurity.
This identification is done by thin layer chromatography on silica gel with a fluorescent indicator and detection of the free primary amine group by formation of a diazo dye on the plate.
All reagents should be of analytical purity.
:
sodium nitrite: 1 g in 100 ml of 1 M hydrochloric acid (prepared immediately before use);
2-naphthol: 0,2 g in 100 ml of 1 M potassium hydroxide.
alpha-monoglyceryl 4-aminobenzoate: 0,05 g in 100 ml of mixed solvent 3.1;
ethyl 4-aminobenzoate: 0,05 g in 100 ml of mixed solvent 3.1.
Weigh 1,5 g of the product to be analyzed in a 10 ml stoppered graduated flask. Make up to the mark with the solvent 3.1. Stopper and leave for one hour at room temperature in an ultrasonic vibrator (4.2). Filter through a Millipore filter (4.3) and use the filtrate for chromatography.
Deposit 10 μl of sample solution (5.1) and of each standard solution (3.4) on the plate (3.5).
Develop the chromatogram to a height of 150 mm in a tank previously saturated with solvent 3.2. Allow the plate to dry at ambient temperature.
Allow to dry at room temperature for 1 minute and immediately spray with the solution 3.3 (b).
Dry the plate in an oven at 60 oC. The spots appear an orange colour. Alpha-monoglyceryl 4-aminobenzoate: RF 0,07; ethyl 4-aminobenzoate: RF 0,55.
This method determines alpha monoglyceryl 4-aminobenzoate. It will also determine ethyl 4-aminobenzoate. It cannot determine more than 5 % (m/m) of alpha monoglyceryl 4-aminobenzoate and 1 % (m/m) of ethyl 4-aminobenzoate.
The alpha monoglyceryl 4-aminobenzoate and ethyl 4-aminobenzoate contents measured by this method are expressed as percentage by mass (% m/m) of the product.
The product to be analyzed is suspended in methanol and after appropriate treatment of the sample it is determined by high-performance liquid chromatography (HPLC).
All reagents should be of analytical purity and should be suitable for HPLC where appropriate.
The composition of the mobile phase may be changed in order to achieve a resolution factor R ≥ 1,5.
where
=
retention times, in minutes, of the peaks,
=
peak widths at half height, in milimetres,
=
the chart speed, in millimetres per minute.
a These values are given as an indication and correspond to the exact masses of 4.11, 4.12 and 4.13. | ||||||
NB: These solutions may be prepared in a different way. | ||||||
Standard Solution | Alpha-monoglyceryl 4-aminobenzoate | Ethyl 4-aminobenzoate | Ethyl 4-hydroxybenzoate | |||
---|---|---|---|---|---|---|
(μg/ml)a | ml (4.11)a | (μg/ml)a | ml (4.12) | (μg/ml)a | ml (4.13) | |
I | 8 | 2 | 8 | 2 | 50 | 10 |
II | 16 | 4 | 12 | 3 | 50 | 10 |
III | 24 | 6 | 16 | 4 | 50 | 10 |
IV | 40 | 10 | 20 | 5 | 50 | 10 |
Add in succession 1 ml of Carrez I solution (4.15) and 1 ml of Carrez II solution (4.16) and mix after each addition. Make up to the mark with eluant (4.10), re-mix and filter through a pleated filter paper.
=
mass of alpha-monoglyceryl 4-aminobenzoate/mass of ethyl 4-hydroxybenzoate,
=
mass of ethyl 4-aminobenzoate/mass of ethyl 4-hydroxybenzoate.
=
quantity of ethyl 4-hydroxybenzoate (internal standard) weighed, in milligrams, in 4.12,
=
quantity of sample, in grams, weighed in 6.1.1.
This method is suitable for the determination of chlorobutanol (INN) up to a maximum concentration of 0,5 % (m/m) in any cosmetic product, except aerosols.
The content of chlorobutanol measured by this method is expressed as percentage by mass (% m/m) of product.
After appropriate treatment of the product to be analyzed the determination is done by gas chromatography using 2,2,2-trichloroethanol as the internal standard.
All the reagents should be of analytical purity.
Weigh accurately between 0,1 and 0,3 g (p g) of the sample. Place in 100 ml volumetric flask. Dissolve it in ethanol (4.3), add 1 ml of the internal standard solution (4.5) and make up to the mark with ethanol (4.3).
Where
=
retention times, in minutes, of the peaks,
=
peak widths at half height, in millimetres,
=
the chart speed, in millimetres per minute.
Column | I | II |
---|---|---|
Material | Glass | Stainless steel |
Length | 1,80 m | 3 m |
Diameter | 3 mm | 3 mm |
Stationary phase | 10 % Carbowax 20 M TPA on Gaschrom Q 80-100 mesh | 5 % OV 17 on Chromosorb WAW DMCS 80-100 mesh |
Conditioning | 2 to 3 days at 190 oC | |
Temperature: | ||
— injector | 200 oC | 150 oC |
— column | 150 oC | 100 oC |
— detector | 200 oC | 150 oC |
Carrier gas | Nitrogen | Argon/methane (95/5 v/v) |
Flowrate | 35 ml/min | 35 ml/min |
Using five 100 ml volumetric flasks, add 1 ml of the standard solution (4.5) and 0,2, 0,3, 0,4, 0,5, and 0,6 ml of solution 4.4 respectively, and make up to the mark with ethanol (4.3) and mix. Inject 1 μl of each of these solutions into the chromatograph in accordance with the operating conditions described in 6.2.2 and construct a calibration curve by plotting as the abscissa the ratio of the mass of chlorobutanol to that of 2,2,2-trichloroethanol and as the ordinate the ratio of the corresponding peak areas.
For a chlorobutanol content of 0,5 % (m/m) the difference between the results of two determinations in parallel carried out on the same sample should not exceed 0,01 %.
If the result is equal to or exceeds the maximum permitted concentration it is necessary to check the absence of interferences.
This method is intended to detect the presence of quinine in shampoo and hair lotions.
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.
All reagents should be of analytical purity.
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.
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).
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.
Alkaloid | RF |
---|---|
Quinine | 0,20 |
Quinidine | 0,29 |
Cinchonine | 0,33 |
Cinchonidine | 0,27 |
Hydroquinidine | 0,17 |
Detection limit: 0,1 μg of quinine.
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.
The quinine content determined by this method is expressed as a percentage by mass (% m/m) of the product.
After appropriate treatment of the product to be analyzed the determination is done by high-performance liquid chromatography (HPLC).
All reagents should be of analytical purity and suitable for HPLC.
The composition of this mobile phase may be changed in order to achieve a resolution factor R ≥ 1,5.
where
=
retention times, in minutes, of the peaks,
=
peak widths at half height, in millimetres,
=
the chart speed, in millimetres per minute.
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).
Flowrate: 1,0 ml/min.
Detector wavelength (5.3): 332 nm.
Injection volume: 10 μl of filtered solution (6.1).
Measurement: peak area.
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.
where
is the quantity, in micrograms, of anhydrous quinine determined in the 10 microlitres of the filtered solution (6.1).
is the mass of the sample in grams (6.1).
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 %.
The method describes the identification and determination of inorganic sulphites and hydrogen sulphites in cosmetic products. It is only applicable to products that have an aqueous or alcoholic phase and for concentrations up to 0,2 % sulphur dioxide.
The sample is heated in hydrochloric acid, and sulphur dioxide liberated is identified either by its odour or its effect on an indicator paper.
All reagents should be of analytical purity.
The sulphite or hydrogen sulphite content of the sample as determined by the method is expressed as percentage by mass of sulphur dioxide.
After acidification of the sample, sulphur dioxide liberated is distilled into a solution of hydrogen peroxide. Sulphuric acid formed is titrated against a standardized sodium hydroxide solution.
All reagants should be of analytical purity.
Calculate the content of sulphite or hydrogen sulphite by mass in the sample by the expresssion:
where
=
molar concentration of sodium hydroxide solution (3.4),
=
volume of sodium hydroxide (3.4) required for titration (5.8), in millilitres,
=
mass of sample (5.1) in grams.
For a content of 0,2 % m/m of sulphur dioxide the difference between two parallel determinations done on the same sample should not be greater than 0,006 %.
The method describes the identification and determination of chlorates in toothpastes and other cosmetic products.
Chlorates are separated from other halates by thin layer chromatography and identified by the oxidation of iodide to form iodine.
All reagents should be of analytical purity.
Normal equipment for thin layer chromatography.
If the chlorate is present a blue spot (possibly a brown spot) will appear after half an hour with RF value approximately 0,7 to 0,8.
Halates | RF |
---|---|
Iodate | 0 to 0,2 |
Bromate | 0,5 to 0,6 |
Chlorate | 0,7 to 0,8 |
It should be noted that bromates and iodates give immediate reaction. Care should be taken not to confuse spots from bromates and chlorates.
The chlorate content of the sample determined by this method is expressed as percentage by mass of chlorate.
Chlorate is reduced by zinc powder under acid conditions. The formed chloride is measured by potentiometric titration using a silver nitrate solution. A similar determination before reduction permits the possible presence of halides.
All reagents should be of analytical purity.
Weigh accurately a quantity ‘m’ of approximately 2 g in centrifuge tube. Add about 15 ml acetic acid (3.1) and mix carefully. Wait 30 minutes and centrifuge for 15 minutes at 2 000 rev/min. Transfer the supernatant solution to a 50 ml volumetric flask. Repeat centrifuging twice by adding 15 ml acetic acid (3.1) to the residue. Collect the solution containing chlorate in the same volumetric flask. Fill to the mark with acetic acid (3.1).
Take 20 ml of solution 5.1 and add 0,6 g of zinc powder (3.2). Bring to the boil in a flask fitted with a condenser tube. After 30 minutes boiling, cool and filter. Rinse the flask with water. Filter and combine the filtrate with the rise's.
Titrate 20 ml solution 5.2 with silver nitrate (3.3) by using the potentiometer (4.2). Titrate in the same way 20 ml of solution 5.1 with silver nitrate (3.3).
NB: If the product contains bromine or iodine derivatives which can release bromides or iodides after reduction, the titration curve will have several inflexion points. In this case the volume of the titrated solution (3.3) corresponding to chloride is the difference between the last and the penultimate inflexion points.
The content of chlorate of the sample (% m/m) is calculated by the formula:
where
=
volume in millilitres, of silver nitrate solution (3.3) used to titrate solution 5.2,
=
volume, in millilitres, of silver nitrate solution (3.3) used to titrate 20 millilitres of solution 5.1,
=
molality of silver nitrate standard solution (3.3),
=
mass of sample, in grams.
For a chlorate content of 3 to 5 % m/m the difference between the results of two determinations carried out in parallel on the same sample should not exceed 0,07 % m/m.
The method describes the procedure for identifying and determining rinse of cosmetic products containing sodium iodate.
Sodium iodate is separated from other halates by thin layer chromatography and identified by the oxidation of iodide to form iodine.
All reagents should be of analytical purity.
Ammonia solution (28 % m/v) /acetone /butanol (60/130/30 v/v/v).
If iodate is present a blue spot (the colour may be brown or become brown on standing) will appear immediately with an RF value approximately 0 to 0,2.
It should be noted that bromates give immediate reactions at RF values approximately at 0,5 to 0,6 and chlorates, after about 30 minutes, at RF values of 0,7 to 0,8 respectively.
The sodium iodate content as determined by this method is expressed as a percentage by mass of sodium iodate.
Sodium iodate is dissolved in water and determined by means of high-performance liquid chromatography, using in series, a reverse-phase C18 column and an anion-exchange column.
All reagents should be of analytical purity and especially suitable for high-performance liquid chromatography (HPLC).
Prepare a stock solution containing 50 mg sodium iodate per 100 ml water.
The pH of the resulting solution is 6.2.
Weigh accurately a test portion of approximately 1,0 g sample in a 10 ml glass stoppered calibrated tube or measuring flask.
Fill up to the mark with water and mix.
If necessary, filter the solution.
Determine the iodate in the solution by means of HPLC as described in section 5.2.
Finely divide part of the sample and weigh accurately a test portion of approximately 1,0 g into a 100 ml glass stoppered measuring cylinder. Fill up to 50 ml with water and shake vigorously for one minute. Centrifuge and filter through a filter paper (4.1) or allow the mixture to stand for at least one night.
Shake the jellylike solution vigorously and filter it through a filter paper (4.1).
Determine the iodate in the filtrate by means of HPLC as described in section 5.2.
Flowrate: 1 ml/min.
Detector wavelength (4.2): 210 nm.
Injection volume: 10 µl.
Measurement: peak area.
Pipette respectively 1,0, 2,0, 5,0, 10,0 and 20,0 ml of the sodium iodate stock solution (3.3) into 50 ml volumetric flasks. Fill to the mark and mix.
The solutions thus obtained, contain 0,01, 0,02, 0,05, 0,10 and 0,20 mg sodium iodate per ml respectively.
Inject a 10 µl portion of each standard iodate solution into the liquid chromatograph (4.2) and obtain a chromatogram. Determine the peak area for iodate and plot a curve relating the peak area to the sodium iodate concentration.
Calculate the sodium iodate content, in percentage by mass (% m/m), using the formula:
where
is the mass, in grams, of the test portion (5.1),
is the total volume of the sample solution, in millilitres, obtained as described in 5.1,
is the concentration, in milligrams per millilitre of sodium iodate, obtained from the calibration curve (5.3).
For a sodium iodate content of 0,1 % (m/m) the difference between the results of two parallel determinations carried out on the same sample must not exceed 0,002 %.
In an acidified solution of a cosmetic product, iodate (IO3-) is reduced to iodide (I—) by sulphite and the resulting solution is investigated by means of HPLC. If a peak having a retention time corresponding to the retention time of iodate disappears after treatment with sulphite, the original peak can most probably be attributed to iodate.
Pipette a 5 ml portion of the sample solution obtained as described in section 5.1 into a conical flask.
Adjust the pH of the solution to a value of 3 or lower with hydrochloric acid (3.1); universal indicator paper (3.7).
Add three drops of sodium sulphite solution (3.2) and mix.
Inject a 10 µl portion of the solution into the liquid chromatograph (4.2).
Compare this chromatogram with the chromatogram obtained as described in paragraph 5 for the same sample.
ISO 5725.
ISO 5725.
ISO 5725.
ISO 5725.
ISO 5725.
ISO 5725.