- Latest available (Revised)
- Original (As adopted by EU)
After exit day there will be three versions of this legislation to consult for different purposes. The legislation.gov.uk version is the version that applies in the UK. The EU Version currently on EUR-lex is the version that currently applies in the EU i.e you may need this if you operate a business in the EU.
The web archive version is the official version of this legislation item as it stood on exit day before being published to legislation.gov.uk and any subsequent UK changes and effects applied. The web archive also captured associated case law and other language formats from EUR-Lex.
EU Directives are being published on this site to aid cross referencing from UK legislation. After IP completion day (31 December 2020 11pm) no further amendments will be applied to this version.
Mercaptoacetic acid is separated from the excipient by precipitation with cadmium di(acetate) solution. After methylation with diazomethane, prepared either in situ or in advance in a diethyl ether solution, the methyl derivative of the mercaptoacetic acid is measured by gas/liquid chromatography, methyl octanoate being used as the internal standard.
All the reagents must be of analytical quality.
Sodium acetate trihydrate, 77 g.
Acetic acid (glacial), 27,5 g.
Demineralized water to give a final volume of one litre.
The solution obtained contains about 1,5 g of diazomethane in 100 ml of diethyl ether. As diazomethane is a toxic and very unstable gas, all experiments must be carried out under a powerful hood and the use of ground-glass apparatus must be avoided (there are special kits for this purpose).
Weigh accurately into a 50 ml centrifuge tube enough of the sample to give a presumed quantity of 50 to 70 mg of mercaptoacetic acid. Acidify with a few drops of hydrochloric acid (5.2.2.2) to obtain a pH of about 3.
Add 5 ml of demineralized water and 10 ml of acetate buffer solution (5.2.2.6).
Check with pH paper that the pH value is about 5. Then add 5 ml of cadmium di(acetate) solution (5.2.2.4).
Wait 10 minutes and then centrifuge for at least 15 minutes at 4 000 g. Remove the supernatant liquid which may contain an insoluble fat (in the case of cream products). This fat cannot be confused with the thiols which collects in a compact mass at the bottom of the tube. Check that no precipitation occurs when a few drops of cadmium di(acetate) solution (5.2.2.4) are added to the supernatant.
Where earlier identification revealed no reducing agents other than the thiols, check by iodometry that the thiol present in the supernatant liquid does not exceed 6 to 8 % of the initial quantity.
Introduce 10 ml of methanol (5.2.2.3) into the centrifuge tube containing the precipitate and finely disperse the precipitate with a stirring rod. Centrifuge again for at least 15 minutes at 4 000 g. Pour off the supernatant and check for the absence of thiols.
Wash the precipitate a second time by the same procedure.
Still using the same centrifuge tube, add:
2 ml of methyl octanoate solution (5.2.2.5),
5 ml of hydrochloric acid in methanol (5.2.2.7).
Completely dissolve the thiols (a little insoluble matter may persist from the excipient). This is solution ‘S’.
With an aliquot of this solution, check iodometrically that the thiols content is at least 90 % of that obtained in 5.1.
The methylation is carried out either by in situ preparation (5.2.5.1) or with previously prepared diazomethane solution (5.2.5.2).
Into the methylation apparatus (5.2.3.2) containing 1 ml of ether (5.2.2.11) introduce 50 μl of solution ‘S’ and methylate by the method (5.2.3.2) with about 300 mg of l-methyl-3 nitro-1-nitrosoguanidine (5.2.2.8). After 15 minutes (the ether solution should be yellow to indicate excess diazomethane) transfer the sample solution to a 2 ml bottle having an airtight stopper. Place in the refrigerator overnight. Methylate two samples simultaneously.
Introduce, into a 5 ml stoppered flask, 1 ml of diazomethane solution (5.2.2.12) then 50 μl of solution ‘S’. Leave in the refrigerator overnight.
Prepare a standard solution of mercaptoacetic acid (5.2.2.1) of known strength containing about 60 mg of pure mercaptoacetic acid (5.2.2.1) in 2 ml.
This is solution ‘E’.
Precipitate, assay and methylate as described in 5.2.4 and 5.2.5.
Type: stainless steel.
Length: 2 m.
Diameter: 3 mm.
20 % didecyl phthalate/chromosorb, WAW 80 to 100 mesh.
Flame ionization. A suitable sensitivity setting for the electrometer of the flame ionization detector is 8 × 10-10 A.
Carrier gas: nitrogen.
pressure: 2,2 bar,
flow: 35 ml/min.
Auxiliary gas: hydrogen.
pressure: 1,8 bar,
flow: 15 ml/min.
Detector supplies: as specified by the makers of the apparatus.
Injector: 200 oC
Detector: 200 oC
Column: 90 oC
5 mm/min.
3 μl Carry out five injections.
let:
=
retention times (in minutes),
=
peak widths at half height (in millimetres),
=
the chart speed (in millimetres per minute).
It is recommended that chromatography be terminated by regulating the tempera-ture from 90 to 150 oC at a rate of 10 oC per minute so as to eliminate substances liable to interfere with subsequent measurements.
This is calculated with respect to methyl octanoate on the basis of a standard mixture.
If ‘t’ represents mercaptoacetic acid:
let:
=
its response factor,
=
its mass (in milligrams) in the mixture,
=
its peak area.
If ‘c’ represents methyl octanoate:
let:
=
its mass (in millegrams) in the mixture,
=
its peak area,
This coefficient varies according to the apparatus used.
If ‘t’ represents mercaptoacetic acid:
let:
its response factor,
=
its peak area.
If ‘c’ represents methyl octanoate:
let:
=
its mass (in mill grams) in the mixture,
=
its peak area,
=
the mass (in milligrams) of the initial test portion,
then the % (m/m) mercaptoacetic acid present in the sample is:
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?
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?
Latest Available (revised):The latest available updated version of the legislation incorporating changes made by subsequent legislation and applied by our editorial team. Changes we have not yet applied to the text, can be found in the ‘Changes to Legislation’ area.
Original (As adopted by EU): The original version of the legislation as it stood when it was first adopted in the EU. No changes have been applied to the text.
Geographical Extent: Indicates the geographical area that this provision applies to. For further information see ‘Frequently Asked Questions’.
Show Timeline of Changes: See how this legislation has or could change over time. Turning this feature on will show extra navigation options to go to these specific points in time. Return to the latest available version by using the controls above in the What Version box.
Access essential accompanying documents and information for this legislation item from this tab. Dependent on the legislation item being viewed this may include:
This timeline shows the different versions taken from EUR-Lex before exit day and during the implementation period as well as any subsequent versions created after the implementation period as a result of changes made by UK legislation.
The dates for the EU versions are taken from the document dates on EUR-Lex and may not always coincide with when the changes came into force for the document.
For any versions created after the implementation period as a result of changes made by UK legislation the date will coincide with the earliest date on which the change (e.g an insertion, a repeal or a substitution) that was applied came into force. For further information see our guide to revised legislation on Understanding Legislation.
Use this menu to access essential accompanying documents and information for this legislation item. Dependent on the legislation item being viewed this may include:
Click 'View More' or select 'More Resources' tab for additional information including: