Azocolorants in Textiles and Toys

Enclosure 4
Test Method and Discussion

The Danish Method

Reductive cleavage of azodyes

Approx. 1 g of sample was cut into smaller pieces and placed in a 50 ml Pyrex-flask containing 17 ml preheated basic-sweat simulator; intemal standard (isotope labelled aniline) was then added. As some of the samples were very water absorbent another 2-3 ml of simulator was added to cover the sample.

The samples were heated for 30 min at 70°C, at which point 3 ml of a freshly prepared solution of sodium dithionite in water (approx. 0.2 g/l) was added. The samples were then heated for another 30 min at 70°C. After cooling to laboratory temperature the samples were worked up on Extrelut-columns and concentrated by the same procedure as the German Method with the modification that the samples were evaporated on a Kuderna-Danish evaporator to approx. 2 ml, i.e. they were not evaporated and blown to dryness and then redissolved in 2 ml methanol.

A blind sample and a control standard for recovery analysis at low levels (corresponding to 0. 5 -1 mg/kg) were prepared in the same way.

Free arylamines (grown up articles)

Approx. 1 g of sample was cut into smaller pieces and placed in a 50 ml Pyrex-flask containing 17 ml preheated acidic-sweat simulator; internal standard (isotope labelled aniline) was then added.

The samples were heated for 1 hour at 70° C. After cooling to laboratory temperature the samples were worked up by the same procedure as for the reductive cleavage.

A blind sample and a control standard for recovery analysis at low levels (Corresponding to 0.5-1 mg/kg) were prepared in the same way.

Free arylamines (baby articles)

Approx. 1 g of sample was cut into smaller pieces and placed in a 100 ml Pyrex-flask, added 5 0 ml 0.07 M hydrochloric acid and internal standard (isotope labelled aniline).

The samples were shaken for 1 hour at laboratory temperature and then subsequently heated for 1 hour at 37°C.

The extracts were basified by addition of approx. 2.5 ml 1 M potassium carbonate in water, and 10 g of sodium chloride was then added and dissolved. The samples were subsequently extracted with 5 ml of teri-butyl methylether and the organic phase dried over sodium sulfate.

A blind sample and a control standard for recovery analysis at low levels (corresponding to 0. 5 -1 mg/kg) were prepared in the same way.

Analysis and quantification

The analyses were carried out by capillary column gas chromatography combined with mass spectrometric detection in full scan rnode (GC-MS). Samples, blind sample and control standard were analysed together with external standards of arylamines prepared in pure solvent containing internal standard. The quantification has been carried out against the external standards using the internal standard as a correcting factor.

Comments

Under the conditions of the reductive cleavage, o-aminoazotoluene and 2-amino-4-nitrotoluene are cleaved to o-toluldine and 2,4-diaminotoluene respectively.

The detection limits has been estimated from the recovery tests and external standards at low levels.

Experimental conditions

Table 11.1
link to tabel
Detection limits for the analysis for aromatic amines

Discussion of the Danish Method versus the German method

The Danish Method (described under »The Danish Method«) is a modification of the German Method (Amtliche Sammlung von Untersuchungsverfahren nach § 35 LMBG, B 82.02-2, Sept. 1996). The two major modifications are:

1. the Danish Method uses a different buffer-solution and at different pH from what is used in the German Method
    
2. the Danish method uses an intemal standard

The established detection limits for the Danish Method has been based on two considerations:

1. the analytical detection limit, i.e. the instrument determined detection limit of the individual aromatic amine in pure solvent
    
2. the recovery of aromatic amines in the extraction step based on a standard at a low concentration level

The uncertainty in the quantification step lies in time fact that the content is quantified against an external standard, i.e. a standard that has not undergone time same extraction procedure as the samples. This means that there has been made no corrections for recoveries less than 100% and at the low concentration levels, the recoveries typically appears between 40 an 80%, highest for the smaller aromatic amines (e.g. o-toluidine, p-chloroaniline etc.) and lowest for the larger aromatic amines (e.g. the benzidines). At higher concentration levels the recoveries typically are 70-90%. Thus, the quantification procedure tends to underestimate the content of aromatic amines.

Ideally, the quantlfication should be carried out against standards that have undergone the same extraction procedure as the samples. Standards at several concentration levels would be required as the recovery tends to become larger at higher concentrations. The general trend of recoveries increasing with concentrations could also mean that one calibration curve has to be established for low concentrations and another calibration curve for higher concentrations. However, the only commercially available solution containing all relevant aromatic amines also contains o-aminoazotoluene and 2-amino-4-nitrotoluene which are reduced to o-toluidine and 2,4-diaminotoluene respectively, already being present in the solution.

In order to gain more knowledge about the uncertainty of the method, especially the reproducibility of the recoveries, it would be necessary to perform a multiple number of recovery experiments with a standard at low concentration level (or more at different, low levels) so that standard deviations can be calculated. For comparison, a table of recoveries of selected aromatic amines are given (* taken from the German Method); it should be noted that the results given are single determination performed by 6 different laboratories. The standards has been at contrition levels 30 mg/kg sample or more. The detection method is not specified (can be GC-FID, GC-MS, UPLC-DAD, CE-DAD or densitometry).

Table 11.2 is taken from »Bundesgesundheitsblatt, Nr. 2, 3 9. Jahrgang, Februar 1996« (here given in English):

Table 11.2
link to tabel
Recoveries (The recovery of the amines 2, 5, 7, 8, 9, 10, 16 and 17 in the calibration solutions were determined in 6 laboratories)

In B 82.02-2 September 1996 it is stated:

The recovery of the amines must fulfil the following minimum requirements:

The numbers of the amines referes to the following list in the same paper:

*Azo-dyes which can split off these two aromatic amines are by the analyse method B 82.02-2 detected as o-toluid ine and 2,4-toluylenediamine respectively.