Trends in Analytical Chemistry, Vol. 24, No.

1, 2005

Trends

Determination of banned azo dyes in consumer goods

Lars-Henric Ahlstrom , Cecilia Sparr Eskilsson, Erland Bjorklund
Azo dyes, extensively used for coloring a variety of consumer goods, such as leather, clothes, food, and toys, can under certain conditions be reduced to form conrmed or suspected carcinogenic aromatic amines. This article gives an overview of the state of development of analytical procedures for the determination of such azo dyes, which are banned by the European Commission. 2004 Elsevier Ltd. All rights reserved.
Keywords: Analytical procedures; Aromatic amines; Azo dyes; Consumer goods

1. Introduction
Lars-Henric Ahlstrm*, o Cecilia Sparr Eskilsson, Erland Bjrklund o Department of Analytical Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, Sweden

*Corresponding author. Tel.: +46-46-222-8176; Fax: +46-46-222-4544; E-mail: lars-henric. ahlstrom@analykem.lu.se

Since the second half of the 19th century, azo dyes have been extensively used in numerous industrial applications mainly because of their colorfastness and low price. Azo dyes are synthetic organic colorants, characterized by chromophoric azo groups (N@N). Currently, there are over 3000 azo dyes in use worldwide and they account for 65% of the commercial dye market. These dyes offer a wide spectrum of colors and are used for coloring a variety of consumer goods, such as leather, clothes, food, toys, plastics and cosmetics [1,2]. However, whereas azo dyes are relatively resistant to degradation under aerobic conditions, they can be readily reduced to form aromatic amines under anaerobic conditions (see Fig. 1). The reduction primarily occurs through cleavage of the azo group by azoreductases in intestinal bacteria, liver cells, and skin surface micro ora [3,4]. A number of the aromatic amines formed have been found to be carcinogenic in experimental animals and thus pose a risk for consumers [5,6]. The carcinogenic risk of aromatic amines in humans was rst observed in 1895 by German surgeon Ludwig Rehn, who reported high rates of urinary bladder

cancer among men employed in the dyestuff industry, who were chronically exposed to large amounts of aniline [7]. Over the years, subsequent epidemiological studies have shown additional evidence that long-term occupational exposure to certain aromatic amines (e.g., benzidine, 4-aminobiphenyl, and 2-naphthylamine) that are used in the dye industry increases the risk of developing cancer [8,9]. The main routes of exposure of consumers to azo dyes and their degradation products are oral ingestion (e.g., young children sucking on toys that contain dyed textile or leather garments) and dermal absorption (e.g., sweat and friction cause dyes, contained in clothes worn near the skin, to elute). In July 1994, the German Government, against this background, introduced the 2nd amendment to their Consumer Goods Ordinance [10], which prohibited the use of certain azo colorants (i.e., dyes and pigments) in dened articles that come into direct, prolonged contact with human skin. The azo dyes affected by this legislation are those that, after reduction, may form one or more of the 20 conrmed or suspected carcinogenic aromatic amines listed in the Ordinance in detectable concentrations (i.e., >30 ppm) [10]. Similar restrictions regarding azo dyes were applied shortly afterwards in other Member States of the European Union (EU), but, in the interests of transparency and maintenance of the Single Market, the 19th amendment of the Council Directive 76/769/EEC (relating to restrictions on the marketing and use of certain dangerous substances and preparations) was recently accepted by the European Parliament. This EU Directive was published in September 2002 [11], and all EU 49

0165-9936/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2004.09.004

Trends

Trends in Analytical Chemistry, Vol. 24, No. 1, 2005

Acid Black 077

NH2
SO2 N H

NH2 N N HO3S

OH N N SO3H

N N

Azo cleavage Chemical or enzymatic reduction

H2N

NH2

Benzidine

Figure 1. Illustration of the reductive cleavage of a banned azo dye (Acid Black 077) into the carcinogenic amine (Benzidine).

member states had until 11 September 2003 to enact appropriate legal restrictions in their countries. This amendment, largely based on the German legislation, represents an attempt to ensure a high level of health and consumer protection on harmful azo dyes that are used in specic textile and leather products. The main changes from the German legislation are: azo pigments are not addressed; articles that come in contact with the oral cavity are covered; and, two amines (2-methoxyaniline and 4-aminoazobenzene) have been added to the list of 20 amines. The European Commission (EC) emphasizes that harmonized test methods are necessary for the application of the Directive and should preferably be developed at a European level [11]. Although several analytical methods for the determination of aromatic amines in azo dyed products have been described in the literature, the ofcial German DIN 53316 method [12] is currently the most accepted. Concerning leather, the appropriate analytical method to be used is the European Standard CEN ISO/TS 17234 [13], recently approved by the European Committee for Standardization (CEN). However, since this method is almost identical to the DIN 53316 method, it will not be discussed any further. It is well known that the DIN 53316 method suffers from major drawbacks, including low accuracy and precision. Furthermore, sample preparation employs conventional liquid-liquid extraction, which is timeconsuming and requires large consumption of hazardous organic solvents. As a consequence, two EU-funded projects has been initiated since 1998: SMT4-CT972194 (19982000) and G6RD-CT-2001-00600620 (20022004). The focuses of these projects have been development of alternative analytical procedures and feasibility studies for the preparation of certied reference materials containing certain harmful aromatic amines known to originate from some azo dyes used in the leather industry. This article gives an overview of analytical procedures for the determination of banned 50

azo dyes, capable of formation upon reductive cleavage of any of the 22 aromatic amines (see Table 1) covered by the EU Directive in consumer goods. The chemical structures presented in Table 1 reveal that there are great differences in physical properties between these substances (e.g., water solubility and volatility), which adds to the complexity of developing analytical procedures. Apart from leather and textile applications, the literature covered also includes other consumer goods of importance, such as nger paints, hard candies, and soft drinks. A summary of the covered analytical procedures can be found in Table 2.

2. Conventional determination Determination of azo dyes generally comprises four steps: pre-treatment; reduction, where the relevant azo dyes react with a reducing agent; extraction, where the formed amines are collected; and, nally, determination with an analytical technique. 2.1. Pre-treatment Pre-treatment of samples can often be grinding or cutting into small pieces to increase the surface area of the sample in order to enhance the following reduction step. In the case of leather samples, a degreasing step is often necessary before the reduction and extraction step to facilitate sample wetting. Commonly, this is accomplished using hazardous organic solvents. 2.2. Reduction and extraction The two most frequently used reducing agents for azo dyes are sodium dithionite and tin(II) chloride, where the former is the predominant. 2.2.1. Sodium dithionite. Early papers by German research groups describe the determination of more than

http://www.elsevier.com/locate/trac

Trends in Analytical Chemistry, Vol. 24, No. 1, 2005

Trends

Table 1. List of the 22 aromatic amines covered by EU Directive 76/769/EEC [11]

Amine CAS pKa
H2N H3C

Amine
NH2 CH3

CAS

pKa

NH2

1 4-Aminobiphenyl

92-67-1

4.3

12 3,3-Dimethylbenzidine
CH3

119-93-7

4.6

H2N

NH2

H2N CH3

NH2

2 Benzidine
CH3
NH2

92-87-5

4.7

13 4,4-Methylenedi-o-toluidine 838-88-0
H 2N

5.2

OMe

CH3

Cl 3 4-Chloro-o-toluidine

95-69-2

3.8

14 p-Cresidine
Cl
H2N Cl

120-71-8

4.7

NH2

NH2

4 2-Napthylamine
CH3

91-59-8

4.2

15 2,2-Dichloro-4,4methylenedianiline
O

101-14-4

3.3

N N

NH2

5 CH3 o-Aminoazotoluene
NH2 O2 N CH3

97-56-3

3.0

16 4,4-Oxydianiline
S
H2N

H2N

NH2

101-80-4

5.5

NH2

6 5-Nitro-o-toluidine

99-55-8

2.3

17 4,4-Thiodianiline

139-65-1

4.6

Planelles et al. [17] presented a study for the determination of amines formed after reductive cleavage of three azo dyes using sodium dithionite. Parameters, such as amount of reducing agent/amount of dye ratio, pH of reduction solution, reduction time and temperature, were studied. It was demonstrated that the most suitable conditions varied from one dye to another. Extraction was in this case performed with diethyl ether. In similar studies, dyes were reduced with sodium dithionite in boiling water and the formed amines were then extracted with chloroform repeatedly before nal determination [18,19]. A recent study optimised a reduction procedure for dyes mostly used in toys [20]. Different parameters for the reduction step, such as temperature, reaction time and dithionite/dye ratio, were investigated by a factorial design. The reduction solution was directly analyzed for the presence of 24 amines. Reduction of dyestuffs with sodium dithionite in aqueous alkaline solution has also been performed, with a following liquidliquid extraction with ethyl acetate [21]. However, no specic reduction conditions were reported. 2.2.2. Tin(II) chloride. A study on 14 dyestuffs used in consumer fabric dye products was performed by reduction with tin(II) chloride dissolved in concentrated hydrochloric acid (HCl) [22]. After reduction, the solution was made alkaline and the amines were extracted with 1,1,1-trichloroethane. In the commercial homedyeing kits tested, the presence of amine-based dyes was conrmed in several samples. 2.2.3. Comparative study. A comparative study between the use of sodium dithionite and tin(II) chloride as reducing agent was published by Voyksner et al. [23]. Procedures were evaluated for the reductive cleavage of 16 commercial azo dyes using both reducing agents. Reduction with sodium dithionite (in boiling methanol) showed that different dyes required different amounts of sodium dithionite to be decolorized. Reduction with tin(II) chloride was also conducted in boiling methanol. After decolarization, sodium carbonate was added, and the amines were extracted with ethyl acetate. The authors stated that tin(II) chloride was preferable for this application, since it was more powerful, yielding a greater number of products. However, further evaluation of these reducing agents should be carried out to facilitate an equitable comparison of their potential in this eld. 2.2.4. Other reducing agents. Besides the two abovementioned reducing agents, there are other published approaches for the reduction of azo dyes. By using commercial zinc dust and ammonium format or formic acid in methanol at room temperature, azo compounds were cleaved to form amines [24].
http://www.elsevier.com/locate/trac

Cl

NH2

NH2

CH3

7 4-Chloroaniline
NH2

106-47-8

4.0

18 o-Toluidine
NH2

95-53-4

4.5

NH2 OMe

NH2 CH3

8 2,4-Diaminoanisole

615-05-4

5.3

19 2,4-Diaminotoluene
H3C NH2 CH3

95-80-7

5.1

H2N

NH2

H3C

9 4,4-Methylenedianiline
Cl H2N Cl NH2

101-77-9

5.3

20 2,4,5-Trimethylaniline
H2N MeO

137-17-7

5.0

10 3,3-Dichlorobenzidine
MeO OMe

91-94-1

2.7

21 2-Methoxyaniline

90-04-0

4.5

H2N

NH2

H2N

N N

11 3,3-Dimethoxybenzidine

119-90-4

4.7

22 4-Aminoazobenzene

60-09-3

3.1

20 amines in textiles [14,15]. In these studies, the samples were blended with sodium dithionite in 1 M sodium hydroxide. The alkaline solution was transferred to Extrelut columns and the amines were eluted with ethyl acetate. Some textile samples showed detectable amounts of amines. Lancaster and Lawrence [16] determined amines in soft drinks and hard candies by direct addition of solid sodium dithionite to the soft drink or dissolved candy solution followed by extraction with chloroform.

51

52
http://www.elsevier.com/locate/trac

Trends

Table 2. Summary of analytical procedures for the determination of banned azo dyes in consumer goods
Sample Conventional methodology Leather Amine No.a 120 Pre-treatment Degreasing 1 g of sample using 2x20 mL of nhexane. Decantation followed by evaporation of the residue n-hexane overnight. Cutting the sample into small pieces. Reduction Reduction of the degreased sample in 17 mL of citrate buffer (pH 6.0) using 23x1.5 mL of sodium dithionite (0.2 g/mL water). Reduction of 2 g of sample in 25 mL of 1 M sodium hydroxide using 1 g of sodium dithionite. Adjustment of pH to 2.12.6 prior to reduction of 200 mL of sample using 60 mg of sodium dithionite. Adjustment of pH to 2.12.6 prior to reduction of 125 mL of sample solution using 60-240 mg of sodium dithionite. Addition of 50 mL of water to a solution consisting of 1050 mg of sample (0.11.25 % w/w) dissolved in water/methanol (5/1 or 2/1, w/w) prior to reduction using 100500 mg of sodium dithionite. Reduction of 0.5 g of sample in 10 mL of boiling water using 0.52.0 g of sodium dithionite. Reduction of 100 mg of sample in 10 mL of boiling water using 1 g of sodium dithionite. Reduction of 5 g of preheated sample solution (equivalent to 40 g of dye) using 20 l of aqueous sodium dithionite. Reduction of 150 mg of sample dissolved in 10 mL of methanol using 1mL of tin (2) chloride (25% w/v in conc. hydrochloric acid). Reduction of a solution of 1 mmol of sample in boiling methanol using 98 mmol of sodium dithionite (25% w/v in water) under nitrogen atmosphere. Reduction of a solution of 1 mmol of sample in boiling methanol using 14 mmol of tin (2) chloride (40% w/v in conc. hydrochloric acid).
b

Extraction Alkalinization prior to liquid-liquid extraction using Extrelut columns and 40 mL of methyl tert-butyl ether. Alkalinization prior to liquid-liquid extraction using Extrelut columns and 40 mL of ethyl acetate.

Final determination HPLC-DAD, GCFID/MS, TLC, or CE-DAD GC-MS, HPLCDAD

Reference [12]

Textiles

122

[14,15]

Soft drinks,

Hard candies

1, 4

Dissolving 75 g of sample in 250 mL of distilled water in a sealed cylinder overnight.

b

Adjustment of pH to 8.5 prior to liquid-liquid extraction using 3x25 mL of chloroform.

Ion Pair HPLC-UVc

[16]

Dyes

2, 11, 18

Adjustment of pH to 10 prior to liquid-liquid extraction using 3x20 mL of diethyl ether.

HPLC-UV

[17]

Dyes

2, 11, 12

Alkalinization (pH>10) prior to liquid-liquid extraction using 5mL of chloroform. Alkalinization (pH>10) prior to liquid-liquid extraction using 5mL of chloroform.
b

TLC

[18]

Dyes Dyes

2 17, 820

GC-MS HPLC-DAD

[19] [20]

Dyes

2, 11, 12

Alkalinization prior to liquid-liquid extraction using 3x2 mL of 1,1,1-trichloroethane. Liquid-liquid extraction using ethyl acetate.

TLC and IR

[22]

Dyes

18

Separating the sample from diluents using Soxhlet extraction with methylene chloride.

HPLC-MS

[23]

Trends in Analytical Chemistry, Vol. 24, No. 1, 2005

Dyes

18

Separating the sample from diluents using Soxhlet extraction with methylene chloride. Drying the sample in a microwave oven subsequent to cutting it into sheets.

Adjustment of pH to 78 prior to liquid-liquid extraction using ethyl acetate. Addition of methanol, as modifier, to the extraction vessel containing 0.2 g of dry sample prior to extraction with carbon dioxide using SFE. MAE and Soxhlet extraction using methanol. SFE using methanol-modified carbon dioxide.

HPLC-MS, GC-MS

[23,26]

Recent methodology

Finger-paints

14

GC-FID

[42]

Plastics

Leather

2, 7, 1012, 18

Mixing PVC resins with azo dyes and calcium carbonate followed by homogenization, addition of plasticizer, and oven curing. Cutting the sample into sheets prior to extraction. Degreasing the sample with neat carbon dioxide using SFE. Cutting the sample into small pieces.

HPLC-DAD

[43]

Textiles and leather Te xtiles and synthetic leather

a

14, 720

Reduction of 0.3 g of degreased sample (in 20 mL of citrate buffer, pH 6.0) using 1 mL of aqueous sodium dithionite (0.1 g/mL). Reduction of 1 g of sample in 17 mL of citrate buffer (pH 6.0) using 3mL of aqueous sodium dithionite (0.2 g/mL).
b

SFE using neat or methanol-modified carbon dioxide. MAE using citrate buffer (pH 6.0) and/or methanol. SPME (direct immersion) of the reduction solution. Thermal extraction by means of pyrolysis.

HPLC-DAD

[44]

GC-MS

[45]

14, 11, 12, 22

GC-MS

[46]

The numbers refer to the amines listed in Table 1. Not used or reported. After diazotation of the amines formed with sodium nitrite subsequent to derivatization with 2-naphtol-3,6-disulphonic acid. d Direct determination of the dyes.
b c

Trends in Analytical Chemistry, Vol. 24, No. 1, 2005

Trends

In another study, several azo dyes commonly used in textiles were exposed to electrochemical treatment using a diamond electrode [25]. Procedures have been evaluated for reductive cleavage of eight commercial azo dyes using hydrogen (H2 ) and palladium (Pd) [26]. The reduction was accomplished directly in a heated injection port liner in a gas chromatograph (GC). This method was compared with reduction with tin(II) chloride in solution as in [24]. Similar or better results were obtained with the H2 /Pd method. 2.3. Final determination Several analytical techniques, such as thin layer chromatography (TLC), gas chromatography (GC), and highperformance liquid chromatography (HPLC), have been employed for the determination of low levels of aromatic amines. Whereas TLC and GC have mostly been used for qualitative analysis, HPLC has been the method of choice for quantication purposes. 2.3.1. TLC. In an early paper by Pinder and Tinsley [22], TLC was used as a screening method with further examination of the extracts by infrared spectroscopy (IR), while, in a study by Narvekar and Srivastava, modern TLC was used for quantitative analysis of several amines and their isomers [27]. 2.3.2. GC. Conrmation and in some cases quantication of either free amines [14,19,21,26] or after derivatization [2830] has frequently been performed utilizing GC coupled to mass spectrometry (MS). Longo and Cavallaro stated that, in general, the determination of underivatized amines by GC allows for the determination of low levels but is not selective enough for the simultaneous identication of a wide series of compounds [28]. They therefore derivatized the amines with hepta uorobutyric anhydride (HFBA) to decrease the limit of determination. Narvekar and Srivastava [30] have also described a GC-MS method for identication of amines and their isomers by derivatization with pentauoropropionic anhydride (PFPA). Derivatization was performed in tetrahydrofuran and the amide derivates formed were analyzed with good resolution between the amines and their isomers. The limits of detection of underivatized amines were somewhat higher than for the amine-PFPA derivatives. 2.3.3. HPLC. The most widespread analytical technique for quantitative determination of aromatic amines is HPLC. Already in 1984, Radzik et al. [31] determined the metabolite (4-nitroaniline) of a commonly used textile dye (Disperse Orange 3) in liver microsomal incubation media by direct injection of the incubation

solution into the LC system equipped with an electrochemical detector. Several groups have shown that 20 amines or more can be separated and quantied using LC with some sort of octadecyl silane (ODS) column and a gradient mobile phase consisting of mixtures of water or aqueous buffer, methanol and/or acetonitrile [15,20,23,25,32,33]. Detection is usually done at a single wavelength with a UV [23,32,33] or with a diode array detector (DAD) [15,20,25]. LC-MS has also been utilized for aromatic amine analysis [23,34]. For determination of amines in dyed candies and soft drinks, the amines formed were diazotised with sodium nitrite before coupling with 2-naphthol-3,6-disulphonic acid [16]. The colored derivatives were analyzed using reverse-phase ion-pair HPLC. For the analysis of sulphonated azo dyes in food, triethylamine was added to the mobile phase as an ionpair reagent for the chromatographic separation [34]. 2.3.4. Miscellaneous. Another analytical technique that has been used is capillary zone electrophoresis (CZE). Borrs et al. [35] showed separation of 15 amines for o monitoring azo dye synthesis. In 2001, Barek et al. [36] reviewed modern electroanalytical techniques for the detection of environmental carcinogenic compounds, among them aromatic amines.

3. Modern aspects The least developed part in the overall analytical procedure is probably the extraction step, as is the case in many other analytical schemes. The 1990s saw increased interest in extraction techniques among analytical chemists, as this step was a major reason for low sample throughput. Also, in the case of aromatic amines, a few studies have been dealing with modern extraction techniques during the last decade. Between 1993 and 1996, four studies can be recognized, demonstrating the potential of superuid extraction (SFE) and microwaveassisted extraction (MAE) as alternatives to conventional extraction techniques for the extraction of banned amines from contaminated soils [3740]. After 1996, the focus shifted, presumably as a consequence of the cumulative restrictions on azo dyes, towards other types of matrices, such as nger paints, toy products and leather [20,4146]. 3.1. SFE and MAE In two nearly identical investigations by Garrigs et al. o [41,42], several parameters in SFE was optimised by means of a factorial design for the determination of aromatic amines in nger paints. The parameters optihttp://www.elsevier.com/locate/trac

53

Trends

Trends in Analytical Chemistry, Vol. 24, No. 1, 2005

mised were extraction pressure, temperature, modier content and static extraction time. In the earlier study [41], wet white nger paint was spiked with amines. The best SFE conditions differed between the amines with maximum recoveries of 5565%. However, these recoveries were better than those obtained with conventional procedures based on Soxhlet extraction (recovery of 2530%). The SFE methodology also resulted in a substantial decrease in extraction time needed. In the later study, a nearly identical approach was tested [42], but the outcome was not the same, since optimal conditions differed and recoveries were changed. Unfortunately, no explanation was given for these differences and no general method was suggested, since maximum recoveries were obtained at different conditions for the various amines. However, an interesting nding was that methanol was the best modier out of ve tested [42]. Other observations showed that there could be some variability in extraction efciencies, depending on matrix. Additionally, the time the amine is allowed to interact with the matrix prior to the extraction dramatically decreases the recoveries. After 24 h, the recoveries in some cases were less than half of those reported after direct extraction. Finally, ve real paints were extracted and, in all cases, 4-chloro-o-toluidine was found. However, considering the great variability in recoveries because of matrix effects and the time allowed for amines to interact with the matrix, these data are very uncertain, and the method can by no means be considered nal. Additionally, the papers did not address the problem of reduction, since the paints were never spiked with azo dyes. Even so, the work performed so far by Garrigs and o co-workers gives new insight into the complex matter of extraction of aromatic amines. In 2002, Garrigs and colleagues extended their work o on consumer goods by utilizing both SFE and MAE for the extraction of azo dyes from plastics [43]. In this work, polyvinyl-chloride (PVC) resins were mixed with calcium carbonate and single-solvent dyes. Optimisation of both the SFE and the MAE procedures was done by factorial designs. For both techniques, the harshest extraction conditions gave the highest recoveries. An overall comparison between MAE, SFE and Soxhlet showed that MAE is the best method. However, the drawback with MAE was less selectivity in the extraction, since citrate was co-extracted to a greater extent with this method. Another important consumer goods application is leather samples. Eskilsson et al. recently demonstrated the possibilities of replacing the DIN 53316 method with new methods based on SFE or MAE. Two separate procedures were developed for the determination of aromatic amines formed from azo dyed leather [44]. All amines investigated in the study were extractable as 54
http://www.elsevier.com/locate/trac

pure substances with SFE using neat and methanolmodied carbon dioxide. Degreasing with solvent extraction is not very satisfactory, since a large amount of the azo dyes co-extracts, and consequently degreasing was performed with SFE leaving the azo dyes in the leather while all fat was removed. Additionally, the leather is completely dry after SFE, while, in solvent extraction, the leather has to be dried externally. Even though the SFE procedure only incorporated a few manual steps, the MAE procedure, despite being more labor-intensive and less selective, was considered to be the best choice for this application since it yielded the highest recovery values (see Fig. 2). 3.2. Solid-phase microextraction An solid-phase microextraction (SPME) based method combined with GC-MS has been developed to detect the usage of banned azo dyes in colored textiles and leather [45]. The azo dyes were determined by quantifying the corresponding aromatic amines, which were formed after a reductive cleavage step performed in citrate buffer. In total, 18 aromatic amines were investigated, together with two internal standards that were added to each extract. These were 2,4,5-trichloroaniline for monocyclic amines and 2-methyl-1-naphthylamine for amines with two aromatic rings. The recoveries of the various analytes were good at four tested levels (3090 lg/g) with values in the range 80100% for most analytes. However, these recoveries were based on standard solutions of aromatic amines and not on real samples. It is therefore difcult to evaluate the recoveries for the method developed. An interesting future investigation would be to perform reductive cleavage of azo dyes from certied reference leather samples combined with SPME to test the overall efciency against certied data. 3.3. Pyrolysis Recently, Plum et al. utilized pyrolysis GC coupled with MS detection (Py-GC-MS) as a solvent-free sample preparation for qualitative determination of seven aromatic amines formed from azo dyes in textile products [46]. In this method, the amines are thermally extracted directly into the analysis system and a preceding reduction step is therefore not required. In the initial experiments with three pure azo dyes, the pyrolyser temperature was varied to examine the inuence on recovery of the amines (measured as peak area). Although the dyes responded differently to the changes in temperature, it was found that moderate temperature (500C) was preferred since higher temperatures led to a decrease in recovery because of further degradation of the amines. This was also conrmed in the subsequent experiments by which contaminated samples (0.5 mg) of cotton, wool, synthetic leather, and polyamide were analyzed at different pyrolyser temperatures. Textiles products made from synthetic bre and wool gave

Trends in Analytical Chemistry, Vol. 24, No. 1, 2005

Trends

Figure 2. Recovery (%) (n 2) for different methods in the determination of aromatic amines from real leather samples (data from [45]).

reasonable or good results whereas samples of cotton failed in the detection of amines. However, the two main drawbacks of this technique are that the recovery of the amines is highly matrix-dependent and that larger sample amounts tend to accelerate soiling of the injector tube, which might affect the detection. Even so, Py-GC-MS is a simple technique that could be attractive for rapid screening (30 min/sample) of amines in certain textiles; and, in cases of positive ndings, conrmation and quantication can be performed according to conventional methodology. 4. Conclusions The ban on azo dyes recently imposed by the EU demands fast, cost-effective, environmentally friendly, accurate and precise analytical procedures for detecting low levels of azo dye degradation products (i.e., aromatic amines) in consumer goods. Although it is hard to meet all those criteria, the recent introduction of modern extraction techniques, such as MAE and SFE, is clearly a step in this direction. The main drawback of such techniques is the inevitably high investment costs. It should be stressed that the lack of certied reference materials in this eld complicates method validation, especially in terms of accuracy. In most studies, referred to in this paper, accuracy is based on the recovery of known amounts of amines spiked into the sample matrix. Even though this approach is widely used for assessing accuracy, the spiking might not simulate the natural behavior of the amines.

Furthermore, method development that emphasizes reduction and extraction is challenging, in view of the wide disparity in physical properties between the amines, together with complexity of the matrices. References
[1] K.T. Chung, Environ. Carcin. Ecotox. Rev. C 18 (2000) 51. [2] F. Rai, J.D. Hall, C.E. Cerniglia, Food. Chem. Toxicol. 35 (1997) 897. [3] S. Hildenbrand, F.W. Scmahl, R. Wodarz, R. Kimmel, P.C. Dartsch, Int. Arch. Occup. Environ. Health 72 (1999) M52. [4] T. Platzek, C. Lang, G. Grohmann, U.S. Gi, W. Baltes, Hum. Exp. Toxicol. 18 (1999) 552. [5] J.H. Weisburger, Mutat. Res. 506507 (2002) 9. [6] P. Vineis, R. Pirastu, Cancer Causes Control 8 (1997) 346. [7] L. Rehn, Arch. Klin. Chir. 50 (1895) 588. [8] M.C. Yu, P.L. Skipper, S.R. Tannenbaum, K.K. Chan, R.K. Ross, Mutat. Res. 506507 (2002) 21. [9] R.I. Freudenthal, E. Stephens, D.P. Anderson, Int. J. Toxicol. 18 (1999) 353. [10] Second amendment to the German Consumer Goods Ordinance, Bundesgesetzblatt, Part 1, 1994, p. 1670. [11] European Commission, Off. J. Eur. Commun. L 243 (2002) 15. [12] DIN 53316, DIN Deutsches Institut f r Normung e.V., 1997. u [13] CEN ISO/TS 17234, Leather Chemical tests Determination of certain azo colorants in dyed leathers, Beuth Verlag, Berlin, Germany, 2003. [14] K. Friedrichs, H.D. Winkeler, G. Prior, GIT Fachz. Lab. 39 (1995) 901. [15] H.D. Winkeler, GIT Spezial Chromatograph. 16 (1996) 6. [16] F.E. Lancaster, J.F. Lawrence, Food Addit. Contam. 9 (1992) 171. [17] F. Planelles, E. Verdu, D. Campello, N. Grane, J.M. Santiago, J. Soc. Leather Technol. Chem. 82 (1998) 45. [18] A. Puntener, D. Mausezahl, C. Page, J. Soc. Leather Technol. Chem. 77 (1993) 1.

http://www.elsevier.com/locate/trac

55

Trends
[19] D. Muralidharan, V.S. Sundara Rao, J. Soc. Leather Technol. Chem. 78 (1994) 139. [20] M.C. Garrigs, F. Reche, M.L. Mar A. Jimnez, J. Chromatogr. o n, e A 976 (2002) 309. [21] S.W. Oh, M.N. Kang, C.W. Cho, M.W. Lee, Dyes Pig. 33 (1997) 119. [22] A.G. Pindar, H.M. Tinsley, Analyst (Cambridge, UK) 109 (1984) 1101. [23] R.D. Voyksner, S. Rolf, J.T. Keever, H.S. Freeman, W.N. Hsu, Environ. Sci. Technol. 27 (1993) 1665. [24] S. Gowda, K. Abiraj, C. Gowda, Tetrahedron Lett. 43 (2002) 1329. [25] M.M. Dvila-Jimnenz, M.P. Elizalde-Gonzlez, A. Gutirreza e a e Gonzlez, J. Chromatogr. A 889 (2000) 253. a [26] R.F. Straub, R.D. Voyksner, J.T. Keever, Anal. Chem. 65 (1993) 2131. [27] M.S. Narvekar, A.K. Srivastava, J. Planar Chromatogr. 14 (2001) 360. [28] M. Longo, A. Cavallaro, J. Chromatogr. A 753 (1996) 91. [29] N. Lichtenstein, W. Paumbaum, K. Quellmalz, M. Bernards, M. Henning, Gefahrstoffe Reinhalt. Luft 57 (1997) 139. [30] M.S. Narvekar, A.K. Srivastava, Chromatographia 55 (2002) 729. [31] D.M. Radzik, J.S. Brodbelt, P.T. Kissinger, Anal. Chem. 56 (1984) 2927. [32] C.S. Lu, S.D. Huang, J. Chromatogr. A 696 (1995) 201. [33] E. Verd , D. Planelles, D. Campello, N. Gran, J.M. Santiago, u e AQEIC Bol. Tec. 48 (1997) 53. [34] M.R. Fuh, K.J. Chia, Talanta 56 (2002) 663. [35] S. Borrs, G. Barber, J. Biada, N. Agull, Dyes Pigm. 43 (1999) o a o 189. [36] J. Barek, J. Cvacka, A. Muck, V. Quaiserov, Fresenius J. Anal. a Chem. 369 (2001) 556. [37] T.S. Oostdyk, R.L. Grob, J.L. Snyder, M.E. McNally, Anal. Chem. 65 (1993) 596. [38] V. Lopez-Avila, R. Young, N. Teplitsky, J. AOAC Int. 79 (1996) 142. [39] T.S. Oostdyk, R.L. Grob, J.L. Snyder, M.E. McNally, J. Cell Sci. 31 (1993) 177.

Trends in Analytical Chemistry, Vol. 24, No. 1, 2005

[40] T.S. Oostdyk, R.L. Grob, J.L. Snyder, M.E. McNally, J. Environ. Sci. Health, Part A 30 (1995) 783. [41] M.C. Garrigs, F. Reche, K. Pern o ias, A. Snchez, A. Jimnez, a e J. Chromatogr. A 819 (1998) 259. [42] M.C. Garrigs, F. Reche, K. Pern A. Jimnez, J. Chromatogr. A o ias, e 896 (2000) 291. [43] M.C. Garrigs, F. Reche, M.L. Mar K. Pern o in, ias, A. Jimnez, e J. Chromatogr. A 963 (2002) 427. [44] C.S. Eskilsson, R. Davidsson, L. Mathiasson, J. Chromatogr. A 955 (2002) 215. [45] F. Cioni, G. Bartolucci, G. Pieraccini, S. Meloni, G. Moneti, Rapid Commun. Mass Spectrom. 13 (1999) 1833. [46] A. Plum, W. Engewald, A. Rehorek, Chromatographia 57 (2003) S243. Lars-Henric Ahlstr m is a PhD student at the Department of o Analytical Chemistry, Lund University, Sweden. His research is focused on determination of banned azo dyes utilizing modern extraction techniques. Cecilia Sparr Eskilsson obtained her PhD in analytical chemistry at Lund University, Sweden, in 2003. Her research activities involved sample preparation using modern extraction techniques, such as SFE, MAE and PLE. She is currently working as a researcher at AstraZeneca, Lund, Sweden. Erland Bjrklund obtained his PhD in analytical chemistry in 1998 o at Lund University, Sweden. His work involved extraction of environmental contaminants from solid matrices using SFE and PLE. He spent 6 months at the Energy & Environmental Research Center in Grand Forks, ND, USA, studying selective SFE as a means to measure bioavailability. He also worked 12 months as an analytical chemist for the European Commission, Ispra, Italy, developing fast extraction methods for pollutants in fatty food and feed. At present, he is an Assistant Professor at the Department of Analytical Chemistry, Lund University.

56

http://www.elsevier.com/locate/trac