Determination of Banned Azo Dyes in Consumer Goods

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

Determination of banned azo dyesin consumer goods
Lars-Henric Ahlstr€om, Cecilia Sparr Eskilsson, Erland Bj€orklund
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 confirmed 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.

ª 2004 Elsevier Ltd. All rights reserved.

Keywords: Analytical procedures; Aromatic amines; Azo dyes; Consumer goods

*Corresponding author.

Tel.: +46-46-222-8176;

Fax: +46-46-222-4544;

E-mail: lars-henric.

[email protected]

Lars-Henric Ahlstr€om*,

Cecilia Sparr Eskilsson,

Erland Bj€orklund

Department of Analytical

Chemistry, Lund University,

P.O. Box 124, S-221 00 Lund,

Sweden

0165-9936/$ - see front matter ª 200

1. Introduction

Since the second half of the 19th century,azo dyes have been extensively used innumerous industrial applications mainlybecause of their colorfastness and lowprice. Azo dyes are synthetic organic col-orants, characterized by chromophoric azogroups (–N@N–). Currently, there are over3000 azo dyes in use worldwide and theyaccount for 65% of the commercial dyemarket. These dyes offer a wide spectrumof colors and are used for coloring a vari-ety of consumer goods, such as leather,clothes, food, toys, plastics and cosmetics[1,2].

However, whereas azo dyes are rela-tively resistant to degradation under aer-obic conditions, they can be readilyreduced to form aromatic amines underanaerobic conditions (see Fig. 1).

The reduction primarily occurs throughcleavage of the azo group by azoreductasesin intestinal bacteria, liver cells, and skinsurface micro flora [3,4]. A number of thearomatic amines formed have been foundto be carcinogenic in experimentalanimals and thus pose a risk for consum-ers [5,6].

The carcinogenic risk of aromaticamines in humans was first observed in1895 by German surgeon Ludwig Rehn,who reported high rates of urinary bladder

4 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2004.09.004

cancer among men employed in the dye-stuff industry, who were chronicallyexposed to large amounts of aniline [7].Over the years, subsequent epidemiologi-cal studies have shown additional evi-dence that long-term occupationalexposure to certain aromatic amines (e.g.,benzidine, 4-aminobiphenyl, and 2-naph-thylamine) that are used in the dyeindustry increases the risk of developingcancer [8,9].

The main routes of exposure of con-sumers to azo dyes and their degradationproducts are oral ingestion (e.g., youngchildren sucking on toys that contain dyedtextile or leather garments) and dermalabsorption (e.g., sweat and friction causedyes, contained in clothes worn near theskin, to elute).

In July 1994, the German Government,against this background, introduced the2nd amendment to their Consumer GoodsOrdinance [10], which prohibited the useof certain azo colorants (i.e., dyes andpigments) in defined articles that comeinto direct, prolonged contact with humanskin. The azo dyes affected by this legisla-tion are those that, after reduction, mayform one or more of the 20 confirmed orsuspected carcinogenic aromatic amineslisted in the Ordinance in detectable con-centrations (i.e., >30 ppm) [10].

Similar restrictions regarding azo dyeswere applied shortly afterwards in otherMember States of the European Union(EU), but, in the interests of transparencyand maintenance of the Single Market, the19th amendment of the Council Directive76/769/EEC (relating to restrictions onthe marketing and use of certain danger-ous substances and preparations) wasrecently accepted by the European Par-liament. This EU Directive was publishedin September 2002 [11], and all EU

49

mail to: lars-henric.�[email protected]

mail to: lars-henric.�[email protected]

NH2NH2

SO2 N N N N N N

NH2 OH

HO3S SO3H

NH

NH2

Azo cleavage Chemical or enzymatic reduction

Acid Black 077

Benzidine

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

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

member states had until 11 September 2003 to enactappropriate legal restrictions in their countries. Thisamendment, largely based on the German legislation,represents an attempt to ensure a high level of healthand consumer protection on harmful azo dyes that areused in specific textile and leather products. The mainchanges from the German legislation are: azo pigmentsare not addressed; articles that come in contact with theoral cavity are covered; and, two amines (2-meth-oxyaniline and 4-aminoazobenzene) have been added tothe list of 20 amines.

The European Commission (EC) emphasizes that har-monized test methods are necessary for the application ofthe Directive and should preferably be developed at aEuropean level [11]. Although several analytical meth-ods for the determination of aromatic amines in azo dyedproducts have been described in the literature, the offi-cial German DIN 53316 method [12] is currently themost accepted. Concerning leather, the appropriateanalytical method to be used is the European StandardCEN ISO/TS 17234 [13], recently approved by theEuropean Committee for Standardization (CEN). How-ever, since this method is almost identical to the DIN53316 method, it will not be discussed any further.

It is well known that the DIN 53316 method suffersfrom major drawbacks, including low accuracy andprecision. Furthermore, sample preparation employsconventional liquid-liquid extraction, which is time-consuming and requires large consumption of hazardousorganic solvents. As a consequence, two EU-fundedprojects has been initiated since 1998: SMT4-CT97-2194 (1998–2000) and G6RD-CT-2001-00600620(2002–2004). The focuses of these projects have beendevelopment of alternative analytical procedures andfeasibility studies for the preparation of certified referencematerials containing certain harmful aromatic aminesknown to originate from some azo dyes used in theleather industry. This article gives an overview ofanalytical procedures for the determination of banned

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

azo dyes, capable of formation upon reductive cleavageof any of the 22 aromatic amines (see Table 1) coveredby the EU Directive in consumer goods.

The chemical structures presented in Table 1 revealthat there are great differences in physical propertiesbetween these substances (e.g., water solubility andvolatility), which adds to the complexity of developinganalytical procedures.

Apart from leather and textile applications, the liter-ature covered also includes other consumer goods ofimportance, such as finger paints, hard candies, and softdrinks. A summary of the covered analytical procedurescan be found in Table 2.

2. Conventional determination

Determination of azo dyes generally comprises four steps:pre-treatment; reduction, where the relevant azo dyesreact with a reducing agent; extraction, where theformed amines are collected; and, finally, determinationwith an analytical technique.

2.1. Pre-treatmentPre-treatment of samples can often be grinding orcutting into small pieces to increase the surface area ofthe sample in order to enhance the following reductionstep. In the case of leather samples, a degreasing step isoften necessary before the reduction and extraction stepto facilitate sample wetting. Commonly, this is accom-plished using hazardous organic solvents.

2.2. Reduction and extractionThe two most frequently used reducing agents for azodyes are sodium dithionite and tin(II) chloride, where theformer is the predominant.

2.2.1. Sodium dithionite. Early papers by Germanresearch groups describe the determination of more than

Table 1. List of the 22 aromatic amines covered by EU Directive76/769/EEC [11]

Amine CAS pKa Amine CAS pKa

NH2

NH2

CH3CH3

NH2

1 4-Aminobiphenyl 92-67-1 4.3

12 3,3´-Dimethylbenzidine 119-93-7 4.6

NH2NH2

CH3

NH2

CH3

NH2

2 Benzidine 92-87-5 4.7

13 4,4´-Methylenedi-o-toluidine 838-88-0 5.2

CH3

NH2

Cl

NH2

CH3OMe

3 4-Chloro-o-toluidine 95-69-2 3.8

14 p-Cresidine 120-71-8 4.7

NH2

Cl

NH2

Cl

NH2

4 2-Napthylamine 91-59-8 4.2

15 2,2´-Dichloro-4,4´- methylenedianiline

101-14-4 3.3

N

CH3

N

CH3

NH2

O

NH2NH25 o-Aminoazotoluene 97-56-3 3.0

16 4,4´-Oxydianiline 101-80-4 5.5

NH2

CH3O2N NH2

S

NH2

6 5-Nitro-o-toluidine 99-55-8 2.3

17 4,4´-Thiodianiline 139-65-1 4.6

NH2ClNH2

CH3

74-Chloroaniline 106-47-8 4.0

18 o-Toluidine 95-53-4 4.5

NH2

NH2

OMe

NH2

CH3

NH2

8 2,4-Diaminoanisole 615-05-4 5.3

19 2,4-Diaminotoluene 95-80-7 5.1

NH2NH2

NH2CH3

CH3CH3

9 4,4´-Methylenedianiline 101-77-9 5.3

20 2,4,5-Trimethylaniline 137-17-7 5.0

Cl

NH2

Cl

NH2

NH2

MeO

10 3,3´-Dichlorobenzidine 91-94-1 2.7

21 2-Methoxyaniline 90-04-0 4.5

NH2 NH2

OMeMeO

NNH2 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, thesamples were blended with sodium dithionite in 1 Msodium hydroxide. The alkaline solution was transferredto Extrelut columns and the amines were eluted withethyl acetate. Some textile samples showed detectableamounts of amines.

Lancaster and Lawrence [16] determined amines insoft drinks and hard candies by direct addition of solidsodium dithionite to the soft drink or dissolved candysolution followed by extraction with chloroform.

Planelles et al. [17] presented a study for the deter-mination of amines formed after reductive cleavage ofthree azo dyes using sodium dithionite. Parameters, suchas amount of reducing agent/amount of dye ratio, pH ofreduction solution, reduction time and temperature,were studied. It was demonstrated that the most suitableconditions varied from one dye to another. Extractionwas in this case performed with diethyl ether.

In similar studies, dyes were reduced with sodiumdithionite in boiling water and the formed amines werethen extracted with chloroform repeatedly before finaldetermination [18,19].

A recent study optimised a reduction procedure fordyes mostly used in toys [20]. Different parameters forthe reduction step, such as temperature, reaction timeand dithionite/dye ratio, were investigated by a factorialdesign. The reduction solution was directly analyzed forthe presence of 24 amines.

Reduction of dyestuffs with sodium dithionite inaqueous alkaline solution has also been performed, witha following liquid–liquid extraction with ethyl acetate[21]. However, no specific reduction conditions werereported.

2.2.2. Tin(II) chloride. A study on 14 dyestuffs used inconsumer fabric dye products was performed by reduc-tion with tin(II) chloride dissolved in concentratedhydrochloric acid (HCl) [22]. After reduction, the solu-tion was made alkaline and the amines were extractedwith 1,1,1-trichloroethane. In the commercial home-dyeing kits tested, the presence of amine-based dyes wasconfirmed in several samples.

2.2.3. Comparative study. A comparative study betweenthe use of sodium dithionite and tin(II) chloride asreducing agent was published by Voyksner et al. [23].Procedures were evaluated for the reductive cleavage of16 commercial azo dyes using both reducing agents.Reduction with sodium dithionite (in boiling methanol)showed that different dyes required different amounts ofsodium dithionite to be decolorized. Reduction withtin(II) chloride was also conducted in boiling methanol.After decolarization, sodium carbonate was added, andthe amines were extracted with ethyl acetate. Theauthors stated that tin(II) chloride was preferable for thisapplication, since it was more powerful, yielding a greaternumber of products. However, further evaluation of thesereducing agents should be carried out to facilitate anequitable comparison of their potential in this field.

2.2.4. Other reducing agents. Besides the two above-mentioned reducing agents, there are other publishedapproaches for the reduction of azo dyes. By usingcommercial zinc dust and ammonium format or formicacid in methanol at room temperature, azo compoundswere cleaved to form amines [24].

http://www.elsevier.com/locate/trac 51

Table 2. Summary of analytical procedures for the determination of banned azo dyes in consumer goods

Sample Amine No.a Pre-treatment Reduction Extraction Final determination Reference

Conventional methodology

Leather 1–20 Degreasing 1 g of sample using 2x20 mL of n-hexane. Decantation followed by evaporationof the residue n-hexane overnight.

Reduction of the degreased sample in 17 mLof citrate buffer (pH 6.0) using 2–3x1.5 mL ofsodium dithionite (0.2 g/mL water).

Alkalinization prior to liquid-liquid extractionusing Extrelut columns and 40 mL of methyl tert-butyl ether.

HPLC-DAD, GC-FID/MS, TLC, orCE-DAD

[12]

Textiles 1–22 Cutting the sample into small pieces. Reduction of 2 g of sample in 25 mL of 1 Msodium hydroxide using 1 g of sodiumdithionite.

Alkalinization prior to liquid-liquid extractionusing Extrelut columns and 40 mL of ethylacetate.

GC-MS, HPLC-DAD

[14,15]

Soft drinks,

Hard candies

4

1, 4

b

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

Adjustment of pH to 2.1–2.6 prior to reductionof 200 mL of sample using 60 mg of sodiumdithionite.

Adjustment of pH to 2.1–2.6 prior to reductionof 125 mL of sample solution using 60-240 mgof sodium dithionite.

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

Ion Pair HPLC-UVc [16]

UVDyes 2, 11, 18 b Addition of 50 mL of water to a solutionconsisting of 10–50 mg of sample (0.1–1.25 %w/w) dissolved in water/methanol (5/1 or 2/1,w/w) prior to reduction using 100–500 mg ofsodium dithionite.

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

HPLC- [17]

Dyes 2, 11, 12 b Reduction of 0.5 g of sample in 10 mL ofboiling water using 0.5–2.0 g of sodiumdithionite.

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

TLC [18]

S2 b Reduction of 100 mg of sample in 10 mL ofboiling water using 1 g of sodium dithionite.

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

GC-M [19]

8–20 Dyes

Dyes

1–7, b Reduction of 5 g of preheated sample solution(equivalent to 40 µg of dye) using 20 µl ofaqueous sodium dithionite.

b HPLC-DAD [20]

Dyes 2, 11, 12 b Reduction of 150 mg of sample dissolved in10 mL of methanol using 1mL of tin (2)chloride (25% w/v in conc. hydrochloric acid).

Alkalinization prior to liquid-liquid extractionusing 3x2 mL of 1,1,1-trichloroethane.

TLC and IR [22]

Dyes 18 Separating the sample from diluents using Soxhlet extraction with methylene chloride.

Reduction of a solution of 1 mmol of sample inboiling methanol using 98 mmol of sodiumdithionite (25% w/v in water) under nitrogenatmosphere.

Liquid-liquid extraction using ethyl acetate. HPLC-MS [23]

Dyes 18 Separating the sample from diluents using Soxhlet extraction with methylene chloride.

Reduction of a solution of 1 mmol of sample inboiling methanol using 1–4 mmol of tin (2)chloride (40% w/v in conc. hydrochloric acid).

Adjustment of pH to 7–8 prior to liquid-liquid extraction using ethyl acetate.

HPLC-MS, GC-MS [23,26]

Recentmethodology

Finger-paints 1–4 Drying the sample in a microwave ovensubsequent to cutting it into sheets.

b Addition of methanol, as modifier, to theextraction vessel containing 0.2 g of drysample prior to extraction with carbon dioxide using SFE.

GC-FID [42]

Plastics d Mixing PVC resins with azo dyes and calciumcarbonate followed by homogenization,addition of plasticizer, and oven curing.Cutting the sample into sheets prior toextraction.

b MAE and Soxhlet extraction using methanol.SFE using methanol-modified carbon dioxide.

HPLC-DAD [43]

Leather 2, 7, 10–12, 18 Degreasing the sample with neat carbondioxide using SFE.

Reduction of 0.3 g of degreased sample (in20 mL of citrate buffer, pH 6.0) using 1 mL ofaqueous sodium dithionite (0.1 g/mL).

SFE using neat or methanol-modified carbondioxide. MAE using citrate buffer (pH 6.0)and/or methanol.

HPLC-DAD [44]

STextiles andleather

1–4, 7–20 Cutting the sample into small pieces. Reduction of 1 g of sample in 17 mL of citrate buffer (pH 6.0) using 3mL of aqueous sodiumdithionite (0.2 g/mL).

SPME (direct immersion) of the reductionsolution.

GC-M [45]

Te xtiles andsynthetic leather

1–4, 11, 12, 22 b b Thermal extraction by means of pyrolysis. GC-MS [46]

a The numbers refer to the amines listed in Table 1. b Not used or reported. c 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.

Tren

ds

Tren

dsin

Analytical

Chem

istry,Vol.24,No.1,2005

52

http

://www.elsevier.co

m/lo

cate/trac


In another study, several azo dyes commonly used intextiles were exposed to electrochemical treatment usinga diamond electrode [25].

Procedures have been evaluated for reductive cleav-age of eight commercial azo dyes using hydrogen (H2)and palladium (Pd) [26]. The reduction was accom-plished directly in a heated injection port liner in a gaschromatograph (GC). This method was compared withreduction with tin(II) chloride in solution as in [24].Similar or better results were obtained with the H2/Pdmethod.

2.3. Final determinationSeveral analytical techniques, such as thin layer chro-matography (TLC), gas chromatography (GC), and high-performance liquid chromatography (HPLC), have beenemployed for the determination of low levels of aromaticamines. Whereas TLC and GC have mostly been used forqualitative analysis, HPLC has been the method of choicefor quantification purposes.

2.3.1. TLC. In an early paper by Pinder and Tinsley [22],TLC was used as a screening method with furtherexamination of the extracts by infrared spectroscopy(IR), while, in a study by Narvekar and Srivastava,modern TLC was used for quantitative analysis of severalamines and their isomers [27].

2.3.2. GC. Confirmation and in some cases quantifica-tion of either free amines [14,19,21,26] or afterderivatization [28–30] has frequently been performedutilizing GC coupled to mass spectrometry (MS). Longoand Cavallaro stated that, in general, the determina-tion of underivatized amines by GC allows for thedetermination of low levels but is not selective enoughfor the simultaneous identification of a wide series ofcompounds [28]. They therefore derivatized the amineswith hepta fluorobutyric anhydride (HFBA) to decreasethe limit of determination.

Narvekar and Srivastava [30] have also described aGC-MS method for identification of amines and theirisomers by derivatization with pentafluoropropionicanhydride (PFPA). Derivatization was performed intetrahydrofuran and the amide derivates formed wereanalyzed with good resolution between the amines andtheir isomers. The limits of detection of underivatizedamines were somewhat higher than for the amine-PFPAderivatives.

2.3.3. HPLC. The most widespread analytical techniquefor quantitative determination of aromatic amines isHPLC. Already in 1984, Radzik et al. [31] determinedthe metabolite (4-nitroaniline) of a commonly used tex-tile dye (Disperse Orange 3) in liver microsomal incu-bation media by direct injection of the incubation

solution into the LC system equipped with an electro-chemical detector.

Several groups have shown that 20 amines or morecan be separated and quantified using LC with some sortof octadecyl silane (ODS) column and a gradient mobilephase 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 aUV [23,32,33] or with a diode array detector (DAD)[15,20,25].

LC-MS has also been utilized for aromatic amineanalysis [23,34].

For determination of amines in dyed candies and softdrinks, the amines formed were diazotised with sodiumnitrite before coupling with 2-naphthol-3,6-disulphonicacid [16]. The colored derivatives were analyzed usingreverse-phase ion-pair HPLC.

For the analysis of sulphonated azo dyes in food,triethylamine was added to the mobile phase as an ion-pair reagent for the chromatographic separation [34].

2.3.4. Miscellaneous. Another analytical technique thathas been used is capillary zone electrophoresis (CZE).Borr�os et al. [35] showed separation of 15 amines formonitoring azo dye synthesis.

In 2001, Barek et al. [36] reviewed modern electro-analytical techniques for the detection of environmentalcarcinogenic compounds, among them aromatic amines.

3. Modern aspects

The least developed part in the overall analytical pro-cedure is probably the extraction step, as is the case inmany other analytical schemes. The 1990s sawincreased interest in extraction techniques among ana-lytical chemists, as this step was a major reason for lowsample throughput. Also, in the case of aromatic amines,a few studies have been dealing with modern extractiontechniques during the last decade. Between 1993 and1996, four studies can be recognized, demonstrating thepotential of superfluid extraction (SFE) and microwave-assisted extraction (MAE) as alternatives to conventionalextraction techniques for the extraction of bannedamines from contaminated soils [37–40].

After 1996, the focus shifted, presumably as a con-sequence of the cumulative restrictions on azo dyes,towards other types of matrices, such as finger paints,toy products and leather [20,41–46].

3.1. SFE and MAEIn two nearly identical investigations by Garrig�os et al.[41,42], several parameters in SFE was optimised bymeans of a factorial design for the determination ofaromatic amines in finger paints. The parameters opti-



mised were extraction pressure, temperature, modifiercontent and static extraction time.

In the earlier study [41], wet white finger paint wasspiked with amines. The best SFE conditions differedbetween the amines with maximum recoveries of55–65%. However, these recoveries were better thanthose obtained with conventional procedures based onSoxhlet extraction (recovery of 25–30%).

The SFE methodology also resulted in a substantialdecrease in extraction time needed. In the later study, anearly identical approach was tested [42], but the out-come was not the same, since optimal conditions differedand recoveries were changed. Unfortunately, no expla-nation was given for these differences and no generalmethod was suggested, since maximum recoveries wereobtained at different conditions for the various amines.

However, an interesting finding was that methanolwas the best modifier out of five tested [42]. Otherobservations showed that there could be some variabilityin extraction efficiencies, depending on matrix. Addi-tionally, the time the amine is allowed to interactwith the matrix prior to the extraction dramatically de-creases the recoveries. After 24 h, the recoveries in somecases were less than half of those reported after directextraction.

Finally, five real paints were extracted and, in allcases, 4-chloro-o-toluidine was found. However, con-sidering the great variability in recoveries because ofmatrix effects and the time allowed for amines to interactwith the matrix, these data are very uncertain, and themethod can by no means be considered final. Addition-ally, the papers did not address the problem of reduction,since the paints were never spiked with azo dyes. Evenso, the work performed so far by Garrig�os andco-workers gives new insight into the complex matter ofextraction of aromatic amines.

In 2002, Garrig�os and colleagues extended their workon consumer goods by utilizing both SFE and MAE forthe extraction of azo dyes from plastics [43]. In thiswork, polyvinyl-chloride (PVC) resins were mixed withcalcium carbonate and single-solvent dyes.

Optimisation of both the SFE and the MAE procedureswas done by factorial designs. For both techniques, theharshest extraction conditions gave the highest recov-eries. An overall comparison between MAE, SFE andSoxhlet showed that MAE is the best method. However,the drawback with MAE was less selectivity in theextraction, since citrate was co-extracted to a greaterextent with this method.

Another important consumer goods application isleather samples. Eskilsson et al. recently demonstratedthe possibilities of replacing the DIN 53316 method withnew methods based on SFE or MAE. Two separateprocedures were developed for the determination ofaromatic amines formed from azo dyed leather [44]. Allamines investigated in the study were extractable as


pure substances with SFE using neat and methanol-modified carbon dioxide. Degreasing with solventextraction is not very satisfactory, since a large amountof the azo dyes co-extracts, and consequently degreasingwas performed with SFE leaving the azo dyes in theleather while all fat was removed. Additionally, theleather is completely dry after SFE, while, in solventextraction, the leather has to be dried externally. Eventhough the SFE procedure only incorporated a fewmanual steps, the MAE procedure, despite being morelabor-intensive and less selective, was considered to bethe best choice for this application since it yielded thehighest recovery values (see Fig. 2).

3.2. Solid-phase microextractionAn solid-phase microextraction (SPME) based methodcombined with GC-MS has been developed to detect theusage of banned azo dyes in colored textiles and leather[45]. The azo dyes were determined by quantifying thecorresponding aromatic amines, which were formedafter a reductive cleavage step performed in citratebuffer. In total, 18 aromatic amines were investigated,together with two internal standards that were added toeach extract. These were 2,4,5-trichloroaniline formonocyclic amines and 2-methyl-1-naphthylamine foramines with two aromatic rings. The recoveries of thevarious analytes were good at four tested levels (30–90lg/g) with values in the range 80–100% for most ana-lytes. However, these recoveries were based on standardsolutions of aromatic amines and not on real samples. Itis therefore difficult to evaluate the recoveries for themethod developed. An interesting future investigationwould be to perform reductive cleavage of azo dyes fromcertified reference leather samples combined with SPMEto test the overall efficiency against certified data.

3.3. PyrolysisRecently, Plum et al. utilized pyrolysis GC coupled withMS detection (Py-GC-MS) as a solvent-free samplepreparation for qualitative determination of seven aro-matic amines formed from azo dyes in textile products[46]. In this method, the amines are thermally extracteddirectly into the analysis system and a precedingreduction step is therefore not required. In the initialexperiments with three pure azo dyes, the pyrolysertemperature was varied to examine the influence onrecovery of the amines (measured as peak area).Although the dyes responded differently to the changesin temperature, it was found that moderate temperature(500�C) was preferred since higher temperatures led to adecrease in recovery because of further degradation ofthe amines. This was also confirmed in the subsequentexperiments by which contaminated samples (0.5 mg) ofcotton, wool, synthetic leather, and polyamide wereanalyzed at different pyrolyser temperatures. Textilesproducts made from synthetic fibre and wool gave

Figure 2. Recovery (%) (n ¼ 2) for different methods in the determination of aromatic amines from real leather samples (data from [45]).


reasonable or good results whereas samples of cottonfailed in the detection of amines. However, the two maindrawbacks of this technique are that the recovery of theamines is highly matrix-dependent and that largersample amounts tend to accelerate soiling of the injectortube, which might affect the detection. Even so,Py-GC-MS is a simple technique that could be attractivefor rapid screening (�30 min/sample) of amines incertain textiles; and, in cases of positive findings,confirmation and quantification can be performedaccording to conventional methodology.

4. Conclusions

The ban on azo dyes recently imposed by the EUdemands fast, cost-effective, environmentally friendly,accurate and precise analytical procedures for detectinglow levels of azo dye degradation products (i.e., aromaticamines) in consumer goods. Although it is hard to meetall those criteria, the recent introduction of modernextraction techniques, such as MAE and SFE, is clearly astep in this direction. The main drawback of such tech-niques is the inevitably high investment costs.

It should be stressed that the lack of certified referencematerials in this field complicates method validation,especially in terms of accuracy. In most studies, referredto in this paper, accuracy is based on the recovery ofknown amounts of amines spiked into the samplematrix. Even though this approach is widely used forassessing accuracy, the spiking might not simulate thenatural behavior of the amines.

Furthermore, method development that emphasizesreduction and extraction is challenging, in view of thewide 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. Rafii, 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. 506–507 (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. 506–507 (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€ur Normung e.V., 1997.

[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.



[19] D. Muralidharan, V.S. Sundara Rao, J. Soc. Leather Technol.

Chem. 78 (1994) 139.

[20] M.C. Garrig�os, F. Reche, M.L. Mar�ın, A. Jim�enez, J. Chromatogr.

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. D�avila-Jim�enenz, M.P. Elizalde-Gonz�alez, A. Guti�errez-

Gonz�alez, J. Chromatogr. A 889 (2000) 253.

[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. Pflaumbaum, 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�u, D. Planelles, D. Campello, N. Gran�e, J.M. Santiago,

AQEIC Bol. Tec. 48 (1997) 53.

[34] M.R. Fuh, K.J. Chia, Talanta 56 (2002) 663.

[35] S. Borr�os, G. Barber�a, J. Biada, N. Agull�o, Dyes Pigm. 43 (1999)

189.

[36] J. Barek, J. Cvacka, A. Muck, V. Quaiserov�a, Fresenius’ J. Anal.

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.


[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. Garrig�os, F. Reche, K. Pern�ias, A. S�anchez, A. Jim�enez,J. Chromatogr. A 819 (1998) 259.

[42] M.C. Garrig�os, F. Reche, K. Pern�ias, A. Jim�enez, J. Chromatogr. A

896 (2000) 291.

[43] M.C. Garrig�os, F. Reche, M.L. Mar�in, K. Pern�ias, A. Jim�enez,

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€om is a PhD student at the Department of

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 Bj€orklund obtained his PhD in analytical chemistry in 1998

at Lund University, Sweden. His work involved extraction of environ-

mental 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.

Determination of Banned Azo Dyes in Consumer Goods

Documents

Transcript of Determination of Banned Azo Dyes in Consumer Goods