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Validation of a liquid chromatography-mass spectrometry method fordetermining the migration of primary aromatic amines from cookingutensils and its application to actual samplesRaquel Sendóna; Juana Bustosb; Jose Juan Sánchezb; Perfecto Paseiroa; Ma Eugenia Cirugedab

a Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Pharmacy, University ofSantiago de Compostela, 15782 Santiago de Compostela, Spain b Monomers and Additives Section,Chemical Area, National Food Centre, 28220 Majadahonda, Madrid, Spain

First published on: 10 September 2009

To cite this Article Sendón, Raquel , Bustos, Juana , Sánchez, Jose Juan , Paseiro, Perfecto and Cirugeda, Ma Eugenia(2010)'Validation of a liquid chromatography-mass spectrometry method for determining the migration of primary aromaticamines from cooking utensils and its application to actual samples', Food Additives & Contaminants: Part A, 27: 1, 107 —117, First published on: 10 September 2009 (iFirst)To link to this Article: DOI: 10.1080/02652030903225781URL: http://dx.doi.org/10.1080/02652030903225781

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Food Additives and ContaminantsVol. 27, No. 1, January 2010, 107–117

Validation of a liquid chromatography–mass spectrometry method for determining the migration

of primary aromatic amines from cooking utensils and its application to actual samples

Raquel Sendona, Juana Bustosb*, Jose Juan Sanchezb, Perfecto Paseiroa and Ma Eugenia Cirugedab

aDepartment of Analytical Chemistry, Nutrition and Bromatology, Faculty of Pharmacy, University of Santiago de Compostela,15782 Santiago de Compostela, Spain; bMonomers and Additives Section, Chemical Area, National Food Centre, Spanish FoodSafety and Nutrition Agency, 28220 Majadahonda, Madrid, Spain

(Received 2 June 2009; final version received 1 August 2009)

Many cooking utensils are made of nylon, a material that may incorporate azodyes and where primary aromaticamines (PAAs) are the starting substances. Moreover, aromatic amines may also be present as technicalimpurities. Another source of PAAs could be aromatic isocyanates used as monomers in the productionof polyurethanes. The aim of this work was to validate a simple LC–MS/MS method for the determination ofeight primary aromatic amines (m-phenylenediamine, 2,6- and 2,4-toluenediamine, 1,5-diaminonaphthalene,aniline, 4,40-diaminonaphenylether, 4,40-methylenedianiline and 3,30-dimethylbenzidine) in the aqueous foodsimulant 3% acetic acid (w/v). The detection limits calculated were adequate with respect to present legislation.The method was validated at four concentration levels (2, 5 10 and 20 mg kg�1). Global internal reproducibilitywas in the range 5.6–21.4% (RSDR) depending on the compound and concentration. Mean recoveries for alllevels varied between 89 and 100%, depending on the amine. A total of 39 samples of cooking utensils wereanalyzed using the described method and the results obtained after the third migration test were not compliantin approximately half of the samples.

Keywords: chromatography, LC/MS; method validation; food contact materials; food simulants; migration

Introduction

Food contact materials are all materials and articlesintended to come into contact with foodstuffs, includ-ing packaging materials but also cutlery, dishes,processing machines, containers, etc. In recent years,the use of plastic utensils, such as turners, whisks and

spoons, for cooking and frying has increased becausethey are cheap, unbreakable (resistant to high tem-peratures) and do not scratch other surfaces.

Most of these utensils are made of nylon, thegeneric name for polyamides which have characteristicamide groups in the backbone chain. These amidegroups are very polar and can interact with each otherby hydrogen bonding. Therefore, and because thepolyamide backbone is regular and symmetrical,nylons are often crystalline and make excellent fibers.

There are two classes of synthetic nylons. One isformed from cyclic monomers; for example, nylon 6,which has six carbon atoms per repeat unit andis normally manufactured from the monomer"-caprolactam. Another example in this class is nylon

12, having 12 atoms in the repeat unit and made fromthe lactam of 12-amino dodecanoic acid. The singleindex used in describing these nylons indicates thenumber of carbon atoms in the repeat unit. The second

class of synthetic nylon is formed from various

diamines and diacids, and their nomenclature involves

two indices indicating the number of carbon atoms in

the diamine and diacid units, respectively, e.g. nylon

6,6, made from hexamethylene diamine and adipic acid

(Gupta 1989). These materials could incorporate

azodyes (synthetic organic colorants with characteristic

chromophoric azo groups), where primary aromatic

amines (PAAs) are the starting substances. Moreover,

pigments and dyes may contain aromatic amines as

technical impurities.Another source of PAAs could be the aromatic

isocyanates used as monomers in the production of

polyurethanes. Polyurethanes are used as adhesivesin laminated films but also in printing inks and

lacquers. If the ratio between the monomers is wellbalanced and the reaction is complete, all monomers

disappear to form the polymeric network. However,

if free isocyanates are present and reach the food, thiscan lead to the formation of PAAs.

In brief, PAAs can occur in food contact articles as

residual monomers, as hydrolysis products of isocya-

nates or as contaminants of azodyes. They maymigrate or be chemically formed in foodstuffs coming

into contact with these articles. Furthermore, if these

*Corresponding author. Email: [email protected]

ISSN 0265–203X print/ISSN 1464–5122 online

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DOI: 10.1080/02652030903225781

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food contact materials are badly manufactured ormade with inappropriate raw material, chemicalsubstances can migrate from the articles to thefood and thus contribute to food contamination.Consequently, PAAs have been implicated in severalalerts related to the migration from kitchen utensilsmade of nylon imported mainly from China(RASFF 2008).

Some PAAs are classified as ‘‘possibly carcinogenicto humans’’ by the International Agency for Researchon Cancer and, thus, their presence in foodstuffsshould be avoided (Brede et al. 2003). EuropeanLegislation stipulates that the levels of PAAs infood or in food simulant should not be detectable(i.e. 0.01mg kg�1 of food or food stimulant; form-PDA, the specified level is 0.02mgkg�1 analyticaltolerance included) (EC 2007). Thus, the EuropeanCommittee for Standardization has proposed a pho-tometric method (based on diazotisation of free PAA)for screening purposes only; if PAAs levels are above2 mg aniline equivalents kg�1, a confirmation methodis required (Mortensen et al. 2005).

The published literature shows that a number ofdifferent analytical methods have been employed fordetection of PAAs, although not necessarily appliedto food contact materials. Aniline was analyzed byBornick et al. (2001) using solid-phase extraction andliquid chromatography (LC), while Reddy-Nooneet al. (2007) determined aniline and other aromaticamines as their iodo-derivatives by liquid-phasemicroextraction and gas chromatography GC).LC/MS was applied by Sakai et al. (2002) to determinetoluenediamine, and LC–MS/MS by Sutthivaiyakitet al. (2005) to analyze 2,4-toluendiamine, 4,40-diami-nodiphenylether 4,40-methylenedianiline and 3,30-dimethylbenzidine. With respect to food contactmaterials, Brede et al. (2003) applied solid-phaseanalytical derivatization followed by GC/MS for thedetermination of PAAs migration in a water foodsimulant. LC with ultraviolet detection was used in theanalysis of aniline in plastic materials subjected torepeated testing (Brede et al. 2003; Brede and Skjeurak2004) and for the determination of migration of eightPAAs in laminates (Ellendt et al. 2003).

Previous studies has demonstrated the potential ofelectrospray ionization MS/MS for the direct analysisof methylene-dianiline (MDA) and 2,4-toluenediamine(TDA) in some food simulants (Palibroda et al.2004), and subsequent developments have enhancedthe capabilities of LC methods coupled to MS.Mortensen et al. (2005) developed an LC–MS/MSmethod to determine the migration of several PAAs inaqueous food simulants. A recent Food StandardsAgency project (Burch and Cooper 2008) describesanother LC-MS/MS method and its applicabilityto the quantification of PAAs in food simulants,foodstuffs and in the evaluation of migration models.

The aim of this study was to develop a simpleLC-MS/MS method for the determination of eightprimary aromatic amines and to validate the methodin the aqueous food simulant 3% acetic acid (w/v).This simulant is considered to be the most aggressivefor nylon. The selected amines were: m-phenylenedia-mine (m-PDA), 2,6- and 2,4-toluenediamine (2,6-TDA, 2,4-TDA), 1,5-diaminonaphthalene (1,5-DAN),aniline (ANL), 4,40-diaminonaphenylether (4,40-DPE),4,40-methylenedianiline (4,40-MDA) and 3,30-dimethyl-benzidine (3,30-DMB). Method validation shoulddemonstrate that it is fit for purpose, taking intoconsideration the restrictions or specific migrationlimit (SML) established in Directive 2002/72/EC andamendments (EC 2002), which, as mentioned pre-viously for all amines selected, was not detectable.Furthermore, and in view of the numerous alerts overthe last few years (RASFF 2008), the developedmethod was applied to a number of actual samples toevaluate the potential migration of these PAAs fromnylon cooking utensils.

Materials and methods

Chemicals and reagents

Methanol was LC–MS Chromasolv grade, supplied byRiedel-de Haen (Seelze, Germany) and ultrapure waterwas prepared using a Milli-Q filter system (Millipore,Bedford, MA, USA). Standards of m-phenylenedia-mine [m-PDA; CAS Registry No. 108-45-2, (99.4%)],4,40 methylenedianiline [4,40-MDA; CAS RegistryNo. 101-77-9, (99.2%)], 1,5 diaminonaphthalene [1,5-DAN; CAS Registry No. 2243-62-1, (99.6%)], and 3,30

dimethylbenzidine [3,30-DMB; CAS Registry No. 119-93-7, (�98%)] were purchased from Fluka Chemie AG(Buchs, Switzerland); 2,6 toluenediamine [2,6-TDA;CAS Registry No. 823-40-5, (99.9%)], 2,4 toluendia-mine [2,4-TDA, CAS Registry No. 95-80-7, 99.8%)],and 4,40 diaminophenylether [4,40-DPE, CAS RegistryNo. 101-80-4, (99.9%)], were purchased from Riedel-de Haen (Seelze, Germany), and aniline hydrochloride[ANL, CAS Registry No. 142-04-1, (99.6%)] was fromSigma (Schnelldorf, Germany).

Glacial acetic acid was purchased to Probus(Barcelona, Spain), and ammonium hydroxide(solution �25% in H2O) was from Fluka ChemieAG (Buchs, Switzerland). PTFE filters (0.2 mm inter-nal diameter) used were from Waters (Milford,MA, USA).

PAAs individual stock solutions containing500mg l�1 were prepared in methanol. These solutionswere kept protected from light in the refrigeratorfor up to six months. Mixed intermediate standardsolutions in methanol and 3% acetic acid (w/v)were prepared by dissolving appropriate amountsof each solution to yield concentrations of 5

108 R. Sendon et al.


and 0.1mg l�1, respectively. The 5mg l�1 mix solutionwas prepared monthly while the 0.1mg l�1 solutionwas prepared weekly. Calibration solutions in 3%acetic acid (w/v) in the range 2–20 mg l�1 were prepareddaily. All solutions were kept at 4�C and protectedfrom light.

Equipment and chromatographic conditions

The LC–MS/MS system comprised an Alliance 2795liquid chromatograph coupled to a Micromass QuattroPremier mass detector controlled by Mass Lynxsoftware. Chromatographic separation was performedwith an XTerra RP18 column (150� 4.6mm, 5 mmparticle size) at 22�C, all from Waters, (Milford, MA,USA).

The mobile phase was ammonium acetate 10mM/methanol (95 : 5, v/v) in isocratic mode for 0.8min,followed by a gradient to 25% methanol for 2min andanother gradient to 67% methanol for 40min andfinally isocratic elution for 7min. The flow-rate was

0.6ml min�1 and the injection volume was 50 ml.MS/MS detector settings: positive electrospray ioniza-tion (ESI) mode, probe temperature 400�C, ionizationsource temperature 120�C, electron multiplier voltage660V, desolvation gas nitrogen at 700 l h�1, conegas nitrogen at 50 l h�1 and collision gas argon at0.6mlmin�1. The acquisition was in multiple reactionmonitoring (MRM). See Table 1 for cone voltages andcollision energies.

Infrared spectrometry (Fourier transformed IR)

For all samples, FTIR was performed on a Perkin-Elmer (Boston, MA, USA) Spectrum One apparatusrunning under Spectrum� (v. 6.1.0) software andequipped with an universal attenuated total reflectance(UATR). ATR spectra were measured with a resolu-tion of 4 cm�1 in the range 4000–650 cm�1. Eachspectrum was the result of 16 co-added scans. Foridentification purposes, sample spectra were comparedagainst IR libraries (KnowItAll Informatics System,

Table 1. Experimental data for quantification.

Name Abbreviation CAS No. StructureQuantificationMRM traces

Cone(V)

Collision(eV)

m-Phenylenediamine m-PDA 108-45-2 108.7! 91.7 20 15

2,6-Toluenediamine 2,6-TDA 823-40-5 122.8!107.8 20 18

2,4-Toluenediamine 2,4-TDA 95-80-7 122.8!107.8 20 18

1,5-Diaminonaphthalene 1,5-DAN 2243-62-1 158.8!142.8 25 20

Aniline ANL 62-53-3 93.8!76.8 20 18

4,40-Diaminodiphenylether 4,40-DPE 101-80-4 201.1!107.8 20 20

4,40-Methylenedianiline 4,40-MDA 101-77-9 199.2!105.9 25 25

3,30-Dimethylbenzidine 3,30-DMB 119-93-7 213.2!196.1 25 25

Food Additives and Contaminants 109


IR edition, v. 7.9 and HaveItAll� IR databases, allfrom Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Samples and method procedure

Different cooking utensils were bought in several localsupermarkets. A brief description is given in Table 2,in which all information displayed was obtained fromthe label (if included) or from the utensil itself. Thesamples were bought in two different periods: The firstbatch was acquired at the end of 2005 (Samples 1–22)and the second in the middle of 2007 (Samples 24–41).Migration experiments were carried out in the sameway for all samples. PAA migration was determinedby immersing the cooking utensils in 3% acetic acid(w/v). Each sample was placed in a beaker or flask andfilled with a given volume of simulant, covered withaluminum foil and transferred to a preheated oven.

The aqueous simulant was previously heated and thenput into contact with the sample, ensuring that thesample surface intended for food contact wasimmersed in the simulant (including 2 cm of handlein some cases). The contact area was estimated bygeometric calculation. Migration conditions were 2 hat 100�C following those established in Directive 97/48/EC (EC 1997) and assuming a 45 and 530mincontact time in the worst case scenario. Taking intoconsideration that these articles are intended forrepetitive uses, three migration experiments wereperformed using new simulant each time, and at least24 h were allowed to pass between each exposure.

For LC analysis, an aliquot of the migrationsolution was neutralized to pH 6–7 with ammonia,filtered through a 0.2 mm PTFE syringe filter andinjected into the chromatograph. In all cases, no clean-up was applied.

Table 2. Description of analyzed cooking utensils.

Sample Utensil Specifications Food symbol Country of origin

1 Slotted turner Black nylon Yes Portugal2 Slotted turner Heat resistance 210�C No –3 Soup Spoon Without specification Yes RPC (China)4 Ladle Nylon. Heat resistance 210�C Yes Spain5 Slotted Spatula Without specification No Spain6 Ladle Nylon. Heat resistance 210�C Yes Spain7 Ladle Nylon. Heat resistance 210�C Yes Spain8 Slotted turner Without specification No Spain9 Sauce spoon Black nylon No RPC (China)10 Soup Spoon Polymer with coating. Heat resistance 210�C No Spain11 Ladle Without specification No China12 Ladle Without specification No China13 Serving Tong 25 cm Nylon-INOX 18/8 Yes China14 Skimmer Nylon. Heat resistance 220�C Yes China15 Ladle Nylon No Spain16 duplicate of 1 Slotted turner Black nylon Yes Portugal17 duplicate of 2 Slotted turner Heat resistance 210�C No –18 duplicate of 3 Soup Spoon Without specification Yes RPC (China)19 duplicate of 4 Ladle Nylon. Heat resistance 210�C Yes Spain21 Ladle Nylon Yes China22 Soup Spoon Nylon Yes China24 Soup Spoon Heat resistance 220�C Yes China25 Ladle Heat resistance 220�C Yes China26 duplicate of 25 Ladle Heat resistance 220�C Yes China27* Fork PAþGF. Heat resistance 220�C Yes Sweden28* Spoon PAþGF. Heat resistance 220�C Yes Sweden29* Slotted turner PAþGF Heat resistance 220�C Yes Sweden30 Ladle PA66þGF Yes Spain31 duplicate of 30 Ladle PA66+GF Yes Spain32 Ladle Inox-nylon Yes Spain33 Soup Spoon Nylon. Heat resistance 210�C Yes China34 Sauce spoon Nylon. Heat resistance 210�C No China35 Ladle Nylon. Heat resistance 200�C Yes Slovenia36 Sauce spoon Nylon. Heat resistance 220�C Yes Spain37 Serving fork Nylon No RPC (China)38 Spatula Nylon No Spain39 Skimmer Nylon. Heat resistance 200�C Yes Spain40 Slotted turner Nylon. Heat resistance 200�C Yes Spain41 Ladle Nylon Yes –

Note: *Set of three different items.



Validation experiments

Calibration was performed by external calibrationusing a series of standards of known concentrationsprepared in fresh simulant, neutralized in the sameway as samples and injected in duplicate or triplicate.Each calibration curve was constructed at four cali-bration levels ranging from 2 to 20 mg l�1 and assessedby linear regression. The curves were prepared in threenon-consecutive days, spread over 20 days. Residualplots were examined to detect obvious patterns and anANOVA lack-of-fit test was used to assess linearity.Other parameters estimated were the correlationcoefficient and linearity, calculated according to thefollowing equation:

Cm% ¼ 100� 1�Sb

b

� ��

where Sb is the standard deviation of the slope and bis the slope of the calibration curve.

Detection limits (DLs) were determined in accor-dance with the guidelines of the American ChemicalSociety (MacDougall and Crummett 1980), analyzingsolutions of decreasing concentration until the mini-mum concentration required to generate a signal-to-noise ratio of 3 was achieved. Trueness and precisionof the method were determined by running spikingexperiments on migration solutions with each batch ofsamples. Data from 12 days, over a period of threeand a half months, were collected for validation. Thenumber of within-day replicates ranged from two to10. Four fortification levels (2, 5, 10 and 20 mg kg�1)were assayed using several migration solutions fromdifferent samples. Between eight and 22 data sets,depending on the amine and concentration, were takenfor validation. Precision (internal reproducibility) andtrueness are expressed in terms of relative standarddeviation and recovery for each level and amine,respectively.

Selectivity was based on a comparison of thechromatograms obtained for PAAs standard solutions,PAAs-free migration solutions from nylon utensilsand spiked migration solutions. Finally, the stability ofPAAs in the food simulant was determined by analysisof solutions of 3% acetic acid (w/v) spiked with acombination of PAAs (n¼ 5 replicates) followed bystorage for 2 h at 100�C and 2 h at 70�C. The meanresults in each case were compared with those obtainedfor the same spiked solutions not subjected to storageconditions (control solutions).

Results and discussion

Mass spectrometry

To obtain more characteristic ions (parent and daugh-ter ions; Table 1), individual standard solutions of eachPAA (5mgml�1) were infused directly into the mass

spectrometer using a built-in syringe pump andacquired in full scan mode (range m/z 60–300). Conevoltages were adjusted to the maximum sensitivity foreach parent ion. ESI source parameters were alsooptimized, i.e. probe temperature, ionization sourcetemperature, electron multiplier voltage, desolvationand cone gas. Once the best conditions for the parentions were selected, daughter ion scans were acquired.Several collision voltages were tested and those yield-ing the most intense and stable fragments were chosen.In this way, the cone and collision voltages were fixedto obtain the highest signal/noise ratio for eachtransition.

The multiple reaction monitoring transitions(MRM) selected for each PAA did not differ fromthose reported in the literature (Mortensen et al. 2005;Burch and Cooper 2008). In all cases, the moreintensive parent ion corresponded to the protonatedmolecular ion [MþH]þ, and no other ions of signif-icant intensities were observed. For each PAA, twodifferent daughter ions were selected; one for quanti-fication purposes (Table 1) and the second for confir-mation only. For m-PDA, ANL and 3,3-DMB, thequantifier daughter ion corresponded to the loss of theprimary amine group; 2,6 and 2,4-TDA lost the methylgroup [MþH�CH3]

þ; 4,40-DPE and 4,40-MDAyielded the [MþH�C6H7N]þ ion, i.e. lost one anilinegroup. 1,5-DAN was different; its ionization yielded[MþH]þ, but an intense [M.]þ was also observedwhen the daughter ion scan was acquired (Figure 1),and the same occurred when the molecule lost oneof its primary amine group.

The specificity of the transitions used for quanti-fication was examined by analyzing individual stan-dard solution of each PAA. Thus, not all transitionswere used at all times as this greatly influencedsensitivity; therefore, ions selected as qualifiers wereonly monitored in those samples that exhibited resultsover the restriction limit. For 2,6 and 2,4-TDA, thequalifier transition was 122.8! 105.8, which corre-sponds to the [MþH�NH3]

þ ion; for ANL it was93.8! 50.7, i.e. [MþH�C2H5N]þ due to rupture ofthe aromatic ring; for 4,40-MDA it was 199.2! 76.8(complete break down of the molecule forming thearomatic rings) and for 3,30-DMB it was213.2! 181.1, that is [MþH�CH3�NH3]

þ. In allcases, higher collision voltages were required thanthose needed for the quantification transitions.

Chromatography

Initially, a previous validated method (Mortensen et al.2005) was checked for in-house validation to analyzethe samples, but unsatisfactory results were obtainedfor some amines. The separation of 2,6 and 2,4-TDAwas inadequate when simulant 3% acid acetic (w/v)



was used and the sensitivity of 3,30-DMB was alsocompromised. After several trial runs with different

combinations of mobile phase/column, ammonium

acetate 10mM/methanol using a C18 column wereselected as they produced better peak resolutions for all

PAAs. The gradient used was based on the proposed

method for the LC–UV determination of these PAAs(Brauer and Funke 2002), but some adjustments were

made to make it suitable for the column used in the

MS/MS method. Figure 2 shows the chromatogramsobtained. Despite the possibility of a scheduled acqui-

sition for each individual PAA, the acquisition of all

min2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0

%

0

100

MRM of 9 channels,ES+213.16 > 196.06

1.701e+004

PAAs07_847

PAA 8 (3,3’ DMB)25.86

min

%

0

100


1.344e+005

PAAs07_847 PAA 7 (4,4’ MDA)21.10

min

%

0

100


1.168e+005

PAAs07_847 PAA 6 (4,4’ DPE)16.53

min

%

0

100


4.347e+002

PAAs07_847 PAA 5 (Aniline)9.40

7.876.333.270.83

10.9717.33

15.4020.30 33.5322.33

23.8629.36

26.43

min

%

0

100


4.469e+004

PAAs07_847 PAA4 (1,5 diaminonaph)9.77

min

%

-1

99


6.607e+004

PAAs07_847 PAA 3 (2,4-TDA)6.93

6.00

min

%

-1

99


6.607e+004

PAAs07_847

6.93PAA 2 (2,6-TDA)6.00

min

%

-1

99

MRM of 9 channels,ES+108.7 > 91.72.935e+004

PAAs07_847 PAA 1 (m-PDA)5.40

4.33

min5.0 10.0 15.0 20.0 25.0 30.0 35.0

%

0

100


3.102e+002

PAAs07_846

min

%

0

100


7.464e+002

PAAs07_846

min

%

0

100


3.830e+002

PAAs07_846

min

%

0

100


1.868e+002

PAAs07_846

min

%

0

100


3.813e+002

PAAs07_846

min

%

0

100


4.907e+002

PAAs07_846

min

%

0

100


4.907e+002

PAAs07_846

min

%

0

100

MRM of 9 channels,ES+108.7 > 91.71.460e+003

PAAs07_846

(b)(a)

Figure 2. Chromatogram of third migration extract of sample 16. (a) Spiked with the PAA mix (10mg l�1) showing thequantification traces used. (b) Without spiking.

m/z50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

%

0

100 159143

142

11599

55

93

132

117

158

[M+H]+

[M•]+

[M+H-NH3]+

[M•-NH3]+

Figure 1. Daughter scan of 1,5-DAN (m/z159).



compounds in a complete run-time was preferred aspreliminary experiments on some samples exhibitedunknown peaks with the transitions selected but atdifferent retention time of the respective PAA. It maybe possible to identify other migrating compounds orthose formed in the simulant, but this was not withinthe scope of the present study. Nevertheless, it is asubject of further research in this field.

Method validation

Linear regression was applied to the calibration curvesobtained on three different days. Examination ofresidual plots showed no obvious pattern. Residualswere normally distributed when tested by the Shapiro–Wilks normality test (p4 0.05). ANOVA test showed ahighly significant regression (p5 0.001) and the lack offit was not significant (p4 0.05) for all amines in allthe curves, which demonstrates the validity of thelinear model at the working range. In addition, thecorrelation coefficients of the calibration curves werealways 40.994 and linearity, calculated according tothe equation given above, was 496%.

Detection limits for the individual PAAs are givenin Table 3. DLs were calculated as the amount ofanalyte present in the food simulant (mg kg�1), but theyalso refer to the cooking utensil surface. Due to thedifferent shapes of cooking utensils, each sample had aspecific contact-surface/simulant-volume ratio. Thus,in two opposing situations, these DLs were calculated.The lowest ratio was represented by a utensil with lowcontact surface (a slotted turner, 1.114 dm2) but forwhich a high volume of simulant was needed (425ml)to ensure that all contact surface was tested. On theother hand, the highest ratio was represented by autensil with a medium surface (a sauce spoon,1.3781 dm2), but for which less simulant was required(134ml). In Table 3, DLs were also calculated using thestandard assumption of 6 dm2 in contact with 1 kg of

food/food simulant (ratio 6 dm2 l�1). In all cases, the

detection limits calculated were adequate regarding

the present legislation. Previously published studies

also recorded these levels, but only in one case was

no pre-concentration step required (Mortensen et al.

2005). The present work achieves lower DLs for all

amines selected (except for ANL), because less

compounds are monitored at the same time, thus

increasing sensitivity. Brede et al. (2003) obtained

lower DLs (5–10 times lower) employing GC–SPE

derivatization or with LC–UV–SPE (Brauer and

Funke 2002), but these techniques are costly and

time-consuming.It has been noted (Mortensen et al. 2005) that once

the individual determination of PAAs has been

facilitated, expressing the DLs as ANL equivalents is

inappropriate due to differences in the molecular

weight of amines. The fourth amendment of

Directive 2002/72/EC, Directive 2007/19/EC

(EC 2007), setting restrictions for PAAs no longer

refers to aniline equivalents.Global internal reproducibility, including all con-

centration levels and all amines (Table 4) was in the

range 5.6–21.4% (RSDR) depending on the compound

and concentration. These values are appropriate since

Table 3. Detection limits.

PAAs

mg l�1 mg dm�2

Standardsolution

Standard ratio6 dm2 kg�1

Lowest ratio2.62 dm2 kg�1

Highest ratio(10.28 dm2 kg�1)

m-PDA 0.5 0.083 0.191 0.0492,6-TDA 0.5 0.083 0.191 0.0492,4-TDA 0.5 0.083 0.191 0.0491,5-DAN 1.0 0.167 0.382 0.097ANL 1.0 0.167 0.382 0.0974,40-DPE 0.5 0.083 0.191 0.0494,40-MDA 0.5 0.083 0.191 0.0493,30-DMB 0.5 0.083 0.191 0.049TOTAL* 4.5 0.750 1.718 0.438

Note: *Total expresses the sum of all amines shown except m-PDA (i.e. it has a separate SML).

Table 4. Precision expressed as internal reproducibility(RSDR%) of the method in migration solutions from nyloncooking utensils.

PAAs 2 ngml�1 5 ngml�1 10 ngml�1 20 ngml�1

m-PDA 12.3 7.9 9.4 9.32,6-TDA 13.6 9.7 11.9 10.32,4-TDA 21.4 11.3 16.5 9.71,5-DAN 18.4 9.0 11.6 12.1ANL 9.8 20.0 11.3 20.34,40-DPE 12.2 6.8 10.3 5.64,40-MDA 8.4 7.4 12.8 9.93,30-DMB 15.2 8.9 13.1 13.1



they are below the value estimated by the Horwitz

function modified by Thompson (Thompson 2000)

i.e. RSD 22% for concentrations below 1.2 10�7.

Mean recoveries were good for all the amines and at

all concentrations, ranging between 93 and 100%

(including all levels), and are also according to CEN

TS 15356 (CEN 2006). The lowest validation levelguarantees the method utility for routine analysis

regarding compliance or not with Directive 2002/72/

EC (EC 2002). The lowest validation level guarantees

the method utility for routine analysis regarding

compliance or not with Directive 2002/72/EC

(EC 2002).No certified reference material is available for

the determination of PAAs migrating from cooking

utensils. To prove the validity and also to support the

trueness of the method, the laboratory participated in a

proficiency test organized by FAPAS�. The test

sample was 3% acetic acid containing 2,4-TDA,

ANL and 4,4-MDA with assigned values of 1.02,

16.0 and 2.00mg aniline equivalents kg�1 simulant,respectively. Thus, the trueness of the method was

assessed regarding these compounds, with z-score,

according to FAPAS protocol (FAPAS 2002), of 0.1

for 2,4-TDA, 0.3 for ANL and �0.3 for 4,4-MDA.

These z-scores confirm the method suitability for the

proposed objectives.Regarding selectivity, no relevant interferences

were detected from analysis of blank migration

solutions (3% acetic acid) obtained from nylon

cooking utensils. Representative chromatograms of

PAAs standards and a blank migration solution are

given in Figure 2. The stability of PAAs under two

possible migration conditions for nylon utensils wasstudied for the eight PAAs included in the method.

These conditions were 2 h at 100�C and 2 h at 70�C

in 3% acetic acid (w/v). The mean results (n¼ 5

replicates) in each case were compared with the control

solutions (not subjected to storage conditions). For all

amines tested, the recoveries found after 2 h/100�C

were �83%, except for 1,5-DAN (6%). Statisticalevaluation of the results (t-test, 95% confidence level)

showed no difference between means before and after

2 h at 100�C for ANL, 4,4-DPE, 4,4-MDA and

3,3DMB. After 2 h/70�C, recoveries were �92%,

except for 1,5-DAN (65%). Statistical evaluation of

the results (t-test, 95% confidence level) showed nodifference between means before and after 2 h at 70�C

for 2,4-TDA � ANL, 4,4-DPE, 4,4-MDA or 3,3-DMB.

Previous work reported that most of the PAAs were

stable to heating (121�C for 2 h) in water, 3% acetic

acid and 10% ethanol, but not in olive oil (Burch and

Cooper 2008), except for 1,5-DAN. After heating

and storage (121�C plus 10 days, 40�C) only the earlyeluting PAAs (m-PDA, 2,6- and 2,4-TDA) were poorly

recovered, which agrees with our results.

Sample analysis

To the best of our knowledge, only a couple of studieshave investigated the migration of PAAs from cookingutensils (Mortensen et al. 2005; Perharic et al. 2006),while various national enforcement campaigns onPAAs have detected several cases of non-compliancewith EU limits. RASFF (2008) has reported a numberof food alerts over the last 4 years (2005: 22notifications; 2006: 30 notifications; 2007: 23 notifica-tions; 2008: 28 notifications). Thus, several sampleswere purchased from different retail stores in Madrid(Spain). The first batch was obtained at the beginningof method development; the second at the end,complementing the first selection.

Initially, and before migration experiments werecarried out, the contact area of each utensil wascalculated and IR spectra were also obtained for allsamples. IR spectroscopy is the most direct way toidentify polymers; when using the UATR accessory nosample preparation is needed. FTIR spectra werecollected by simply placing the cooking utensil overthe diamond and lowering the compression arm. Abackground spectrum of air was recorded each time.The results were compared with those in commercialdatabase libraries (BioRad�), and showed that themajority of samples were made of polyamide 6 (77% ofsamples tested) or polyamide 66 (23% of samples). Thestrongest peaks in the IR spectra were those corre-sponding to the amide I and amide II bands found at1630 (C¼O stretch) and 1530 cm�1 (C�N stretch andCO�N�H bend), respectively. Also, the N�H stretchin 3300 cm�1 was very intense (Enlow et al. 2005).

The next step was to carry out migration experi-ments. As mentioned in Materials and methodssection, samples were exposed for 2 h at 100�C to theaqueous food simulant 3% acetic acid. Mortensenet al. (2005) also used these conditions. Owing to thehydrophilic nature of these compounds and the type ofpolymer, 3% acetic acid was selected for migrationexperiments as the worst case scenario regarding PAAmigration. Migration in olive oil has been reported(Brede and Skjeurak 2004; Burch and Cooper 2008) asbeing difficult. Lower migration values were obtainedcompared with those in aqueous simulants, perhapsdue to degradation of PAAs or reaction with oilcomponents.

The utensils were intended for cooking and, thus,to be used with hot food. Furthermore, some of themare labeled ‘‘heat resistance 210/220�C’’. EU legislation(EEC 1982) has established a contact temperature of100�C for aqueous food simulants, when the actualcontact temperature is �100�C. The migration timeselected was based on the assumption that actualcontact time (worst case scenario) would be 30min;therefore, and according to established principles, themigration time should be 4� 30min, i.e. 2 h.



Moreover, as nylon utensils are intended forrepeated use, for compliance purposes only the resultof the third migration was taking into account. Theresults are summarized in Table 5. In some samples,very high levels of PAAs were found and dilution ofthe migration solutions was necessary prior to analysis.A total of 39 samples were analyzed and 54% did notcomply with current legislation. Furthermore, from thefirst to third migration, samples 11, 18, 19, 36, 37, 38,40 and 41 imparted a brown color to the simulant.These samples corresponded to those exhibiting highermigration values. The two main compounds identifiedwere 4,40-MDA and ANL, as reported previously(Brede and Skjeurak 2004; Mortensen et al. 2005;Perharic et al. 2006) and in several RASFF notifica-tions (RASFF 2008). In some samples, 2,4-TDA and3,3-DMB were also identified, but in lesser amounts

than 4,40-MDA and ANL. Higher values were alwaysattributed to 4,40-MDA. This compound is used in themanufacture of some types of polyamide, apparentlyas a comonomer, to increase the stability of the plasticat high temperatures. It has also been suggested that4,40-MDA is used to produce the azodyes that give theutensils their black color (Petersen et al. 2004). Slightdifferences were found regarding the number ofsamples that exceeded the SML, depending on theyear of purchase. Samples acquired during 2005 were57% non-compliant, while those acquired in 2007recorded a 50% non-compliance.

Of all samples giving PAAs values above therestriction limit, only one was identified by IR asPA66 (sample 5), which represented 11% of all samplesidentified as PA66 versus 67% of samples identified asPA6. Samples labeled as ‘‘duplicated’’ in Table 2 were

Table 5. Results of migration experiments. Data shown correspond to the third exposure.All results are expressed as the sum of all compounds identified.

Sample UtensilContact

surface (dm2)Ratio

(dm2 l�1)Migration(mg dm�2) PAAs identified

1 Slotted turner 1.3 3.752 Slotted turner 1.1 2.62 909 4 40-MDA & ANL3 Soup spoon 2.4 5.49 559 4 40-MDA & ANL4 Ladle 1.5 3.36 230 4 40-MDA & ANL5 Slotted spatula 1.6 4.26 74.3 4 40-MDA6 Ladle 1.2 2.98 2.0 ANL7 Ladle 1.5 3.288 Slotted turner 1.5 3.989 Sauce spoon 1.5 4.32 107 4 40-MDA & ANL

10 Soup spoon 1.9 3.7111 Ladle 1.5 3.44 2470 4,40-MDA & ANL12 Ladle 1.8 3.5313 Serving tong 0.9 6.4214 Skimmer 2.1 5.5515 Ladle 1.4 3.1716 Slotted turner 1.3 3.5017 Slotted turner 1.1 2.68 751 4 40-MDA & ANL18 Soup spoon 2.4 5.25 926 4 40-MDA & ANL19 Ladle 1.4 4.33 315 4 40-MDA & ANL21 Ladle 1.6 3.55 7.6 ANL & 4,40-MDA22 Soup spoon 2.7 7.10 140 4 40-MDA & ANL24 Soup spoon 2.6 8.9825 Ladle 2.1 7.2626 Ladle 2.1 7.1427 Fork 0.8 3.28 68.1 ANL28 Spoon 0.9 3.46 105 ANL29 Slotted turner 0.9 3.92 80.9 ANL30 Ladle 1.6 4.7931 Ladle 1.6 5.6632 Ladle 1.5 5.6733 Soup spoon 2.6 6.9334 Sauce spoon 1.4 8.7235 Ladle 1.4 3.8936 Sauce spoon 1.4 10.28 329 4,40-MDA & ANL37 Serving fork 0.7 4.70 369 4,40-MDA & ANL38 Spatula 2.1 6.38 798 4,40-MDA & ANL39 Skimmer 2.4 4.11 179 4,40-MDA & ANL40 Slotted turner 1.1 2.90 860 4,40-MDA & ANL41 Ladle 1.3 4.70 562 4,40-MDA & ANL



those bought at the same time, in the same store,sometimes sold as a set and apparently identical, butno information was available as regards batch numberconfirming that the two samples came from the samelot. Duplicate samples gave quite different results,e.g. samples 3 and 18. This suggests a lack ofhomogeneity in these types of materials.

Although only the result of the third exposure wastaking into account for comparison with legal limits,the first and second migration solutions were alsoanalyzed, as shown in Figure 3. For sample 11, theresult for the first migration is not completely showndue to the high value (6416mg dm�2). In most cases,migration levels decreased between the first and secondmigration; but, for samples 17 and 22, the levels in thesecond migration were slightly higher. A decrease inPAA migration was observed in most samples (73%)between the second and third migration; however, thiswas not the case for samples 4, 11, 18, 19, 37 and 39,and, in particular, samples 18 and 37, where theincrease was especially high.

It is worth noting, that in 20 of the 22 samplesexceeding the restriction limit, the values were sig-nificantly above the limit. Therefore, it is expected thatthese utensils will be non-compliant after severalexposures. Mortensen et al. (2005) reported that,after simulating 1–2 years of household use, samplescontinued to release PAA.

Conclusions

A simple LC–MS/MS method capable of directlyanalyzing (without clean-up) a food simulant has

been developed and the low validation level achievedguarantees the method suitability for routine analysisand monitoring for compliance with EU Directive2002/72/EC. Several unknown compounds have alsobeen detected that could be related to PAAs; thus,further investigation is needed in this field. Mostnotifications relating to cooking utensils concernedproducts originating from third-world countries. Theproposed method has proved to be adequate for testingcompliance with European Union legislation. Thoughthe first RASFF notification concerning PAAs migra-tion from cooking utensils was in 2004, the issueis unresolved, as demonstrated by the fact that, insubsequent years, the number of alerts did notdecrease: 22 notifications in 2005, 30 in 2006, 23 in2007, 28 in 2008 and 15 notifications during the firstsweeks of 2009.

Acknowledgements

The contents of this paper are the responsibility of theauthors alone and should not be taken to representthe opinions of the supporting organizations. Authors aregrateful to the ‘‘Angeles Alvarino’’ Program financed by‘‘Consellerıa de Innovacion e Industria, Xunta de Galicia’’for the postdoctoral contract of R. Sendon. The authors aregrateful to Carmen Tejedor Roman and Libertad GarcıaMartın for their excellent technical assistance.

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