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Multi-analyte methods for the detection of photoinitiators and amine synergists in food contact materials and foodstuffs – Part I: HPLC-DAD screening of materials ab

T. Jung , C. Browatzki a

ab

& T.J. Simat

b

CVUA Stuttgart, Fellbach, Germany

b

TU Dresden, Chair of Food Science and Food Contact Materials, Dresden, Germany Accepted author version posted online: 10 Jan 2014.Published online: 04 Mar 2014.

Click for updates To cite this article: T. Jung, C. Browatzki & T.J. Simat (2014) Multi-analyte methods for the detection of photoinitiators and amine synergists in food contact materials and foodstuffs – Part I: HPLC-DAD screening of materials, Food Additives & Contaminants: Part A, 31:3, 512-536, DOI: 10.1080/19440049.2013.877600 To link to this article: http://dx.doi.org/10.1080/19440049.2013.877600

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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 3, 512–536, http://dx.doi.org/10.1080/19440049.2013.877600

Multi-analyte methods for the detection of photoinitiators and amine synergists in food contact materials and foodstuffs – Part I: HPLC-DAD screening of materials T. Junga,b, C. Browatzkia,b and T.J. Simatb* a

CVUA Stuttgart, Fellbach, Germany; bTU Dresden, Chair of Food Science and Food Contact Materials, Dresden, Germany

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(Received 28 May 2013; accepted 13 December 2013) The objective of this work was to develop a HPLC-DAD method suitable for the screening of food contact materials for a total of 63 monomeric and polymeric photoinitiators and amine synergists. Such multi-analyte methods are worthwhile for official control laboratories, where normally no information about the composition of the applied inks or varnishes on the printed or lacquered materials is available and thus target analyses are not feasible. The polymeric analytes were each separated in a multitude of substance peaks, which largely overlaid those of the other compounds. Thus, for 13 polymeric photoinitiators and amine synergists a hydrolysis method was developed that reduced the number of ultraviolet (UV) detectable peaks to only one. This allowed easier identification and – preliminary – semi-quantification of these polymeric substances with adequate limits of detection. The remaining 50 photoinitiators and amine synergists were combined in one HPLC-DAD method. But since many of these substances are structurally related, partly retention times and spectra did not differ significantly. Thus selectivity was enhanced by preparing a database containing all spectra and retention times of the investigated compounds. Furthermore, the retention times of those 50 substances were calculated relative to two internal standards to overcome variances of retention from run to run or due to matrix effects. The developed method was tested for the analysis of food contact materials. Extractions of these were performed with acetonitrile and partially the extracts were subsequently concentrated in a steam of nitrogen. Limits of detection of photoinitiators and amine synergists in concentrated packaging extracts were in the range between 0.02 and 5.5 µg dm−2. Keywords: UV printing inks and varnishes; food contact materials; photoinitiators; amine synergists; HPLC-DAD screening; polymeric photoinitiators; polymeric amine synergists

Introduction Ultraviolet (UV) inks and varnishes are used to print and lacquer all kinds of food contact materials as, for instance, cartonboard, rigid and flexible plastics, and multilayer materials such as beverage cartons. They consist usually of oligomeric acrylates on basis of polyesters, polyurethanes, polyethers and epoxy resins, low molecular weight acrylates, pigments and photoinitiators, among others. Under UV radiation photoinitiators start the polymerisation of the monomers and oligomers, thereby causing curing of the ink (Papilloud & Baudraz 2002; Yoo et al. 2004; Glöckner et al. 2008). Substances that may be transferred from materials printed with these kinds of inks to the foodstuffs are mainly photoinitiators, amine synergists, which are used together with some types of photoinitiators, and low molecular weight acrylates (Papilloud & Baudraz 2002; Yoo et al. 2004; Green 2010; BMELV 2011; Food Standards Agency 2011). Two of the best known contamination incidents of foodstuffs with photoinitiators from printed packaging materials are that of isopropylthioxanthone in 2005 and 4-methylbenzophenone in 2009. While the former was detected in beverages, e.g. baby milk and cloudy juices, the latter was found in breakfast cereals (EFSA 2005, 2009). Since then some *Corresponding author. Email: [email protected] © 2014 Taylor & Francis

efforts have been undertaken by the printing ink industry to avoid similar contaminations. One of the approaches was the development of so-called polymeric photoinitiators (PPI) and polymeric amine synergists (PAS). Here, the reactive monomeric photoinitiator or amine synergist, respectively, is bound to an oligomeric backbone, e.g. polyethylene glycol or hyperbranched polyglycerol (Chen et al. 2007; Jiang et al. 2009), to raise the molecular weight, and thus lowering the migration potential and toxicological relevance. Substances with a molecular mass above 1000 Daltons are very unlikely to be absorbed by the gastrointestinal tract when ingested with the food (EFSA 2008). The widespread detection of photoinitiators and amine synergists in packaged foodstuffs has led some research activities on this issue. Many analysis methods for photoinitiators and amine synergists using different techniques, e.g. GC-MS or MS/MS, HPLC coupled to a diode array (DAD) and/or a fluorescence detector (FLD) or a MS/MS, have been developed. Table 1 shows a selection for several types of food contact materials and foodstuffs with most diverse extraction procedures and subsequently cleaning and concentration steps. It can be seen that the trend is towards the development of multi-analyte methods

6 5 7 9

11

17

1, 77, 5, 10, 14

77, 10, 1, 5, 15, 26, 29

77, 12, 2-hydroxybenzophenone, 13, 19, 18, 6, 5, BPacr

9, 10, 11, 6, 5, 14, 77, 18, 3, 1, 41

77, 8, 12, 21, 18, 2-hydroxybenzophenone, 13, 19, 10, 1, 41, 11, 3, 15, 36, 5, 14

14

10, 11, 15, 9, 23, 1, 6, 14, 36, 77, 26, 40, 5, 29

10, 77, 11, 15, 1, 5

6

77, 10, 11, 5, 15, 1

2

3

7, 6, 22

1, 3

No.

Analytes

GC-MS

LC-MS/MS

HPLC-DAD, confirmation by GC-MS

LC-MS/MS

HPLC-DAD, confirmation by GC-MS GC-MS, confirmation by LC-MS/MS

HPLC-DAD/FLD

GC-MS

HPLC-DAD, confirmation by GC-MS; GC-MS for number 7 in food GC-MS

Method used

Paper, board and foodstuffs

Beverage cartons and foodstuffs

Packaging materials for milk and the milk Paperboard food packaging materials and foodstuffs

Beverage cartons and foodstuffs

Packaging materials suitable for migration tests in cells Intended for screening of packaging extracts Packaging materials (e.g. beverage cartons, plastic cups/foils) and foodstuffs Beverage cartons and foodstuffs

Paper, cartonboard and foodstuffs

Matrix

Acetonitrile; acetonitrile/ dichloromethane (1:1) for food

Dichloromethane; acetonitrile for food

Acetonitrile

Acetonitrile

Dichloromethane; hexane for food

Acetonitrile

Hexafluoro-2propanole; acetonitrile for food

Water, 3% (w/v) acetic acid, 95% (v/v) ethanol and iso-octane –

Ethanol containing 0.4% triethylamine

Extraction solvent

LOD: 200–300 µg kg−1 packaging; 20–30 µg l−1 food LOD: 0.4–2 ng dm−2 packaging; 0.2–1 µg l−1 food LOQ: < 4 µg dm−2; LOD: 0.05– 2.5 µg kg−1 food LOD: 1.8 µg dm−2 for numbers 77, 12 and 18 and 49 µg dm−2 for BPacr in packaging material LOQ: 0.06–30 µg l−1 solvent; 0.2– 666 µg kg−1 baby food LOQ: 0.25– 2.5 mg kg−1 packaging; analyte dependent in food

LOD for number 1: 0.25 µg dm−2; 0.4 µg kg−1 food

–

LOD: 0.05– 0.2 mg kg−1 packaging; 2 µg kg−1 for number 7 in food –

LOD or LOQ

Table 1. Selection of methods for the determination of photoinitiators and amine synergists in different food contact materials and foodstuffs.

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(continued )

Food Standards Agency (2011)

Gallart-Ayala et al. (2011)

Koivikko et al. (2010)

Shen et al. (2009)

Sagratini et al. (2008)

Sanches-Silva et al. (2008)

Rothenbacher et al. (2007)

Yoo et al. (2004)

Papilloud and Baudraz (2002)

Castle et al. (1997)

Reference

Food Additives & Contaminants: Part A 513

28

12

32, 10, 29, 2-(dimethylamino)ethylbenzoate, 5, 9, 33, 1, 15, 11, 3, 36, 18, 13, 12, 6, 7, 77, 39, 38, 26, 44, 40, 14, 46, 47, 19, 25

12, 9, 26, 58, 14, 5, 18, 52, 54, 46, 10, 40, 25, 29, 47, 77, 11, oxy-phenyl acetic acid 2-[2-oxo-2-phenylacetoxy-ethoxy]-ethyl ester, oxy-phenyl acetic acid 2-[2-hydroxyethoxy]-ethyl ester 15, 1, 41, 40, 19, 61, 53, 39, 38, 36, 59, 63, 60, 32, 23, 56, 44, 55, 57, 72, 74

GC-MS, GC-FID, LC-MS, HPLCDAD, HPLC-CAD, DART

LC-MS/MS

Method used

Cartonboard, beverage cartons, plastic laminates (PE/Al/PE) and containers for migration tests, migration simulants and foodstuffs

Various paper, board and plastic food packaging materials and therein packaged foodstuffs

Matrix LOQ food: ≤ 10 µg kg−1 or ≤ specific migration limit (combination of LC-MS/MS, LC-TOF-MS and GC-MS methods used)

Acetonitrile or hexafluoro-2propanole; food: acetonitrile or acetonitrile/citrate buffer, cleaning up by QuEChERS or Extrelut® columns; Tenax®, 50% (v/v) ethanol Chloroform, dioxane, iso-octane, 95% (v/v) ethanol, 3% (w/v) acetic acid, Tenax®; foodstuffs: tomato soup, orange juice and breakfast cereals (food extracted with acetonitrile or dichloromethane/ acetonitrile (1:1), Tenax® with diethyl ether)

LOD: ≤ 1.7 µg dm−2 for monomeric photoinitiators and amine synergists; 7 µg dm−2 for some polymeric ones, for others no adequate LOD achievable; LOD for food not given

LOD or LOQ

Extraction solvent

Lord et al. (2012)

BMELV (2011)

Reference

Note: The assignment of the numbers in the column “Analytes” to the chemical identity and the structures of the photoinitiators and amine synergists is given in Tables 2 and 3. The numbers in the column “No.” refer to the quantity of photoinitiators and amine synergists, respectively, which can be detected simultaneously with the corresponding detection method. LOD = limit of detection; LOQ = limit of quantification; BPacr = benzophenone acrylate (CAS no. 59626-79-8); PE/Al/PE = Polyethylene/Aluminum/Polyethylene multilayer material.

No.

Analytes

Table 1. Continued .

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514 T. Jung et al.

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Food Additives & Contaminants: Part A capable of simultaneous detection of up to 28 photoinitiators and amine synergists, respectively, in a single run. This saves analysis time and money. In addition, multianalyte methods are in particular worthwhile for official control laboratories, where normally no information about the composition of the applied inks on the packaging materials is available. The latest and by far most extensive study in terms of the number of photoinitiators and amine synergists was performed by Lord et al. (2012). In total 39 photoinitiators and amine synergists, respectively, including polymeric ones, were characterised by them with different analysis techniques, i.e. GC-MS, GC coupled to a flame ionisation detector (FID), LC-MS, HPLC-DAD, HPLC coupled to a charged aerosol detector (CAD) and also with direct analysis in real time (DART) coupled to a time-of-flight (TOF) MS. Their work resulted in an inventory of reference chromatograms and UV and mass spectra to allow easier identification of photoinitiators and amine synergists in packaging analysis. They learned that not all photoinitiators and amine synergists were determinable with one method. Especially the analysis of PPI and PAS was found to be problematic. Many were not detectable using GC-MS and with HPLC-DAD these showed a multitude of peaks, while the LC-MS with positive atmospheric pressure chemical ionisation (APCI) responded to only minor components of the polymers. Furthermore, due to molecular weight variations between different batches they observed a distribution in the peak allocation and response, which was greater in the LC-MS analysis than with UV detection. The developed methods allow the concurrent determination of up to 12 photoinitiators and amine synergists, respectively (Lord et al. 2012). Bentayeb et al. (2013) used DART/TOF-MS to analyse in total nine monomeric photoinitiators and amine synergists as well as three PPIs on the outside and the food contact side of multilayer packaging materials. Together with Koivikko et al. (2010), who analysed a polymeric benzophenone acrylate with HPLC-DAD, as well as Kreitel (2010) and BMELV (2011), who both integrated a polymeric benzophenone and a polymeric thioxanthone derivative into a LC-MS/MS method, these are – to the best of our knowledge – by now the only available published methods for the determination of PPIs and PASs, respectively. The aim of this investigation was to develop a HPLC-DAD screening method for the detection of, in total, 63 photoinitiators and amine synergists in food contact materials. An easy and simple performable extraction procedure including a concentration step was used for the analysis of food contact materials to demonstrate the performance of the developed method. In addition, for PPIs and PASs a hydrolysis method was tested.

515

Materials and methods Chemicals The reference substances are given in Tables 2–4 as well as in Table 7. Most of these have a specified purity but others are only of technical grade. Stock solutions were prepared both in a mixture of isopropanol and tetrahydrofuran (50/50; v/v) and in acetonitrile at a concentration of 1 mg ml−1 and stored in a refrigerator at approximately 4°C. HPLC-grade acetonitrile, isopropanol and methanol were purchased from Merck KGaA (Darmstadt, Germany), HPLC-grade tetrahydrofuran from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Water was purified by a Milli-Q® system (Millipore, Schwalbach, Germany). All other chemicals, i.e. formic acid (98–100%), ammonium formate (>99%), ammonia solution (25%), ammonium acetate (≥98%), ethanol (99%) and potassium hydroxide (85%), were at least of analytical grade.

HPLC-DAD analysis All analyses were performed on an Agilent 1100 HPLC system (Agilent Technologies, Waldbronn, Germany) coupled with a DAD. Details of the developed methods can be taken from Tables 5 and 6.

LC-MS/MS analysis A Prominence® HPLC system from Shimadzu (Duisburg, Germany) coupled with a triple quadrupole mass spectrometer API 3200 Q Trap® system (AB Sciex, Darmstadt, Germany) equipped with a TurboIonSpray® probe was used for analyses. Details of the developed method are given in Table 8. Confirmation was achieved, were possible, by comparing the retention times and the relative ion intensities (quantifier–qualifier ratio) of the hydrolysed solutions with those of the reference standards. LC-MS/MS was used to develop the method, especially for the PPIs and PASs respectively. The use of MS/MS detection is not mandatory in carrying out the presented method. “Monomeric” photoinitiators and amine synergists Relative retention times, range of linearity and limits of detection Identification of photoinitiators and amine synergist in samples measured with HPLC-DAD is done by comparing the retention times and spectra of the signals in a sample with that of the reference substances. As shown below, the retention times of the photoinitiators and amine synergists largely did not differ significantly due to the high number of – even structure related – compounds. In order to minimise run-to-run variances of the retention times these were calculated relatively to the two standards given in Table 4, i.e. the retention times of the photoinitiators and amine synergists were divided by those of the internal standards (IStd). IStd 1 was detectable at all

Analyte

Benzophenone

3-Methylbenzophenone

2,4,6-Trimethylbenzophenone

4-(4´-Methylphenylthio)-benzophenone

4-Hydroxybenzophenone

No.

77

8

42

36

13

83846-85-9 C20H16OS 304.09 > 98% TCI Europe N.V. (Zwijndrecht, Belgium) 1137-42-4 C13H10O2 198.07 98% Sigma-Aldrich

119-61-9 C13H10O 182.07 > 99% Merck 643-65-2 C14H12O 196.09 99% Sigma-Aldrich 954-16-5 C16H16O 224.12 > 99% Chemos GmbH (Regenstauf – Germany)

CAS Formula Molecular weight Purity Producer/distributor

OH

19

O

17

18

O

O

12

O

S

21

No.

O

Structure

2-Hydroxy-4methoxybenzophenone

Methyl-o-benzoylbenzoate

131-57-7 C14H12O3 228.08 > 99% Merck

606-28-0 C15H12O3 240.08 > 99% TCI Europe

O

O

O

O

O

O

(continued )

O

OMe

OH

CAS Structure Formula Molecular weight Purity Producer/distributor

2-Methylbenzophenone 131-58-8 C14H12O 196.09 98% Sigma-Aldrich 4-Methylbenzophenone 134-84-9 C14H12O 196.09 99% Sigma-Aldrich 4-Phenylbenzophenone 2128-93-0 C19H14O 258.10 98% Acros Organics (Geel, Belgium)

Analyte

Table 2. Reference substances – “monomeric” photoinitiators and amine synergists, whereby the term “monomeric” includes also polymeric compounds, which showed only one or two peaks on analysis with HPLC-DAD.

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516 T. Jung et al.

2-Hydroxy-4-noctyloxy-benzophenone

4,4′Bis(dimethylamino)benzophenone (Michler’s ketone)

Thioxanthone

1-Chloro-4propoxythioxanthone

4-Isopropylthioxanthone

2-Hydroxy-2-methyl propiophenone

1-[4-(1,1Dimethylethyl) phenyl]-2-hydroxy2-methylpropan-1one

33

7

4

32

41

9

73

Table 2. Continued.

83846-86-0 C16H14OS 254.08 99.5% Fluka (SigmaAldrich) 7473-98-5 C10H12O2 164.08 97% Sigma-Aldrich 68400-54-4 C14H20O2 220.15 >90% IGM Resins

492-22-8 C13H8OS 212.03 >98.5% Fluka (SigmaAldrich) 142770-42-1 C16H13ClO2S 304.03 97% Sigma-Aldrich

90-94-8 C17H20N2O 268.16 >97% Merck

1843-05-6 C21H26O3 326.19 98% Sigma-Aldrich

N

O

S

O

S

O

S

O

O

O

OH

O

Cl

O

O

OH

OH

N

(CH2)7CH3

23

10

3

1

28

6

22

1-[4-(2Hydroxyethoxy) phenyl]-2-hydroxy2-methyl-1propanone

1-Hydroxycyclohexylphenylketone

2,4-Diethylthioxanthone

2-Isopropylthioxanthone

2-Chlorothioxanthone

4,4′Bis(diethylamino)benzophenone (Michler’s ethyl ketone)

4-Dimethylaminobenzophenone

947-19-3 C13H16O2 204.12 99% Sigma-Aldrich 106797-53-9 C12H16O4 224.10 98% Sigma-Aldrich

5495-84-1 C16H14OS 254.08 > 98% IGM Resins B.V. (Waalwijk, the Netherlands) 82799-44-8 C17H16OS 268.09 98% Sigma-Aldrich

86-39-5 C13H7ClOS 245.99 98% Sigma-Aldrich

530-44-9 C15H15NO 225.12 98% abcr GmbH & Co. KG (Karlsruhe, Germany) 90-93-7 C21H28N2O 324.22 > 98% Merck

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HO

O

S

O

S

O

S

O

HO

N

O

O

O

OH

N

(continued )

O

Cl

N

Food Additives & Contaminants: Part A 517

2-Hydroxy-1-(4-(4(2-hydroxy-2methylpropionyl) benzyl)phenyl)-2methyl-1-propanone

Oligo-[2-hydroxy-2methyl-1-[4-(1methyl vinyl)phenyl] propanone]

2-Benzyl-2(dimethyl amino)-4´morpholino butyrophenone

Bis(2,6-dimethoxy benzoyl)-2,4,4trimethyl pentyl phosphine oxide

Diphenyl-(2,4,6trimethylbenzoyl) phosphine oxide

Methylbenzoylformate

2,2-Diethoxyacetophenone

46

57

29

75

26

27

34

Table 2. Continued.

75980-60-8 C22H21O2P 348.13 97% Sigma-Aldrich 15206-55-0 C9H8O3 164.05 98% Sigma-Aldrich 6175-45-7 C12H16O3 208.11 95% Sigma-Aldrich

119313-12-1 C23H30N2O2 366.23 97% Sigma-Aldrich 145052-34-2 C26H35O7P 490.21 65-75% BASF

474510-57-1 C21H24O4 340.17 – BASF SE (Ludwigshafen, Germany) 163702-01-0 (C13H16O2)n (n = 2–5) 800 (average) – Lamberti O

CH3

HO

O

O

N

OH

O

O

O

O

O

O P

O

O

OMe

O

n

N

O

O

CH2

O O O P

O

R

O

OH

11

31

40

25

2,2-Dimethoxy-2phenyl acetophenone

Ethylbenzoyl-formate

2,4,6Trimethylbenzoyl phenylphosphinic acid ethyl ester

Phenyl bis(2,4,6trimethylbenzoyl) phosphine oxide

2-(4-Methylbenzyl)2-dimethylamino-1(4-morpholinophenyl)-1-butanone

2-Methyl-1-(4methylthio) phenyl2-morpholino propan-1-one

15

47

Esacure® ONE (difunctional-αhydroxy ketone; mixture of isomers)

56

84434-11-7 C18H21O3P 316.12 > 95% IGM Resins 1603-79-8 C10H10O3 178.06 95% Sigma-Aldrich 24650-42-8 C16H16O3 256.11 99% Sigma-Aldrich

119344-86-4 C24H32N2O2 380.25 – BASF 162881-26-7 C26H27O3P 418.17 97% Sigma-Aldrich

71868-10-5 C15H21NO2S 279.13 98% Sigma-Aldrich

115055-18-0 C26H32O4 408.23 95% Lamberti S.p.A. (Gallarate, Italy)

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N

O

O

O

O

P O

O

O

OH

(continued )

N

N

O

O

O

O P

OO

O

MeO OMe

O

CH3S

HO

O

518 T. Jung et al.

2-Ethylanthraquinone

4,4′,4′′Methylidynetris (N,Ndimethylaniline)

Tribromomethyl phenyl sulfone

Ethyl-4(dimethylamino)benzoate

Isoamyl 4-(N,Ndimethylamino) benzoate

30

24

48

14

76

17025-47-7 C7H5Br3O2S 389.76 97% abcr 10287-53-3 C11H15NO2 193.11 > 98% Fluka (SigmaAldrich) 21245-01-2 C14H21NO2 235.16 > 98% Chemos

603-48-5 C25H31N3 373.25 – Sigma-Aldrich

84-51-5 C16H12O2 236.08 > 97% Sigma-Aldrich

59867-68-4 C14H10Cl2O2 280.01 > 99% Chemos

N

N

N

Cl2CH

O S

O

O

O

O

O

O

O

O

Br

BrBr

N

O

N

71

5

35

44

51

37

Omnipol™ ASE (polymeric aminobenzoate)

2-Ethylhexyl-4dimethylaminobenzo ate

1-(4-[(4Benzoylphenyl) thio]phenyl)-2methyl-2-[(4methylphenyl) sulfonyl]-1-propan1-one Butoxyethyl-4(dimethylamino) benzoate

Bis(eta(5)cyclopentadienyl)bis(2,6-difluoro-3[pyrrol-1-yl]phenyl)titanium

2,2′-Bis-(2chlorophenyl)4,4′,5,5′tetraphenyl-1,2′biimidazole

– – 1040 (average) – IGM Resins

67362-76-9 C15H23NO3 265.17 > 95% TCI Europe 21245-02-3 C17H27NO2 277.20 > 98.5% Merck

272460-97-6 C30H26O4S2 514.13 – Lamberti

125051-32-3 C30H22F4N2Ti 534.12 – BASF

7189-82-4 C42H28Cl2N4 658.17 >97% TCI Europe

–

O

O

N

N

Cl

O

F

F

N

Ti

O

S

O

Cl

N

O

F F

O

O S O

N

N

N

N

Note: Substances were numbered according to an internal laboratory system. The substances can be grouped into the following classes referring to their structure or function: benzophenones (numbers 77, 21, 8, 12, 42, 18, 36, 19, 13, 17, 33, 22, 7 and 6), thioxanthones (numbers 4, 28, 32, 1, 41 and 3), α-hydroxyketones (numbers 9, 10, 73, 23, 46, 56 and 57), α-aminoketones (numbers 15, 29 and 47), phosphine oxides (numbers 75, 25, 26 and 40), acetophenones (numbers 27, 31, 34, 11 and 43), miscellaneous (numbers 37, 30, 51, 24, 44 and 48) and amine synergists (numbers 35, 14, 5, 76 and 71), whereby, for example, some of the benzophenones can also act as a synergist. Information given here was collected from technical as well material safety data sheets of the producers and/or distributors and from Green (2010).

4-Phenoxy-2,2′dichloro acetophenone

43

Table 2. Continued.

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Food Additives & Contaminants: Part A 519

Ebecryl® P 39 (polymeric benzophenone derivative)

Mixture of: 1,3-di({a-2-(phenyl carbonyl)benzoylpoly[oxy(1methylethylene)]}oxy)-2,2bis({a-2-phenylcarbonyl) benzoylpoly[oxy(1methylethylene)]}oxymethyl)pro pane and {a-2-(phenylcarbonyl) benzoylpoly(oxyethylene)poly[oxy(1-methylethylene)]poly (oxyethylene)}2-(phenyl carbonyl)benzoate (polymeric benzophenone) Omnipol™ SZ (polyethylene glycol(200)di(β-(4(pacetylphenyl)piperazine)) propionate)

58

59

Omnipol™ TX (diester of carboxy-methoxy thioxanthone and polytetramethyleneglycol 250) Genopol® TX-1 (polymeric thioxanthone derivative)

38

55

Omnipol™ 910 (polyethylene glycol(200)di(β-4[4-(2dimethylamino-2-benzyl) butanoylphenyl]piperazine) propionate

61

72

54

Analyte Omnipol™ BP (diester of carboxy-methoxy benzophenone and polytetramethyleneglycol 250) Genopol® BP-1 (polymeric benzophenone derivative)

No. 39

–

81345237-8

88646310-1

–

1003567 -82-5/ 1003557 -16-1

–

–

CAS 51513648-8

–

–

–

O

N

O

S

O

N

O

N

n

n

O

O

O

O

n

O

O

O

O

O

O

O O

O

OO

n

O

O

O

N

O

O

N

n

O

O

O

O O

O

Structure

O O

n

O

O

O

O

nO

O n

n O

O

O

O

O

nO

nO

O

N

O

N

O

N

N

O

O

S

O

O

N

Table 3. Reference substances – polymeric photoinitiators (PPIs) and amine synergists. (PASs).

–

(C4H8O)nC30H18O7S2

(C2H4O)nC52H68N6O5

(C2H4O)nC30H38N4O5

(C3H6O)4nC61H44O12 (C2H4O)2n(C3H6O)n C28H18O5

–

–

Formula (C4H8O)n.C30H22O7

820 (average)

790 (average)

1033 (average)

716 (average)

1196 (average)

–

960 (average)

Molecular weight 730 (average)

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>98

94

75(diester)

>94

–

–

>98

Purity (%) >95

Rahn

(continued )

IGM Resins

IGM Resins

IGM Resins

Rahn AG (Zurich, Switzerland) Cytec Industries Inc. (Woodland Park, NJ, USA) Lambson Ltd (Wetherby, UK)

Producer/distributor IGM Resins

520 T. Jung et al.

Genopol AB-1 (polymeric aminobenzoate derivative) Butoxy(poly(oxy(methyl-1,2ethandiyl)))-4-dimethylaminobenzoate 22346345-4

–

7151290-8

1003567 -84-7 / 1003557 -17-2

1003567 -83-6

O

–

O

N

Cl

N

N

N

O

S

n

O

O

O

O

O

O C H2

O

O

O O

n

n

O

O

N

O

O

O

S

O

Cl

O

CH 2

O

n

O

O

O

O

O

n

nO

O

O

n

n

n

O

S

O

O

O

O

O

n

O

N

n

O

CH 2

O

Cl

O

O

N

N

O

n

O O

n

N

C O H2 S

Cl O

860 (average) –

(C3H6O)nC13H19NO2

488-532

1066 (average)

1899 (average)

–

(C2H4O)nC18H20N2O3 n = 3-4

(C3H6O)4nC41H48N4O8 (C2H4O)2n(C3H6O)n C18H20N2O3

(C3H6O)4n C65H40Cl4O16S4

–

>98

>98

–

10-30

Lambson

Rahn

IGM Resins

Lambson

Lambson

Note: Compounds were numbered according to an internal laboratory system. The compounds can be grouped into the following classes referring to their structure or function: benzophenones (numbers 39, 54, 58 and 59), α-aminoketones (numbers 72 and 61), thioxanthones (numbers 38, 55 and 63) and amine synergists (numbers 60, 53, 52 and 70). Information given here was collected from technical as well material safety data sheets of the producers and/or distributors and from Green (2010).

70

52

®

Mixture of: 1,3-di({a-4(dimethyl amino)benzoyl poly[oxy(1methyl ethylene)]}oxy)-2,2bis({a-4-(dimethylamino)benzoylpoly[oxy(1methylethylene)]}oxymethyl) propane and {a-4(dimethylamino)benzoyl poly(oxyethylene)-poly[oxy(1methyl ethylene)]poly(oxyethylene)}4-(dimethylamino)benzoate Omnipol™ ASA poly(ethylene glycol)bis(pdimethylamino)benzoate

60

53

1,3-Di({a-[1-chloro-9-oxo-9Hthioxanthen-4-yl)oxy]acetylpoly [oxy(1-methyl ethylene)]}oxy)2,2-bis({a-[1-chloro-9-oxo-9Hthioxanthen-4-yl)oxy]acetyl poly[oxy(1methylethylene)]} oxy methyl)propane (polymeric thioxanthone)

63

Table 3. Continued.

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Table 4. Internal standards (IStd) used for the calculation of the relative retention times of the 50 compounds given in Table 2. No.

Chemical name

CAS Formula Molecular weight Purity Producer/distributor

Structure

IStd 1

4-(Dimethylamino)benzaldehyde

100-10-7 C9H11NO 149.08 99% Merck

O

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IStd 2

Nonylbenzene

wavelengths given in Table 6, had a mean retention time of 12.10 min and was used at a concentration of 1 µg ml−1. IStd 2 was only visible at 260 nm, had a mean retention time of 53.57 min and was used at a concentration of 100 µg ml−1 due to its low UVabsorption. Table 6 summarises the retention times and the relative retention times for the 50 photoinitiators and amine synergists listed in Table 2. The LODs and range of linearity for these 50 compounds were determined using calibrations with 11 levels in a concentration range between 0.1 and 10 µg ml−1 acetonitrile. In case of unknown purity of the reference substance the concentration was set to 100%. For calibration, the photoinitiators and amine synergists were divided into different groups because of the co-elutions described below and impurities, respectively. All calibrations were performed by linear regression whereby the intercept was not forced through zero. Visually all calibrations were linear with correlation coefficients ≥ 0.9928, except for number 48 with 0.9765. The LODs were calculated from the signal-to-noise ratios of the substances dissolved in acetonitrile by using equation (1). The 0.1 or 0.5 µg ml−1 level was used for the calculations, except for the following photoinitiators: numbers 48 (1.5 µg ml−1), 51–1 (1.5 µg ml−1), 51–2 (1 µg ml−1) and 75–2 (1 µg ml−1) due to their lower UV absorption. For the photoinitiators with more than one peak the concentration for each peak was set to 100% due to the unknown distribution ratios. LODs as well as the wavelengths used for the quantification of each of these substances are given in Table 6. To calculate the LOD:   cSubstance 3 (1) cLOD ¼ S=N

H

N

1081-77-2 C15H24 204.19 97% TCI Europe

where cLOD is the concentration of the LOD (ng ml−1); cSubstance is the concentration of the reference substance (ng ml−1); and S/N is the signal-to-noise ratio at cSubstance.

Polymeric photoinitiators and amine synergists Hydrolysis and HPLC-DAD analysis Hydrolysis of the 13 PPIs and PASs given in Table 3 was performed according to the method of Schaefer et al. (2004), with minor modifications. A total of 1 ml of a solution of the polymeric reference substance in acetonitrile or of the food contact material extract, respectively, was pipetted into a 10 ml volumetric flask and 4 ml of a 2 molar ethanolic potassium hydroxide solution were added. The mixture was then incubated for 1 h in a water bath at 60°C. After that 1.7 ml formic acid were added and the flask was filled with ethanol. Prior to analysis with the HPLC-DAD method given in Table 5, the hydrolysed solutions were diluted 1:1 (v/v) with a mixture of 8% (v/v) acetonitrile and 92% (v/v) water pH 2 (adjusted with formic acid), as direct analysis of the ethanolic solutions resulted partially in peaks with strong fronting.

Conversion rates To gain information about the completeness of the hydrolysis, conversion rates were determined for those PPIs and PASs, for which a hydrolysis reference standard (HStd) was available (cf. Table 7). Numbers 38, 39, 52, 53, 54, 59, 60 and 70 were hydrolysed each at three concentrations. Prior to HPLC-DAD analysis, the hydrolysed solutions were diluted as described above, resulting in concentrations of 2, 5 and 10 µg ml−1 in the measuring solutions with regard to the PPIs and PASs.

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Table 5. HPLC-DAD method for the analysis of the hydrolysis products.

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Column Flow (ml min−1) Injection volume (µl) Temperature (°C) Detection wavelengths (nm) Mobile phase Gradient

Synergi™ Polar RP 80A, 100 mm, 3 mm I.D., 4 µm; Phenomenex® 0.6 10 30 260, 300, 330, 370; spectra were recorded from 200 to 500 nm Acetonitrile (A)/water, adjusted to pH 2 with formic acid Linear gradient from 8% (v/v) A to 25% (v/v) A in 7 min, linear gradient to 50% (v/v) A in 23 min, linear gradient to 90% (v/v) A in 10 min, 10 min isocratic at 90% (v/v) A, linear gradient to 8% (v/v) A in 0.1 min, 9.9 min isocratic at 8% (v/v) A (stop time).

For quantification of the hydrolysis products calibration solutions were prepared from stock solutions of the HStds 3–6 with 1 mg ml−1 in acetonitrile by diluting them with a mixture of 58% (v/v) acetonitrile and 42% (v/v) water pH 2 (adjusted with formic acid). Six calibration levels at 1, 2, 3, 4, 5 and 10 µg ml−1 were prepared and calculated using linear regression whereby the intercept was not forced through zero. Visually all calibrations were linear with correlation coefficients ≥ 0.9997. The concentrations of the hydrolysis products in the hydrolysed solutions of the PPIs and PASs were then translated into the conversion rates according to equation (2) and are summarised in Table 9. Equation (2). Calculation of the conversion rate in %:  conversion rateð%Þ ¼

chydrolysis product cPPI or PAS

  100

(2)

where chydrolysis product is the concentration of the hydrolysis product (µg ml−1); and cPPI or PAS is the concentration of the PPI or of the PAS, respectively (µg ml−1). For those PPIs for which no HStds were available, i.e. numbers 55, 58, 61, 63 and 72, solutions of these in acetonitrile were hydrolysed at the same three concentration levels as given above and analysed by HPLC-DAD. From these calibrations were performed by linear regression, whereby the intercept was not forced through zero. Visually all calibrations were linear with correlation coefficients ≥ 0.9932. Limits of detection for the hydrolysis products The calibration data of the HStds 3–6 were also used to estimate the LODs for these. In each case the signal-tonoise ratio of the first calibration point with 1 µg ml−1 was inserted in Equation (1). Preparation of food contact material extracts Eight different food contact materials were analysed for their photoinitiator and amine synergist content, all of which were printed and/or lacquered with UV printing

inks and/or varnishes: two plastic cups for dairy products (received from a manufacturer), two disposable plastic drinking cups (obtained from retail), one reusable plastic drinking bottle (obtained from retail), one paper label used on plastic bags for pastries (received from a manufacturer) and two cartonboard packaging for dry food (obtained from retail). The latter were the only two samples that had already been in contact with foodstuffs prior to analysis. Extraction of paper-or cartonboard-based material was performed in brief as follows: 0.5 dm2 of the material were cut into small pieces and then extracted with 20 ml acetonitrile in a shaking water bath at 70°C for 24 h. After cooling the extract in a refrigerator it was filtered through filter paper and, if still cloudy, additionally through a 0.45 µm PET filter. An aliquot was then filled into a brown vial and subjected to HPLC-DAD analysis (Pastorelli et al. 2008; Jung et al. 2013). This extraction procedure was also used here for the food contact materials made from plastic. Due to different printing coverage, i.e. fully versus partially printed materials, diverse amounts of photoinitiators and amine synergists were expected. Thus either 0.5 dm2 of the printed material or the entire item was extracted with 20 ml acetonitrile. In case of the two cartonboards, yoghurt cup 1, drinking cup 1 and the drinking bottle 0.5 dm2 were extracted from each (cf. Table 10). For the extraction of yoghurt cup 2, drinking cup 2 as well as the label in each case the whole article was used due to the low printed area. In addition, to reach sufficient LODs, the extracts of yoghurt cups 1 and 2 and drinking cup 1 were concentrated up by a factor of 10, i.e. 10 ml of the extract were reduced to a final volume of 1 ml under a gentle steam of nitrogen at 60°C. In order to check the extraction and concentration method for the photoinitiators and amine synergists contained in these materials (cf. Table 10), two mixtures of these at concentrations of 1 and 10 µg ml−1 in acetonitrile were prepared. Each 20 ml of these solutions were put into a shaking water bath at 70°C for 24 h. The mix with 10 µg ml−1 was directly analysed while 10 ml of the 1 µg ml−1 solution were concentrated

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up to a volume of 1 ml prior to analysis. In addition, numbers 5, 10, 12, 18, 19 and 77 were also tested under these conditions. Comparison of the not concentrated with the concentrated solution showed no significant differences for the photoinitiators and amine synergists and recoveries in the concentrated solution lay between 95% and 112%. Thus during extraction and concentration no significant loss of the analysed photoinitiators and amine synergist was detected. Prior to all extractions the IStds were added at a concentration that led to 1 and 100 µg ml−1 for IStd 1 and IStd 2, respectively, in the final analysis solutions (cf. Table 4). Analyses were done with the HPLC-DAD method given in Table 6. Quantification was performed using an external calibration of standard solutions in acetonitrile at concentration levels of 1, 5, 10, 15 and 20 µg ml−1. Confirmation was achieved, were possible, by comparing the retention times and the UV spectra of the samples with those of the reference substances.

Results Development of a HPLC-DAD screening method In order to achieve a chromatographic separation of all 63 monomeric and polymeric photoinitiators and amine synergists given in Tables 2 and 3, an approach proposed by Kromidas (2006) was followed. Here the parameters for the separation, i.e. column material, mobile phases etc., are defined in only a few steps. At first several combinations of column materials and mobile phases in terms of different selectivities, pH values and solvents were tested. A mix containing all photoinitiators and amine synergists at a concentration of 10 µg ml−1 in isopropanol–tetrahydrofuran (50:50, v/v) was used. During the tests similar injection volumes, flows and gradients were maintained, i.e. these parameters were adapted for different column dimensions. The HPLC was run at 30°C in linear gradient mode from 10% to 95% (v/v) solvent in 60 min for a column of 150 mm length. Detection wavelengths of the DAD were set to 260, 300, 330 and 370 nm, which are in the range of the main wavelengths of a standard mercury lamp used for the hardening of UV printing inks and lacquers (Glöckner et al. 2008). For evaluation, only the total number of peaks was counted for the different combinations of columns and mobile phases. The best result with in total 126 peaks was obtained on a Zorbax Eclipse XDB-C8 column (150 mm, 4.6 mm I.D., 5 µm; Agilent Technologies, Waldbronn, Germany) with acetonitrile and a 25 mmol ammonium formate buffer adjusted with formic acid to pH 3. This combination was taken for further optimisation. In the next steps, the gradient and the flow rate as well as the length, internal diameter and particle size of the

column were varied. Reference substances were dissolved in acetonitrile as with this solvent the best separation was obtained when used as mobile phase. The pH of the mobile phase was raised to 4 because it was shown that some of the photoinitiators, e.g. numbers 29 and 47, were not stable at pH 3 (Freudenberg 2010). In the end this effort resulted in the HPLC-DAD method summarised in Table 6. In case of the polymeric analytes, PPIs and PASs, for each several single peaks and/or humps of unresolved peaks were observed under the chromatographic conditions chosen (cf. Figure 2). In contrast, for PAS number 71 just one and for the PPI number 57 only two signals, respectively, were detected. The multiple signals of the polymeric analytes co-eluted to a great extent with the other reference substances. This problem was also formerly described by Koivikko et al. (2010). Thus these were excluded here from the mixtures used for calibration. A hydrolysis method was developed for these PPIs and PASs to allow easier identification and particularly quantification (cf. the next sections). But of course these are still detectable by the screening method if present in extracts of food contact materials. Also for some of the other monomeric photoinitiators two or three peaks were observed, further labelled here as “−1”, “−2” and “−3”. This is valid for numbers 51 and 56, which had each two signals, as well as for number 75, for which even three peaks were detected. For the α-hydroxyketone number 56 this could be explained by its structure: it is a mixture of isomers. LC-MS/MS analysis revealed that in case of number 51 only the second peak was the active substance while the first was probably an impurity; this photoinitiator was of technical grade only. For number 75 the third peak could be identified as the target substance and the second one as the photoinitiator number 10 while the first peak could not be assigned to a structure. Despite the optimisation efforts a separation could not be achieved for all substances. Due to the high number of compounds, which are partly structure related, complete chromatographic separation has not been possible. The 2and 3-isomers of methylbenzophenone (numbers 8 and 21), the benzophenones numbers 12 and 17 and the αhydroxyketones numbers 56 and 57, as well as the not structure related pairs of numbers 43 and 76, 19 and 34, 48 and 77, 15 and 22, and 3 and 24 resulted each in one unresolved peak. But the four last mentioned pairs can be distinguished by their characteristic spectra. In addition, the phosphine oxides numbers 26 and 75–3 as well as the thioxanthone number 28 formed one peak. Number 28 can be identified by its characteristic spectrum but the spectra of the two phosphine oxides are nearly identical. Usually up to five to seven different photoinitiators and amine synergists are used in one printing ink. Thus it is unlikely that overlaps will occur often. But for quantification

525

3 + 24

37 33

IStd 2

5 44

36 25

42

18

57–2 + 56–2

57–1 + 56–1

30 28 75–3 + 26

71 35 40

32 6

43 + 76

21 + 8 12 + 17

7 73 11

46 31 10 + 75–2

27 29 47

9 75–1 IStd 1

23

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19 + 34 14 77 + 48 15 + 22

13

41

4

1

Food Additives & Contaminants: Part A

Figure 1. Chromatogram of a mixture of the 50 photoinitiators and amine synergists given in Table 2, except number 51, which is missing here, and the two IStds (Table 4). The concentration of each reference substance was 10 µg ml−1. All wavelengths are overlaid. The chromatogram is only shown up to 55 min as afterwards no more “monomeric” photoinitiators or amine synergists were detected.

purposes as well as due to the above described impurities it is therefore required to calibrate with different mixtures of the photoinitiators and amine synergists. Figure 1 shows a chromatogram of a mixture of the “monomeric” substances, whereby the term “monomeric” includes also the two polymeric substances numbers 71 and 57, which showed only one or two peaks, respectively. Furthermore it was shown that the retention times of several substances depended highly on the pH value of the mobile phase. For numbers 13, 15, 24, 29 and 47 a retention time shift of up to 29.8 min was observed between pH values of 4 and 9 of the mobile phase. Thus for the correct identification of these it is very important to keep the pH constant with an adequate buffer. All other photoinitiators and amine synergists had at least stable retention times, independently from the pH value of the mobile phase. The retention times of all 50 “monomeric” photoinitiators and amine synergists were calculated relative to two internal standards in order to compensate retention time shifts. Table 6 summarises the retention times as well as the relative retention times. In addition, it also contains the LODs and the wavelengths used for the quantification of each of these substances. LODs varied between 5 and 1365 ng ml−1, thus representing a wide range of sensitivity of the components to the UV detector.

Polymeric photoinitiators and amine synergists Hydrolysis As already stated above most of the PPIs and PASs showed several humps of unresolved peaks, which coeluted with other monomeric reference substances. For example, Figure 2 shows the chromatogram of the PPI

number 60 in acetonitrile. It is obvious that beside the problem of co-elution with other photoinitiators and amine synergists, quantification of all these peaks would almost be impossible, and quantification using one or more “quantifier peaks” could vary due to differences in the composition of the mixtures between different lots and manufactures, respectively. In addition, a sufficient LOD would hardly be reached because of the distribution over many peaks. Since in many PPIs and PASs the chromophore is bound by an ester bond to a polymeric backbone, hydrolysis should ideally release the chromophore containing substance. This would simplify identification and quantification a lot.

Analysis of the hydrolysis products Hydrolysis reduced the number of peaks to one, except for the PASs numbers 52, 53, 60 and 70. These showed two peaks, which were identified as 4-(dimethylamino)benzoic acid and its ethyl ester by HPLC-DAD and LC-MS/MS analysis (cf. Tables 5 and 8). Tests showed that the formation ratio of these is influenced by the hydrolysis conditions, e.g. by the reaction time and the concentration of the ethanolic potassium hydroxide solution. Under the hydrolysis conditions given here, the 4-(dimethylamino)benzoic acid is formed preferably. Figure 3 shows an HPLC-DAD chromatogram of a mixture of all hydrolysed polymeric substances and Table 7 the assignment of the peaks of the hydrolysis products (H) to the corresponding PPIs and PASs, respectively. The exact structures of PAS number 52 and of PPI number 54 are unknown. After hydrolysis, the chromophore of number 52 was identified as 4-(dimethylamino) benzoic acid (H 3) and that of number 54 as

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Table 6. HPLC-DAD screening method. Retention times, relative retention times, quantification wavelengths and LODs for the “monomeric” photoinitiators and amine synergists given in Table 2. Column Flow (ml min−1) Injection volume (µl) Temperature (°C) Detection wavelengths (nm) Mobile phase Gradient

Zorbax Eclipse XDB-C8, 150 mm, 2.1 mm I.D., 3.5 µm; Agilent Technologies 0.49 5 30 260, 300, 330, 370; spectra were recorded from 200 to 500 nm Acetonitrile (A)/25 mmol ammonium formate buffer pH 4 3.5 min isocratic at 8% (v/v) A, linear gradient to 40% (v/v) A in 6.5 min, 17 min isocratic at 40% (v/v) A, linear gradient to 85% (v/v) A in 28 min, 10 min isocratic at 85% (v/v) A, linear gradient to 100% (v/v) A in 10 min, 10 min isocratic at 100% (v/v) A, linear gradient to 8% (v/v) A in 0.1 min (stop time), 9.9 min isocratic at 8% (v/v) A.

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Retention times (min) No. 23 75–1b 9 29 13 27 47 51–1a 46 31 10 75–2b 19 34 14 77 48 15 22 4 7 73 11 12 17 21 8 40 71 30 35 28 75–3b 26 57–1 56–1 57–2 56–2 42 43 76 18 41 1 32

“Absolute”

Relative to IStd 1

Relative to IStd 2

Wavelength for quantification (nm)

LOD (ng ml−1)

9.542 11.445 11.637 13.091 13.234 13.513 14.139 14.879 15.522 15.660 16.167 16.193 16.927 17.009 17.529 18.446 18.467 18.703 18.863 19.734 20.009 21.106 21.382 22.828 22.996 23.480 23.572 24.271 27.582 29.360 29.696 30.262 30.669 30.799 32.372 32.388 33.388 33.400 36.140 37.103 37.171 37.470 37.959 38.314 39.158

0.789 0.946 0.962 1.082 1.094 1.117 1.169 1.230 1.283 1.294 1.336 1.338 1.399 1.406 1.449 1.524 1.526 1.546 1.559 1.631 1.654 1.744 1.767 1.887 1.900 1.940 1.948 2.006 2.280 2.426 2.454 2.501 2.535 2.545 2.675 2.677 2.759 2.760 2.987 3.066 3.072 3.097 3.137 3.166 3.236

0.178 0.214 0.217 0.244 0.247 0.252 0.264 0.278 0.290 0.292 0.302 0.302 0.316 0.318 0.327 0.344 0.345 0.349 0.352 0.368 0.374 0.394 0.399 0.426 0.429 0.438 0.440 0.453 0.515 0.548 0.554 0.565 0.573 0.575 0.604 0.605 0.623 0.623 0.675 0.693 0.694 0.699 0.709 0.715 0.731

260 260 260 370 300 260 370 260 260 260 260 260 260 260 300 260 260 300 370 260 370 260 260 260 300 260 260 260 300 260 300 260 300 260 260 260 260 260 260 300 300 300 260 260 260

222 205 15 13 7 27 21 658 7 34 44 70 10 31 41 53 1365 55 14 5 30 14 25 16 26 21 15 280 20 39 73 47 105 292 48 98 24 19 21 19 20 48 6 5 76 (continued )

Food Additives & Contaminants: Part A

527

Table 6. Continued . Retention times (min) No.

“Absolute”

Relative to IStd 1

Relative to IStd 2

Wavelength for quantification (nm)

LOD (ng ml−1)

39.767 41.858 42.063 43.794 44.346 45.779 46.745 47.778 51.807 52.337

3.287 3.459 3.476 3.619 3.665 3.783 3.863 3.949 4.282 4.325

0.742 0.781 0.785 0.818 0.828 0.855 0.873 0.892 0.967 0.977

370 260 260 260 260 260 300 260 260 300

9 17 17 20 42 31 15 305 29 36

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6 3 24 36 25 44 5 51–2a 37 33

Notes: aThe structure given in Table 2 for this photoinitiator could only be assigned to the second peak, i.e. 51–2, by LC-MS/MS analysis. The first signal was probably an impurity; the reference substance was of technical grade only. b Only the third peak of number 75 could be assigned to the structure given in Table 2 for this photoinitiator. The second signal was identified as number 10 by LC-MS/MS analysis while for the first one no structure proposal could be made. This was probably an impurity; the purity was specified as 65–75% for the phosphine oxide (number 75–3).

2-benzoylbenzoic acid (H 4) (cf. Table 7). The identity of the hydrolysis products of the PPIs numbers 38 and 39 was confirmed as 2-(carboxymethoxy)thioxanthone (H 5) and 4-(benzoylphenoxy)acetic acid (H 6), respectively, by HPLC-DAD and LC-MS/MS analysis of the corresponding reference standards. Since no reference standards for the hydrolysis products of the PPIs numbers 61, 63 and 72 were available and the structures of those of numbers 55 and 58 were even completely unknown, for these only the hydrolysed solutions were analysed by LC-MS/MS. In case of numbers 61, 63 and 72 the molecular ions of the presumed hydrolysis products with a mass-to-charge ratio of 438, 321 and 277, respectively, were identified. Unfortunately, full-scan measurements of the hydrolysed solutions of the PPIs numbers 55 and 58 showed no significant peaks. As no information about the structures was given, it was not 2.00

possible to include their hydrolysis products into the LCMS/MS method, which is summarised in Table 8. Conversion rates To gain information about the completeness of the hydrolysis, conversion rates were determined for those PPIs and PASs, for which a HStd was available (cf. Tables 7 and 9). Concentrations of the hydrolysis products in the hydrolysed solutions were determined by HPLC-DAD analysis. From these the conversion rates were calculated, which are summarised in Table 9. Mean conversion rates were in the range between 44% and 69%, with mean variation coefficients in the range between 9.3% and 25%, except for number 70. For this PAS a conversion rate of 25% and a variation coefficient of 3.8% were determined. The reasons for these low

3 mAU

WVL:260 nm IStd 1

IStd 2

1.50

1.00

0.50

1 2 –0.00 3

–0.50

–1.00 min

–1.50 0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

80.0

85.0

Figure 2. Chromatogram of photoinitiator number 60 at a concentration of 10 µg ml−1 in acetonitrile. Overlay of the 260 nm (bottom line), 300 nm (centre line) and 330 nm (top line) wavelengths. The HPLC-DAD conditions are given in Table 6.

528

T. Jung et al. 30.0

mAU

WVL:260 nm

H3

25.0

20.0

15.0

H1

10.0

H2

H5

5.0

H 10

H6 H7

H4

H8 H9

0.0

1 3 4 2

min

-4.0

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0.0

1.3

2.5

3.8

5.0

6.3

7.5

8.8

10.0

11.3

12.5

13.8

15.0

16.3

17.5

18.8

20.0

21.3

22.5

23.8

25.0

Figure 3. (colour online). Chromatogram of the hydrolysis products (H) of the PPIs and PASs at a concentration of 2 µg ml−1 each (overlay of all wavelengths). Assignment of the peaks to the reference substances is given in Table 7. The chromatogram is only shown up to 25 min as afterwards no more peaks were detected.

conversion rates and high variation coefficients were not yet examined further. On the one hand, it is unknown if conversion rates of 100% are generally achievable for all substances, as for numbers 59, 60 and 70 the purity was not given. For these indeed the lowest conversion rates were determined. On the other hand, deduced from the structures, it should theoretical be possible to obtain more than one of the corresponding hydrolysis products from one molecule of some PPIs and PASs. For example, numbers 38, 39 and 53 possess each two chromophores and numbers 59 and 60 each six (assuming a 1:1 ratio of the two substances present in these mixtures). Thus it should be tested if hydrolysis conditions are strong enough. As Table 9 shows, for the 2 µg ml−1 concentration almost always the lowest conversion rates were obtained. If these values are excluded from the calculations, conversion rates and especially the variation coefficients are much better for most of the substances. This could be a hint for insufficient hydrolysis conditions but also for lacking stability of the hydrolysis products in the solution. Thus, optimisation of the hydrolysis method is needed. At the moment only semi-quantification is possible by performing a standard addition. LODs for the HStds 3–6 were 7, 7, 20 and 8 ng ml−1. Taking into account the mean conversion rates these are in the range between 13 and 28 ng ml−1 for the PASs numbers 52, 53, 60 and 70. For the PPIs numbers 38, 39, 54 and 59 the LODs are then 30, 12, 15 and 11 ng ml−1, respectively. For those PPIs for which no HStds were available, i.e. numbers 55, 58, 61, 63 and 72, the analysis results of the hydrolysed solutions were used to create calibration curves. All these were visually linear, which indicates that for these hydrolysis leads to comparable yields independent from concentration. Despite the above described limitations hydrolysis is already now a useful tool for the identification of these

polymeric substances in food contact material extracts as is shown in the next section with an example. Application of the HPLC-DAD method Extraction of food contact materials The HPLC-DAD method was developed for the screening of food contact materials. Thus, an adequate extraction method as well as an approach for the concentration of the extracts was needed. As, for example, plastic cups for dairy products are mostly only partially printed otherwise sufficient LODs would hardly be reached. To test the extraction several time and temperature modifications of the acetonitrile extraction described by Pastorelli et al. (2008) were compared for food contact materials made from plastic (Figure 4). Acetonitrile was used as all substances were soluble in it. In each case 0.5 dm2 of two different plastic cups were extracted with 20 ml acetonitrile and subsequently 10 ml of the extracts evaporated to a final volume of 1 ml. All analyses were performed in duplicate. It turned out that the extraction at 70°C for 24 h in a shaking water bath yielded on average the highest amounts of photoinitiators and amine synergists, followed by the extraction at 70°C for 5 h (Figure 4). Screening results of food contact materials The results of the analyses of eight food contact materials for their photoinitiator and amine synergist content are summarised in Table 10. In addition, for each sample here those substances are given, which should be detectable in the extracts. These were either known from previous analyses or from the formulations of the used printing inks. Nearly all known photoinitiators and amine synergists could be determined by the proposed screening method

Food Additives & Contaminants: Part A

529

Table 7. Hydrolysis products (H) of the PPIs and PASs given in Table 3. For H 3, H 4, H 5 and H 6 reference standards (HStd) were available. H 9 corresponds to the amine synergist number 14 (cf. Table 2). Peak Reference number substance number

Retention Structure of the hydrolysis time (min) product

Chemical name

CAS Formula Molecular weight pKa UV maximum Purity of the HStd Producer/distributor

H1

5.4

3-[4-(4-Acetylphenyl) piperazin-1-yl]propanoic acid

– C15H20N2O3 276.15 3.80/6.74 234/319 nm

[(4-(2-Dimethylamino-2-benzyl) butanoylphenyl) piperazine] propionate

– C26H35N3O3 437.27 – 344 nm

4-(Dimethylamino)benzoic acid

619-84-1 C9H11NO2 165.08 2.56/4.90 315 nm 98% Sigma-Aldrich 85-52-9 C14H10O3 226.06 3.54 249 nm 98% Sigma-Aldrich 84434-05-9 C15H10O4S 286.03 3.06 288 nm 98% Chemos 6322-83-4 C15H12O4 256.07 3.04 271/254 nm 98.7% International Laboratory USA (San Francisco, CA, USA) – 257/314 nm – 260 nm

72

O

N

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N

H2

61

O OH

6.2 N O N N

H3

52, 53, 60, 70 9.7

OH O

O N OH

O

H4

54, 59

15.4

H5

38

16.1

O

OH

2-Benzoylbenzoic acid

O

O O

2-(Carboxymethoxy)thioxanthone OH

S

H6

39

O

18.7

4-(Benzoylphenoxy)acetic acid O

O OH

H7

55

19.1

–

–

H8

58

19.9

–

–

(continued )

530

T. Jung et al.

Table 7. Continued . Peak Reference number substance number

H9

Retention Structure of the hydrolysis time (min) product

52, 53, 60, 70 20.5

O

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63

21.8

O

Cl

O

S O

CAS Formula Molecular weight pKa UV maximum Purity of the HStd Producer/distributor

4-(Dimethylamino)benzoic acid ethyl ester

O

N

H 10

Chemical name

OH

and in case of the drinking bottle and the cartonboard 2 also further ones were identified. The false-negative results (number 44 in yoghurt cups 1 and 2 as well as numbers 9 and 15 in the label) could be traced back to contents near or below the LOD, which were known in the case of yoghurt cup 2 and the label, respectively. However, at these low concentrations contamination of the foodstuffs is expected to be well below the legally acceptable limits of 50 µg kg−1 food for number 44 and of 10 µg kg−1 food in case of numbers 9 and 15 (EDI 2005). The chromatogram of the extract of cartonboard 2 (Figure 5A) showed two peak humps, which were assumed to belong to PPI number 54. For confirmation and estimation of the amount 10 ml of the extract were concentrated to 1 ml and subsequently subjected to hydrolysis. Figure 5B depictures the chromatogram of the hydrolysed sample extract. Comparison of the main signal with the spectrum and retention time of the HStd 2-benzoylbenzoic acid (cf. Table 7) confirmed the presence of PPI number 54 in the cartonboard at an amount of approximately 170 or 270 µg dm−2, respectively, when taking into account the mean conversion rate of 63% (cf. Table 9). Nevertheless, in each case the correct main photoinitiators and amine synergists have been detected. These results indicate that the developed HPLC-DAD method is in principle suitable for the analysis of food contact materials for the contained photoinitiators and amine synergists. Discussion A HPLC-DAD screening method for the identification and quantification of a total of 63 photoinitiators and amine synergists in extracts of food contact materials was developed. This analysis technique has been widely used for the screening of food contact materials for their photoinitiator

10287-53-3 C11H15NO2 193.11 2.56 315 nm [(1-Chloro-9-oxo-9H– thioxanthen-4-yl)oxy]acetic acid C15H9ClO4S 319.99 2.91 259 nm

and amine synergist content and also the extraction with acetonitrile at 70°C for 24 h (Pastorelli et al. 2008; Sanches-Silva et al. 2008; Koivikko et al. 2010; Lago et al. 2012; Jung et al. 2013). But here it was for the first time applied to the analysis of such a large number of photoinitiators and amine synergists. In addition, with the developed HPLC-DAD method also screening for photoinitiators and amine synergists which are not among the 63 reference substances should be possible. Since the detection wavelengths of the DAD are in the range of the main wavelengths of a standard mercury lamp used for the hardening of UV printing inks and lacquers, also further photoinitiators and amine synergists absorb UV light in the same range. Moreover, for several compounds, i.e. those contained in the concentrated extracts of the analysed food contact materials (cf. Table 10) as well as for numbers 5, 10, 12, 18, 19 and 77, it was shown that a concentration of the extracts by a factor of 10 is possible without significant loss. By the analysis of eight food contact materials, of which the contained photoinitiators and amine synergists were known, it could be demonstrated that the analytes have been determined within the limitations of the developed method. The described extraction procedure is simple and easy performable without high technical effort. In combination with the HPLC-DAD method it can thus be conducted by many laboratories. However, the extraction and concentration method was not validated exhaustively. Especially for plastic materials extraction yields should also be tested by using other solvents and/or techniques, e.g. Soxhlet. Despite a great effort in optimisation, not all “monomeric” photoinitiators and amine synergists could be separated chromatographically. While some of these peak pairs can be distinguished by their characteristic spectra, i.e. numbers 19 and 34, 48 and 77, 15 and 22, 3 and 24, 26 and 28, and 75–3 and 28, others cannot be differentiated,

176.2 261.1 365.2 46 29 39 31

217.2 174.1 70.3 36 27 37 33

Retention time (min) Precursor ion [M + H]+ (m/z) Product ion (m/z) 151.1 150.0 134.1 46 27 37 37

9.7 166.1

H3

Ion spray voltage: 5000 V

Notes: LC conditions are the same as those given in Table 5. Product ions are listed according to their intensity. DP, declustering potential; CE, collision energy.

DP (V) CE (eV)

6.2 438.3

5.4 277.1

Analyte

Compound-specific settings

H2

H1

MRM mode

MS/MS

Curtain gas pressure (N2): 20 psi

Nebuliser gas pressure (N2): 60 psi

Spray parameters

Turbo heater gas pressure (N2): 50 psi

ESI, positive-ion mode

Source

Table 8. LC-MS/MS method for the analysis of the hydrolysis products (H) of the PPIs and PASs (cf. Table 7).

152.0 153.0 77.1 16 47 37 19

15.4 227.1

H4

Turbo heater temperature: 700°C

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213.1 241.0 200.1 51 35 29 67

16.1 287.0

H5

Collision gas pressure: 3 psi

179.1 105.0 77.0 46 27 29 57

18.7 257.1

H6

151.1 134.1 166.1 41 33 41 25

20.5 194.2

H9

35 5

–

21.8 321.0

H 10

Food Additives & Contaminants: Part A 531

532

T. Jung et al.

Table 9. Conversion rates obtained by HPLC-DAD analysis. 2 µg ml−1 No.

10 µg ml−1

45 42 39 25 49 52 42 35

59 55 49 26 76 80 65 53

Conversion rate (%)

Vc (%)

Conversion rate (%)

Vc (%)

53 51 44 25 67 69 63 47

11.4 11.6 9.3 3.8 18.8 18.0 25.3 17.8

57 55 47 25 76 78 73 53

2.7 0.5 4.0 4.5 0.6 1.9 10.9 0.4

56 55 45 24 75 77 81 53

i.e. numbers 8 and 21, 12 and 17, 56 and 57, 43 and 76, and 26 and 75–3. In these cases the HPLC-DAD screening method provides only evidence for two or three substances. But as usually only up to five to seven different photoinitiators and amine synergists are used in one printing ink, it is unlikely that overlaps will occur often. However, separation may be achieved in isocratic mode or by using GC. Due to the better separation properties of GC compared with LC the analysis of, for example, isomers is very easy performable. For the methyl-isomers of benzophenone, i.e. numbers 8, 12 and 21, this was described, for example, by the Food Standards Agency (2011). Another measure to enhance selectivity in most cases may be the use of a mass selective detector. The analysis time of 95 min per sample is comparatively long. For the “monomeric” photoinitiators and amine synergists this can be reduced to approximately 60 min and also for the polymeric ones the main peaks are detected within 60 min. This is sufficient for identification and to subject the corresponding sample to hydrolysis. Further reduction of the analysis time, i.e. to approximately 35 min, can be achieved by using an ultra-HPLC (data not shown). Selectivity and perhaps also LODs may be enhanced by using additionally a fluorescence detector. For example, thioxanthones are known to respond to this detector (Rothenbacher et al. 2007).

Beside identification, the developed HPLC-DAD method can also be applied for the quantification of the “monomeric” photoinitiators and amine synergists. From the amounts of these in the food contact material and the food mass, the maximum possible concentration in the food can be calculated by assuming 100% transfer. These values may then be compared with the maximum acceptable migration limits in food as a basis for a decision whether the packaged foodstuff has to be analysed, or migration tests with simulants should be performed, respectively. But the achieved LODs in combination with the applied extraction and concentration method are not for all substances sufficient to monitor a concentration in the food contact material that would theoretical lead to a contamination below the often applied LOD for non-evaluated substances of 10 µg kg−1 food (1.7 µg dm−2). By application of the European Union model for packages, i.e. 1 kg of food is packaged in a cube with a surface of 6 dm2, and the above described extraction methods, i.e. 0.5 dm2 packaging are extracted with 20 ml solvent and concentrated up by a factor of 10, and assuming 100% migration, it is not possible to achieve this LOD for numbers 48 and 51–1. For cartonboard materials even a much higher mean surface-to-volume ratio of 34 dm2 kg−1 foodstuff was determined (Jung et al. 2013). By using this ratio, in the case of seven photoinitiators and amine Drinking cup

Yoghurt cup

100

4

80 area (mAu*min)

3 2 1 0

60 40 20

No. 29

No. 47

1 h ultrasonic bath

Figure 4.

Mean without values of the 2 µg ml−1 solution

Mean

Conversion rate (%)

area (mAu*min)

Downloaded by [Eindhoven Technical University] at 11:34 17 February 2015

52 53 60 70 38 39 54 59

5 µg ml−1

24 h 40°C

No. 46 5 h 70°C

No. 26 24 h 70°C

0

No. 3 1 h ultrasonic bath

No. 14 24 h 40°C

Photoinitiator and amine synergist yields obtained with different extraction methods.

No. 15 5 h 70°C

24 h 70°C

Food Additives & Contaminants: Part A 13.0

mAU

533

WVL:260 nm

A

IStd 2

10.0

8.0

PPI no. 54

6.0

No. 12 4.0

IStd 1 2.0

0.0

min

–2.0 5.0

0.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

80.0

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30.0

85.0

WVL:260 nm

mAU

B

25.0

H 4: 2–benzoylbenzoic acid

20.0

15.0

10.0

5.0

min

–2.5 0.0

1.3

2.5

3.8

5.0

6.3

7.5

8.8

10.0

11.3

12.5

13.8

15.0

16.3

17.5

18.8

20.0

21.3

22.5

23.8

25.0

26.3

27.5

28.8

30.0

Figure 5. (A) Chromatogram of the extract of cartonboard 2 with the photoinitiators numbers 12 and 54 before hydrolysis. The latter forms two humps of unresolved peaks; and (B) chromatogram of the hydrolysed extract of cartonboard 2, confirming the PPI number 54.

synergists, respectively, it is not possible to control the LOD. A solution therefore could be to use a larger quantity of the packaging material for extraction and/or to concentrate the extract more. For the PPIs and PASs it could be demonstrated that hydrolysis reduces the multitude of signals to only one. This allows easier identification. At the moment quantification might only be possible via standard addition due to very low conversion rates and high variation coefficients. Here optimisation is needed. Furthermore, Lord et al. (2012) have already reported differences in the composition of PPIs and PASs within different batches and producers. However, it is assumed that the influence of these variations is negligible with the hydrolysis method. In addition, it should also be tested which influence the hydrolysis has on the monomeric photoinitiators and amine synergists. As it is common praxis to use PPIs and PASs in combination with monomeric ones, it is possible that these are overlaid by the peaks of the polymeric substances during screening. In this case possibly the hydrolysis could be a solution to identify both, the

monomeric and the polymeric photoinitiators and amine synergists used. Conclusions The HPLC-DAD method reported here allows the screening of packaging materials for a total of 63 photoinitiators and amine synergists. For 13 PPIs and PASs a hydrolysis method was developed that reduces their multitude of UV-detectable substance peaks to only one. This allows the secure identification of these. For the remaining 50 “monomeric” photoinitiators and amine synergists selectivity was enhanced by preparing a database containing all spectra and retention times of the investigated compounds. Furthermore, the retention times of those 50 substances were calculated relative to two internal standards to overcome variances of retention from run to run or due to matrix effects. The developed method was tested for the analysis of food contact materials. Extractions of these were performed with acetonitrile and partially the extracts were subsequently concentrated in a steam of nitrogen. LODs of photoinitiators and amine synergists in concentrated packaging extracts were in the range

534

T. Jung et al.

Table 10. Screening results of eight food contact materials for their photoinitiator and amine synergist content.

Sample number (extraction procedure) Yoghurt cup 1 (plastic, 0.5 dm2 extracted, concentrated)

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Yoghurt cup 2 (plastic, completely extracted, concentrated)

Drinking cup 1 (plastic, 0.5 dm2 extracted, concentrated) Drinking cup 2 (plastic, completely extracted, direct analysis) Drinking bottle (plastic, 0.5 dm2 extracted, direct analysis)

Label (paper, completely extracted, direct analysis)

Cartonboard 1 (0.5 dm2 extracted, direct analysis) Cartonboard 2 (0.5 dm2 extracted, direct analysis and concentrated for hydrolysis)

Known photoinitiator and amine synergist numbers

Detected with the HPLC-DAD method?

29 47 46 26 44 29 47 46 25 44 14 15 3 14 15 3 9 41 1 – – – – 9 10 19 77 15 11 12 18 3 5 10 77 18 5 12 –

Yes Yes Yes Yes No Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes 29 15 11 26 No Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 54

between 0.02 and 5.5 µg dm−2. In addition to the analysis of food contact materials the developed method may also be used for the analysis of food simulants like, for example, ethanol or poly(2,6-diphenyl-p-phenylene oxide) (Tenax®). Thus, compared with other by now available methods, e.g. the UHPLC-MS/MS method published by BMELV (2011) with 28 photoinitiators and amine synergists, nearly two times more “monomeric” photoinitiators and amine synergists can now be analysed in a single run. Fifty-four of the substances included in the method here are listed in Annex 6 of the Swiss Ordinance of the Federal Department of Home Affairs on Materials and Articles, which contains at present the most extensive and latest provisions on printing

Concentrations obtained with the screening method 6.9 9.3 14 41