Article pubs.acs.org/JAFC
Evaluation of Long-Term Migration Testing from Can Coatings into Food Simulants: Polyester Coatings Rafael Paseiro-Cerrato,* Gregory O. Noonan, and Timothy H. Begley Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, College Park, Maryland 20740, United States ABSTRACT: FDA guidance for food contact substances recommends that for food packaging intended for use at sterilized, high temperature processed, or retorted conditions, a migration test with a retort step at 121 °C for 2 h followed by a 10 day migration test at 40 °C should be performed. These conditions are in intended to simulate processing and long-term storage. However, can coatings may be in contact with food for years, and there are very few data evaluating if this short-term testing accurately simulates migration over extended time periods. A long-term migration test at 40 °C with retorted and non-retorted polyester cans using several food simulants (water, 3% acetic acid, 10% ethanol, 50% ethanol, and isooctane) was conducted to verify whether traditional migration testing protocols accurately predict migration from food contact materials used for extended time periods. Time points were from 1 day to 515 days. HPLC-MS/MS was used to analyze polyester monomers, and oligomer migration was monitored using HPLC-DAD/CAD and HPLC-MS. Concentrations of monomers and oligomers increased during the migration experiments, especially in ethanol food simulants. The data suggest that current FDA migration protocols may need to be modified to address changes in migrants as a result of long-term storage conditions. KEYWORDS: food packaging, can coatings, polyesters, oligomers, terephthalic acid, isophthalic acid, nadic acid, long-term migration, stability, hydrolysis, HPLC-DAD/CAD, HPLC-MS/MS
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INTRODUCTION During past years, consumers have become increasingly concerned about the use of bisphenol A (BPA) in food packaging. BPA is a starting substance commonly used in the manufacture of polymeric materials with food contact applications, particularly in epoxy−resin-based coatings. A number of reported toxic effects has been associated with this chemical. However, regulatory bodies, including the FDA and EFSA, continue to conclude that BPA poses no health risk at exposure concentrations.1,2 In an effort to satisfy consumers’ demands and replace food contact materials (FCMs) based on BPA, the food industry is innovating and developing new food packaging, including polyester-based coatings for food cans among others. Polyesters have been extensively used, in particular, polyethylene terephthalate (PET), for food-packaging applications. They are employed in several types of bottles for foods and beverages as well as trays for foods. Unreacted substances from PET migrate into food or food simulants in very small amounts.3,4 Although migrants from PET have been studied, very little has been reported on the relatively new polyester coatings used for canned foods. To evaluate migration from food cans into food, FDA guidance for safety evaluations recommends that when cans have been retorted, pasteurized, or sterilized, a retort step at 121 °C for 2 h followed by a migration test for 10 days at 40 °C should be performed.5 Traditionally, migration testing at 49 °C for 10 days has been considered appropriate to simulate migration from FCMs into food; the temperature was later reduced to 40 °C to harmonize testing protocols between the United States and Europe. However, canned foods may be stored for years (typical “use by” dates are 2−5 years), and This article not subject to U.S. Copyright. Published 2016 by the American Chemical Society
there are very few data evaluating migration over extended time periods even for conventional coatings6−8 and, to the best of our knowledge, none for the newest coatings. During the manufacture of coatings, substances such as monomers, additives, and oligomers may remain unreacted in the polymeric network and potentially migrate into food or food simulants. Monomers and additives must be approved for use in food packaging, and they are relatively easy to track in a migration experiment because there are usually available as standards. However, oligomers formed in can coatings may vary from one coating formulation to another, and there are not available as standards, making them difficult to identify and quantitate throughout a migration experiment. There are limited studies identifying polyester oligomer identification.9,10 In a previous study, we tentatively identified several oligomers from polyester can coatings employed in the presented migration experiments.11 These identified oligomers may be useful to monitor migration and to evaluate the stability of polyester oligomers during a migration experiment. The aim of this work is to monitor the migration of monomers and identified oligomers of polyester can coatings into five food simulants during short- and long-term migration experiments (from 1 day to almost 1.5 years (515 days)) at 40 °C. Data obtained in this investigation will be used to evaluate if current FDA migration testing protocols for safety evaluations accurately predict migration from food cans for extended time periods. Received: Revised: Accepted: Published: 2377
December 11, 2015 February 19, 2016 February 25, 2016 February 25, 2016 DOI: 10.1021/acs.jafc.5b05880 J. Agric. Food Chem. 2016, 64, 2377−2385
Journal of Agricultural and Food Chemistry
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Article
reduced to 40% in 6 min, followed by a reduction to 0% in 0.1 min, and held for 4.9 min. Mobile phase was returned to the starting ratio and held for 5 min of equilibration for a total run time of 45 min. The total run time was 25 min. The MS data were acquired using an electrospray source (ESI) in the negative mode and the Qtrap was set as follows: curtain gas, 55; ion spay voltage, −3500; temperature, 450 °C; ion source gas 1, 40; ion source gas 2, 70; declustering potential, −55. MS/MS transitions were as follows: for IPA and TA, m/z 165.1 to 121.1 (collision energy (CE −18) and 77.1 (CE −25); for NA, m/z 181.0 to 115.3 (CE −18) and 71.5 (CE −30); and for TA-d4, m/z 169.1 to 124.9 (CE −21). Migration Experiment. Polyester can coatings (height × diameter dimensions, 11.2 × 7.6 cm) were supplied by an industrial partner. Five food simulants were tested in this experiment: water, 3% acetic acid, 10% ethanol, 50% ethanol, and isooctane. Cans containing polar food simulants (water, 3% acetic acid, and 10% ethanol) were filled and sealed in an industry partner facility. A set of cans containing the polar food simulants were subjected to a retort step at 121 °C for 2 h. Cans with nonpolar simulants (50% ethanol and isooctane) were filled in the laboratory. For 50% ethanol, to simulate inert conditions (reduced oxygen content), the simulant was conditioned by sonicating for 15 min followed by sparging the solution with a stream of nitrogen for 5 min; afterward, cans were filled and sealed. All cans with simulants were placed at 40 °C for 1, 10, 30, 330, and 515 days. All migration times, except for 10 days, were sampled in triplicate. Upon opening, all samples were immediately transferred into glass vials (endcapped) with polypropylene caps and PTFE septa and stored at 4 °C until analysis. A few retorted cans containing water and 10% ethanol had a yellow color and were found to have some small damage and were discarded. For the 3% acetic acid, all cans were rusted inside and the coating/can interface was obviously degraded. Consequently, the data are not presented here. Control samples were collected prior to filling the cans as well as a zero time point for retorted cans. In addition to migration experiments, extracts of two unused polyester cans were performed by five consecutive extractions with ACN at 40 °C for 24 h. Method Development. For monomer determination, a concentration step of simulants was performed. For the water, 25 mL was spiked with formic acid, to reach a final acid concentration of 0.1%, and then shaken. Simulant was loaded on a conditioned (3 mL of methanol and 3 mL of water) Agilent Bond Elut C18, 500 mg, 3 mL column mounted on a vacuum manifold (pressure was maintained below 2 mmHg). The cartridge was eluted with 2 mL of ACN, which was concentrated to 1 mL under a N2 stream at 40 °C using a Techne Dry Block (Staffordshire, UK) sample concentrator. For concentrations of 10% ethanol, 50% ethanol, and isooctane, 25 mL was concentrated almost to dryness using a sample concentrator Rapivap (Labconco Corp., Kansas City, MO, USA). After concentration, 5 mL of ACN was added to the glass vials, which contained the remaining simulant (except for the isooctane, where 1 mL of THF and 4 mL of ACN were added). Afterward, the solutions were evaporated almost to dryness under inert conditions and redissolved in 1 mL of ACN. ACN concentrated solution (0.2 mL) was diluted with 0.8 mL of water and spiked with TA-d4 used as internal standard. Vials were shaken and then analyzed by HPLC-MS/MS. For analysis of oligomers, in the water and 10% ethanol retorted cans and 50% ethanol, the pure simulants were analyzed by HPLCDAD/CAD and HPLC-MS. For oligomers with low concentration in samples, especially in the non-retorted cans, the same concentration step described above for monomers was performed. Concentrated solutions were both analyzed directly and further diluted with water (80%) prior to analysis. The dilution was required to produce better chromatographic resolution of the more polar compounds. To determine the recovery of the concentration steps, water-based simulants were spiked with an acetonitrile solution containing PET cyclic trimer, BHET, 1/2MET, 1/2DET, MET, and DET; the spiking levels were between 0.02 and 0.2 μg/mL. The isooctane food simulant was spiked with a THF solution of the same compounds at 0.04 μg/ mL. Recovery of monomers was evaluated with a similar protocol as
MATERIALS AND METHODS
Chemicals and Standard Solutions. Acetonitrile (ACN) (Optima for LC-MS), methanol (Optima for LC-MS), isooctane (Optima), and water (Optima for LC-MS) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Tetrahydrofuran (THF) for HPLC and ethyl alcohol (absolute) were purchased from Acros (Morris Plains, NJ, USA). N,N-Dimethylacetamide (NNDA) chromasolv Plus for HPLC ≥99.9% was purchased from Sigma-Aldrich. Acetic acid (glacial) was from Fisher Scientific. 1,1,1,3,3,3-Hexafluoro-2-propanol(HFP) 99.9%, bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic anhydride (NAD) ≥95%, isophthalic acid (IPA) 99%, terephthalic acid (TA) 98%, and terephthalic -2,3,5,6-d4 acid (TA-d4) 98 atom % D 98% were purchased from Aldrich. Bis(2-hydroxyethyl) terephthalate (BHET) was acquired from Polysciences Inc. PET cyclic trimer was purchased from Eastman Chemical Products Inc. Terephthalic acid ethyl ester (DET), terephthalic acid methyl ester (MET), terephthalic acid monoethyl ester (1/2DET), and terephthalic acid mono methyl ester (1/2MET) were synthesized in our laboratory. Formic acid for LC-MS 98% was purchased from Fluka (St. Louis, MO, USA). Stock standard solutions of IPA, TA, 1/2MET, 1/2DET, MET, and DET were prepared in 20% NNDA/ACN. Nadic acid (NA) solution was prepared by dissolving NAD in water. Stock solution of BHET was prepared in ACN, and PET cyclic trimer was prepared in HFP. Working solutions were obtained by volumetric dilution in 20% ACN/ H2O except for PET cyclic trimer, which was diluted in ACN. Instrumentation. HPLC-DAD/CAD. An Agilent 1100 HPLC (Agilent Technologies, Wilmington, DE, USA) with an Agilent Zorbax Extend C18, 80 Å (2.1 × 150 mm i.d., 3 μm) column was coupled in series to a DAD (Agilent Technologies) and to a Dionex Corona CAD (Thermo Scientific ultra RS charged aerosol detector, Germering, Germany), respectively. The mobile phases consisted of water (A) and acetonitrile (B). The gradient, starting at 80% of A, was held for 2 min. Then A was reduced to 50% over 3 min and then A was reduced to 0% in 25 min and held for 10 min. Mobile phase was returned to the starting ratio and held for 5 min of equilibration for a total run time of 45 min. The flow rate was 0.3 mL/min, and the injection volume was 10 μL. The DAD was set in the range of 200− 400 nm with 225, 242, 254, 288, and 330 nm. For Corona CAD, full scan range was at 100 pA, power function was 1.00, and filter was set to 4. For nebulizer gas, pressure was 35 psi, temperature set point was 25 °C, and ion trap volt was 20.5 V. Nitrogen was used as gas. Data analysis was performed using an Agilent Chem-Station software. HPLC-MS. High-performance liquid chromatography−mass spectrometry was performed using a Shimadzu UFLC XR (Shimadzu Corp.) coupled to an AB Sciex 5500 Qtrap controlled by Analyst software (Applied Biosystems). An Agilent Polaris 3 (C18) (150 × 2.0 mm) column thermostated at 30 °C was used for separation with a flow rate of 0.3 mL/min. Autosampler temperature was 15 °C. The injection volume was 5 μL. The mobile phases consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient, starting at 80% of A, was held for 10 min. Then A was reduced to 50% in 3 min, followed by a reduction to 0% in 25 min, and held for 10 min. Mobile phase was returned to the starting ratio and held for 5 min of equilibration for a total run time of 45 min. The total run time was 45 min. The MS data were acquired using an electrospray source (ESI) in the positive (+) and negative (−) modes, and the Qtrap was set as follows: full scan mode (from m/z 100 to 1000); scan rate, 1000 Da/s; curtain gas, 30; ion spay voltage, −4000 V for negative mode and 4000 V for positive mode; temperature, 350 °C; ion source gas 1, 40; ion source gas 2, 70; declustering potential, 35; and entrance potential, 10. HPLC-MS/MS. Analysis of carboxylic acids was achieved by using a Shimadzu UFLC XR coupled to an AB Sciex 4000 Qtrap controlled by Analyst software. Agilent Polaris 3 (C18) (150 × 2.0 mm) thermostated at 30 °C was used as stationary phase. Autosampler temperature was 15 °C. The injection volume was 5 μL. Flow rate was 0.1 mL/min. The mobile phases consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient, starting at 90% of A, was decreased to 80% in 10 min. Then A was 2378
DOI: 10.1021/acs.jafc.5b05880 J. Agric. Food Chem. 2016, 64, 2377−2385
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Table 1. Oligomers Tracked by Using LC-MS and Compounds Tracked by Using HPLC-DAD during the Migration Experiment HPLC-MS
a
HPLC-DAD
compound
mode
m/z
tRa
IPA + MBO IPA + 2MBO NA + COH IPA + COH NA + COH + MBO IPA + COH + MBO IPA + NA + 2MBO 2IPA + 2MBO IPA + NA + COH + MBO IPA + TOH + COH 2IPA + 2MBO + COH 3IPA + 2MBO + COHc IPA + NA + 2COHc
− + + − + + + − + + + + +
237.0 311.0 331.0 291.0 381.0 387.0 497.0 457.0 551.2 425.0 607.0 715.0 587.0
5.9 6.9 6.5 7.3 8.0 8.1 8.7 9.0 11.2 11.1 12.4 19.7 23.3
compound unknown unknown unknown unknown unknown unknown
1 2 3 4 5 6
(U1) (U2) (U3) (U4) (U5) (U6)
maxb (nm) 225, 225 214, 242 225, 225,
266, 342
tRa
266, 342 266, 342
2.3 2.7 6.6 8.0 9.4 17
IPA + 2MBO
214, 236, 288
7.4
IPA + COH + MBO
214, 236, 288
9.2
2IPA + 2MBO
214, 236, 288
11.5
236, 288
Retention time in minutes. bWavelengths with maximal coefficient of molar absorption in the spectra. cCyclic oligomers.
Figure 1. Spectra of the unknowns tracked by HPLC-DAD: (A) unknown 2; (B) unknowns 1, 5, and 6; (C) unknown 4, which could correspond with a TA derivative; (D) unknown 3, which corresponds with an IPA derivative. oligomers; however, the spiking concentrations were from 0.1 to 0.2 μg/mL. Stability tests were performed on BHET and PET cyclic trimer and on monomers, in 50% ethanol held at 40 °C for 10, 30, and 120 days. The spiking concentration was from 0.04 to 0.4 μg/mL, and all testing was performed in duplicate. Acetonitrile can extracts were performed in duplicate. Extracts were analyzed using the same methods applied to the food simulants, except the concentration step from 25 to 1 mL was performed under a N2 stream at 40 °C. Acetonitrile extract samples were not analyzed by HPLC-MS.
during the long-term storage experiment. The monitored oligomers were identified in a previous study11 and are products of the reaction of carboxylic acids such as TA, IPA, and NA with polyols such as 1,4-cyclohexanedimethanol (COH) and 2-methyl-1,3-propanediol (MBO) and one chain stopper, 4-tert-butylphenol (TOH). However, there are no standards of the oligomers available to determine concentrations migrating into food simulants. Therefore, BHET (m/z 255.0) and PET cyclic trimer (m/z 577.0) were used to quantitate linear and cyclic oligomers, respectively, and 1/ 2DET (m/z 193.0) was used as a proxy during mass spectrometry in negative mode analysis. Although the use of these proxy compounds may not give accurate concentrations for each of the oligomers, it does provide relative differences
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RESULTS AND DISCUSSION Development of Analytical Methods and Data Processing of Migrants. The aim of the work was to track the migration of monomers (IPA, TA, NA) and oligomers 2379
DOI: 10.1021/acs.jafc.5b05880 J. Agric. Food Chem. 2016, 64, 2377−2385
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Table 2. Recovery of Polyester Derivatives and Monomers in Food Simulantsa simulant water 3% acetic acid 10% ethanol 50% ethanol isooctane a
BHET 93 92 92 98 88
± ± ± ± ±
1.7 1.5 2.8 2.1 8.7
trimer 65 71 106 92 100
± ± ± ± ±
7.6 7.7 1.4 0.3 4.4
1/2MET 96 100 102 109 88
± ± ± ± ±
5.8 1.7 7.3 12.3 4.2
1/2DET 38 120 101 103 88
± ± ± ± ±
1.8 10.9 1.5 6.6 4.1
MET
DET
± ± ± ± ±
85 ± 7.3 88 ± 2.4 0 0 87 ± 6.1
93. 91.7 32.5 38.5 66
6.5 9.3 5.3 26.7 20.5
TA 94 88 91 86 66
± ± ± ± ±
IPA
1.2 7.6 1.4 3.1 15.9
96 100 85 80 85
± ± ± ± ±
NA 0.8 9.9 2.8 2.8 9.8
94 88 85 85 84
± ± ± ± ±
1.5 4.5 2.5 2.9 7.6
Recovery (mean (%) ± SD (n = 3−5)). Spiking level between 0.02 and 0.2 μg/mL.
between time points, allowing the study to determine what changes in oligomer concentrations take place at extended time points. Additionally, the proxy compounds give confidence in the different employed detectors’ performance in terms of variability, coefficient correlation, accuracy, LOD, and LOQ. Several authors have used similar approaches in previous studies.12,13 It is necessary to state that what is relevant in this study is the monitoring of the relative change in intensity of the tracking compounds during the migration time. The migration of oligomers from days 1 to 515 was monitored using a long (40 min) chromatographic separation and three different detectors for aromatic polyesters such as DAD, CAD, and MS detectors. The use of a long chromatographic runtime in the method will permit the analysis of polar and nonpolar migrants. The different detectors were used with the aim of a comprehensive detection of possible migrants that may have different responses in different detectors and to observe how the profile of the migrants behaves during the long-term migration test. In addition, the good correlation among data obtained with different detectors will give confidence in the migration results during the experiment. The mass spectrometer was used to monitor 13 compounds during the migration experiments (Table 1). These oligomers were selected for analysis due to their different polarities, structure (linear or cyclic compound), and higher concentration in the samples. The employed standard had acceptable precision (2.5−4.5% RSD (n = 7−9)), good correlation coefficient (r2 > 0.99) across the concentration range (from 0.05/0.1 to 1 μg/mL), and LOD from 0.01 to 0.03 μg/mL. The advantage of the MS detector is that it is more selective than DAD or CAD and can supply valuable information tracking selected compounds. The diode array detector is a universal detector for aromatic polyesters with a good % RSD = 2.6 and sensitivity (LOD = 0.08 μg/mL) for BHET at 225 nm. This wavelength was selected as a compromise to quantify oligomers based on the studies conducted by Schaefer 2004 and also taking into account that most of oligomers in this polyester can coating are based on isophthalic acid.11 Of the identified oligomers, only IPA + 2MBO, 2IPA + 2MBO and IPA + COH + MBO were identified in the simulants using the DAD. However, several “unknown” compounds that have a significant response were also monitored; these compounds are labeled as unknowns in Table 1. Unknown 2 does not belong to the family of phthalate polyester because it has a higher response at 225 nm and lacks a maximum in its spectra particularly at 236 and 242 nm, which typically correspond to TA and IPA oligomers respectively (Figure 1A). Unknowns 1, 5, and 6 have also different spectra as polyester derivatives with maxima at 225, 266, and 342 nm (Figure 1B). Unknown 4 has a spectrum similar to that of a phthalate polyester derivative based on TA (Figure 1C). Unknown 3 has a relevant response in the detector, and it does
have the same spectrum as phthalate polyester derivatives based on IPA (Figure 1D); however, its structure was not identified. These compounds will be monitored during the migration experiments to observe how they behave during the long-term migration experiment. With regard to the CAD, no significant improvement in sensitivity was observed in comparison to DAD, so only UV data are presented. Standards were obtained for the target monomers (TA, IPA, and NA). Because of confirmation requirements, it was decided to analyze them by using HPLC-MS/MS. The performance of the method was r2 ≥ 0.99, % RSD (between 1 and 2), and limit of detection (0.005, 0.03, and 0.06 μg/mL for NA, TA, and IPA respectively). The LOD was calculated as 3 times the S/N (n = 7) of the blanks. LOQ was calculated as 3 times the LOD. For analyte confirmation, the two transitions for each monomer should match with ±20% regard to the ratio of the standards. Sample Treatment and Recovery. On the basis of intralaboratory experience, some compounds would not have appropriate sensitivity, particularly in the non-retorted cans; consequently, a concentration step from 25 to 1 mL was required. To prove that the concentration step was appropriate for oligomers and monomers, recoveries of available standards of polyester oligomers BHET, 1/2DET, and PET cyclic trimer and monomers (NA, TA, and IPA) were conducted. In addition, it was decided to include 1/2DET, MET, and DET as polyester derivatives to give more confidence in the recovery results. An SPE C18 column was used to concentrate samples in water food simulant. To retain the monomers in the SPE C18 column, simulant was spiked with formic acid to achieve 0.1%. To prove that no hydrolysis occurs by adding the acid, a pretest was conducted comparing the results with water sample concentrated by using a sample concentrator. No differences were observed for monomers and oligomers by using the acid at this concentration. Acceptable recoveries were obtained for most of compounds (Table 2). Low recoveries were obtained for MET in ethanol solution, this was expected since this compound is not stable in ethanol simulants.14 For DET, recoveries were zero in ethanol food simulants, most likely due to it has similar behavior as MET in these types of simulants. Recovery for PET cyclic trimer was from 65 to 71% in water and 3% acetic acid most likely due to low solubility in the most polar simulants. Recovery analyses were performed by using HPLC-DAD for the polyester derivatives and HPLC-MS/MS for monomers. Stability Test of Polyester Derivatives and Monomers. Separate stability tests were carried out for oligomeric (BHET, PET cyclic trimer) and monomeric (TA, IPA, and NA) analytes. The tests were conducted in 50% ethanol with sampling at 10, 30, and 120 days. This simulant was selected as intermediate simulant among polar simulants and nonpolar simulants. For the monomers, acceptable recoveries were obtained (87−103%) in 50% ethanol throughout the 120 days. 2380
DOI: 10.1021/acs.jafc.5b05880 J. Agric. Food Chem. 2016, 64, 2377−2385
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However, for the oligomers, decreases in concentration were observed during the experiments. Recovery for BHET was 55% at 10 days and 21% at 120 days. For PET cyclic trimer, recovery at 10 days was 30% and was 0% at 30 days. As the concentration decreased for the test oligomers, new peaks appeared at shorter tR than for the PET cyclic trimer. These new peaks had spectra similar to those of the oligomers, suggesting structural similarities between the oligomers and these new compounds. On the basis of the structural similarity, shorter retention times, and solvent system, it is likely that the oligomers were undergoing hydrolysis to produce smaller, more polar, products. To evaluate this potential hydrolysis, samples were analyzed with an UHPLC-HRMS using the same method as PaseiroCerrato et al.11 The identified compounds corresponded with PET cyclic trimer, BHET, and the proposed product of hydrolysis are presented in Table 3. All of them were in the
COH increase over the same time period. One likely explanation for these differing trends is that oligomers IPA + 2MBO and IPA + COH + MBO are suffering hydrolysis, which produced IPA + MBO and IPA + COH (Figure 2). In the three
Table 3. Identified Compounds in PET Cyclic Trimer and BHET Stability Test in 50% Ethanol
Figure 2. Migration of some polyester derivatives in water-retorted cans.
compound b
3TA + 3EG 3TA + 3EG 2TA + 2EG TA + 2EGc TA + EG TA
mode
exact mass
tRa
positive positive negative positive negative negative
577.1341 595.1446 401.0877 255.0863 209.0455 165.0193
12.8 10.6 9.1 6.8 6.6 6.1
cans containing water and stored for 515 days, the simulant became yellow, and two of the cans showed evidence of oxide sediments in the water simulant. The hydrolysis of polyester resins is described in the literature,15 and it has been demonstrated here in using stability tests that resulted in yields of oligomers of lower molecular weight. These results show that hydrolysis of some polyester compounds may occur. The results of the migration of monomers into water are similar to those noted for the oligomers, with the majority showing little to no change in concentration from days 1 to 515 (Table 5). The one exception is NA, which exhibits a drop in concentration at day 330 and an even more dramatic drop at day 515. Given the stability of the other compounds and no repetition of this trend in 10% ethanol, it is unclear what produced this decrease in concentration. Retorted Cans Containing 10% Ethanol. For 10% ethanol food simulants in retorted cans, there is an increase in the concentration of some of the migrants in the simulant from 30 days to 515 days (Table 6). However, from 10 to 330 days high molecular weight oligomers such as 2IPA + 2MBO + COH, cyclic 3IPA + 2MBO + COH, and cyclic IPA + NA + 2COH, decrease in concentration from 9 to 3 μg/dm2, from 1.6 to 0.4 μg/dm2, and from 0.9 to 0.1 μg/dm2, respectively (data not presented in the table). This suggests that high molecular weight oligomers could undergo hydrolysis and yield monomers and low molecular weight oligomers. In terms of
a c
Retention time in minutes. bPET cyclic trimer. EG is ethylene glycol. BHET.
range of the acceptable 5 ppm mass accuracy agreement. This indicates that these polyester oligomers undergo a degradation process in the selected simulant over time. This may have an impact on the migration experiments and the interpretation of the migration results, because the migrating oligomers may have behaviors similar to those of BHET and PET cyclic trimer. Long-Term Migration Test of Retorted Cans. Retorted Cans Containing Water. Oligomer migration results for retorted cans containing water are presented in Table 4. The concentrations of a majority of the compounds show very little change from 1 to 330 days, but then exhibit a greater change from 330 to 515 days. A number of the oligomers (IPA + 2MBO and IPA + COH + MBO) show a significant decrease at the 515 day point. This trend was noted for both the MS and DAD results, suggesting it is not the result of an interfering compound suppressing ionization in the later time points. Alternatively, the concentrations for IPA + MBO and IPA +
Table 4. Compound Migration Results for HPLC-MS and HPLC-DAD for Tracked Compound in Water Retorta HPLC-MS IPA + MBO
IPA + COH
NA + COH
IPA + 2MBO
2IPA + 2MBO
NA + COH + MBO
IPA + COH + MBO
IPA + NA + 2MBO
IPA + TOH + COH
U2
1 10 30 330 515
39 ± 3 37 37 ± 2 48 ± 4 75
47 ± 3 44 46 ± 8 50 ± 1 91
91 ± 7 88 80 ± 8 72 ± 7 77
94 ± 13 95 90 ± 4 75 ± 22 26
16 ± 1 16 16 ± 1 16 ± 1 15
22 ± 3 24 21 ± 2 18 ± 1 22
77 ± 9 69 68 ± 9 54 ± 15 23
26 ± 1 28 22 ± 2 21 ± 4 19
61 ± 5 50 58 ± 10 40 ± 9 31
61 ± 9 57 66 ± 6 147 ± 1 131
PRc
40
64
96
70
16
23
58
31
61
time
a
HPLC-DAD
b
Data expressed in μg/dm2.
b
57
U5
IPA + 2MBO
IPA + COH + MBO
132 ± 2 123 123 ± 3 124 ± 3 107
74 ± 5 68 68 ± 1 61 ± 4 11
93 ± 4 89 85 ± 4 76 ± 8 28
128
68
90
Times are in days. cPR, post-retort time zero. 2381
DOI: 10.1021/acs.jafc.5b05880 J. Agric. Food Chem. 2016, 64, 2377−2385
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to prove the hypothesis for the hydrolysis of these particular polyester derivatives. Similar results were observed for polyester derivatives in the DAD, where compounds increase from 117 to 139 μg/dm2 (increase of 19%) for compound IPA + 2MBO and from 167 to 210 μg/dm2 (increase of 26%) for IPA + MBO + COH. On the other hand, unknown 2 has an increase of 152% with respect to the 10 day migration point. For the analyzed monomers (Table 5), an increase in concentration occurs after 10 day migration experiments, when increments above 59% were achieved (i.e., from 17.1 to 27.3 μg/dm2 for IPA). These also may be attributed to a hydrolysis process. Additionally, when the concentrations of some compounds are plotted versus the square root of time (Figure 3), a trend is seen similar to that of
Table 5. Monomer Migration Times for Retort Cans with 10% Ethanol, for Retort Cans with Water, for Non-retorted Cans with 50% Ethanol, and for Total Extract with Acetonitrile (ACN) at 40 °C for 24 ha LC-MS/MS simulant
time (days)
NA
IPA
TA
1 10 30 330 515
3 ± 0.2 2.6 2.7 ± 0.2 1.4 ± 0.7 0.5
14.5 ± 0.3 15.2 16.6 ± 1.2 14.6 ± 4.7 12.7
1.6 ± 0.1 1.8 2 ± 0.1 2.3 ± 0.6 1.8
PRb
2.7 ± 0.1
16.7 ± 1.4
1.9 ± 0.5
1 10 30 330c 515c
4.1 ± 0.1 4.4 4.7 ± 0.7 6.5 7.6
13.8 ± 1.7 17.1 17 ± 1.5 21.1 27.3
1.7 ± 0.3 1.7 2.2 ± 0.1 2.4 4.3
PRb
4.4 ± 0
18.1 ± 0.5
1.9 ± 0.3
50% ethanol
1 10 30 330 515
ND ND ND 1.5 ± 0.1 3.2 ± 0.04
ND ND 9.2 ± 0.3 16.6 ± 0.6 18.3 ± 0.6
ND 0.3 0.3 1.2 ± 0.2 1.9 ± 0.1
ACN
extract
0.3
water
10% ethanol
6.4
Figure 3. Migration of two polyester derivatives in 10% ethanol retorted cans.
0.5
Data expressed in μg/dm2. bPost-retort time zero. cResults for this time are an average of two samples. a
the results obtained in 50% ethanol in non-retorted cans (Figure 4). This increase may suggest that interaction between polymer and simulant could also happen, but this hypothesis will be discussed later. The increase in concentration of some compounds during the migration experiment shows that the migration test at 10 days could not address exposure to long storage times for some migrants.
mass transfer, hydrolysis would not explain the total increase of the low molecular weight oligomers. However, not all high molecular weight oligomers were determined, and these oligomers may form several low molecular weight products (i.e., 2IPA + 2MBO + COH may yield IPA + MBO + COH and IPA + MBO). Standards of oligomers would be necessary
Table 6. Compound Migration Results for HPLC-MS and HPLC-DAD for Tracked Compounds in 10% Ethanola HPLC-MS
a
time (days)
IPA + MBO
IPA + COH
NA + COH
IPA + 2MBO
2IPA + 2MBO
NA + COH + MBO
IPA + COH + MBO
IPA + NA + 2MBO
IPA + TOH + COH
IPA + COH + NA + MBO
1 10 30 330b 515b
47 ± 1 55 55 ± 6 68 73
73 ± 6 90 82 ± 7 106 109
125 ± 8 141 134 ± 7 134 152
153 ± 11 133 149 ± 12 203 204
22 ± 0.2 24 23 ± 1 24 27
37 ± 9 40 37 ± 13 60 64
190 ± 8 163 172 ± 11 236 267
48 ± 6 54 49 ± 5 51 54
44 ± 4 44 40 ± 5 34 39
147 ± 1 137 150 ± 4 116 143
PRc
51
74
137
141
22
43
52
43
157
time (days)
U1
U2
U3
IPA + 2MBO
2IPA + 2MBO
U4
IPA + COH + MBO
U5
U6
1 10 30 330b 515b
165 ± 7 174 176 ± 6 214 219
46 ± 3 48 59 ± 3 94 121
49 ± 1 48 46 ± 2 41 43
117 ± 7 117 114 ± 3 135 139
110 ± 6 112 114 ± 8 116 122
12 ± 1 13 14 ± 0.4 18 19
162 ± 4 167 166 ± 4 199 210
169 ± 4 171 176 ± 2 170 170
66 ± 1 65 66 ± 2 77 81
PRc
166
53
53
113
116
12
154
176
67
180 HPLC-DAD
Data expressed in μg/dm2. bResults for this time are an average of two samples. 2382
c
Post-retort time zero. DOI: 10.1021/acs.jafc.5b05880 J. Agric. Food Chem. 2016, 64, 2377−2385
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Article
in increased migration and more potential for hydrolysis products. When data were plotted versus the square root of time (Figure 4), behavior very similar to the results presented by Begley et al.17 was observed for the migration of a UV stabilizer from PET. Begley explains this phenomenon as a consequence of ethanol most likely interacting with the PET polymer. This may also have an impact on the current migration results. On the other hand, coatings are cross-linked polymers with a glass transition temperature (Tg) probably higher than the temperature of the migration experiment. Diffusion may slow when the migration temperature is below the Tg. Consequently, the slow diffusion of the analyzed compounds into the simulant has also to be considered when estimating the final concentration after many years of storage in contact with a food. Is important to state that results obtained in 50% ethanol at the 515 day point are close to results obtained in retort cans with 10% ethanol and water, which means that during the retort step the interaction, polymer−simulant and/or hydrolysis, as well as coefficient diffusion of migrants is accelerated in the heating process. Non-retorted Cans Containing Water. With regard to testing water non-retorted cans, very little migration occurred after the 10 day migration test. Only in concentrated samples (from 25 to 1 mL) were quantifiable amounts determined in LC-MS from 0.6 μg/dm2 for IPA + 2MBO to 1.6 μg/dm2 for IPA + MBO + COH at 1 year migration point. For the rest of the analytical techniques no detected levels of compounds were found. Non-retorted Cans Containing 10% Ethanol. For tests using 10% ethanol in non-retorted cans, migration did not stop after 10 days; however, the amount of migration was low. Only when test samples were concentrated 25 times were quantifiable concentrations found in DAD for IPA + 2MBO (levels of 12 ± 1 μg/dm2 for 330 days and 15 ± 1 μg/dm2 at 515 days), IPA + MBO + COH (from 21 ± 1 μg/dm2 for 330 days and 24 ± 1 μg/dm2 at 515 days), and unknown 5 (levels of 6 ± 1 μg/dm2 for 330 days and 8 ± 1 μg/dm2 at 515 days). By using LC-MS, detectable amounts of IPA + COH and IPA +
Figure 4. Migration of some polyester derivatives in 50% ethanol nonretorted cans.
It is important to remark that the majority of compounds analyzed by using the different detection techniques remain stable to 30 days of migration, which means that the increases in concentration occur at a slow rate during the migration test time in 10% ethanol. Besides, in the worst-case scenario, migrations for TA and IPA are well below the specific migration limits proposed in the European Union.16 Long-Term Migration Test of Non-retorted Cans. Nonretorted Cans Containing 50% Ethanol. Cans with 50% ethanol were not subjected to a retort step because of laboratory safety reasons, and only results for non-retorted cans are available. In 50% ethanol food simulant, all tracked compounds increase significantly in concentration during the migration experiment from 10 to 515 days. This was true for oligomers as well as for monomers (Tables 5 and 7). The increase in monomers is consistent with the hydrolysis discussed earlier. The increase in oligomers is associated with the migrations of large compounds (>1000 Da), because an increased ethanol concentration can reduce the resistance to mass transfer for higher molecular weight oligomers, resulting
Table 7. Compound Migration Results for HPLC-MS and HPLC-DAD for Tracked Compounds in 50% Ethanol and Total Extract with Acetonitrile (ACN) at 40 °C for 24 ha HPLC-MS
simulant
time (days)
IPA + MBO
IPA + COH
NA + COH
IPA + 2MBO
2IPA + 2MBO
NA + COH + MBO
1 10 30 330 515
1 1 15 ± 0.1 45 ± 5 63 ± 4
1 11 15 ± 0.2 62 ± 7 102 ± 3
ND 4 4 111 ± 11 156 ± 11
ND 4 29 ± 7 193 ± 7 299 ± 6
ND 1 1 19 ± 0.3 30 ± 2
ND ND ND 36 ± 4 73 ± 4
times (days)
U1
U2
U3
IPA + 2MBO
2IPA+2MBO
IPA + COH + MBO
U4
U5
U6
1 10 30 330 515
ND ND ND ND ND
ND 11 61 ± 3 80 ± 9 133 ± 5
ND ND ND 11 52 ± 3
ND 11 11 131 ± 6 199 ± 3
ND ND ND 61 ± 1 120 ± 4
11 11 11 170 ± 8 292 ± 8
ND ND 4 4 54 ± 1
ND ND 11 91 ± 4 148 ± 2
ND ND 11 72 ± 1 108 ± 4
extract
21
17
ND
26
47
ND
9
20
50% ethanol
simulant
50% ethanol
ACN a
22
IPA + COH + MBO ND 4 18 ± 3 194 ± 4 348 ± 29 HPLC-DAD
IPA + NA + 2MBO
IPA + TOH + COH
IPA + COH + NA + MBO
2IPA + 2MBO + COH
ND ND 4 18 ± 1 31 ± 1
ND ND ND 31 ± 0.3 69 ± 6
ND ND 21 ± 4 128 ± 14 176 ± 40
ND ND ND 32 ± 6 81 ± 2
Data expressed in μg/dm2. 2383
DOI: 10.1021/acs.jafc.5b05880 J. Agric. Food Chem. 2016, 64, 2377−2385
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limit of concerns, short test times could lead to underestimating exposure to some reaction products that result only from long storage times. These results suggest that FDA migration protocols may need to be revised to address changes in migrants as a result of long-term storage. Future analysis in real food would be required to guide decisions on the guidance modification.
MBO were achieved at 30 days of migration and IPA + COH + MBO migration was of 1 ± 0.2 μg/dm2. In addition, migration amounts to 1 year increased to 16 ± 1 and 33 ± 1 μg/dm2 for IPA + 2MBO and IPA + COH + MBO, respectively. For the rest of the compounds except for cyclic 3IPA + 2MBO + COH and cyclic IPA + NA + 2COH, migration was from 1 to 6 μg/ dm2 at 330 days point. Migration amounts for monomers for 330 and 515 days were for NA and IPA from 0.5 to 1.6 μg/dm2 and from 1.5 to 2.2 μg/dm2, respectively, and for TA a detected level of 0.3 μg/dm2 was found after 30 days of migration. For non-retorted cans results into water, 10% ethanol, and 50% ethanol indicate that migration does not stop after 10 days of migration at 40 °C in the test polyester can coatings. Non-retorted Cans Containing Isooctane. With regard to the isooctane food simulant, potential migrants were not detected. These results were expected because polyesters are polar polymers and are most likely not soluble in a nonpolar simulant such as isooctane. Acetonitrile Can Extraction of Migrants. To prove the hypothesis of hydrolysis, cans were extracted with ACN at 40 °C for 1 day and for 5 consecutive days. It was observed that all compounds were extracted in the first day. Results are presented in Table 5 for monomers and Table 7 for oligomers (results for oligomers were determined only using DAD). Data show that compounds are in lower concentration in the acetonitrile extract than in the simulants. This supports the hypothesis, presented earlier, that compounds might suffer from hydrolysis and/or that simulants interact with polymer. Because compound concentrations increase in 50% ethanol during time, an additional test was performed extracting the coating with ACN to 10 days to see if concentration increased over time in acetonitrile as well, but the same result for 1 and 10 day extracts were obtained. It is important to remark that some compounds, such as U1 and U3, were not detected in the ACN extraction; this is probably because these compounds are not present as unreacted compounds in the coating, and they are formed from the degradation of other compounds with a higher molecular weight. Moreover, in the ACN extract, compounds with higher concentration elute in the last part of the chromatogram; these compounds have the same spectra as an IPA derivative and most likely are oligomers with higher molecular weight (>1000 Da). These compounds may also migrate into the simulant and suffer hydrolysis into smaller oligomers. That could also explain why simulants have higher concentrations of migrants than in the can ACN extract. Briefly, a long-term migration study was conducted in polyester food can coatings to evaluate current FDA migration protocols. The migration experiment was performed at 40 °C from 1 to 515 days using water, 3% acetic acid, 10% ethanol, 50% ethanol, and isooctane as food simulants. The 3% acetic acid results suggest that this simulant is not appropriate for testing this particular type of polyester coating using the type of can fill conditions in this study. Nondetectable concentrations of polyester derivatives and monomers were obtained in isooctane. In water, 10% ethanol, and 50% ethanol, increases in concentration were achieved for several compounds beyond the conventional 10 day migration test; this is especially true for ethanol-based simulants. This increase in concentration may be attributed to a hydrolysis process of polyester oligomers or to the interaction between the simulant and the coating. It is important to note that significantly longer test times are needed to detect increases in hydrolysis reaction products. Although in the worst-case scenario monomer migration is well below the
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AUTHOR INFORMATION
Corresponding Author
*(R.P.-C.) Phone: (240) 402-1370. Fax: (301) 436-2634. Email:
[email protected]. Funding
This project was supported in part by an appointment to the Research Participation Program at the Center for Food Safety and Applied Nutrition administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. Notes
The authors declare no competing financial interest.
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REFERENCES
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