d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
Available online at www.sciencedirect.com
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Determination of homologous distributions of bisEMA dimethacrylates in bulk-fill resin-composites by GC–MS Jürgen Durner a,∗ , Klaus Schrickel b , David C. Watts c , Nicoleta Ilie a a
Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig-Maximilians-University of Munich, Goethestr. 70, 80336 Munich, Germany b Thermo Fisher Scientific, Scientific Instruments, Im Steingrund 4–6, 63303 Dreieich, Germany c School of Dentistry and Photon Science Institute, University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. Ethoxylated bisphenol A dimethacrylate (bisEMA) is a basis monomer in several
Received 9 December 2014
dental resin composites. It was the aim of the present study to develop a method allowing
Received in revised form
detection of bisEMA and its different degrees of ethoxylation eluted from polymerized resin
10 February 2015
composites.
Accepted 10 February 2015
Methods. High-temperature gas chromatography/mass spectrometry (HT-GC/MS) by direct on-column injection was used to identify ethoxylated bisEMA in ethanol/water (3:1) eluates from polymerized specimen of four bulk-fill resin composites – Venus® bulk fill, Surefil®
Keywords:
SDRTM flow, FiltekTM Bulk Fill and Sonic FillTM . Additionally, the unpolymerised pastes were
GC/MS
analysed.
bisEMA
Results. The developed method allowed identification of a homologous series of bisEMA up to
Bisphenol A ethoxylate
twelve ethoxy groups in the unpolymerised materials. The molecular masses of the homolo-
dimethacrylate
gous bisEMA varied between 452 g/mol and 892 g/mol and were detected for retention times
Elution
from 9.43 min to 13.36 min. Analysis of eluates from polymerised materials identified bisEMA
Dental composite
monomers with less than 6 ethoxy groups. Chromatograms showed larger peak areas for the
Bulk fill
lower volatile bisEMA with 4–6 ethoxy groups compared with higher volatile bisEMA with
Resin composite
2 or 3 ethoxy groups, thus indicating that the amounts of these homologues in the pastes were higher. Significance. Ethoxylated bisEMA with up to twelve ethoxy groups can be identified by HTGC/MS. In all eluates bisEMA was found. The higher the number of ethoxy groups the lower are the peak areas from bisEMA in the gas chromatogram. These findings may be significant for toxicological analysis of resin-composites incorporating bis-EMA. © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗
Corresponding author. Tel.: +49 89 44005 9301; fax: +49 89 44005 9302. E-mail address: [email protected] (J. Durner).
http://dx.doi.org/10.1016/j.dental.2015.02.006 0109-5641/© 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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1.
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
Introduction
The chemical composition of dental restoratives influences the material properties, the biocompatibility and the indication of use and is closely related to the identity and quantities of released substances [1–4]. Identifying and quantifying these substances allows for the assessment of the toxicological risk of dental restoratives. However, this cannot be solved by a single analytical method and represents a challenge for analytical chemistry with its extensive range of preparation and measuring methods [5]. Common monomers released from resin based dental materials are bisphenol A glycidyldimethacrylate (bisGMA), ethoxylated bisphenol A dimethacrylate (bisEMA; bisphenol A ethoxylate dimethacrylate), urethane dimethacrylate (UDMA), triethylene glycol dimethacrylate (TEGDMA), or 2hydroxyethyl methacrylate (HEMA) [6,7]. But also substances such as 2-hydroxy-4-methoxy benzophenone (HMBP), camphorquinone (CQ) or impurities from bisGMA synthesis such as triphenyl phosphane (TPP) were identified in eluates from polymerized specimen [8]. Moreover, ions such as Cu2+ , Fe2+ , Zn2+ , Ca2+ , Sr2+ , Al3+ and F− can be released from resins, glass ionomer or glass polyalkenoate cements [9–11]. To identify all these compounds, different analytical methods are needed. Larger molecular size monomers, such as bisGMA, UDMA or bisEMA, are well detected by high-performance liquid chromatography (HPLC) or HPLC mass spectrometry (HPLC/MS; the method is also called liquid chromatography/mass spectrometry (LC/MS)). After derivatisation of bisEMA, the different degrees of ethoxylation can be also be separated and determined by capillary zone electrophoresis (CZE) [12]. Due to its complexity this method is less used in the analysis of dental resin composites [12]. The main deficiency of this method compared with LC/MS or gas chromatography/mass spectrometry (GC/MS) techniques is the need for intermediate preparation steps of the eluates, prior to analysis of lower molecular size monomers, such as TEGDMA, HEMA, HMBP, CQ or TPP with detection and identification by GC/MS. For identifying metal ions, such as Zn2+ , Cu2+ , Al3+ and Ca2+ , atomic spectroscopy techniques, inductively coupled plasma mass spectrometry (ICP-MS) or some photometric techniques after complexation to a colored complex, are needed. However, it would be desirable to have one technique to identify and quantify all the substances in the eluates from dental restoratives. The importance of GC/MS in detecting elutable and toxicological relevant substances is steadily increasing. The method allows detection of a variety of the elutable and toxicological relevant substances (an overview is given in [8]). But it was not possible until now to establish an analytical procedure to detect low volatile monomers like bisEMA (Fig. 1 and Table 1) and its different degrees of ethoxylation. However, their identification is important since they have been shown to have a cyto- and genotoxic potential [13]. The regular GC/MS methods, described so far in the dental literature for identifying larger and low volatile methacrylate based monomers, include the insertion of the eluates in the injector, before reaching the column. The drawback of this method is that these molecules are only detectable as fragments and not as parent molecules, leaving doubts in the interpretation of the measured data.
Moreover, the detection of a molecule as fragments does not allow a quantification of the intact substance. Therefore, methods for detection of parent molecules are required. This might be achieved by impeding the fragmentation of the molecules prior to reaching the column. The techniques to avoid a fragmentation include either a direct introduction of the substances on the column or derivatisation of the parent molecule. The last method includes an additional preparation step and must consequently take into account reduced detection sensitivity due to incomplete derivatisation of the initial molecules [14–16]. Moreover, the derivatisation reaction might involve, besides the target molecules of interest, reaction with other low volatile molecules present in the eluates, making the interpretation of the chromatograms more difficult. Therefore the aim of the present study was to develop a new GC/MS method allowing for a direct introduction of the eluated substances on the column, by eliminating the risk of fragmentation in the injector and thus the identification of bisEMA as a parent molecule. Additionally, the method should also be able to separate and identify the different degrees of ethoxylation in bisEMA.
2.
Methods
2.1.
Specimen preparation
Three low-viscosity and one high viscosity bulk-fill resin composites, known to contain bisEMA, were analysed (Table 1). Cylindrical specimens were prepared in Teflon molds of 4 mm height and 3 mm diameter (n = 6 from each material). The molds were filled in one (bulk) increment, covered by a thin glass slide and cured by applying a high irradiance light curing unit (Bluephase 20i, High power mode, 20s, Ivoclar Vivadent, Schaan, Liechtenstein) placed directly on the specimen surface. Specimens were incubated 5 min after initiating the polymerisation in an ethanol/water (3:1) solution at 37 ◦ C for 24 h (Ethanol absolute EMSURE® , water for chromatography LiChrosolv® ; both solvents were obtained from Merck, Darmstadt, Germany). Each aliquot was analyzed with a gas chromatography unit, coupled with mass spectrometry (GC/MS). Moreover from each uncured material paste 100 mg were dissolved in 1000 ml ethanol/water (3:1) and measured (n = 6).
2.2.
GC/MS analysis
The analysis of the eluates was performed on a Trace GC 1310 gas chromatograph connected to an ISQ mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany). A MXT® -HT SimDist capillary column (length 15 m, inner diameter 0.25 mm, coating 0.1 m; Restek Corporation, Bellefonte, PA, USA) and MXT® Retention Gap (length 5 m, inner diameter 0.53 mm, Restek Corporation) was used as the capillary column for GC separations. The GC oven was heated from 50 ◦ C (1 min isotherm) to 400 ◦ C (1 min isotherm) with a rate of 30 ◦ C/min and 1.0 l of the solution was injected directly oncolumn. Helium was used as carrier gas at a constant flow rate of 1.5 ml/min. The aliquot from the eluate was directly applied on the column by a programmed temperature vaporization
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d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
Fig. 1 – Chemical structure of ethoxylated bisphenol A dimethacrylate (bis-EMA). n and m are the numbers of repeats of the ethoxy unit.
Table 1 – Materials, manufacturer, chemical composition of matrix and filler as well as filler content by weight (wt.) and volume (vol.) %. Materials ®
Venus bulk fill
Manufacturer color, batch
Resin matrix
Kulzer, Universal 010030 Dentsply Caulk, Universal, 01082
UDMA, bisEMA
FiltekTM Bulk Fill
3M-ESPE, A3 N414680
Sonic FillTM
Kerr, A3 3815160
bisGMA,UDMA, bisEMA, Procrylat resins bisGMA, TEGDMA, bisEMA
Surefil® SDR® flow
Filler
Filler wt./vol.
Ba–Al–F–Si–glass and SiO2 Ba–Al–F–B–Si–glass and St–Al–F–Si–glass ZrO2 /SiO2 , YbF3
Modified UDMA, TEGDMA, bisEMA
SiO2 , Glass, oxide
65/38 68/44
64.5/42.5
83.5/n.s
Abbreviations: bisEMA, ethoxylated bisphenol A dimethacrylate; bisGMA, bisphenolA diglycidyl ether dimethacrylate; TEGDMA, triethyleneglycol dimethacrylate; UDMA, urethane dimethacrylate. Data are provided by manufacturers; n.s = not specified.
(PTV) injector system at 50 ◦ C. The temperature of the direct coupling (transferline) from the GC to the mass spectrometer was 350 ◦ C. The MS was operated in electron ionisation mode (EI, 70 eV), with the ion source operated at 350 ◦ C; only positive ions were scanned. Scans were made over the range m/z 40–1100 at a scan rate of 3 scan/s for scans operated in full scan mode for identification and quantitation of the analytes. In the single ion mode (SIM) targeted analysis of the bisEMA homologues was performed for the following m/z: 69, 113, 437, 481, 525, 563, 569, 613, 657, 701, 745, 789, 833 and 877. The integration of the chromatograms was carried out over the base peak or other characteristic mass peaks of the compounds, typically M+ and M+ -15. Identification of the various substances was achieved by interpretation of their fragmentation patterns [6,17,18]. For each degree of ethoxylation of bisEMA, the ratio between the area of the molecule specific mass peak detected in eluates of the polymerised and unpolymerised material was calculated and presented in parts per mille (0/00). x=
2.3.
Area bisEMA(i) polymerised Area bisEMA(i) unpolymerised
Table 2 – Molecular mass of different degrees of ethoxylation of ethoxylated bisphenol A dimethacrlyate (bisEMA) (Fig. 1). Moreover the identifying mass signal (molecular mass – 15) and single ion monitoring (SIM) mass in the mass spectrum is shown. n+m=
∗ 1000(0/00)
Calculations and statistics
The results are presented as means (standard deviation) (SD).
3.
were identified, with up to twelve ethoxy groups (Table 2 and Fig. 2). The molecular masses of the homologous bisEMAs varied from 452 g/mol to 892 g/mol and were detected at retention times varying from 9.43 min to 13.36 min (Table 2). The results of the measurements are presented as the area ratios of the mass specific signal at a specific retention time in the eluate from polymerized and unpolymerized dental resins and are listed in Table 3. High ethoxylation degrees (eleven or twelve ethoxy groups) were only detected in the solutions of the unpolymerised materials. In the eluates obtained from polymerised resin composites, only ethoxylation degrees
Results
The new developed method with on column injection allowed identifying bisEMA as a complete molecule, that means detection – not only by fragment patterns – but also as a parent peak. Also, different degrees of ethoxylated bisEMA homologues
2 3 4 5 6 7 8 9 10 11 12
Retention time (min)
9.43 10.04 10.60 11.09 11.48 11.82 12.12 12.41 12.69 13.00 13.36
Molecular weight (g/mol)
Molecular weight-15 (identification and SIM)
452 496 540 584 628 672 716 760 804 848 892
437 481 525 569 613 657 701 745 789 833 877
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d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
Fig. 2 – Mass spectrogram from different materials (a) Venus® bulk fill, (b) Surefil® SDR® flow) at a retention time of 11.5 min. The substance at this retention time can be identified as bisEMA(6). Characteristic mass signals are the mother peak at 628 m/z, the loss of a methyl group (leading to the signal at 613 m/z). Further characteristic signals for methacrylate based materials are the signals at 69 and 113 m/z. In both mass spectrograms the loss of ethoxy groups (loss of 44 m/z) can be seen.
lower than 10, and lower than 7 in Surefil® SDR® flow, were detected. Related to the initial amount of bisEMA homologue (from the unpolymerised material), the greatest quantity of ethoxylated bisEMA was identified in the eluates obtained from polymerised specimens of: (i) FiltekTM Bulk Fill (12.77 per mille for the threefold ethoxylated bisEMA (bisEMA(3)) and (ii) Sonic FillTM (8.62 per mille for the twofold ethoxylated bisEMA(2)) (Table 3). Within a given material, it was apparent that the amount of eluted bisEMA homologue decreased with increased degree of ethoxylation (n + m). This result was derived from the area ratio from a specific bisEMA homologous (e.g. n + m = 4) in the eluate and in the solved paste as well as from the area under
the mass signal from this bisEMA homologeus in the eluate. For the solved pastes the greatest peak areas were found for BisEMA(4) to bisEMA(6) for most of the materials investigated. The distributions of bisEMA homologues in the eluates for each material are shown in Fig. 4.
4.
Discussion
The hydrophobic basic monomer bisEMA is used in dental restoratives to reduce viscosity of the material due to the absence of free hydroxyl groups [19]. A lower viscosity of the monomers allows incorporation of a greater amount of
Table 3 – Area ratio of the mass specific signal at one retention time in the eluate from polymerized and unpolymerized material. Data are presented in per mille as mean (n = 6) (SD). n+m= 2 3 4 5 6 7 8 9 10 11 12
FiltekTM Bulk Fill
Sonic FillTM
– 12.77 (0.63) 8.08 (2.63) 3.16 (1.13) 1.44 (0.92) 1.29 (0.68) 0.83 (0.68) 1.19 (0.01) 0.67 (0.69) – –
8.62 (4.91) 3.20 (1.49) 0.97 (0.58) 0.60 (0.25) 0.30 (0.25) 0.30 (0.14) 0.23 (0.10) 0.26 (0.08) 1.16 (0.01) – –
Surefil® SDR® flow 0.14 (0.03) 0.84 (0.24) 0.30 (0.13) 0.11 (0.06) 0.10 (0.02) 0.05 (0.03) – – – – –
–, no ratio could be built because no signal was detected in the eluate of polymerized materials.
Venus® bulk fill 1.88 (0.66) 2.90 (0.53) 1.74 (0.24) 0.57 (0.17) 0.21 (0.17) 0.17 (0.05) 0.05 (0.06) 0.06 (0.01) 0.03 (0.01) – –
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
477
Fig. 3 – Gas chromatogram from the solved paste from Venus® bulk fill. It is shown that the different ethoxylated BisEMA homologous can be separated (but because of the scaling of the ordinate it is only possible to show the results up to n + m = 9).
inorganic filler, thus enhancing the mechanical properties of the resin composite [20]. Compared to bisGMA or TEGDMA, bisEMA in dental materials is not a single monomer, but rather a generic name for a large homologous series of ethoxylated bisphenol A based dimethacrylate molecules. An ethoxylation reaction is generally achieved by means of ethylene oxide (oxirane), the simplest epoxide [21]. Ethylene oxide is an alkane with an oxygen atom bonded to two carbon atoms forming a three membered ring. Due to the high ring tension, it is very reactive [21]. Therefore the ethoxylation reaction is unselective and difficult to control, leading to different ethoxylated products and byproducts, which must be separated analytically. The degree of ethoxylation is indicated by the indexes n and m (Fig. 1) and mentioned in brackets in the chemical term (bisEMA(4) means n + m = 4). While monomers with different (m + n) degrees of ethoxylation can be more easily separated (n + m = 4 can be more easy separated from n + m = 12), isomers (similar n + m index) cannot. This is, however, not a unique characteristic of bisEMA. Different extents of ethoxylation are also known for polyethylene glycol (PEG). PEG is a polymerisation product of water, mono ethylene glycol or diethylene glycol (as starter molecules) and ethylene oxide. During the polymerization process different amounts of ethylene oxide molecules react with the starter molecule to a polymer. In combination with dimethacrylates, PEG is also used in dental resin composites [22].
The present study used the cold on-column injection technique in combination with a retention gap to analyse bisEMA and its homologous. The cold on-column injection offers some advantages for the analyses of low volatile substances, since sample discrimination and alteration can be reduced. A higher analytical precision and accuracy results [23]. In combination with a retention gap, on-column injection is commonly employed for large volume injections [23]. Furthermore, to our best knowledge, high-temperature GC (HT-GC) was used here for the first time to analyse eluates from dental materials. Regular temperatures used so far in GC methods have been below 340 ◦ C [24–26]. Because of the use of a high temperature column it was possible to heat up to 400 ◦ C and to vaporize the low volatile bisEMA homologues. In very general terms, the higher the mass the lower the volatility of a substance (the volatility also depends upon other chemical characteristics such as the quantity of hydroxyl groups) [14]. It was demonstrated that up to twelve ethoxy groups in the bisEMA molecule were detectable with HT-GC/MS and without further preparation step such as derivatisation of bisEMA. To quantify substances in the mass spectrum of GC/MS, either a calibration curve (external standardization) or a labelled (often multiple deuterated) analyte (internal standardization) might be used. An external standardization was not possible in the present study because a reference material should consider two aspects: firstly that it must clearly differentiate within homologous series (ascending order of the
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Fig. 4 – Distribution of the bisEMA homologues in the eluates for each material. The ordinate shows the area ratio of the mass specific signal at one retention time (that means at one specific n + m or in other words for one specific homologue) in the eluate from polymerized and unpolymerized material. The data are presented in per mille. The abscissa shows the number (n + m) of ethoxy groups in the bisEMA molecules.
total number of ethoxy groups: n + m = 1, 2, 3, 4 etc.) and secondly differentiate between the isomers corresponding to the same homologous formula. To our knowledge, no commercial reference standards are available, and an individual product synthesis or separation of homologous and isomers would then be needed. Both solutions are expensive and technically challenging. The use of an internal standard (labelled bisEMA) failed due to the same limitations. Therefore quantification of the different forms of ethoxylated bisGMA was not possible in the classical manner. Instead of using such an approach, we calculated the ratio of the peak areas corresponding to each bisEMA homologues in the eluate from the polymerised and non-polymerised materials (area bisEMA(i) polymerised/area bisEMA(i) non-polymerised). This area ratio for bisEMA(2) can be compared with that of bisEMA(5). The main advantage of this procedure consists in minimizing the effect of different sensitivities of the mass spectrometer and different volatilities. As in fractional arithmetic, these influencing factors can be ‘cancelled out’ because they influence e.g. bisEMA(5) in the eluate in the same manner as for bisEMA(5) in the solved paste [27]. The lack of a control material causes a second problem. No reference spectra are available for the homologous bisEMA molecules in mass spectral libraries such as the NIST (National Institute of Standards and Technology) MS database nor could they be created by us. In this case only the chemical analysis of the mass spectrum in combination with the
retention time was possible. The parent peak can be assigned to the mass of the corresponding bisEMA and its number of ethoxy groups. One of the main peaks was the parent peak minus 15 mass units (CH3 group). This fragmentation is often seen in branched hydrocarbons. Furthermore a frequently occurring fragmentation pattern of methacrylate based dental monomers was seen (m/z = 69 in combination with m/z = 113). The fragment m/z = 69 can be identified as C4 H5 O (methacrylic acid without the hydroxyl group), m/z = 113 can be identified as C6 H9 O2 (methacrylic acid with the two carbon atoms from the ethoxy group). Besides, the mass difference between some main peaks in the mass spectra was 44 mass units. This mass can be assigned to the loss of one ethoxy group in the molecule. Furthermore, the main peaks of bisphenol A (m/z = 119 and 213; according to the reference spectra from NIST mass spectral library) were identified in our mass spectra. Similar to the CZE method, it was possible to determine by GC/MS the total amount of the ethoxy groups in bisEMA but not to differentiate between the different isomers [12]. To further exemplify, in the case of 6 ethoxy groups (n + m = 6) in the molecule, three isomers might exist: one isomer with symmetric distribution of the ethoxy groups (n = 3 and m = 3) and two isomers with an asymmetrical distribution of the ethoxy groups (n = 1 and m = 5; n = 2 and m = 4). According to the unspecific method of bisEMA(4) synthesis (see above) all isomers can exist in the matrix of the paste. It must however be considered, that, for a given molecular mass (isomer), the greater the
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
asymmetrical distribution of the ethoxy groups, the greater the differences in their volatility and accordingly their chromatographical behaviour. Therefore it could be assumed that this asymmetrical distribution might influence the shape of the peak in the gas chromatogram, e.g. the formation of twin peaks or shoulders, as observed in Fig. 3. The different degrees of ethoxylation of bisEMA form a homologous series, that means molecules varying by a fixed molecular grouping, in this case an ethoxy group, with the expectation that the higher the mass of each member of the series, the lower its volatility. For most of the examined materials, the integrated area in the gas chromatogram from bisEMA(4) to bisEMA(6) was maximal compared to other bisEMA homologues, especially relative to peak areas for members of lower molecular mass (and therefore higher volatility) in the homologous series: bisEMA(2) and bisEMA(3). This is an indicator that greater proportions of bisEMA(4) to bisEMA(6) was present in the paste, compared to bisEMA(2) and bisEMA(3) (Fig. 3). The distributions of bisEMA homologues in the eluates for each material are shown in Fig. 4. To elute most of the unpolymerized (co)monomers and additives, ethanol:water 3:1 was used. This follows the recommendation of the United States Food and Drug Administration (US FDA) as a food/oral simulating liquid of clinical relevance [28]. It was determined that the ratio of lower molecular mass bisEMA molecules (such as bisEMA(2)) was greater relative to higher molecular mass bisEMA homologues such as bisEMA(10). This may be explained by the concept of gaps existing in the polymer network. Durner et al. have suggested that such gap sizes can influence the amounts of elutable substances from polymerized specimens [29]. In those previous experiments, bleaching led to some degradation of the threedimensional polymer network and therefore more substances became elutable. Higher molecular mass bisEMA homologues cannot so readily elute from the polymer network, as can lower molecular mass bisEMA molecules, because of their steric hindrance. The larger the molecule the larger the gap required in the network for simple elution to occur. Musenga et al. [12] used CZE to differentiate between the bisEMA homologues. With this analytical technique they found up to n + m = 30 bisEMA homologues in a pure bisEMA. In a further step they analysed the eluate of a dental resin. They found bisEMA homologues up to n + m = 7. This is in good agreement with our findings of n + m = 10. Furthermore it demonstrates that the more widely available GC/MS technics is an appropriate method to identify these analytes in the eluates of dental materials.
5.
Conclusions
HT-GC/MS is a suitable method for detection of bisEMA homologues in eluates of dental restoratives. BisEMA was found in all eluates from polymerized specimen from all examined bulk fill materials. It can be assumed that the greater the number of (m + n) ethoxy groups in the bisEMA molecule, the lower is the amount that is eluted. These findings may be significant
479
for toxicological analysis of resin-composites incorporating bis-EMA.
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
Determination of homologous distributions of bisEMA dimethacrylates in bulk-fill resin-composites by GC–MS Jürgen Durner a,∗ , Klaus Schrickel b , David C. Watts c , Nicoleta Ilie a a
Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig-Maximilians-University of Munich, Goethestr. 70, 80336 Munich, Germany b Thermo Fisher Scientific, Scientific Instruments, Im Steingrund 4–6, 63303 Dreieich, Germany c School of Dentistry and Photon Science Institute, University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. Ethoxylated bisphenol A dimethacrylate (bisEMA) is a basis monomer in several
Received 9 December 2014
dental resin composites. It was the aim of the present study to develop a method allowing
Received in revised form
detection of bisEMA and its different degrees of ethoxylation eluted from polymerized resin
10 February 2015
composites.
Accepted 10 February 2015
Methods. High-temperature gas chromatography/mass spectrometry (HT-GC/MS) by direct on-column injection was used to identify ethoxylated bisEMA in ethanol/water (3:1) eluates from polymerized specimen of four bulk-fill resin composites – Venus® bulk fill, Surefil®
Keywords:
SDRTM flow, FiltekTM Bulk Fill and Sonic FillTM . Additionally, the unpolymerised pastes were
GC/MS
analysed.
bisEMA
Results. The developed method allowed identification of a homologous series of bisEMA up to
Bisphenol A ethoxylate
twelve ethoxy groups in the unpolymerised materials. The molecular masses of the homolo-
dimethacrylate
gous bisEMA varied between 452 g/mol and 892 g/mol and were detected for retention times
Elution
from 9.43 min to 13.36 min. Analysis of eluates from polymerised materials identified bisEMA
Dental composite
monomers with less than 6 ethoxy groups. Chromatograms showed larger peak areas for the
Bulk fill
lower volatile bisEMA with 4–6 ethoxy groups compared with higher volatile bisEMA with
Resin composite
2 or 3 ethoxy groups, thus indicating that the amounts of these homologues in the pastes were higher. Significance. Ethoxylated bisEMA with up to twelve ethoxy groups can be identified by HTGC/MS. In all eluates bisEMA was found. The higher the number of ethoxy groups the lower are the peak areas from bisEMA in the gas chromatogram. These findings may be significant for toxicological analysis of resin-composites incorporating bis-EMA. © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗
Corresponding author. Tel.: +49 89 44005 9301; fax: +49 89 44005 9302. E-mail address: [email protected] (J. Durner).
http://dx.doi.org/10.1016/j.dental.2015.02.006 0109-5641/© 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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1.
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
Introduction
The chemical composition of dental restoratives influences the material properties, the biocompatibility and the indication of use and is closely related to the identity and quantities of released substances [1–4]. Identifying and quantifying these substances allows for the assessment of the toxicological risk of dental restoratives. However, this cannot be solved by a single analytical method and represents a challenge for analytical chemistry with its extensive range of preparation and measuring methods [5]. Common monomers released from resin based dental materials are bisphenol A glycidyldimethacrylate (bisGMA), ethoxylated bisphenol A dimethacrylate (bisEMA; bisphenol A ethoxylate dimethacrylate), urethane dimethacrylate (UDMA), triethylene glycol dimethacrylate (TEGDMA), or 2hydroxyethyl methacrylate (HEMA) [6,7]. But also substances such as 2-hydroxy-4-methoxy benzophenone (HMBP), camphorquinone (CQ) or impurities from bisGMA synthesis such as triphenyl phosphane (TPP) were identified in eluates from polymerized specimen [8]. Moreover, ions such as Cu2+ , Fe2+ , Zn2+ , Ca2+ , Sr2+ , Al3+ and F− can be released from resins, glass ionomer or glass polyalkenoate cements [9–11]. To identify all these compounds, different analytical methods are needed. Larger molecular size monomers, such as bisGMA, UDMA or bisEMA, are well detected by high-performance liquid chromatography (HPLC) or HPLC mass spectrometry (HPLC/MS; the method is also called liquid chromatography/mass spectrometry (LC/MS)). After derivatisation of bisEMA, the different degrees of ethoxylation can be also be separated and determined by capillary zone electrophoresis (CZE) [12]. Due to its complexity this method is less used in the analysis of dental resin composites [12]. The main deficiency of this method compared with LC/MS or gas chromatography/mass spectrometry (GC/MS) techniques is the need for intermediate preparation steps of the eluates, prior to analysis of lower molecular size monomers, such as TEGDMA, HEMA, HMBP, CQ or TPP with detection and identification by GC/MS. For identifying metal ions, such as Zn2+ , Cu2+ , Al3+ and Ca2+ , atomic spectroscopy techniques, inductively coupled plasma mass spectrometry (ICP-MS) or some photometric techniques after complexation to a colored complex, are needed. However, it would be desirable to have one technique to identify and quantify all the substances in the eluates from dental restoratives. The importance of GC/MS in detecting elutable and toxicological relevant substances is steadily increasing. The method allows detection of a variety of the elutable and toxicological relevant substances (an overview is given in [8]). But it was not possible until now to establish an analytical procedure to detect low volatile monomers like bisEMA (Fig. 1 and Table 1) and its different degrees of ethoxylation. However, their identification is important since they have been shown to have a cyto- and genotoxic potential [13]. The regular GC/MS methods, described so far in the dental literature for identifying larger and low volatile methacrylate based monomers, include the insertion of the eluates in the injector, before reaching the column. The drawback of this method is that these molecules are only detectable as fragments and not as parent molecules, leaving doubts in the interpretation of the measured data.
Moreover, the detection of a molecule as fragments does not allow a quantification of the intact substance. Therefore, methods for detection of parent molecules are required. This might be achieved by impeding the fragmentation of the molecules prior to reaching the column. The techniques to avoid a fragmentation include either a direct introduction of the substances on the column or derivatisation of the parent molecule. The last method includes an additional preparation step and must consequently take into account reduced detection sensitivity due to incomplete derivatisation of the initial molecules [14–16]. Moreover, the derivatisation reaction might involve, besides the target molecules of interest, reaction with other low volatile molecules present in the eluates, making the interpretation of the chromatograms more difficult. Therefore the aim of the present study was to develop a new GC/MS method allowing for a direct introduction of the eluated substances on the column, by eliminating the risk of fragmentation in the injector and thus the identification of bisEMA as a parent molecule. Additionally, the method should also be able to separate and identify the different degrees of ethoxylation in bisEMA.
2.
Methods
2.1.
Specimen preparation
Three low-viscosity and one high viscosity bulk-fill resin composites, known to contain bisEMA, were analysed (Table 1). Cylindrical specimens were prepared in Teflon molds of 4 mm height and 3 mm diameter (n = 6 from each material). The molds were filled in one (bulk) increment, covered by a thin glass slide and cured by applying a high irradiance light curing unit (Bluephase 20i, High power mode, 20s, Ivoclar Vivadent, Schaan, Liechtenstein) placed directly on the specimen surface. Specimens were incubated 5 min after initiating the polymerisation in an ethanol/water (3:1) solution at 37 ◦ C for 24 h (Ethanol absolute EMSURE® , water for chromatography LiChrosolv® ; both solvents were obtained from Merck, Darmstadt, Germany). Each aliquot was analyzed with a gas chromatography unit, coupled with mass spectrometry (GC/MS). Moreover from each uncured material paste 100 mg were dissolved in 1000 ml ethanol/water (3:1) and measured (n = 6).
2.2.
GC/MS analysis
The analysis of the eluates was performed on a Trace GC 1310 gas chromatograph connected to an ISQ mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany). A MXT® -HT SimDist capillary column (length 15 m, inner diameter 0.25 mm, coating 0.1 m; Restek Corporation, Bellefonte, PA, USA) and MXT® Retention Gap (length 5 m, inner diameter 0.53 mm, Restek Corporation) was used as the capillary column for GC separations. The GC oven was heated from 50 ◦ C (1 min isotherm) to 400 ◦ C (1 min isotherm) with a rate of 30 ◦ C/min and 1.0 l of the solution was injected directly oncolumn. Helium was used as carrier gas at a constant flow rate of 1.5 ml/min. The aliquot from the eluate was directly applied on the column by a programmed temperature vaporization
475
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
Fig. 1 – Chemical structure of ethoxylated bisphenol A dimethacrylate (bis-EMA). n and m are the numbers of repeats of the ethoxy unit.
Table 1 – Materials, manufacturer, chemical composition of matrix and filler as well as filler content by weight (wt.) and volume (vol.) %. Materials ®
Venus bulk fill
Manufacturer color, batch
Resin matrix
Kulzer, Universal 010030 Dentsply Caulk, Universal, 01082
UDMA, bisEMA
FiltekTM Bulk Fill
3M-ESPE, A3 N414680
Sonic FillTM
Kerr, A3 3815160
bisGMA,UDMA, bisEMA, Procrylat resins bisGMA, TEGDMA, bisEMA
Surefil® SDR® flow
Filler
Filler wt./vol.
Ba–Al–F–Si–glass and SiO2 Ba–Al–F–B–Si–glass and St–Al–F–Si–glass ZrO2 /SiO2 , YbF3
Modified UDMA, TEGDMA, bisEMA
SiO2 , Glass, oxide
65/38 68/44
64.5/42.5
83.5/n.s
Abbreviations: bisEMA, ethoxylated bisphenol A dimethacrylate; bisGMA, bisphenolA diglycidyl ether dimethacrylate; TEGDMA, triethyleneglycol dimethacrylate; UDMA, urethane dimethacrylate. Data are provided by manufacturers; n.s = not specified.
(PTV) injector system at 50 ◦ C. The temperature of the direct coupling (transferline) from the GC to the mass spectrometer was 350 ◦ C. The MS was operated in electron ionisation mode (EI, 70 eV), with the ion source operated at 350 ◦ C; only positive ions were scanned. Scans were made over the range m/z 40–1100 at a scan rate of 3 scan/s for scans operated in full scan mode for identification and quantitation of the analytes. In the single ion mode (SIM) targeted analysis of the bisEMA homologues was performed for the following m/z: 69, 113, 437, 481, 525, 563, 569, 613, 657, 701, 745, 789, 833 and 877. The integration of the chromatograms was carried out over the base peak or other characteristic mass peaks of the compounds, typically M+ and M+ -15. Identification of the various substances was achieved by interpretation of their fragmentation patterns [6,17,18]. For each degree of ethoxylation of bisEMA, the ratio between the area of the molecule specific mass peak detected in eluates of the polymerised and unpolymerised material was calculated and presented in parts per mille (0/00). x=
2.3.
Area bisEMA(i) polymerised Area bisEMA(i) unpolymerised
Table 2 – Molecular mass of different degrees of ethoxylation of ethoxylated bisphenol A dimethacrlyate (bisEMA) (Fig. 1). Moreover the identifying mass signal (molecular mass – 15) and single ion monitoring (SIM) mass in the mass spectrum is shown. n+m=
∗ 1000(0/00)
Calculations and statistics
The results are presented as means (standard deviation) (SD).
3.
were identified, with up to twelve ethoxy groups (Table 2 and Fig. 2). The molecular masses of the homologous bisEMAs varied from 452 g/mol to 892 g/mol and were detected at retention times varying from 9.43 min to 13.36 min (Table 2). The results of the measurements are presented as the area ratios of the mass specific signal at a specific retention time in the eluate from polymerized and unpolymerized dental resins and are listed in Table 3. High ethoxylation degrees (eleven or twelve ethoxy groups) were only detected in the solutions of the unpolymerised materials. In the eluates obtained from polymerised resin composites, only ethoxylation degrees
Results
The new developed method with on column injection allowed identifying bisEMA as a complete molecule, that means detection – not only by fragment patterns – but also as a parent peak. Also, different degrees of ethoxylated bisEMA homologues
2 3 4 5 6 7 8 9 10 11 12
Retention time (min)
9.43 10.04 10.60 11.09 11.48 11.82 12.12 12.41 12.69 13.00 13.36
Molecular weight (g/mol)
Molecular weight-15 (identification and SIM)
452 496 540 584 628 672 716 760 804 848 892
437 481 525 569 613 657 701 745 789 833 877
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d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
Fig. 2 – Mass spectrogram from different materials (a) Venus® bulk fill, (b) Surefil® SDR® flow) at a retention time of 11.5 min. The substance at this retention time can be identified as bisEMA(6). Characteristic mass signals are the mother peak at 628 m/z, the loss of a methyl group (leading to the signal at 613 m/z). Further characteristic signals for methacrylate based materials are the signals at 69 and 113 m/z. In both mass spectrograms the loss of ethoxy groups (loss of 44 m/z) can be seen.
lower than 10, and lower than 7 in Surefil® SDR® flow, were detected. Related to the initial amount of bisEMA homologue (from the unpolymerised material), the greatest quantity of ethoxylated bisEMA was identified in the eluates obtained from polymerised specimens of: (i) FiltekTM Bulk Fill (12.77 per mille for the threefold ethoxylated bisEMA (bisEMA(3)) and (ii) Sonic FillTM (8.62 per mille for the twofold ethoxylated bisEMA(2)) (Table 3). Within a given material, it was apparent that the amount of eluted bisEMA homologue decreased with increased degree of ethoxylation (n + m). This result was derived from the area ratio from a specific bisEMA homologous (e.g. n + m = 4) in the eluate and in the solved paste as well as from the area under
the mass signal from this bisEMA homologeus in the eluate. For the solved pastes the greatest peak areas were found for BisEMA(4) to bisEMA(6) for most of the materials investigated. The distributions of bisEMA homologues in the eluates for each material are shown in Fig. 4.
4.
Discussion
The hydrophobic basic monomer bisEMA is used in dental restoratives to reduce viscosity of the material due to the absence of free hydroxyl groups [19]. A lower viscosity of the monomers allows incorporation of a greater amount of
Table 3 – Area ratio of the mass specific signal at one retention time in the eluate from polymerized and unpolymerized material. Data are presented in per mille as mean (n = 6) (SD). n+m= 2 3 4 5 6 7 8 9 10 11 12
FiltekTM Bulk Fill
Sonic FillTM
– 12.77 (0.63) 8.08 (2.63) 3.16 (1.13) 1.44 (0.92) 1.29 (0.68) 0.83 (0.68) 1.19 (0.01) 0.67 (0.69) – –
8.62 (4.91) 3.20 (1.49) 0.97 (0.58) 0.60 (0.25) 0.30 (0.25) 0.30 (0.14) 0.23 (0.10) 0.26 (0.08) 1.16 (0.01) – –
Surefil® SDR® flow 0.14 (0.03) 0.84 (0.24) 0.30 (0.13) 0.11 (0.06) 0.10 (0.02) 0.05 (0.03) – – – – –
–, no ratio could be built because no signal was detected in the eluate of polymerized materials.
Venus® bulk fill 1.88 (0.66) 2.90 (0.53) 1.74 (0.24) 0.57 (0.17) 0.21 (0.17) 0.17 (0.05) 0.05 (0.06) 0.06 (0.01) 0.03 (0.01) – –
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
477
Fig. 3 – Gas chromatogram from the solved paste from Venus® bulk fill. It is shown that the different ethoxylated BisEMA homologous can be separated (but because of the scaling of the ordinate it is only possible to show the results up to n + m = 9).
inorganic filler, thus enhancing the mechanical properties of the resin composite [20]. Compared to bisGMA or TEGDMA, bisEMA in dental materials is not a single monomer, but rather a generic name for a large homologous series of ethoxylated bisphenol A based dimethacrylate molecules. An ethoxylation reaction is generally achieved by means of ethylene oxide (oxirane), the simplest epoxide [21]. Ethylene oxide is an alkane with an oxygen atom bonded to two carbon atoms forming a three membered ring. Due to the high ring tension, it is very reactive [21]. Therefore the ethoxylation reaction is unselective and difficult to control, leading to different ethoxylated products and byproducts, which must be separated analytically. The degree of ethoxylation is indicated by the indexes n and m (Fig. 1) and mentioned in brackets in the chemical term (bisEMA(4) means n + m = 4). While monomers with different (m + n) degrees of ethoxylation can be more easily separated (n + m = 4 can be more easy separated from n + m = 12), isomers (similar n + m index) cannot. This is, however, not a unique characteristic of bisEMA. Different extents of ethoxylation are also known for polyethylene glycol (PEG). PEG is a polymerisation product of water, mono ethylene glycol or diethylene glycol (as starter molecules) and ethylene oxide. During the polymerization process different amounts of ethylene oxide molecules react with the starter molecule to a polymer. In combination with dimethacrylates, PEG is also used in dental resin composites [22].
The present study used the cold on-column injection technique in combination with a retention gap to analyse bisEMA and its homologous. The cold on-column injection offers some advantages for the analyses of low volatile substances, since sample discrimination and alteration can be reduced. A higher analytical precision and accuracy results [23]. In combination with a retention gap, on-column injection is commonly employed for large volume injections [23]. Furthermore, to our best knowledge, high-temperature GC (HT-GC) was used here for the first time to analyse eluates from dental materials. Regular temperatures used so far in GC methods have been below 340 ◦ C [24–26]. Because of the use of a high temperature column it was possible to heat up to 400 ◦ C and to vaporize the low volatile bisEMA homologues. In very general terms, the higher the mass the lower the volatility of a substance (the volatility also depends upon other chemical characteristics such as the quantity of hydroxyl groups) [14]. It was demonstrated that up to twelve ethoxy groups in the bisEMA molecule were detectable with HT-GC/MS and without further preparation step such as derivatisation of bisEMA. To quantify substances in the mass spectrum of GC/MS, either a calibration curve (external standardization) or a labelled (often multiple deuterated) analyte (internal standardization) might be used. An external standardization was not possible in the present study because a reference material should consider two aspects: firstly that it must clearly differentiate within homologous series (ascending order of the
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d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
Fig. 4 – Distribution of the bisEMA homologues in the eluates for each material. The ordinate shows the area ratio of the mass specific signal at one retention time (that means at one specific n + m or in other words for one specific homologue) in the eluate from polymerized and unpolymerized material. The data are presented in per mille. The abscissa shows the number (n + m) of ethoxy groups in the bisEMA molecules.
total number of ethoxy groups: n + m = 1, 2, 3, 4 etc.) and secondly differentiate between the isomers corresponding to the same homologous formula. To our knowledge, no commercial reference standards are available, and an individual product synthesis or separation of homologous and isomers would then be needed. Both solutions are expensive and technically challenging. The use of an internal standard (labelled bisEMA) failed due to the same limitations. Therefore quantification of the different forms of ethoxylated bisGMA was not possible in the classical manner. Instead of using such an approach, we calculated the ratio of the peak areas corresponding to each bisEMA homologues in the eluate from the polymerised and non-polymerised materials (area bisEMA(i) polymerised/area bisEMA(i) non-polymerised). This area ratio for bisEMA(2) can be compared with that of bisEMA(5). The main advantage of this procedure consists in minimizing the effect of different sensitivities of the mass spectrometer and different volatilities. As in fractional arithmetic, these influencing factors can be ‘cancelled out’ because they influence e.g. bisEMA(5) in the eluate in the same manner as for bisEMA(5) in the solved paste [27]. The lack of a control material causes a second problem. No reference spectra are available for the homologous bisEMA molecules in mass spectral libraries such as the NIST (National Institute of Standards and Technology) MS database nor could they be created by us. In this case only the chemical analysis of the mass spectrum in combination with the
retention time was possible. The parent peak can be assigned to the mass of the corresponding bisEMA and its number of ethoxy groups. One of the main peaks was the parent peak minus 15 mass units (CH3 group). This fragmentation is often seen in branched hydrocarbons. Furthermore a frequently occurring fragmentation pattern of methacrylate based dental monomers was seen (m/z = 69 in combination with m/z = 113). The fragment m/z = 69 can be identified as C4 H5 O (methacrylic acid without the hydroxyl group), m/z = 113 can be identified as C6 H9 O2 (methacrylic acid with the two carbon atoms from the ethoxy group). Besides, the mass difference between some main peaks in the mass spectra was 44 mass units. This mass can be assigned to the loss of one ethoxy group in the molecule. Furthermore, the main peaks of bisphenol A (m/z = 119 and 213; according to the reference spectra from NIST mass spectral library) were identified in our mass spectra. Similar to the CZE method, it was possible to determine by GC/MS the total amount of the ethoxy groups in bisEMA but not to differentiate between the different isomers [12]. To further exemplify, in the case of 6 ethoxy groups (n + m = 6) in the molecule, three isomers might exist: one isomer with symmetric distribution of the ethoxy groups (n = 3 and m = 3) and two isomers with an asymmetrical distribution of the ethoxy groups (n = 1 and m = 5; n = 2 and m = 4). According to the unspecific method of bisEMA(4) synthesis (see above) all isomers can exist in the matrix of the paste. It must however be considered, that, for a given molecular mass (isomer), the greater the
d e n t a l m a t e r i a l s 3 1 ( 2 0 1 5 ) 473–480
asymmetrical distribution of the ethoxy groups, the greater the differences in their volatility and accordingly their chromatographical behaviour. Therefore it could be assumed that this asymmetrical distribution might influence the shape of the peak in the gas chromatogram, e.g. the formation of twin peaks or shoulders, as observed in Fig. 3. The different degrees of ethoxylation of bisEMA form a homologous series, that means molecules varying by a fixed molecular grouping, in this case an ethoxy group, with the expectation that the higher the mass of each member of the series, the lower its volatility. For most of the examined materials, the integrated area in the gas chromatogram from bisEMA(4) to bisEMA(6) was maximal compared to other bisEMA homologues, especially relative to peak areas for members of lower molecular mass (and therefore higher volatility) in the homologous series: bisEMA(2) and bisEMA(3). This is an indicator that greater proportions of bisEMA(4) to bisEMA(6) was present in the paste, compared to bisEMA(2) and bisEMA(3) (Fig. 3). The distributions of bisEMA homologues in the eluates for each material are shown in Fig. 4. To elute most of the unpolymerized (co)monomers and additives, ethanol:water 3:1 was used. This follows the recommendation of the United States Food and Drug Administration (US FDA) as a food/oral simulating liquid of clinical relevance [28]. It was determined that the ratio of lower molecular mass bisEMA molecules (such as bisEMA(2)) was greater relative to higher molecular mass bisEMA homologues such as bisEMA(10). This may be explained by the concept of gaps existing in the polymer network. Durner et al. have suggested that such gap sizes can influence the amounts of elutable substances from polymerized specimens [29]. In those previous experiments, bleaching led to some degradation of the threedimensional polymer network and therefore more substances became elutable. Higher molecular mass bisEMA homologues cannot so readily elute from the polymer network, as can lower molecular mass bisEMA molecules, because of their steric hindrance. The larger the molecule the larger the gap required in the network for simple elution to occur. Musenga et al. [12] used CZE to differentiate between the bisEMA homologues. With this analytical technique they found up to n + m = 30 bisEMA homologues in a pure bisEMA. In a further step they analysed the eluate of a dental resin. They found bisEMA homologues up to n + m = 7. This is in good agreement with our findings of n + m = 10. Furthermore it demonstrates that the more widely available GC/MS technics is an appropriate method to identify these analytes in the eluates of dental materials.
5.
Conclusions
HT-GC/MS is a suitable method for detection of bisEMA homologues in eluates of dental restoratives. BisEMA was found in all eluates from polymerized specimen from all examined bulk fill materials. It can be assumed that the greater the number of (m + n) ethoxy groups in the bisEMA molecule, the lower is the amount that is eluted. These findings may be significant
479
for toxicological analysis of resin-composites incorporating bis-EMA.
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