Potential confounders of bisphenol-a analysis in dental materials

Highlights

  • A cured experimental dental resin was analyzed for extractable bisphenol A (BPA).

  • Extracted BPA appeared ∼30-fold higher when quantified by LC/UV vs LC/MS/MS.

  • LC/UV results reflect confounding by a co-eluting EDMAB contaminant or derivative.

  • Use of LC/MS/MS enables accurate and unequivocal identification of extracted BPA.

Abstract

Objectives

In the published literature, a variety of analytical methods have been used to quantify and report bisphenol A (BPA) release from dental resins. The objective of this study was to compare results obtained for quantification of BPA in dental resin extracts using an LC/UV analytical method and an LC/MS/MS method.

Methods

A cured Bis-GMA-based resin representative of commercial dental products was extracted according to ISO 10993 guidelines for medical devices. d16BPA was included as an internal standard. Sample processing followed expert recommendations for minimizing BPA sample contamination. Extracts were separated using HPLC methods and analyzed for BPA using LC/UV and LC/MS/MS detection methods.

Results

The reported BPA concentrations were about 30-fold higher using LC/UV vs. LC/MS/MS. Full scan LC/MS/MS in both positive and negative modes showed that the apparent high BPA values seen with LC/UV were caused by co-elution of a previously unidentified chemical, thought to arise from one of the polymerization initiators.

Significance

These results emphasize the potential difficulties in obtaining accurate analyses of BPA in complex mixtures such as dental resins and their extracts. Both good separation methodology and a detection method with high specificity and sensitivity are important to avoid incorrect identification of extractables, and consequent incorrect quantitative assignments for species of interest. Reliable methods are essential for accurate estimation of patient exposure to BPA and development of meaningful health risk assessments.

Introduction

Bisphenol-A (BPA, CAS Number 80-05-7) is a chemical intermediate in the production of numerous materials including epoxy resins, polyester resins, polyacrylate resins, and polycarbonate plastics . Although it is not intentionally added to dental and orthodontic products, BPA is a starting material for three monomers used in dental resins and orthodontic adhesives, namely BPA diglycidyl methacrylate (Bis-GMA), BPA dimethacrylate (Bis-DMA) and BPA ethoxylate dimethacrylate (Bis-EDMA) . As a result, trace amounts of BPA may remain in these resins as a manufacturing residue. The widespread use of BPA as a plasticizer in non-dental applications, and its claimed health effects have caused the chemical to be scrutinized by numerous regulatory authorities . These authorities agree that potential BPA exposure from dental and orthodontic materials is far below any potential exposure from food and from other medical devices and of low potential risk for health effects . However, because of continued media attention to BPA, some dental/orthodontic professionals and their patients remain concerned about the safety of even very low levels of BPA.

As part of product stewardship, manufacturers of dental resins incorporate new BPA research into ongoing assessments of patient risk. Accurate data are essential to evaluate the risk and safety of a chemical exposure, and the usefulness of any published BPA study lies in the completeness of the data (so as to be able to calculate potential human exposure) and the reliability of the experimental methods. Investigators assaying BPA from extracts of cured dental and orthodontic resins have used a number of separation and detection methods including liquid chromatography (LC) with ultraviolet (UV) detection , electrospin ionization (ESI) with MS detection , and gas chromatography (GC) or LC with MS (mass spectrometry) detection. However, some researchers have pointed out disadvantages of UV methods . In our own research, we obtained striking differences in reported BPA content depending on the analytical method used. Here, we present the results of a study comparing UV detection and Tandem Mass Spectrometry (MS/MS) as methods to quantify BPA extracted from a cured dental resin.

Materials and methods

Experimental resin

An experimental light-cured dental resin was prepared, consisting of 83% silanized silica (CAS Number 100402-78-6), 16% BisGMA (CAS Number 1565-94-2), <1% of Camphorquinone (CPQ, CAS Number 10373-78-1), and <1% ethyl 4 – (dimethylamino) benzoate (EDMAB, CAS Number 10287-53-3). The starting materials were the same as used in 3M commercial oral care resins, and were obtained from proprietary sources. Ingredients were processed in a planetary mixer and the mixed resin was transferred to circular stainless steel molds (15 mm ID × 1.7 mm high) for curing. Light-curing was carried out in a Triad 2000 curing unit (Dentsply, York PA, USA) for 10 min per side, using an unfiltered halogen bulb emitting a broad spectrum of visible light, with an irradiance of 150 mW/cm2 and a light source-sample distance of approximately 10 cm. We used the Dentsply unit rather than a hand-held curing light because for this study we wished to eliminate the oxygen inhibition layer as a variable in the extraction. During irradiation, poly films were used to cover the top and bottom surfaces of the samples. The cured discs were removed from the molds and lightly cleaned with a low-lint laboratory wipe to remove any dust generated during disassembly. Cured discs were stored at room temperature until the time of extraction (total of 7 weeks of storage).

Resin extraction

Resin extraction was carried out using guidance in ISO 10993-12 . Cured discs were placed in glass vials with polypropylene caps (Fisher Scientific, Pittsburg PA, USA) and extracted in HPLC grade methanol (VWR Scientific, Radnor PA, USA) at a ratio of 3.3 cm 2 disc/mL methanol, equivalent to 0.42 g disc/mL. Methanol was used because it is a suggested extraction solvent for generation of medical device samples for testing in biological systems and because our experience indicates that methanol is a highly aggressive solvent for BisGMA-based resins.

The methanol was spiked with 30 ng/mL d16 BPA (Cambridge Isotope Laboratories, Tewksbury, MA, USA) as an internal standard (IS). The purity of d16 BPA was listed as 98% d16 by the manufacturer. Extraction was conducted at 37 °C with mild agitation (100 rpm) for 24 h. A method blank consisting of methanol alone was included with all extraction steps and subsequent analyses.

BPA quantitation

BPA separation and quantitation were carried out on an Agilent 1200 SL with UV diode ray detection (DAD) (Agilent Technologies, Santa Clara, CA, USA) coupled to an AB/Sciex Qtrap 3200 Liquid Chromatograph with tandem mass spectrometry capability (AB/Sciex LLC, Framingham, MA, USA). Separation was performed with an Agilent Eclipse XDB-C18 column (Agilent Technologies, Santa Clara, CA, USA) with 2.1 × 150 mm dimensions and 3.5 micron packing. All chromatography solvents were HPLC grade from EMD Millipore, Billerica, MA, USA. The mobile phase was a mixture of unbuffered water (channel A) and methanol (channel B). The gradient was applied as follows: 20% B (preinjection reequilibration, 5 min); 70% B (0−0.5 min); 75% B (0.5−6 min); 99% B (6.1 min followed by a 4 min hold). The flow rate was 0.5 mL/min and the injection volume was 5 μL. Column effluent was split between the diode array detector and the Qtrap mass spectrometer. A wavelength of 225 nm was used to plot the UV response, but full DAD spectra were acquired during all LC/MS experiments. The Qtrap was operated in standard triple quadrupole multiple reaction monitoring mode. The Turbo IonSpray source of the Qtrap was optimized for BPA detection with ESI voltage of -4500 V, nebulizer gas of 80 psi, desolvation gas pressure and temperature of 70 psi and 700 °C, respectively, and counter-current gas of 30 psi. A declustering potential of -50 V was optimized for generation of the deprotonated BPA molecular ion, and the entrance potential and collision cell entrance potential were optimized at -10 and -20 V, respectively. Two fragment ions, one involving loss of methyl radical (m/z 212) and one involving loss of phenol (m/z 133) were used to monitor BPA. The 227/212 transition was optimized using a collision energy of -25 V and collision exit potential of -2.0 V, while the 227/133 transition employed a collision energy of -36 V and a collision exit potential of -1.0 V. Each of the three transitions monitored during the entire chromatographic run was assigned a 100 ms dwell time.

The same d16 BPA used in the resin extraction was employed as an IS in all cases to correct for any losses during sample preparation or variability injection volume or detection efficiency. The d16 IS was monitored using a 241/223 transition involving loss if d3 methyl radical was used and the same tuning employed for the analogous transition if d0 BPA was used. Tests of the d16 IS in our LC/MS/MS assay showed that it gave no signal at d0, and as such did not contribute to the BPA signal during the assay. The level of detection was 3 ppb BPA.

Identification of co-eluting chemical(s)

Identification of co-eluting chemicals was carried out on an Agilent 1260 HPLC coupled to an Agilent 6224 Time of Flight (TOF) LC/MS (Agilent Technologies, Santa Clara, CA, USA) with Dual ESI source operated in positive and negative modes. Chromatography was performed as for the initial BPA analyses except that some samples were also run using water and acetonitrile (both containing 6 mM ammonium acetate to aid ionization), to enhance detection of less polar analytes in positive mode for the TOF analyses. The dual ESI was set at nebulizer gas pressure of 45 psi, desolvation gas flow and temperature of 11 lpm and 325 °C respectively, capillary voltage of 400 V, skimmer voltage of 65 V, fragmenter voltage of 155 V, and octopole radiofrequency of 750 V. All scans were automatically recalibrated with the standard Agilent Reference Mixture using 121.0509 and 922.0098 ions for recalibration. The full scan mass range employed was 105−1700 atomic mass units (amu) at 1 scan per second.

Materials and methods

Experimental resin

An experimental light-cured dental resin was prepared, consisting of 83% silanized silica (CAS Number 100402-78-6), 16% BisGMA (CAS Number 1565-94-2), <1% of Camphorquinone (CPQ, CAS Number 10373-78-1), and <1% ethyl 4 – (dimethylamino) benzoate (EDMAB, CAS Number 10287-53-3). The starting materials were the same as used in 3M commercial oral care resins, and were obtained from proprietary sources. Ingredients were processed in a planetary mixer and the mixed resin was transferred to circular stainless steel molds (15 mm ID × 1.7 mm high) for curing. Light-curing was carried out in a Triad 2000 curing unit (Dentsply, York PA, USA) for 10 min per side, using an unfiltered halogen bulb emitting a broad spectrum of visible light, with an irradiance of 150 mW/cm2 and a light source-sample distance of approximately 10 cm. We used the Dentsply unit rather than a hand-held curing light because for this study we wished to eliminate the oxygen inhibition layer as a variable in the extraction. During irradiation, poly films were used to cover the top and bottom surfaces of the samples. The cured discs were removed from the molds and lightly cleaned with a low-lint laboratory wipe to remove any dust generated during disassembly. Cured discs were stored at room temperature until the time of extraction (total of 7 weeks of storage).

Resin extraction

Resin extraction was carried out using guidance in ISO 10993-12 . Cured discs were placed in glass vials with polypropylene caps (Fisher Scientific, Pittsburg PA, USA) and extracted in HPLC grade methanol (VWR Scientific, Radnor PA, USA) at a ratio of 3.3 cm 2 disc/mL methanol, equivalent to 0.42 g disc/mL. Methanol was used because it is a suggested extraction solvent for generation of medical device samples for testing in biological systems and because our experience indicates that methanol is a highly aggressive solvent for BisGMA-based resins.

The methanol was spiked with 30 ng/mL d16 BPA (Cambridge Isotope Laboratories, Tewksbury, MA, USA) as an internal standard (IS). The purity of d16 BPA was listed as 98% d16 by the manufacturer. Extraction was conducted at 37 °C with mild agitation (100 rpm) for 24 h. A method blank consisting of methanol alone was included with all extraction steps and subsequent analyses.

BPA quantitation

BPA separation and quantitation were carried out on an Agilent 1200 SL with UV diode ray detection (DAD) (Agilent Technologies, Santa Clara, CA, USA) coupled to an AB/Sciex Qtrap 3200 Liquid Chromatograph with tandem mass spectrometry capability (AB/Sciex LLC, Framingham, MA, USA). Separation was performed with an Agilent Eclipse XDB-C18 column (Agilent Technologies, Santa Clara, CA, USA) with 2.1 × 150 mm dimensions and 3.5 micron packing. All chromatography solvents were HPLC grade from EMD Millipore, Billerica, MA, USA. The mobile phase was a mixture of unbuffered water (channel A) and methanol (channel B). The gradient was applied as follows: 20% B (preinjection reequilibration, 5 min); 70% B (0−0.5 min); 75% B (0.5−6 min); 99% B (6.1 min followed by a 4 min hold). The flow rate was 0.5 mL/min and the injection volume was 5 μL. Column effluent was split between the diode array detector and the Qtrap mass spectrometer. A wavelength of 225 nm was used to plot the UV response, but full DAD spectra were acquired during all LC/MS experiments. The Qtrap was operated in standard triple quadrupole multiple reaction monitoring mode. The Turbo IonSpray source of the Qtrap was optimized for BPA detection with ESI voltage of -4500 V, nebulizer gas of 80 psi, desolvation gas pressure and temperature of 70 psi and 700 °C, respectively, and counter-current gas of 30 psi. A declustering potential of -50 V was optimized for generation of the deprotonated BPA molecular ion, and the entrance potential and collision cell entrance potential were optimized at -10 and -20 V, respectively. Two fragment ions, one involving loss of methyl radical (m/z 212) and one involving loss of phenol (m/z 133) were used to monitor BPA. The 227/212 transition was optimized using a collision energy of -25 V and collision exit potential of -2.0 V, while the 227/133 transition employed a collision energy of -36 V and a collision exit potential of -1.0 V. Each of the three transitions monitored during the entire chromatographic run was assigned a 100 ms dwell time.

The same d16 BPA used in the resin extraction was employed as an IS in all cases to correct for any losses during sample preparation or variability injection volume or detection efficiency. The d16 IS was monitored using a 241/223 transition involving loss if d3 methyl radical was used and the same tuning employed for the analogous transition if d0 BPA was used. Tests of the d16 IS in our LC/MS/MS assay showed that it gave no signal at d0, and as such did not contribute to the BPA signal during the assay. The level of detection was 3 ppb BPA.

Identification of co-eluting chemical(s)

Identification of co-eluting chemicals was carried out on an Agilent 1260 HPLC coupled to an Agilent 6224 Time of Flight (TOF) LC/MS (Agilent Technologies, Santa Clara, CA, USA) with Dual ESI source operated in positive and negative modes. Chromatography was performed as for the initial BPA analyses except that some samples were also run using water and acetonitrile (both containing 6 mM ammonium acetate to aid ionization), to enhance detection of less polar analytes in positive mode for the TOF analyses. The dual ESI was set at nebulizer gas pressure of 45 psi, desolvation gas flow and temperature of 11 lpm and 325 °C respectively, capillary voltage of 400 V, skimmer voltage of 65 V, fragmenter voltage of 155 V, and octopole radiofrequency of 750 V. All scans were automatically recalibrated with the standard Agilent Reference Mixture using 121.0509 and 922.0098 ions for recalibration. The full scan mass range employed was 105−1700 atomic mass units (amu) at 1 scan per second.

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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Potential confounders of bisphenol-a analysis in dental materials
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