2.1

Solvents and reagents

All solvents in this study were HPLC grade. Water, methanol, acetonitrile, caffeine, BisEMA, DEGDMA, EBPADA, and MMA were from Sigma–Aldrich, UK. BisGMA, TEGDMA, UDMA, HEMA were supplied by Röhm GmbH, Germany ( Table 2 ).

2.2

Characterization of uncured resin

Fourteen commercial resin-composite materials including six bulk-fill materials and eight conventional resin-composite materials were tested. A list of the resin-composites studied is given in Table 1 . Three samples of each uncured resin-composite material were prepared. For each sample 0.1 g of uncured resin-composite was dissolved in 1.5 ml methanol (containing 0.1 mg/ml caffeine as an internal standard). The samples were shaken for 10 min until the composite was completely dissolved in the methanol solution. The samples were then centrifuged at 15,000 g for one minute to separate the filler particles from the solution (IEC MicroCL 17 centrifuge, Thermo Electron Corporation, USA). The supernatant methanol solution containing the dissolved resin was then quickly withdrawn with a syringe without disturbing the precipitated fillers, passed through a 0.2 μm membrane filter, and transferred into an HPLC vial. Resulting solutions were then analysed without any further work-up by high performance liquid chromatography and mass spectrometry (LC/MS). Samples of reference monomers (BisGMA, UDMA, TEGDMA, BisEMA, EBPADA, HEMA, MMA, and DEGDMA) dissolved in methanol were also analysed to facilitate their identification in the uncured resin solutions prepared from different composite materials.

The analysis was carried out using an Agilent 1100LC/MSD system. This consisted of an Agilent 1100 high performance liquid chromatograph with diode array detection coupled to an Agilent 1100 ion trap mass spectrometer (Agilent Technologies, Germany). Chromatography was performed using a reverse phase Phenomenex SphereClone 5 μm ODS (2) column of dimensions 4.6 mm × 250 mm (Phenomenex, USA). An isocratic method of acetonitrile and water (50:50) and flow rate of 0.5 ml/min was used and the UV detection was set at 205 nm. The column temperature was 22 °C with a run time of 70 min for each sample. The analytes were ionized using electrospray (ESI) in positive ion mode with an applied capillary voltage of 3 kV. Nebulizing nitrogen gas pressure was 60 psi, drying gas flow was 10 l/min, and the drying gas temperature was 350 °C. The mass range was set at 50–1000 m / z . The LC/MS system was controlled by Chemstation (version B.01.03) and LC/MSD Trap (version 3.3) software.

Identification of the various compounds was achieved by comparison of their mass spectra (mass-to-charge-ratio ( m / z )) and chromatographic retention times with those of reference compounds. For compounds with no available reference, the molecular weight was calculated from the highest intensity m / z value and compared with data in the literature. To compare the quantity of each compound in different resin-composite materials, the chromatogram peak area% of each compound in relation to the total chromatogram area was calculated. Log P values (octanol–water partition coefficient) of identified non-fragmented compounds with known structural formula were computationally obtained using the ACD/ChemSketch (version 11.02) software.

2.3

Statistical analysis

Data were entered into statistical analysis software (SPSS, V20, Chicago, USA), were checked for normality using a Shapiro–Wilk’s test, and analysed using one-way ANOVA to assess differences in specific monomer content between different uncured materials. A Tukey post hoc test was used for multiple comparisons. Pearson correlation and linear regression analyses were carried out to explore the relationship between logP values and reverse phase liquid chromatography retention time. All tests were carried out at α = 0.05.

2.1

Solvents and reagents

All solvents in this study were HPLC grade. Water, methanol, acetonitrile, caffeine, BisEMA, DEGDMA, EBPADA, and MMA were from Sigma–Aldrich, UK. BisGMA, TEGDMA, UDMA, HEMA were supplied by Röhm GmbH, Germany ( Table 2 ).

2.2

Characterization of uncured resin

Fourteen commercial resin-composite materials including six bulk-fill materials and eight conventional resin-composite materials were tested. A list of the resin-composites studied is given in Table 1 . Three samples of each uncured resin-composite material were prepared. For each sample 0.1 g of uncured resin-composite was dissolved in 1.5 ml methanol (containing 0.1 mg/ml caffeine as an internal standard). The samples were shaken for 10 min until the composite was completely dissolved in the methanol solution. The samples were then centrifuged at 15,000 g for one minute to separate the filler particles from the solution (IEC MicroCL 17 centrifuge, Thermo Electron Corporation, USA). The supernatant methanol solution containing the dissolved resin was then quickly withdrawn with a syringe without disturbing the precipitated fillers, passed through a 0.2 μm membrane filter, and transferred into an HPLC vial. Resulting solutions were then analysed without any further work-up by high performance liquid chromatography and mass spectrometry (LC/MS). Samples of reference monomers (BisGMA, UDMA, TEGDMA, BisEMA, EBPADA, HEMA, MMA, and DEGDMA) dissolved in methanol were also analysed to facilitate their identification in the uncured resin solutions prepared from different composite materials.

The analysis was carried out using an Agilent 1100LC/MSD system. This consisted of an Agilent 1100 high performance liquid chromatograph with diode array detection coupled to an Agilent 1100 ion trap mass spectrometer (Agilent Technologies, Germany). Chromatography was performed using a reverse phase Phenomenex SphereClone 5 μm ODS (2) column of dimensions 4.6 mm × 250 mm (Phenomenex, USA). An isocratic method of acetonitrile and water (50:50) and flow rate of 0.5 ml/min was used and the UV detection was set at 205 nm. The column temperature was 22 °C with a run time of 70 min for each sample. The analytes were ionized using electrospray (ESI) in positive ion mode with an applied capillary voltage of 3 kV. Nebulizing nitrogen gas pressure was 60 psi, drying gas flow was 10 l/min, and the drying gas temperature was 350 °C. The mass range was set at 50–1000 m / z . The LC/MS system was controlled by Chemstation (version B.01.03) and LC/MSD Trap (version 3.3) software.

Identification of the various compounds was achieved by comparison of their mass spectra (mass-to-charge-ratio ( m / z )) and chromatographic retention times with those of reference compounds. For compounds with no available reference, the molecular weight was calculated from the highest intensity m / z value and compared with data in the literature. To compare the quantity of each compound in different resin-composite materials, the chromatogram peak area% of each compound in relation to the total chromatogram area was calculated. Log P values (octanol–water partition coefficient) of identified non-fragmented compounds with known structural formula were computationally obtained using the ACD/ChemSketch (version 11.02) software.

2.3

Statistical analysis

Data were entered into statistical analysis software (SPSS, V20, Chicago, USA), were checked for normality using a Shapiro–Wilk’s test, and analysed using one-way ANOVA to assess differences in specific monomer content between different uncured materials. A Tukey post hoc test was used for multiple comparisons. Pearson correlation and linear regression analyses were carried out to explore the relationship between logP values and reverse phase liquid chromatography retention time. All tests were carried out at α = 0.05.