Determination of BisphenoI-A and Related

Compounds in Human Saliva by Gas

Chromatography - Mass Spectrometry

A. Zafra 1/M. del Olrno 1/ R. Pulgar2/A. Naval6n ~/J. L.VflcheX*

1 Department of Analytical Chemistry, University of Granada, 18071 Granada, Spain; E-Maih Jvilchez@ugr.es
2 Department of Stomatology, U niversity of Granada, 18071 Granada, Spain

GMA), has been reported to be cytotoxic

KeyWords in some cell culture systems [2, 3].
A recent study by Olea et al. identified
Gas chromatography - mass spectrometry BPA and implicated bis-DMA as estro-
Saliva analysis genic factors in human saliva one hour
BisphenoI-A and related compounds after sealants had been istalled [4].
Triethylenglycol dimethacrylate Resins applied as dental sealants are
polymerised through a self-cure chemical
process or by photoactivation with visible
Summary light but incomplete conversion of mono-
mers during the curing process can lead to
A simple and sensitive method for the determination of trace amounts of bisphenoJ-A (BPA), bi- residual monomers potentially leaking
sphenoI-A diglycidyl dimethacrylate (bis-GMA), bisphenoI-A dimethacrylate (bis-DMA) and out of the cured resin.
triethyleneglycol dimethacrylate (TEGDMA) in human saliva is proposed. These materials are High-performance liquid chromato-
used in dental restorations, as composites and sealants, and are sometimes detected in hu- graphy (HPLC) has been used to analyse
man saliva after dental treatment. The proposed method involves protein precipitation using and characterise dental polymers, includ-
acetonitrile followed by acidification, evaporation of the solvent and dissolution with dichloro- ing residual monomers [5, 6], to study
methane prior to injection into a GC-MS. composites [7, 8] or other dental materials
Thermal derivatization in the injection system was used for the identification and quantifi- such as visible light-cured resinous liners
cation of bis-GMA. Clean-up is not necessary using SIM mode. BisphenoI-F (BPF)was used as [9] and sealants [10]. Direct analysis of re-
internal standard. The linear range was 15 to 1000 I~g 9L 1 for BPA, 50 to 10000 I~g 9L 1 for storative resins also uses N M R [11]. Gas
bis-GMA, 50to 10001~g. L 1 for bis-DMAand 1 to 1001~g. L 1 forTEGDMA. chromatography (GC) coupled with FID
The detection limits were 3,15,10 and 0.3 I~g. L 1 for BPA, bis-GMA, bis-DMA and TEGD- and MS had been used to identify residual
MA, respectively. Validation of the proposed method was carried out by using the standard monomers of bis-GMA and triethylengly-
addition methodology. col dimethacrylate (TEGDMA) migrated
Samples of 10 mLof human saliva collected 1 h aflerdental treatmentwere analysed in or- into water from photo-cured resins [12].
der to assess the applicability of the method to detect and quantify such compounds origi- T E G D M A is used to reduce the initial
nated from methacrylic resins used in odontological treatment. viscosity of the maj or monomers.
A new GC-MS method for detection
and determination of such dental sealants
bis-GMA, bis-DMA, BPA and TEGD-
M A in human saliva after restorative den-
Introduction bis-GMA is synthesised from bisphenol-A tal treatment is presented here.
(BPA) and glycidyl methacrylates having
Dental resin systems based on dimetha- appropriate mechanical properties, che-
crylate monomers like bisphenol-A digly- mical stability and simulates natural tooth
cidyl dimethacrylate (bis-GMA) and bi- colour.
sphenol-A dimethacrylate (bis-DMA) are Bisphenol-A, present as an impurity in
now used because they are an alternative some resins (bis-GMA) and as a degrada-
to the dental amalgams [1]. tion product in others (bis-DMA and bis-

Original Chromatographia 2002, 56, August (No. 3/4) 213

0009-5893/00/02 213- 06 $ 03.00/0 9 2002 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
Experimental
Apparatus and Software
All chromatographic measurements were
performed with a Hewlett Packard (Palo
Alto, CA, USA) system comprised of a
5890 gas chromatograph fitted with an
HP 7673 autosampler, a split-splitless in-
jector for capillary columns and a 5971
mass spectrometer with an EI of 70 eV as
the ionization source and quadrupole
mass filter. This system was modified to
enhance its sensitivity and could be used
in complete scan and selected ion moni-
toring (SIM) modes. The mass spectro-
meter was calibrated every day before use
with perfluorotributylamine (PFTBA).
The column was an HP1-MS fused silica
capillary (30 m • 0.25 mm i. d., 0.25 pm
film thickness) bonded with methyl sili-
cone gum phase. The computer was an
HP-UX Chemsystem with a spectral li-
brary of more than 140,000 compounds,
which was used to control the entire chro-
matographic system. Helium (purity
99.999%) was used as carrier gas.
Thermogravimetric studies were car-
ried out with a Shimadzu (Kyoto, Japan)
TGA-50 thermogravimetric analyser
using an alumina cell and inert atmo-
sphere of nitrogen.
The Statgraphics [13], Quimio [14] and
Alamin [15] software packages were used
for the statistical analysis of data and for
regression analysis (linear model).
Reagents
Stock solutions of BPA [80-05-7] (Sigma,
St. Louis, MO, USA) and T E G D M A
[109-16-0] (Sigma) containing 100pg.
mL 1 were prepared in ethanol 99% (v/v)
(Panreac, Barcelona, Spain). bis-DMA
[3253-39-2] (Sigma) and bis-GMA [1565-
94-2] (R6hm, Darmstadt, Germany) were
dissolved in tetrahydrofuran at a concen-
tration of 100 p g . m L 1 and 1000pg.
mL 1, respectively. All chemicals used in
this study were of analytical reagent grade
except bis-GMA, a gift from R6hm
GmbH Chemische Fabrik, which is not
sold as a standardised reagent.
A standard solution of 100 pg. mL 1
of bisphenol-F (Aldrich, Steinheim, Ger-
many) in ethanol was used as an internal
standard.
The solutions were stored in dark bot-
tles at 4 ~ remaining stable for at least
one month.
Acetonitrile and dichloromethane were
supplied by Panreac.
214
Sample Preparation
The saliva samples were collected from
volunteers having undertaken dental
treatment repairs in the Dentistry Faculty
of Granada (Spain).
20.0 mL of acetonitrile was added to
10.0 mL of each saliva sample in a strong
basic medium in order to precipitate pro-
teins. After vortex mixing for 30 s the sam-
ple was centrifuged for 10 min. at
3000 r.p.m. The supernatant liquid, after
acidification with hydrochloric acid, was
evaporated to dryness at 80 ~ in a heated
cabinet, the residue being dissolved in
10mL of dichloromethane and mechani-
cally shaken for 2 h. Next the sample was
evaporated to near dryness in a rotary va-
cuum evaporator at 40 ~ and transferred
to a 150 pL microvial. After evaporation
to dryness under nitrogen, 50.0 pL of di-
chloromethane were added and the mix-
ture was mechanically shaken for 2 min at
room temperature. At this point, the sam-
ple is ready to be injected into the GC-MS.
GC-MSAnalysis
A 2 pL aliquot of the sample is injected by
the autosampler using the splitless injec-
tion mode with the split valve closed for
2 min. The GC-MS parameters used were:
injector temperature, 200 ~ for BPA and
T E G D M A and 300 ~ for bis-GMA and
bis-DMA; detector temperature, 280~
and oven temperature program: 150~
(2min), 3 0 ~ 1, 270oC (7 min),
5 ~ min 1,300 ~ (1 min).
The selected ions of the compounds for
SIM mode operation were: m/z 344 and
345 for the standard (BPF); m/z 41, 69 y
113 for TEGDMA; m/z 213 and 228 for
BPA; m/z 69, 349 and 364 for bis-DMA
and m/z 325 and 340 for the three com-
pounds derived from the thermal degra-
dation of bis-GMA at 300 ~
The concentrations of the analytes
were calculated by the internal standard
method.
Results and Discussion
ThermogravimetricAnalysis
of BPA,bis-GMA,bis-DMA
and TEGDMA
Although previous studies demonstrated
that the volatility and thermal stability of
Chromatographia 2002, 56, August (No. 3/4)
BPA, bis-DMA and T E G D M A make
them suitable for detection and quantifi-
cation by GC-MS, we found that bis-
GMA could not be detected by this tech-
nique because of its low volatility. How-
ever, four chromatographic peaks ap-
peared for bis-GMA, three of major
abundance and one of lesser abundance.
This may be due to thermal degradation
of this compound with the increments in
the injector temperature. Thermogravi-
metric analysis and IR spectra confirmed
this point. The TGA of the compounds
was carried out between 20 and 950 ~ in
an inert atmosphere of nitrogen.
The D T G curve of standard bisphenol-
A shows an endothermic peak at 335 ~
corresponding to sublimation with de-
composition. In the TGA curve, sublima-
tion starts at 200 ~ and decomposition at
280~ respectively, showing two IR
bands at 2334.9 and 2365.5 cm 1 corre-
sponding to CO2 (Figure lb). BPA re-
mains stable until this temperature.
The D T G curve of standard bis-GMA
shows an endothermic peak at 437 ~ cor-
responding to its thermal decomposition.
In the TGA curve, decomposition starts at
245 ~ Figure la shows the IR spectrum
of the decomposition products. The ap-
pearance of BPA with characteristic bands
at 832.8, 1179.1, 1515.1, 2981.6, 3653.7
cm 1 a n d a w i d e b a n d a t 1739.0cm 1 at-
tributed to the presence of a mixture of ke-
tonic compounds (as corroborated via
MS) related to BPA was observed.
The DTG curve of bis-DMA shows
two endothermic peaks at 314~ and
454 ~ attributed to a mass loss of 21.8%
and 73.2% respectively. The IR spectrum
of decomposition products (Figure lc)
shows the presence of CO2 (2334.9
2365.5 cm 1 ) in the first decomposition step
at 314~ and CO2, BPA and methacrylic
compounds (1734.1 1754.5cm 1) at the
decomposition temperature of 454 ~ We
observed that bis-DMA remains stable at
280 ~
Finally, the DTG curve of T E G D M A
shows two endothermic peaks at 308 ~
and 436 ~ with associated mass losses of
83.9% and 15.2%, respectively. In both
cases the IR signals appear at 944.8,
1168.9, 1301.3 and 2961 cm 1, (Figure
ld). Tentatively these bands could be at-
tributed to 2-hydroxyethyl methacrylate
and ethyl methacrylate. T E G D M A is
stable at 240 ~
Original