Page 6 - Shimadzu Journal vol.2 Issue1
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Human Health                                                                                                                                                                                                              Human Health




                                                                                                                                      2-2 Sample Preparation                             3. Results and Discussion                                                                          being reported in a study of human exposure to BPA through
                Quantitative analysis of bisphenol A in water                                                                         Standard solutions. Stock solutions of BPA and BPA-G were prepared   Since BPA is a constituent of many plastics including some used in               drinking water.  An example of the analysis of BPA in type I
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                                                                                                                                      in methanol at final concentrations of 1 mg/mL each. Working   laboratory plastic ware and cap liners of vials and bottles, the                       purified laboratory water is shown in Fig. 4. It should be noted
                and serum using UHPLC-MS-MS                                                                                           standards were made by serial dilution from stock solutions.   solvents and materials used for BPA and BPA-G extraction and                           that even many laboratory water purification systems produce
                                                                                                                                      Calibration standards were prepared by mixing 1 µL of each working   analysis had to be tested for BPA. Several brands of HPLC-grade                  water contaminated with BPA, since they often contain
                                                                                                                                      standard with 24 µL blank mouse serum or water and vortex mixing.  methanol, methyl-tert-butyl ether and acetonitrile tested positive                 polysulfone filters that leach BPA monomer. 12
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            Richard B. van Breemen , Guannan Li , Yang Yuan , and Ke Huang 1                                                          Serum. Each unknown serum sample (25 µL) or calibration standard   for BPA. Eventually, all laboratory ware and solvents utilized for
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            1 Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, 833 South Wood         (25 µL) was mixed with 100 µL acetonitrile containing the surrogate   sample preparation and analysis were confirmed to be free of BPA
            Street, Chicago, IL 60612 USA                                                                                             standards 5 ng/mL [d6]-BPA and 5 ng/mL  [ C 12]-BPA-G. The mixture   contamination.
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                                                                                                                                      was vortexed for 1 min, centrifuged for 15 min at 13000 x g at 4 C, and   The recoveries of BPA and BPA-G from serum using
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            ABSTRACT                                                                                                                  then the supernatant was removed and evaporated to dryness. The   solvent/solvent extraction were ʙ90% and ʙ87%, respectively.
                                                                                                                                      residue was reconstituted in 25 µL of 50% aqueous methanol, and a 5   The standard curves for BPA and BPA-G in water and serum were
            Endocrine disruptors represent a major toxicological and public health issue, and the xenoestrogen bisphenol A (BPA) has received much
            attention due to its high volume of production for plastics and widespread human exposure. The measurement of BPA in biological and   µL aliquot was injected onto the UHPLC-MS-MS system for analysis.  linear. For example, the standard curves for BPA and BPA-G in
            environmental samples is essential for risk assessment. However, such analyses are challenging due to the trace levels of BPA in these   Water. Each water sample (50 mL) was spiked with [d6]-BPA, acidified   mouse serum shown in Fig. 3 were linear over the range of 0.2 to
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            samples and the possibility of false positive determinations due to background contamination. To overcome these limitations, we   with 50 µL 33% HCl (aq) and loaded onto a Waters (Milford, MA) Oasis   25 ng/mL ( r  = 0.998) and 0.1 to 25 ng/mL (r  =  0.996),
            developed a highly sensitive and selective method using ultrahigh pressure liquid chromatography (Shimadzu Nexera) and a new   HLB 5cc 200 mg LP glass solid phase extraction cartridge that had been   respectively.  The mouse serum used to prepare these curves
            generation triple quadruple mass spectrometer (Shimadzu LCMS-8050).                                                                                                          contained traces of BPA and BPA-G despite attempting to raise
                                                                                                                                      conditioned using 4 mL portions of methyl-t-butyl ether, methanol and
                                                                                                                                      water. The cartridge was washed with 4 mL water, and then BPA was   the animals in a BPA-free laboratory environment. This problem
            Key Words                                                                                                                 eluted using 4 mL methanol and then 4 mL of 10% methanol in   illustrates the ubiquitous nature of BPA in the laboratory and in
            Bisphenol A, bisphenol A-glucuronide, serum, UHPLC-MS-MS, LCMS-8050, water quality                                        methyl-t-butyl ether. The combined methanol and methyl-t-butyl ether   the environment.
                                                                                                                                      eluates were evaporated to dryness under a stream of nitrogen, and
                                                                                                                                      the residue was reconstituted in 100 µL of 50% aqueous methanol for
            1. Introduction
                                                                                                                                      analysis using UHPLC-MS-MS.
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            Used as a plasticizer in epoxy resins such as those lining canned   have been reported in neonates.  To enable risk assessment of   UHPLC-MS-MS. Chromatographic separations were carried out using a
            foods, as a constituent of thermal papers used in cash register   BPA exposure, BPA and BPA-G need to be determined in serum   Shimadzu (Kyoto, Japan) LCMS-8050 triple quadrupole mass
            receipts and as a monomer for polycarbonate plastic used in   not only of adults but also of neonates and infants, from whom   spectrometer equipped with a Shimadzu Nexera UHPLC system. BPA
            production of water bottles, bisphenol A (BPA) is a high volume   serum sample sizes are typically quite small. 5         and BPA-G were separated on a Waters (Milford, MA) Acquity UPLC
            industrial chemical with considerable human and environmental   BPA has been measured in surface water samples using gas   BEH (2.1 x 50 mm, 1.7 µm) C18 column. A 1.5 min linear gradient was
                   1,2
            exposure.  BPA (Fig. 1) has estrogenic effects and has been   chromatography-tandem mass spectrometry (GC-MS),  high      used from 10-100% acetonitrile in water followed by a hold at 100%
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            shown to function as an endocrine disruptor.  There is concern   performance liquid chromatography-tandem mass spectrometry   for 0.4 min at a flow rate of 0.4 mL/min. The total run time including
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            that environmental BPA exposure can adversely impact aquatic life   (HPLC-MS-MS)  and ultrahigh pressure liquid           equilibration was 3.5 min. The column oven temperature was 45 °C,
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            and human health, especially in children who might be more   chromatography-MS-MS (UHPLC-MS-MS).  Most methods for the    and the injection volume was 5 µL.
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            sensitive to endocrine disruptors.
                                                               measurement of BPA and BPA-G involve chemical or enzymatic             Negative ion electrospray mass spectrometry with selected reaction
                                                               deconjugation to convert BPA-G to BPA prior to quantitative            monitoring (SRM) and a dwell time of 50 ms per transition was used for
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                                                               analysis. For example, Kosarac, et al.  enzymatically deconjugated     the measurement of each analyte. Deprotonated molecules were used
                                                               BPA-G prior to derivatization and measurement of total BPA in          as the precursor ions, and the most abundant two product ions (Fig. 2)
                                                               serum using GC-MS-MS. Among the few methods that measure               were used as quantifiers and qualifiers, respectively, for SRM. For BPA,
                                                               both BPA and BPA-G directly in serum, most rely on                     the SRM transitions were m/z 227 to 212 (quantifier) and m/z 227 to
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                                                               HPLC-MS-MS  or UHPLC-MS-MS  and utilize negative ion                   133 (qualifier); and for the surrogate standard [d6]-BPA, the SRM
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                                                               electrospray without need for derivatization.                          transitions were  m/z 233 to 215 (quantifier) and  m/z 233 to 113
                                                               We developed methods based on UHPLC-MS-MS for the analysis             (qualifier). For BPA-G and its surrogate standard [13C12]-BPA-G, the
                                                               of BPA in water and analysis of both BPA and intact BPA-G in           SRM transitions were m/z 403 to 227 (quantifier) and m/z 403 to 113
                                                               serum. Unlike GC-MS-MS assays, our approach requires no                (qualifier); and  m/z 415 to 239 (quantifier) and  m/z 415 to 113   Fig. 3. Standard curves obtained for BPA and BPA-G
                Fig. 1. Chemical structures of BPA and its primary   derivatization of BPA, and unlike GC-MS-MS and some              (qualifier), respectively.                                  in mouse serum using UHPLC-MS-MS with
                     human metabolite, BPA-glucuronide.        HPLC-MS-MS based assays, our method avoids hydrolysis of                                                                           negative ion electrospray and SRM.
                                                               BPA-G. UHPLC was utilized instead of HPLC to shorten
            Human exposure to BPA can result from such diverse sources as   chromatographic separation time (less than 2 min per analysis),                                              The lower limit of quantitation for BPA in water was 0.01 ng/mL,
            consumption of canned foods, drinking water, dental composites,   and when volumes of serum samples are small, our method is                                                 and the lower limits of quantitation for BPA and BPA-G in mouse
            and cash register receipts. The large quantity of BPA produced   unique in that only 25 µL of serum are required.                                                            serum were 0.5 ng/mL and 0.2 ng/mL, respectively. Although
            world-wide each year has resulted in BPA contamination of rivers                                                                                                             these LLOQ values for BPA and BPA-G in serum are not the lowest
            and lakes that can affect wildlife as well as humans using these                                                                                                             in the literature, the serum sample size used in our experiments
            resources for drinking water. Therefore, methods are needed for   2. Experimental                                                                                            (25 µL) was at least 10-fold smaller than previous methods. For
            the quantitative analysis of BPA in water.                                                                                                                                   example, using UHPLC-MS-MS Gerano et al . reported  an LLOQ
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            After absorption, BPA is metabolized in the liver of adults to form   2-1 Materials                                                                                          of 0.1 for both BPA and BPA-G in serum using a sample size of
            primarily BPA-monoglucuronide (BPA-G; Fig. 1) and to a lesser   HPLC-grade solvents (acetonitrile, methanol and water) were                                                  250 µL serum. Therefore, on a sample size basis, our method is as
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            extent, BPA-sulfate, before being excreted in urine.  Since the   purchased from Burdick & Jackson (Honeywell, Muskegon, MI).                                                sensitive as any in the literature.
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            activities of glucuronyltransferases that conjugate BPA with   BPA and [rings- C 12]-BPA-G were purchased from Sigma-Aldrich                                                 The method for measuring BPA in water was applied to the
            glucuronic acid are low at birth and increase for the next several   (St. Louis, MO). Bisphenol A mono-β-D-glucuronide (BPA-G) was                                           analysis of drinking water and surface water samples from the
            weeks, neonates might be exposed to higher concentrations of   obtained from the Midwest Research Institute (Kansas City, MO),   Fig. 2. Negative ion electrospray product ion tandem mass   Chicago area, Jamaica and Ghana. BPA in water in these samples
            unconjugated BPA than adults. While levels of unconjugated BPA   and [d 6]-BPA was purchased from Cambridge Isotope Laboratories   spectra of the deprotonated molecules of BPA (m/z   ranged from none detected to up to 0.186 ng/mL. These data are
            in adult human serum are typically low (≤0.5 ng/mL), higher levels   (Andover, MA).                                            227) and BPA-G (m/z 403). The two most abundant
                                                                                                                                           product ions of each were selected for use during SRM.
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