Journal of Chromatography B, 953–954 (2014) 80–85

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Determination of seven bisphenol analogues in reed and Callitrichaceae by ultra performance liquid chromatography–tandem mass spectrometry Libin Lu a,b , Yunjia Yang a,b , Jing Zhang a,b , Bing Shao a,b,∗ a b

School of Public Health, Capital Medical University, Beijing 100069, China Beijing Key Laboratory of Diagnostic and Traceability For Food Poisoning, Beijing Research Center for Preventive Medicine, Beijing 100013, China

a r t i c l e

i n f o

Article history: Received 5 August 2013 Received in revised form 22 January 2014 Accepted 2 February 2014 Available online 10 February 2014 Keywords: Bisphenol analogues Reed Callitrichaceae Solid-phase extraction UPLC–MS/MS

a b s t r a c t An analytical procedure was developed to simultaneously determine bisphenol S, bisphenol F, bisphenol B, bisphenol A, bisphenol AF, tetrachlorobisphenol A, and tetrabromobisphenol A in reed and Callitrichaceae. Homogenized samples were extracted with acetonitrile and purified using an ENVITM -Carb cartridge followed by an NH2 cartridge. The analytes were separated and quantified by ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The recoveries at three fortified levels in reed and Callitrichaceae were 57–108% and 68–106%, respectively, with relative standard deviations of no more than 15% (n = 6). The method limits of quantification and detection for the seven bisphenol analogues were 0.005–0.500 ␮g/kg and 0.002–0.150 ␮g/kg, respectively. This method was used to analyze the seven compounds in ten reed and Callitrichaceae samples collected from Zhejiang, China.

1. Introduction Bisphenol A (BPA, 2,2-bis (4-hydroxydiphenyl) propane) has been widely used as the primary intermediate in the production of polycarbonate plastics and epoxy resins for several decades [1–3]. BPA can trigger adverse health outcomes, particularly through endocrine disruption, even at doses of 10–100 ng/kg body weight [2–4]. The EU Commission, US Environmental Protection Agency, and Health Canada have set different limits on its application. BPA in commercial products is gradually being replaced with its analogues, such as bisphenol S (BPS, 4,4 -sulfonyldiphenol), bisphenol F (BPF, 4,4 -methylenebisphenol), bisphenol B (BPB, 2,2-bis (4-hydroxyphenyl) butane), and bisphenol AF (BPAF, hexafluorobisphenol A), to comply with these restrictions [5]. BPS is used as an alternative to BPA in the production of thermal paper [6], and BPF, BPAF, BPB, and BPS are used in the production of polycarbonate plastic and resins [7–11]. Another two widely used analogues are tetrachlorobisphenol A (TCBPA) and tetrabromobisphenol A (TBBPA), which are used as reactive flame retardants in resins and polycarbonate plastics.

∗ Corresponding author. Tel.: +86 10 64407191; fax: +86 10 64407210. E-mail address: [email protected] (B. Shao). http://dx.doi.org/10.1016/j.jchromb.2014.02.003 1570-0232/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

Studies have shown that the toxicities (e.g., genotoxicity and estrogenic activity) of BPS, BPB, BPAF and BPF are similar to that of BPA [5,9,12]. The estrogenic effect of BPAF and BPB on gene and protein expression in MCF-7 breast cancer cells is greater than that of BPA [12], and BPAF binds to ␤-estrogen receptors more effectively than BPA [13]. BPAF may cause testosterone reduction by directly affecting testis function in adult male rats [14]. TBBPA and TCBPA could be ligands of peroxisome proliferator-activated receptors and inhibit the binding of T3 to TR␣ [15,16]. Among these bisphenol analogues mentioned above, BPA, TBBPA and TCBPA had been widely studied and found in different environmental matrices [3,17–19]. As emerging environmental contaminants, BPAF, BPS and BPF have been concerned recently on its occurrence in foodstuffs from the United States [20] and environmental matrices [21–23], mainly in water and sediment. Many methods have been developed for the analysis of BPA, TCBPA and TBBPA in environmental and food samples using high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS/MS), gas chromatography–mass spectrometry (GC–MS/MS) and, to a limited extent, immunochemical methods [17–19,24–27]. A few analytical methods of the determination of bisphenol analogues in vegetables or fruits were recently reported. Kang et al. [28] developed an analytical method for determination of BPA in vegetables and fruits based on sample extraction with acetone and HPLC analysis with fluorescence detection. Lu et al. [29] reported the analysis of BPA in vegetables and fruits by GC–MS/MS.

L. Lu et al. / J. Chromatogr. B 953–954 (2014) 80–85

81

CH3 CH2

CH3 HO

C

HO

OH

C

OH

CH3

CH3

Bisphenol A (BPA)

Bisphenol B (BPB)

O HO

S

OH

CH2

HO

OH

O

Bisphenol F (BPF)

Bisphenol S (BPS)

Cl

Br

Cl

Br CH3

CH3 HO

C

HO

OH

C

OH

CH3

CH3 Cl

Br

Cl

Br

Tetrabromobisphenol A (TBBPA)

Tetrachlorobisphenol A (TCBPA) CF3 HO

C

OH

CF3

Bisphenol AF (BPAF) Fig. 1. Chemical structures of target compounds.

Li et al. [30] reported the analysis of TBBPA in cabbage and radish by LC–MS/MS based on Soxhlet extraction. As important biological components of the environment, plants play a significant role in contaminant enrichment and removal. Reed and Callitrichaceae are two common aquatic plants in many regions. Reed and some other riparian buffer zone plants can remediate organic pollutants and decrease contamination of sludge [31,32]. An analytical method for determination of bisphenol analogues in aquatic plants was necessary to study the chemical’s transformation in the whole aquatic ecosystems. Here, we developed a sensitive and precise method using solidphase extraction (SPE) followed by ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for the determination of BPS, BPF, BPB, BPA, BPAF, TCBPA, and TBBPA (chemical structures are shown in Fig. 1) in two common aquatic plants, reed and Callitrichaceae.

The blank samples were collected from river and market of nonindustrial pollution area in Beijing, both of which were ensured free of analytes and used for method development. After the analytical method was developed, some reed and Callitrichaceae were collected from different sampling sites in a region near a BPAF manufacturing plant in Zhejiang, China.

2. Materials and methods

2.3. Sample pretreatment procedure

2.1. Reagents and chemicals

The collected samples were pre-rinsed with Milli-Q water. After removal of the surface water by air drying, the samples were homogenized with a BÜCHI B-400 mixer (BÜCHI Labortechnik AG, Switzerland) and stored at −20 ◦ C until analysis. A total of 2.0 g Callitrichaceae or 1.0 g reed was spiked with 5.0 ng BPA-d4 , TCBPA13 C , and TBBPA-13 C 12 12 as internal standards. The samples were extracted twice with 5 mL acetonitrile, sonicated at room temperature for 20 min, and centrifuged at 9000g for 10 min at 4 ◦ C. The extract was diluted to 40 mL with water and then applied to GCB cartridge, which was conditioned and equilibrated with 18 mL of methanol followed by 6 mL of water. After washed by 6 mL of methanol/water (50:50 v/v), the GCB cartridge was eluted with 6 mL of methanol/acetone (40:60 v/v). Then, the eluate was applied to a NH2 cartridge preconditioned by 6 mL methanol and

BPS (>98.0%), BPF (>99.0%), BPF-d10 (>99.0%), BPA (98.5%), BPAd4 (>97.8%), BPB (>98.0%), and BPAF (98%) were purchased from the Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). TCBPA (>99.0%), TCBPA-13 C12 (>99.0%), TBBPA (>99.0%), and TBBPA-13 C12 (>99.0%) were obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). HPLC-grade methanol, acetonitrile, ethyl acetate, and acetone were supplied by Dickma (Lake Forest, CA, USA). Ultrapure water was obtained from a Milli-Q Ultrapure water system (Millipore, Bedford, MA, USA). Formic acid (99%) was purchased from Acros Organics (Morris Plains, NJ, USA). Stock standard solutions (10 mg/mL) were individually prepared by dissolution in methanol and stored at −20 ◦ C. Intermediate solutions were prepared from

the stock solutions by appropriate dilution in methanol/water (50:50 v/v) and stored at −20 ◦ C. The Supelclean ENVITM -Carb cartridge (GCB, 500 mg, 6 mL) was purchased from Supelco (Bellefonte, PA, USA). Oasis HLB (500 mg, 6 mL) and Sep-Pak C18 (500 mg, 6 mL) cartridges were purchased from Waters (Milford, MA, USA). The Bond Elut LRC-NH2 (100 mg) cartridge was from Agilent Technologies (Lake Forest, CA, USA). 2.2. Sample collection

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L. Lu et al. / J. Chromatogr. B 953–954 (2014) 80–85

Table 1 MS/MS parameters for the analysis of the target compounds. Compound

BPA BPA-d4 b BPS BPF BPF-d10 b BPB BPAF TCBPA TCBPA-13 C12 b TBBPA TBBPA-13 C12 b a b

Precursor ion (m/z) [M − H]−

227.2 231.2 248.9 199.1 208.7 241.1 335.0 365.1 377.1 542.9 554.9

Quantitation

Confirmation

Product ion (m/z)

CEa (eV)

Product ion (m/z)

CE (eV)

212.1 216.0 107.9 92.9 109.8 212.1 265.0 314.0 297.0 445.8 459.8

18 20 28 21 20 20 20 25 30 35 33

133.0 – 155.9 105.0 – 147.0 197.0 286.0 – 419.7 –

26 – 22 21 – 30 35 30 – 40 –

CE: collision energy. Only one fragment ion was selected for the isotope internal standard.

collected in a vial. Additional 1 mL methanol containing 1% formic acid was used to wash the NH2 cartridge, and the eluate was collected in another vial. Two vials of elute were dried under a gentle nitrogen stream. The residuals was reconstituted to 1 mL with methanol/water (50:50 v/v), respectively. The first vial was used for the analysis of BPS, BPF, BPA, BPB, and BPAF, while the second was for TCBPA and TBBPA.

2.4. UPLC–MS/MS analysis A Waters Acquity UPLCTM system with a Waters Acquity UPLCTM BEH C18 column (2.1 mm × 100 mm; particle size, 1.7 ␮m) and coupled to a Waters XevoTM TQ-S triple-quadrupole mass spectrometer was used for target separation and quantification. The mobile phase was methanol and water at a flow rate of 0.4 mL/min. The gradient conditions were as follows: initial conditions of 40% methanol for 1 min, followed by a linear increase to 80% methanol within 5 min. Methanol was then increased to 100% at 6.1 min and held for 2.0 min before returning to the initial state at 8.5 min to equilibrate the column for 1.5 min before the next injection. The injection volume was 5 ␮L, and the compounds were detected in multiple-reaction monitoring mode (MRM) with an electrospray ionization mode (ESI) interface operating in negative ion mode. The capillary voltage was 2.8 kV. The source temperature and desolvation temperature were 150 ◦ C and 400 ◦ C, respectively. Nitrogen gas (99% purity) was used as both the cone gas and desolvation gas at flow rates of 150 L/h and 1000 L/h, respectively. For each analyte, two transitions were selected for identification, and the corresponding cone voltage and collision energy were optimized for maximum detection sensitivity.

of background contamination, and no significant signals (S/N > 3) were present in these blank samples. 2.6. Calibration and method validation Two types of standard calibration curves were prepared for the method assessment. Internal standard calibration curves for BPF, BPA, TCBPA, and TBBPA were obtained by linear regression of standard to internal standard peak area ratio versus the concentration of analytes. Matrix-matched standard curves (plotted using standards spiked in extracts of blank samples before analysis) were created to quantify BPS, BPB, and BPAF. All calibrations were performed in triplicates and the validity of calibration curves was assessed by calculation of RSDs of their slopes and intercepts. Method limits of quantitation (MLOQs) and method limits of detection (MLODs) were defined as the concentration of analyte yielding S/N = 10 and S/N = 3 in spiked blank samples, respectively. The recoveries of bisphenol analytes from reed and Callitrichaceae were calculated at three spiking levels: 1.0 g of reed and 2.0 g of Callitrichaceae were spiked with 0.2, 0.5, and 1.0 ng of BPS and BPAF (1.0, 2.5, and 5.0 ng of BPA and TCBPA; 2.0, 5.0, and 10 ng of BPB, BPF, and TBBPA). At each spiking level, six replicates were prepared to test the accuracy and intra-day precision (expressed by the percent relative standard deviation, RSD) of the method. The inter-day precision was evaluated by spiking 1.0 g reed and 2.0 g Callitrichaceae samples with 0.5 ng of BPS and BPAF (2.5 ng of BPA and TCBPA; 5.0 ng of BPF, BPB, and TBBPA). Three replicates were prepared and analysed over the 5 consecutive days. 3. Results and discussion 3.1. Optimization of UPLC–MS/MS

2.5. Quality control Contamination must be avoided throughout the entire analytical procedure. Previous studies have demonstrated that trace amounts of BPA (

Determination of seven bisphenol analogues in reed and Callitrichaceae by ultra performance liquid chromatography-tandem mass spectrometry.

An analytical procedure was developed to simultaneously determine bisphenol S, bisphenol F, bisphenol B, bisphenol A, bisphenol AF, tetrachlorobisphen...
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