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1 chemistry analytical_methods 1 chemistry analytical_methods Document Transcript

  • Food and Agriculture Organization of the United Nations WHO/HSE/FOS/11.1 Background Paper onChemistry and Analytical Methods for Determination of Bisphenol A in Food and Biological Samples FAO/WHO Expert Meeting on Bisphenol A (BPA) Ottawa, Canada, 2–5 November 2010 Prepared by Xu-Liang Cao Food Research Division, Bureau of Chemical Safety, Health Canada, Ottawa, Ontario, Canada Note to readers: The first draft of this paper was prepared by the named author, and the paper was then revised following discussions at the November 2010 meeting.
  • © World Health Organization 2011All rights reserved. Publications of the World Health Organization are available on the WHO web site(www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia,1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail:bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whetherfor sale or for noncommercial distribution – should be addressed to WHO Press through the WHO website (http://www.who.int/about/licensing/copyright_form/en/index.html).The designations employed and the presentation of the material in this publication do not imply theexpression of any opinion whatsoever on the part of the World Health Organization concerning the legalstatus of any country, territory, city or area or of its authorities, or concerning the delimitation of itsfrontiers or boundaries. Dotted lines on maps represent approximate border lines for which there maynot yet be full agreement.The mention of specific companies or of certain manufacturers’ products does not imply that they areendorsed or recommended by the World Health Organization in preference to others of a similar naturethat are not mentioned. Errors and omissions excepted, the names of proprietary products aredistinguished by initial capital letters.All reasonable precautions have been taken by the World Health Organization to verify the informationcontained in this publication. However, the published material is being distributed without warranty ofany kind, either expressed or implied. The responsibility for the interpretation and use of the materiallies with the reader. In no event shall the World Health Organization be liable for damages arising fromits use. 1
  • Toxicological and Health Aspects of Bisphenol ACONTENTS1. Chemistry ........................................................................................................................................................22. Analytical methods..........................................................................................................................................4 2.1 Sample preparation ...............................................................................................................................4 2.1.1 Deconjugation with enzymes .....................................................................................................4 2.1.2 Solvent extraction ......................................................................................................................4 2.1.3 Solid-phase extraction..............................................................................................................19 2.1.4 Derivatization...........................................................................................................................19 2.1.5 Solid-phase microextraction.....................................................................................................20 2.1.6 Stir bar sorptive extraction .......................................................................................................20 2.1.7 Coacervative microextraction ..................................................................................................20 2.2 Separation and detection .....................................................................................................................20 2.2.1 Liquid chromatography–based methods ..................................................................................20 2.2.2 Gas chromatography–mass spectrometry.................................................................................22 2.2.3 Enzyme-linked immunosorbent assay......................................................................................23 2.3 Method validation ...............................................................................................................................243. Conclusions and recommendations ...............................................................................................................10References ............................................................................................................................................................25Sensitive and reliable analytical methods are available for the determination of bisphenol A (BPA) in both foodand biological samples. Solvent extraction and solid-phase extraction are the most commonly used and mosteffective methods for the extraction of BPA in food and biological samples. Although isotope dilution methodsbased on mass spectrometry and tandem mass spectrometry are the most reliable for the detection of BPA, manyof the results of BPA determination in both food and biological samples have been generated by methods thatare not based on mass spectrometry.The majority of methods used to measure free and total BPA in food and biological samples have been validatedfor certain performance parameters, such as accuracy, precision, recovery and limit of detection. Most methodsfulfil the requirements of single-laboratory validation. For biological samples, however, validation of methodsfor conjugated BPA is very limited. By the current standards of analytical science, findings of BPA in foodsamples and most biological samples are reliable. Nevertheless, care needs to be taken to avoid cross-contamination with trace levels of BPA during sample collection, storage and analysis.1. CHEMISTRYBisphenol A (BPA) is the common name for 4,4′-dihydroxy-2,2-diphenylpropane(International Union of Pure and Applied Chemistry [IUPAC] name). Its chemical structureand physicochemical properties are shown in Table 1. BPA is a white solid (available incrystals or flakes) with a mild phenolic odour under ambient conditions. Its melting point is155 °C, and its specific gravity is 1.060–1.195 g/cm3. BPA is generally considered to be amoderately hydrophobic compound (octanol–water partition coefficient [Kow] of 103.4), with aslight polarity due to the two hydroxyl groups. It is soluble in acetic acid and very soluble inethanol, benzene and diethyl ether (Lide, 2004). Although it was classified as insoluble inwater in the 85th edition of the CRC Handbook of Chemistry and Physics (Lide, 2004), BPAis generally considered to be fairly soluble in water, with a solubility of 300 g/m3 at 25 °C.BPA has a relatively high boiling point (398 °C at 101.3 kPa) and low vapour pressure (5.3 ×10−6 Pa at 25 °C), and its concentration in air will be very low. Its air–water partitioncoefficient (Kaw) is very low (10−9); thus, BPA is unlikely to evaporate from aqueoussolution. The very high value of its octanol–air partition coefficient (Koa; 2.6 × 1012) alsosuggests that BPA in gaseous form will sorb strongly to solid surfaces (Cousins et al., 2002). 2
  • Chemistry and Analytical MethodsThe pKa value of BPA is between 9.59 and 11.30; thus, BPA will be present mainly in itsmolecular form in liquid media with pH lower than 7.Table 1. Physicochemical properties of bisphenol A Property Value Chemical Abstracts Service registry number 80-05-7 Chemical structure Other names 4,4′-dihydroxy-2,2-diphenylpropane 2,2-bis(4-hydroxyphenyl)propane 4,4′-isopropylidenediphenol Formula C15H16O2 Molecular weight 228.29 g/mol Melting point 155 °Ca Boiling point 398 °C at 101.3 kPab 3b Specific gravity 1.060–1.195 g/cm 3 b Water solubility 300 g/m at 25 °C −6 a Vapour pressure 5.3 × 10 Pa at 25 °C a Log Kow 3.40 a Log Kaw −9.01 Log Koa 12.41a a pKa 9.59–11.30a Cousins et al. (2002).b Staples et al. (1998).The BPA molecule has a fairly strong fluorophore due to the conjugated π-electrons in thetwo benzene rings, and thus it can be detected by fluorescence detector. Its chromophore isrelatively weak, and the sensitivity of ultraviolet (UV) detection is much lower than that offluorescence detection.Although BPA is fairly stable in its solid form, it does not persist in the environment. Aerobicbiodegradation is the dominant loss process for BPA in river water and soil, and itsdegradation half-life is about 4.5 days (Cousins et al., 2002). Its loss process in theatmosphere is due to the rapid reaction with hydroxyl radicals, and the photo-oxidation half-life for BPA in air is about 4 hours (Cousins et al., 2002). Chlorinated BPA can be found inboth wastewater and drinking-water, as BPA can be easily chlorinated by sodiumhypochlorite, a bleaching agent in paper factories and a disinfection agent in sewagetreatment plants (Fukazawa et al., 2001; Yamamoto & Yasuhara, 2002), and chlorine, achemical used in the disinfection of drinking-water (Gallard, Leclercq & Croue, 2004).Two major applications of BPA are in the production of polycarbonate plastics and epoxyresins. Polycarbonate is synthesized from BPA and phosgene gas (carbonyl dichloride),whereas epoxy resins are produced from the reaction of BPA with epichlorohydrin. 3
  • Toxicological and Health Aspects of Bisphenol A2. ANALYTICAL METHODSVarious methods have been developed and used to determine BPA in food and biologicalsamples (Tables 2 and 3). Although some of the methods could be used for qualitativescreening purposes, such as the enzyme-linked immunosorbent assay (ELISA), quantitativeresults were reported in all publications. Owing to the complex matrices of the food andbiological samples and the low concentrations of BPA (parts per billion [ppb] or sub–partsper billion levels), extensive sample preparations (extraction, cleanup, concentration,derivatization, etc.) prior to analysis by instruments such as gas chromatography (GC) andliquid chromatography (LC) coupled with various detectors (mass spectrometer [MS], UV,fluorescence detector, electrochemical detector [ECD], etc.) are required, even for qualitativescreening analysis.2.1 Sample preparation2.1.1 Deconjugation with enzymesBPA in biological samples exists as both free BPA and conjugated BPA. The majority ofconjugated BPA is in the form of BPA-glucuronide, whereas only a small portion is in theform of BPA-sulfate. In order to determine the total BPA in biological samples, conjugatedBPA needs to be deconjugated by hydrolysis with enzymes at 37 °C for a period rangingfrom a few hours up to overnight. Among the published results (Table 3), most people usedonly β-glucuronidase for deconjugation, whereas only a few used both β-glucuronidase andsulfatase enzymes for deconjugation. In some of the studies, enzymes were not used(Pedersen & Lindholst, 1999; Sajiki, Takahashi & Yonekubo, 1999; Inoue et al., 2000;Watanabe et al., 2001; Sun et al., 2002, 2004; Kuroda et al., 2003; Mao et al., 2004; Volkel etal., 2005; Xiao et al., 2006; Fernandez et al., 2007; Dirtu et al., 2008; Cobellis et al., 2009;Markham et al., 2010); thus, the results could be for the free BPA only. As the ELISAmethod determines total BPA (free BPA plus conjugated BPA), this deconjugation step withenzyme is not needed in sample preparation. It should be mentioned that, depending on thetypes of β-glucuronidase enzymes (Escherichia coli or Helix pomatia) used, BPA-sulfatecould also be deconjugated (Ye et al., 2005a).2.1.2 Solvent extractionSolvent extraction is one of the most common and effective techniques for the extraction ofBPA from food and biological matrices, and acetonitrile is the most frequently used solventfor this purpose. The other role of acetonitrile is to precipitate the proteins in protein-richsamples, such as infant formula, milk, urine and blood. Acids have also been used for proteinprecipitation (Yoshimura et al., 2002). Non-polar solvents, such as n-hexane, n-heptane andtrimethylpentane, have also been used together with acetonitrile for the extraction of BPAfrom fatty samples (Goodson, Summerfield & Cooper, 2002; Kang & Kondo, 2003;Braunrath et al., 2005; Thomson & Grounds, 2005; Sun, Leong & Barlow, 2006; Fernandezet al., 2007; Podlipna & Cichna-Markl, 2007; Grumetto et al., 2008; Lim et al., 2009).Solvents other than acetonitrile have also been used occasionally for the extraction of BPAfrom biological samples; examples include chloroform (Sun et al., 2002; Kuroda et al., 2003),dichloromethane (Arakawa et al., 2004), methyl tert-butyl ether (MTBE) (Lee et al., 2008),diethyl ether (Ouchi & Watanabe, 2002), dichloromethane and methanol (Pedersen &Lindholst, 1999) and 2-propanol (Ye et al., 2006; Yi, Kim & Yang, 2010). Microwave was 4
  • Chemistry and Analytical MethodsTable 2. Methods for determination of BPA in food samplesSample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precisionFish, meat, fruit, Sample extracted with acetonitrile. Extracts n/a LC-ECD 0.2 ng/ml — 65.5–137.6% Sajiki et al.vegetable, soup, cleaned up with SPE, eluted with ethyl acetate, 2.9% RSD (n = 5) (2007)sauce, dried under nitrogen, reconstituted with LC-MS 0.1 ng/ml — 58.2–129.4%beverage, milk acetonitrile/water (40:60). 3.2% RSD (n = 7) LC-MS/MS 0.1 ng/ml — 1.2% RSD (n = 7)Infant formula Sample spiked with d6-BPA, extracted with n/a LC-MS/MS 0.15 ng/g 0.5 47% Ackerman et acetonitrile, centrifuged. Supernatant cleaned up ng/g 1.4–5.5% RSD for al. (2010) with SPE, eluted with chloroform, reduced to 1.7–9.8 ng/g; 2.9– dryness under nitrogen, reconstituted with 18% RSD for 2.3– methanol/water (50:50). 10.6 ng/gMilk Milk protein precipitated with acetonitrile. n/a LC-MS 0.20 ng/ml — 97.1% (0.6 ng/ml); Yan et al. Supernatant cleaned up further with PSA and 92.4% (15 ng/ml) (2009) online SPE (C30). 15.0% RSD (0.5 ng/ml); 13.2% RSD (15 ng/ml)Honey Honey sample dissolved in water, applied to SPE n/a LC-fluorescence 2.0 ng/g — 103.6% for 5 ng/g; Inoue et al. cartridge (GL-Pak PLS-2, polystyrene divinyl (275/300 nm); 99.9% for 50 ng/g (2003b) benzene), eluted with methanol. LC-MS for 6.6% RSD for 5 confirmation ng/g (n = 6); 5.3% RSD for 50 ng/g (n = 6)Fruit, vegetable Sample extracted with acetonitrile. Extract n/a HPLC-UV (228 — 5–10 84.5–90.1% Yoshida et applied to SPE cartridge, eluted with nm) ng/ml 3.4–4.2% RSD (n = al. (2001) acetone/heptane, evaporated to dryness and 3) reconstituted with mobile phase.Infant formula Sample diluted with water was applied to SPE Chloroform HPLC- 0.9 ppb — 86–104% Biles, cartridge, eluted with chloroform and extract not fluorescence 2–27% RSD (n = 3) McNeal & concentrated. The concentrated extract was derivatized for (235/317 nm); Begley diluted with mobile phase for HPLC analysis. GC-MS GC-MS (for (1997) analysis confirmation) 5
  • Toxicological and Health Aspects of Bisphenol ATable 2 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precisionCoffee drink Sample applied to SPE cartridge, eluted with n/a HPLC- — 2–10 85.3–96.2% Kang & acetonitrile/water (40:60 v/v). fluorescence ng/ml 1.9–6.7% RSD (n = Kondo (275/300 nm) 3) (2002)Milk, dairy Sample blended with acetonitrile and hexane. n/a HPLC- 1–3 ng/ml — 76.9–101.8% Kang &products Hexane phase extracted again with acetonitrile. fluorescence 3.4–24.6% RSD Kondo Acetonitrile phase combined, filtered and (275/300 nm) (n = 5) (2003) evaporated to dryness. Residue dissolved in acetone/n-heptane (3:97 v/v) and applied to Sep- Pak Florisil cartridge for cleanup. BPA eluted with acetone/n-heptane (20:80 v/v), evaporated to dryness and dissolved with mobile phase for HPLC analysis.Beverage Sample loaded onto Oasis HLB SPE cartridge, n/a LC-MS/MS 0.6 ng/l 2.0 ng/l 82.1–96.5% Shao et al. eluted with methanol/dichloromethane (20:80 2.9–7.1% RSD (n = (2005) v/v). 5)Beverage, Beverage sample applied to immunoaffinity n/a HPLC- 0.1–9.3 0.4–0.8 27–103% Braunrath &vegetable, column, eluted with acetonitrile/water (40:60 v/v). fluorescence ng/g ng/g 1.0–31% RSD (n = Cichnafruit, soup, fish Fruit and vegetable sample extracted with (275/305 nm) (fish) 3) (2005); acetonitrile twice, supernatants filtered and Braunrath et applied to immunoaffinity column, eluted with al. (2005); acetonitrile/water (40:60). Podlipna & Fat-containing food sample extracted with Cichna-Markl acetonitrile/hexane (1:1). Acetonitrile extracts (2007) filtered and applied to immunoaffinity column, eluted with acetonitrile/water (40:60).Fish, meat Coacervative microextraction. n/a HPLC- — 15–29 97–111% Bendito et al. 0.2 g decanoic acid dissolved in 2 ml THF in fluorescence ng/g 2.1–7.1% RSD (n = (2009) centrifuge tube; 8 ml water and 140 µl (276/306 nm) 3) hydrochloric acid (0.5 mol/l) added. Mixed with food sample, stirred and centrifuged. Coacervate phase analysed by LC. 6
  • Chemistry and Analytical MethodsTable 2 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precisionFood simulant, Food simulant evaporated to dryness, dry n/a (for HPLC- — 0.12– 89.2–90.6% Munguia-pepper residue redissolved in 5 ml of acetonitrile and HPLC); extract fluorescence 0.2 1.2–5.8% RSD Lopez & filtered. not derivatized (224/310 nm); ng/g Soto-Valdez Pepper sample blended with methanol, filtered. for GC-MS GC-MS for (2001); Liquids evaporated to dryness, residue confirmation Munguia- redissolved with 5 ml of acetonitrile and filtered. Lopez et al. (2002)Vegetable, Coacervative microextraction. n/a HPLC- — 9 ng/g 81–96% Garcia-Prietofruit 0.2 g decanoic acid dissolved in 4 ml THF in fluorescence 3% RSD (n = 6) et al. (2008a) centrifuge tube, 36 ml of hydrochloric acid added (276/306 nm) (1.3 mmol/l). Mixed with food sample, stirred and centrifuged. Coacervate phase analysed by LC.Vegetable, Sample extracted with acetonitrile and hexane. n/a HPLC- 4.5–7.9 13.7– 87.3–105.2% Sun, Leongfruit, fish, meat Acetonitrile extract evaporated, dissolved in fluorescence µg/kg 24.1 0.20–2.96% RSD & Barlow methanol/water (5:95 v/v), loaded onto SPE (235/317 nm) µg/kg (inter-day, n = 5); (2006) cartridge (Oasis HLB), eluted with methanol, 0.04–2.82% RSD methanol:ethyl acetate (50:50) and ethyl acetate. (intra-day, n = 5) Extract evaporated to dryness, reconstituted with acetonitrile/water (90:10 v/v).Fish, Sample spiked with BPA-d14 and extracted with Acetic GC-MS in EI 2 ng/g 7 ng/g 81–103% Goodson,vegetable, acetonitrile (n-heptane also used for fat anhydride mode 4.5% RSD for 11 Summerfieldinfant formula, samples). Extract derivatized with acetic ng/g (n = 6) & Cooperpasta, dessert, anhydride. Derivatized BPA extracted with n- (2002)soup, heptane. Beverage samples derivatized directly.beverageFish, meat, Sample spiked with BPA-d14 and extracted with Acetic GC-MS in EI — 10–20 42–112% Thomson &vegetable, acetonitrile (trimethylpentane also used for fat anhydride mode ng/g 8% RSD for 28.6 Groundsfruit, soup, samples). Extract derivatized with acetic ng/g (n = 8) (2005)dessert, anhydride. Beverage samples derivatizedbeverage directly.Milk Milk sample deproteined with trichloroacetic acid, n/a HPLC- 0.2 ng/ml 0.5 93–102% Liu et al. diluted with water (20-fold), dissolved in fluorescence ng/ml 6.6% RSD (n = 3) (2008) methanol, filtered. Extracted with SPME fibre by (275/315 nm) 7
  • Toxicological and Health Aspects of Bisphenol ATable 2 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precision direct immersion.Fish Sample extracted with acetonitrile. Extract n/a HPLC- 1 ng/g — 70.7–72.9% Tsuda et al. filtered and evaporated to dryness. Residue fluorescence 1.8–4.8% RSD (n = (2000) dissolved in hexane, extracted with acetonitrile (275/300 nm) 5) saturated in hexane. Acetonitrile layer evaporated to dryness, dissolved in hexane, applied to column packed with Florisil PR for cleanup. BPA eluted with acetone and hexane (3:7 v/v). Eluate evaporated to dryness, reconstituted with methanol.Vegetable Canned food (solid or liquid) diluted with water. BPA GC-MS in EI 0.01–0.03 0.033– 84–112% Vinas et al. For derivatization with acetic anhydride, acetic derivatized mode ng/m 0.1 1.96–2.09% RSD (2010) anhydride also added to sample solution with acetic ng/ml (n = 10) together with buffer solution. Derivatized BPA anhydride or extracted with SPME polyacrylate fibre by direct BSTFA immersion. For derivatization with BSTFA, BPA extracted with SPME polyacrylate fibre first, then derivatized in the headspace above BSTFA.Wine Wine sample mixed with PBS, pH adjusted to n/a HPLC- 0.1 ng/ml 0.2 74–81% Brenn- 7.0, filtered, applied to immunoaffinity column fluorescence ng/ml 10–15% RSD (n = Struckhofova and eluted with acetonitrile/water (40:60 v/v). (275/305 nm) 3) & Cichna- Markl (2006)Milk Milk sample diluted with water, applied to C18 No GC-MS in EI 0.15 — 81% Casajuana & SPE cartridge, eluted with derivatization mode µg/kg 5% RSD (n = 3) Lacorte dichloromethane/hexane and ethyl acetate, and for BPA (2004) cleaned up on Florisil column.Tomato Sample extracted with acetonitrile. Acetonitrile n/a HPLC-UV (228 20 µg/kg 66.9 0.14–2.2% RSD Grumetto et phase partitioned with hexane for fat removal. nm) µg/kg (inter-day); 0.04– al. (2008) Extract evaporated, residue dissolved in 1.84% RSD (intra- water/acetonitrile, applied to C18 SPE cartridge, day) eluted with acetonitrile. Eluate evaporated to HPLC- 1.1 µg/kg 3.7 0.2–2.96% RSD dryness, dissolved in hexane/ethyl acetate (96:4 fluorescence µg/kg (inter-day, n = 5); v/v), applied to Florisil cartridge, eluted with ethyl 8
  • Chemistry and Analytical MethodsTable 2 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precision acetate. Eluate evaporated, residue reconstituted (273/300 nm) 0.04–2.82% RSD with acetonitrile. (intra-day, n = 5)Infant formula Sample dissolved in ethanol/water (50:50 v/v), BPA GC-MS in EI — 1.0 79% Kuo & Ding centrifuged. Supernatant filtered, applied to C18 derivatized mode ng/g 9% RSD (n = 5) (2004) SPE cartridge, eluted with methanol. Eluate with BSTFA + derivatized with BSTFA + TMCS. TMCSCoffee, tea, Coffee, tea, fruit, vegetable: sample mixed with n/a HPLC- 3 ng/g — 95.4% Lim et al.fruit, acetonitrile, centrifuged. Supernatant filtered, fluorescence 9.1% RSD (n = 5) (2009)vegetable, dried, dissolved in acetonitrile/water (60:40 v/v). (275/315 nm)fish, meat Fish, meat: sample extracted with acetonitrile and hexane, centrifuged. Solid sample and hexane phase extracted several times with acetonitrile.Milk Sample spiked with BPA-d16, diluted with n/a LC-MS (ESI) 1.7 ng/g 5.1 83–106% Maragou et water/methanol (8:1 v/v), applied to C18 SPE ng/g 2.1–12.5% RSD al. (2006) cartridge and eluted with methanol/water (90:10 (intra-day, n = 6); v/v). Eluate evaporated to dryness and 5.2–17.6% RSD reconstituted with water. (inter-day, n = 6)Water SPME n/a HPLC- 1.1 ng/ml 3.8 22% RSD (n = 4) Nerin et al. fluorescence ng/ml (2002) (275/305 nm)Egg, milk Milk or egg sample mixed with C18 powder, n/a LC-MS/MS 0.1 ng/g — 79.2–86.8% (egg); Shao et al. packed into a column. BPA eluted with methanol. 85.7–93.9% (milk) (2007) Eluate evaporated to dryness, residue 2.86–7.42% RSD redissolved in dichloromethane/hexane (50:50), (egg, n = 5); 3.15– applied to aminopropyl SPE cartridge for 5.29% RSD (milk, cleanup. Eluted with methanol/acetone (50:50 n = 5) v/v). Eluate evaporated to dryness and reconstituted with mobile phase.Infant formula Sample spiked with BPA-d16, mixed with BPA GC-MS in EI — 0.5 85–94% Cao et al. acetonitrile, centrifuged. Supernatant applied to derivatized mode ng/g 2.8–5.0% RSD (n = (2008) C18 SPE cartridge, eluted with acetonitrile in with acetic 6) 9
  • Toxicological and Health Aspects of Bisphenol ATable 2 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precision water (50:50 v/v), evaporated to 3 ml. anhydride Concentrated aqueous extract derivatized with acetic anhydride.Soft drinks Sample spiked with BPA-d16, applied to C18 BPA GC-MS in EI — 0.05 99.9–101% Cao, SPE cartridge, eluted with acetonitrile in water derivatized mode ng/ml 1.3–6.6% RSD (n = Corriveau & (50:50 v/v). Eluate evaporated to 3 ml. with acetic 7) Popovic Concentrated aqueous extract derivatized with anhydride (2009) acetic anhydride.BSTFA, N-O-bis(trimethylsilyl)trifluoroacetamide; ECD, electron capture detector; EI, electron ionization; ESI, electrospray ionization; GC, gas chromatography; HPLC, high-performance liquid chromatography; LC, liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MS, mass spectrometry; MS/MS, tandem massspectrometry; n/a, not applicable; ppb, parts per billion; PBS, phosphate buffered saline; PSA, primary–secondary amine; RSD, relative standard deviation; SPE, solid-phaseextraction; SPME, solid-phase microextraction; THF, tetrahydrofuran; TMCS, trimethylchlorosilane; UV, ultraviolet; v/v, volume per volume 10
  • Chemistry and Analytical MethodsTable 3. Methods for determination of BPA in biological samplesSample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precisionUrine Acetonitrile added to urine sample, centrifuged n/a LC-MS/MS — 1.3–5 — Volkel, to precipitate protein. Supernatant applied to µg/l Kiranoglu online SPE (Oasis HLB). β-Glucuronidase & Fromme added to urine sample for total BPA (2008) determination.Human Sample mixed with hydrochloric acid (0.2 mol/l), BPA derivatized HPLC- 0.04 ppb — 78.6% (serum); 77.7% Kuroda etblood extracted with chloroform, evaporated to with fluorescent fluorescence (ascitic fluid) al. (2003)serum, dryness. Fluorescent reagent DIB-Cl in reagent DIB-Cl (350/475 nm) 4.2% RSD (intra-day,ascitic fluid acetonitrile added to the residue to label BPA. n = 6); 8.0% RSD (inter-day, n = 3)Human Sample mixed with formic acid, diluted with Pentafluoro- GC-MS in ECNI — 280 81.3–83.1% Dirtu et al.serum water. Mixture loaded onto SPE (Oasis HLB) propionic acid mode pg/ml 1.6–5.1% RSD (intra- (2008) cartridge, eluted with methanol/dichloromethane anhydride day); 2.4–14% RSD (1:1 v/v), concentrated to 0.5 ml. Extract (inter-day) cleaned up further on Florisil cartridge, eluted with methanol/dichloromethane (5:1 v/v), derivatized with pentafluoropropionic acid anhydride.Urine Glucuronidase enzyme added to sample, n/a LC-ECD 0.5 µg/l — 115% Liu, Wolff incubated overnight at 37 °C, applied to C18 6.1% RSD (n = 9) & Moline SPE cartridge, eluted with methanol. (2005)Urine Urine sample hydrolysed with hydrochloric acid, BPA derivatized LC-fluorescence 2.7 µg/l — 95.9% Mao et al. eluted on C18 SPE cartridge with dichloro- with fluorescent (228/316 nm) 3.92% RSD (2004) methane. Extracts derivatized with fluorescent reagent p- reagent p-nitrobenzoyl chloride. nitrobenzoyl chlorideUrine β-Glucuronidase and sulfatase added to urine MtBSTFA + 1% GC-MS in 3 ng/ml 7 ng/ml 90–119% Moors et sample for hydrolysis at 37 ΕC overnight. tBDMCS electron impact 4–6% RSD (intra- al. (2007) Hydrolysed urine sample loaded to SPE ionization mode assay, n = 4); 10% cartridge, eluted with acetonitrile/ethyl acetate RSD (inter-assay, n = (1:1 v/v), eluate evaporated to dryness. Residue 6) derivatized with MtBSTFA + 1% tBDMCS. 11
  • Toxicological and Health Aspects of Bisphenol ATable 3 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precisionBlood Blood plasma mixed well with hydrochloric acid BPA derivatized HPLC- 4.6 ppb — 101% Sun et al. (0.2 mol/l), extracted with chloroform. Organic with fluorescent fluorescence 1.0–2.2% RSD (intra- (2002) phase evaporated to dryness, residue reagent DIB-Cl (350/475 nm) day, n = 4); 5.6–6.3% derivatized with DIB-Cl. RSD (inter-day, n = 6)Blood Blood serum mixed with mobile phase n/a HPLC- 0.15 0.50 85.6% Cobellis et (acetonitrile/phosphate buffer at pH 6.0 [35:65 fluorescence ng/ml ng/ml 2 al. (2009) Linearity (r ): 0.989 v/v]). Perchloric acid (25% w/v) added to (273/300 nm); precipitate proteins, centrifuged. Supernatant LC-MS for filtered. confirmationUrine Coacervative microextraction. Urine sample n/a LC-fluorescence 0.197 — 88–95% Garcia- hydrolysed with β-glucuronidase enzyme. 0.1 g (276/306 nm) µg/l 4.5% RSD (n = 3) Prieto et decanoic acid dissolved in 1 ml THF in al. (2008b) centrifuge tube, mixed with hydrolysed urine sample, stirred and centrifuged. Coacervate phase analysed by LC.Blood, Blood sample fortified with BPA-d8, extracted n/a LC-MS/MS 0.05 — 67–109% (urine); 98– Markhamurine with acetonitrile, centrifuged. (NESI) ng/ml 130% (blood) et al. Urine sample fortified with BPA-d8, diluted with 1.4–33.6% RSD (2010) water, loaded onto Oasis HLB SPE cartridge, (urine, n = 5); 4.4–20% eluted with MTBE. Extract evaporated to RSD (blood, n = 5) dryness, reconstituted with acetonitrile/water (50:50 v/v).Urine Urine sample mixed with PBS and centrifuged. n/a HPLC- 0.2 — 78% Schoring- Supernatant applied to enzyme column fluorescence ng/ml 3.4% RSD (n = 4) humer & containing β-glucuronidase and arylsulfatase, (275/305 nm); Cichna- eluted with PBS. Extracts applied to LC-MS (ESI-ion Markl immunoaffinity column, eluted with trap) for (2007) acetonitrile/water (40:60 v/v). confirmationBlood Blood serum diluted with PBS and applied to n/a HPLC- — — 91.8% Zhao et al. immunoaffinity column. BPA eluted with fluorescence 7.1% RSD (n = 6) (2003) methanol/water (80:20 v/v). Extract evaporated (230/315 nm) 12
  • Chemistry and Analytical MethodsTable 3 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precision to dryness, redissolved in acetonitrile/water (60:40).Human Milk sample extracted with acetonitrile, n/a ELISA 0.3 — 102.6% ± 19.0% Kuruto-colostrum centrifuged. Supernatant evaporated, and ng/ml Niwa et al. residue dissolved in phosphate buffer and (2007) applied to SPE cartridge (Oasis HLB). BPA eluted with methanol/acetonitrile (3:1 v/v), evaporated to dryness, reconstituted with phosphate buffer.Urine β-Glucuronidase added to urine sample for BPA derivatized GC-MS in 0.1 — 95–116% Kuklenyik deconjugation overnight. Acetonitrile added to with PFBBr negative ng/ml 6–7% RSD (n = 19) et al. the deconjugated sample. Derivatizing agent chemical (2003) PFBBr in hexane (1:2) loaded onto the SPE ionization mode cartridge (Bond Elute PPL). Deconjugated urine sample loaded onto the SPE cartridge, and derivatized BPA eluted from the cartridge with acetonitrile and ethyl acetate. Extract evaporated to dryness and reconstituted with isooctane.Urine β-Glucuronidase added to urine sample for BPA derivatized GC-MS in EI 0.02 0.1 98.8–101% Kawaguchi deconjugation. Acetic anhydride added for with acetic mode ng/ml ng/ml 1.8–6.7% RSD (n = 6) et al. derivatization. Derivatized BPA extracted into anhydride (2008) the solvent (toluene) contained in the hollow fibre connected to a syringe.Blood, β-Glucuronidase/sulfatase added to sample for BPA derivatized GC-MS in — 0.1–0.05 93-94% (serum); 100– Geens,urine deconjugation. Deconjugated sample applied to with PFBCl electron ng/ml 102% (urine) Neels & SPE cartridge (Oasis HLB), eluted with capture– 9–16% RSD (serum, Covaci methanol/dichloromethane (1:1 v/v). Eluate negative n = 3); 4–10% RSD (2009) evaporated to dryness and derivatized with ionization mode (urine, n = 3) PFBCl.Blood Acetonitrile and hydrochloric acid (1 mol/l) BPA derivatized HPLC- 0.05 — 94.8–95.2% Watanabe added to plasma sample. Centrifuged. with DIB-Cl fluorescence ng/ml 5.8–8.2% RSD (n = 4) et al. Supernatant diluted with water, applied to SPE fluorescent (340/470 nm) (2001) 13
  • Toxicological and Health Aspects of Bisphenol ATable 3 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precision cartridge (Oasis HLB) and eluted with methanol. reagent Eluate evaporated to dryness, reconstituted with acetonitrile, derivatized with fluorescent reagent DIB-Cl.Human milk Milk sample diluted with water, extracted with BPA derivatized HPLC- 0.11 — 70% Sun et al. hexane, centrifuged. Aqueous layer extracted with DIB-Cl fluorescence ng/ml 0.9–8.7% RSD (intra- (2004) with chloroform, organic layer evaporated to fluorescent (350/475 nm) day, n = 5); 4.7–10.4% dryness, residue derivatized with DIB-Cl reagent RSD (inter-day, n = 5) fluorescent reagent.Urine β-Glucuronidase added to sample for BPA derivatized GC-MS in 0.1 — 83% Tsukioka deconjugation. Deconjugated sample applied to with PFBBr negative ion ng/ml 7.4% RSD (n = 5) et al. C18 SPE cartridge, eluted with methanol. chemical (2003) Eluate was concentrated and derivatized with ionization PFBBr. Derivatized sample cleaned up using a Florisil column.Serum, Ammonium acetate buffer, hexane, diethyl ether n/a HPLC- 1.4–2.8 — 78.6–95% Xiao et al.tissues added to serum, centrifuged. Organic layer fluorescence ng/ml 0.1–3.0% RSD (intra- (2006) evaporated to dryness, residue reconstituted (227/313 nm) assay, n = 7); 5.0– with acetonitrile. 11.4% RSD (inter- Tissue sample homogenized with ammonium assay, n = 7) acetate buffer. Methanol and perchloric acid (4 mol/l) added, vortexed and centrifuged. Ammonium acetate buffer added to supernatant, loaded onto C18 SPE cartridge, eluted with methanol. Eluate evaporated to dryness, reconstituted with acetonitrile.Urine β-Glucuronidase added to sample for BPA derivatized GC-MS/MS 0.38 — 62–124% Arakawa et deconjugation. Deconjugated sample spiked with BSTFA ng/ml 9% RSD (n = 5) al. (2004) with BPA-d16, extracted with dichloromethane. Dichloromethane layer evaporated to dryness, residue dissolved in hexane and applied to SPE cartridge. BPA eluted with acetone, eluate evaporated and derivatized with BSTFA. 14
  • Chemistry and Analytical MethodsTable 3 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precisionUrine β-Glucuronidase added to sample for BPA derivatized GC-MS in 0.12 — 101.6% Brock et al. deconjugation. Formic acid and ammonium with PFBBr negative ng/ml 1.1–16% RSD (n = 3) (2001) acetate buffer added to deconjugated sample, chemical applied to C18 SPE column, eluted with ionization mode methanol. Eluate derivatized with PFBBr.Adipose Sample homogenized with hexane and BPA derivatized GC-MS in 0.5 — 95–105% Fernandeztissue acetonitrile. Aqueous phase diluted with water, with BSTFA/TMCS electron impact ng/ml et al. applied to C18 SPE cartridge, eluted with mode (2007) diethyl ether/methanol (9:1 v/v). Eluate derivatized with BSTFA/TMCS (1:1 v/v).Human ELISA n/a ELISA — — — Ikezuki etbiological al. (2002)fluidsHuman Serum sample mixed with hydrochloric acid n/a HPLC- 0.05 79–87.3% Inoue et al.serum (1 mol/l), methanol, water, applied to SPE electrochemical ng/ml 5.1–13.5% RSD (n = (2000) cartridge, eluted with methanol. Eluate detection 6) evaporated to dryness, residue reconstituted HPLC-UV 150 with acetonitrile/water (50:50 v/v). ng/ml HPLC- 10 ng/ml fluorescenceHuman Semen sample acidified with hydrochloric acid, n/a LC-MS — 0.5 100.5% (relative); Inoue et al.semen spiked with BPA-d16 and mixed with water. ng/ml 71.2% (absolute) (2002) Then applied to SPE cartridge, eluted with ELISA — 2.0 4.7% RSD (n = 6) methanol. ng/mlHuman SBSE BPA derivatized GC-MS in 20–100 100–500 95.2–100.7% Kawaguchibody fluids β-Glucuronidase added to urine, plasma or with acetic electron impact pg/ml pg/ml 6.3–9.6% RSD (n = 6) et al. saliva sample buffered with ammonium acetate anhydride ionization mode (2004) for deconjugation. Deconjugated sample diluted with water, derivatized with acetic anhydride, extracted with stir bar coated with PDMS, and then thermally desorbed. 15
  • Toxicological and Health Aspects of Bisphenol ATable 3 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precisionBlood Blood serum hydrolysed with β- n/a HPLC- 0.625 — 91–95% Lee et al. glucuronidase/sulfatase overnight, extracted fluorescence µg/l 3.61–14.83% RSD (2008) with MTBE. MTBE extract evaporated to (227/313 nm) (n = 5) dryness, residue reconstituted with 60% acetonitrile.Human ELISA n/a ELISA 0.3 — 81.9–97.4% Ohkuma etserum ng/ml al. (2002)Urine Urine sample extracted with diethyl ether twice. n/a HPLC-ECD 0.2 — 103% Ouchi & Ether phase evaporated to dryness, residue ng/ml 3–12% RSD (n = 4) Watanabe reconstituted with acetonitrile. (2002) β-Glucuronidase and buffer solution added to urine sample to determine total BPA.Fish tissue Dichloromethane/methanol (2:1 v/v) added to n/a LC-MS (APCI) — 50 ng/g 49–79% Pedersen tissue sample, extracted for 25 min in 2.7–10% RSD (intra- & Lindholst microwave extraction apparatus. assay, n = 6); 3.7– (1999) Dichloromethane phase evaporated to dryness, 14.7% RSD (inter- redissolved in methanol/hexane (1:20), applied assay, n = 6) to SPE cartridge (Sep-Pak NH2), eluted with methanol. Eluate evaporated to dryness, redissolved in methanol.Blood Serum or plasma sample diluted with water, n/a HPLC-ECD 0.2 — 93% Sajiki, applied to SPE cartridge, eluted with ethyl ng/ml 2.9% RSD (n = 5) Takahashi acetate. Eluate evaporated to dryness, & redissolved in acetonitrile/water (40:60 v/v). Yonekubo HPLC-MS (ESI) 0.1 — 93% (1999) ng/ml 7.0% RSD (n = 5)Urine, Sample diluted with methanol, centrifuged, n/a LC-MS/MS 1.14 3.42 Recovery: 92–121% Volkel,blood acetonitrile added, centrifuged again. ng/ml ng/ml (BPA); 90–120% Bittner &plasma (BPA in (BPA in (BPA-gluc) Dekant urine); urine); (2005) 10.1 26.3 ng/ml ng/ml (BPA- (BPA- 16
  • Chemistry and Analytical MethodsTable 3 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precision gluc in gluc in urine) urine)Maternal Serum or fluid applied to SPE cartridge, eluted n/a ELISA 0.2 — 3.5–10.8% RSD (intra- Yamada etserum, with methanol/acetonitrile (3:1). Eluate ng/ml assay); 5.3–8.4% RSD al. (2002)amniotic evaporated to dryness, reconstituted with (inter-assay)fluid phosphate buffer for ELISA analysis.Urine Sample mixed with enzyme solution (β- n/a HPLC-MS/MS 0.4 — 100% Ye et al. glucuronidase/sulfatase in ammonium acetate (negative ion ng/ml 8–17% RSD (n = 60) (2005a) buffer; 1 mol/l; pH 5.0) for deconjugation APCI) overnight. Deconjugated solution diluted with formic acid (0.1 mol/l) and centrifuged, applied to C18 SPE cartridge in the online SPE-HPLC- MS/MS system, eluted with methanol/water (50:50).Urine Sample mixed with ammonium acetate buffer n/a HPLC-MS/MS 0.3 — 98–113% Ye et al. (1 mol/l; pH 5.0), enzyme added for (negative ion ng/ml 8–13% RSD (n = 60) (2005b) deconjugation overnight. Deconjugated solution APCI) diluted with formic acid (0.1 mol/l) and centrifuged, applied to SPE cartridge in the online SPE-HPLC-MS/MS system.Human milk Sample mixed with ammonium acetate buffer n/a HPLC-MS/MS 0.28 — 93.7% Ye et al. (1 mol/l), enzyme added for deconjugation. (negative ion ng/ml 8.2–11.4% RSD (n = (2006) 2-Propanol added to deconjugated solution, APCI) 50) centrifuged. Supernatant diluted with formic acid (0.1 mol/l), applied to SPE cartridge in the online SPE-HPLC-MS/MS system.Human milk Sample mixed with ammonium acetate buffer n/a HPLC-MS/MS 0.3 — 105% Ye et al. (1 mol/l), enzyme added for deconjugation (negative ion ng/ml 6.3–8.3% RSD (n = (2008) overnight. Methanol added to deconjugated APCI) 40) solution, centrifuged. Supernatant diluted with formic acid (0.1 mol/l), applied to SPE cartridge 17
  • Toxicological and Health Aspects of Bisphenol ATable 3 (continued)Sample Extraction/cleanup Derivatization Separation and LOD LOQ Recovery and Reference detection precision in the online SPE-HPLC-MS/MS system.Human milk β-Glucuronidase added to sample for n/a HPLC- 0.6 1.8 65–82% Yi, Kim & deconjugation. Deconjugated sample extracted fluorescence ng/ml ng/ml <15% RSD Yang with 2-propanol, centrifuged. Supernatant (225/305 nm) (2010) evaporated to dryness, reconstituted in 60% LC-MS/MS 0.39 1.3 acetonitrile. ng/ml ng/ml 13Human Serum sample spiked with [ C12]BPA and BPA derivatized GC-MS in 5 pg/ml 15 pg/ml 101–100.9% Yoshimuraserum mixed with formic acid (to prevent BPA with PFBBr negative 4.76–5.42% RSD (n = et al. ionization and protein precipitation). Sample chemical 6) (2002) applied to C18 SPE cartridge, eluted with ionization mode methanol. BPA conjugated with GC-ECD 0.15 — tetrabutylammonium hydrogen sulfate as the pg/ml counter-ion in alkali solution. The ion-paired BPA moved from the aqueous phase to the organic phase as an ion-paired extraction and derivatized with PFBBr.Blood, Placental sample mixed with water and ethyl BPA derivatized GC-MS in — 0.1 — Schön-placental acetate. Plasma sample mixed with ethyl with BSTFA electron impact ng/ml felder et al.tissue acetate. Supernatant derivatized with BSTFA. ionization mode (2002)APCI, atmospheric pressure chemical ionization; BSTFA, N-O-bis(trimethylsilyl)trifluoroacetamide; DIB-Cl, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride; ECD, electroncapture detector; ECNI, electron capture negative ionization; EI, electron ionization; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; GC, gaschromatography; gluc, glucuronide; HPLC, high-performance liquid chromatography; LC, liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MS, massspectrometry; MS/MS, tandem mass spectrometry; MTBE, methyl tert-butyl ether; MtBSTFA, N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide; n/a, not applicable; NESI,negative electrospray ionization; PBS, phosphate buffered saline; PDMS, polydimethylsiloxane; PFBBr, pentafluorobenzylbromide; PFBCl, pentafluorobenzoylchloride; ppb,parts per billion; SBSE, stir bar sorptive extraction; SPE, solid-phase extraction; tBDMCS, tert-butyldimethylchlorosilane; THF, tetrahydrofuran; TMCS, trimethylchlorosilane;UV, ultraviolet; v/v, volume/volume; w/v, weight/volume 18
  • Chemistry and Analytical Methodsalso used to assist the solvent extraction of BPA from fish tissues (Pedersen & Lindholst,1999).2.1.3 Solid-phase extractionFurther cleanup of the extracts from solvent extraction is almost always necessary to removethe co-extracted interferences. Solid-phase extraction (SPE), either alone or in combinationwith solvent extraction, is the technique used most often for the extraction of BPA from bothliquid and solid food and biological samples and further cleanup of the extracts from solventextraction. The C18 (chemically bonded silica) and the Oasis HLB (lipophilic divinylbenzenewith hydrophilic N-vinylpyrrolidone polymer) are the two SPE cartridges used mostfrequently for both food and biological samples (Brock et al., 2001; Watanabe et al., 2001;Yoshimura et al., 2002; Tsukioka et al., 2003; Kuo & Ding, 2004; Mao et al., 2004; Liu,Wolff & Moline, 2005; Shao et al., 2005; Sun, Leong & Barlow, 2006; Xiao et al., 2006;Fernandez et al., 2007; Kuruto-Niwa et al., 2007; Cao et al., 2008; Dirtu et al., 2008;Grumetto et al., 2008; Volkel, Kiranoglu & Fromme, 2008; Cao, Corriveau & Popovic, 2009;Geens, Neels & Covaci, 2009; Markham et al., 2010). Further cleanup with Florisil cartridgeis sometimes also required (Casajuana & Lacorte, 2004; Dirtu et al., 2008). Although solventextraction is always necessary for solid samples, it may not be essential for some liquidsamples. For example, honey (Yan et al., 2009), infant formula (Biles, McNeal & Begley,1997), soft drinks (Shao et al., 2005; Cao, Corriveau & Popovic, 2009), milk (Casajuana &Lacorte, 2004; Maragou et al., 2006), urine (Mao et al., 2004; Liu, Wolff & Moline, 2005;Moors et al., 2007) and blood serum and plasma (Sajiki, Takahashi & Yonekubo, 1999) wereapplied to SPE cartridges directly after dilution with water or deconjugation with enzyme.Immunoaffinity columns were also used to extract BPA and clean up the extracts fromsolvent extraction for food (Braunrath & Cichna, 2005; Braunrath et al., 2005; Brenn-Struckhofova & Cichna-Markl, 2006; Podlipna & Cichna-Markl, 2007), urine(Schoringhumer & Cichna-Markl, 2007) and blood samples (Zhao et al., 2003). Comparedwith the extracts cleaned up by the non-selective C18 SPE cartridges, immunoaffinitycolumns demonstrated better efficiencies in removing matrix interferences as a result of theirselectivity. As the extracts were analysed by an LC-based system, cross-reactivity of othercompounds is not really an issue compared with the ELISA method. However, application ofthe immunoaffinity columns is still very limited. This may be due to 1) the sensitivity of thismethod being similar to that of the conventional methods; 2) the current conventionalmethods working well; and 3) the preparation process of the immunoaffinity column beingvery tedious.2.1.4 DerivatizationExtracts were rarely analysed directly by GC-MS without derivatization (Casajuana &Lacorte, 2004). The additional step of derivatization in sample preparation is almost alwaysrequired for accurate and sensitive quantitative analysis using GC-based methods because ofthe two hydroxyl groups in BPA. This is optional for qualitative GC analysis; extractswithout derivatization have been analysed by GC-MS for confirmation purposes (Biles,McNeal & Begley, 1997; Munguia-Lopez & Soto-Valdez, 2001; Munguia-Lopez et al.,2002). For analysis by GC-MS in electron impact ionization mode, the derivatizationchemicals used most frequently are acetic anhydride (Goodson, Summerfield & Cooper,2002; Kawaguchi et al., 2004, 2008; Thomson & Grounds, 2005; Cao et al., 2008; Cao,Corriveau & Popovic, 2009; Vinas et al., 2010) and N-O-bis(trimethylsilyl)trifluoroacetamide 19
  • Toxicological and Health Aspects of Bisphenol A(BSTFA) (Arakawa et al., 2004; Kuo & Ding, 2004; Fernandez et al., 2007; Vinas et al.,2010), whereas pentafluoropropionic acid anhydride (Dirtu et al., 2008) andpentafluorobenzylbromide (PFBBr) (Brock et al., 2001; Yoshimura et al., 2002; Kuklenyik etal., 2003; Tsukioka et al., 2003) and pentafluorobenzoylchloride (Geens, Neels & Covaci,2009) were used for the derivatization of BPA for GC-MS analysis in electron capturenegative ionization mode.For LC analysis with fluorescence detection, a few publications also reported derivatizingBPA with the fluorescent reagents 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) (Watanabe et al., 2001; Sun et al., 2002, 2004; Kuroda et al., 2003) or p-nitrobenzylchloride (Mao et al., 2004) to improve sensitivity by adding a stronger fluorophore to BPA.2.1.5 Solid-phase microextractionSolid-phase microextraction (SPME) works well for volatile chemicals, but not forsemivolatile and non-volatile chemicals in general, especially in complicated matrices such asfood and biological samples. Most of the applications of SPME for BPA are for simplematrices such as water. Limited applications of SPME were explored for the determination ofBPA in milk (Liu et al., 2008) and the liquids of canned vegetables (Vinas et al., 2010), butmajor issues with this method for BPA, such as the high blank level of BPA in the SPMEfibre, carry-over and matrix effects, were not addressed. The SPME method coupled to GC orLC analysis could be used as a qualitative screening method for BPA, but, again, in simplematrices only, and it is unlikely to see wide application in food and biological samples forquantitative determination of BPA.2.1.6 Stir bar sorptive extractionSimilar to SPME, stir bar sorptive extraction (SBSE) could be used as a qualitative screeningmethod for BPA in simple matrices such as water. Its applications for BPA in food andbiological samples are very limited (Kawaguchi et al., 2004) owing to issues such as carry-over and matrix effects.2.1.7 Coacervative microextractionCoacervative microextraction is almost the same as liquid-phase microextraction and hasbeen investigated for the determination of BPA in foods (Garcia-Prieto et al., 2008a; Benditoet al., 2009) and urine (Garcia-Prieto et al., 2008b). However, the relatively high limits ofdetection (LODs) make this method much less attractive.2.2 Separation and detection2.2.1 Liquid chromatography–based methodsAs BPA can be analysed by LC directly without the derivatization step in sample preparation,LC is the technique used most often for the determination of BPA in both food and biologicalsamples. Various detectors, including UV, fluorescence, ECD, MS and tandem massspectrometry (MS/MS), have been used for the detection of BPA. 20
  • Chemistry and Analytical Methods(a) Liquid chromatography–ultravioletThe chromophore in the BPA molecule is relatively weak, and the sensitivity of UV detectionis low; thus, UV is rarely used for the detection of BPA. The LOD of the UV method forBPA is at least 15 times higher than that of fluorescence detection. The limit of quantification(LOQ) of UV detection at an emission wavelength of 228 nm for BPA ranged from 5–10ng/g to 67 ng/g (3.7 ng/g for fluorescence detection) for food (Yoshida et al., 2001; Grumettoet al., 2008) to 150 ng/ml (10 ng/ml for fluorescence detection) for human serum (Inoue etal., 2000).(b) Liquid chromatography–fluorescenceFluorescence detection is the most frequently used non-MS-based method for LCdetermination of BPA in both food and biological samples. The fluorophore in the BPAmolecule is fairly strong. The most common excitation wavelength used is 275 nm, withslight variation, although lower wavelengths ranging from 224 to 235 nm have also been used(Biles, McNeal & Begley, 1997; Munguia-Lopez & Soto-Valdez, 2001; Zhao et al., 2003;Mao et al., 2004; Sun, Leong & Barlow, 2006; Xiao et al., 2006; Lee et al., 2008; Yi, Kim &Yang, 2010). The emission wavelength used, on the other hand, is more consistent, rangingfrom 300 to 317 nm. Detection limits of the LC-fluorescence methods for BPA varied,depending on the sample matrices and the extraction methods used, from as low as the sub–parts per billion (i.e. sub–nanogram per gram) level for most methods to as high as 15–29ng/g for some other methods (Sun, Leong & Barlow, 2006; Bendito et al., 2009).Fluorescent reagents with stronger fluorophores were also used to derivatize BPA inbiological samples. With excitation and emission wavelengths for BPA derivatized with DIB-Cl at 350 and 475 nm, respectively, LODs as low as 0.04–0.05 ppb were reported by Kurodaet al. (2003) and Watanabe et al. (2001), but an LOD as high as 4.6 ppb was reported by Sunet al. (2002), indicating that this method is still not mature enough for wide application. Theexcitation and emission wavelengths (228 and 316 nm, the same as for non-derivatized BPA)used by Mao et al. (2004) may not be optimized for BPA derivatized with the fluorescentreagent p-nitrobenzoyl chloride, and the LOD (2.7 µg/l) is typical for non-derivatized BPA.Owing to the complex matrices of food and biological samples, non-MS-based methods arelikely to generate false-positive results; thus, confirmation by MS is essential. However,among all the results generated by LC-fluorescence methods, only a few investigatorsconfirmed the results by LC-MS (Inoue et al., 2003a; Schoringhumer & Cichna-Markl, 2007)or GC-MS (Biles, McNeal & Begley, 1997).(c) Liquid chromatography–electrochemical detectorLimited applications of ECD for LC determination of BPA in both food and biologicalsamples were reported (Sajiki, Takahashi & Yonekubo, 1999; Inoue et al., 2000; Ouchi &Watanabe, 2002; Liu, Wolff & Moline, 2005; Sajiki et al., 2007). However, this method hasno more benefit in terms of LOD (sub-ppb levels) than the other non-MS-based methods andthus will be unlikely to find wide application as MS-based instruments become moreaffordable. 21
  • Toxicological and Health Aspects of Bisphenol A(d) Liquid chromatography–mass spectrometry or liquid chromatography–tandem massspectrometryLC-MS or LC-MS/MS is the second most frequently used LC method after LC-fluorescencefor the determination of BPA in both food and biological samples, and it provides much moreconfidence in peak identification based on the mass spectrum. The additional advantage ofMS-based methods is the use of isotope-labelled BPA, such as BPA-d16, BPA-d14 and[13C]BPA. By spiking samples with isotope-labelled BPA at the beginning of the sampleextraction stage, matrix effect, loss of analyte, variations in extract volume, etc. can becorrected; thus, the method will have better precision and accuracy. However, this advantagehas not been fully used in all LC-MS-based methods for BPA, and isotope-labelled BPA wasused in only some of the methods (Inoue et al., 2002; Volkel, Bittner & Dekant, 2005; Ye etal., 2005a,b, 2006; Maragou et al., 2006; Ackerman et al., 2010; Markham et al., 2010).Both negative ion electrospray ionization (ESI) and atmospheric pressure chemical ionization(APCI) have been used to generate gas-phase ions in LC-MS. The most abundant ion in theBPA mass spectrum is m/z 227 ([M-H]−), and it is used for the quantification of BPA in LC-MS analysis in selected ion monitoring mode. In LC-MS/MS, one or more MS/MStransitions of precursor ion m/z 227 to product ion m/z 133 or m/z 212 were monitored for thequantification and confirmation of BPA.Although LC-MS/MS provides more information on product ions and thus more confidencein peak identification compared with LC-MS, the sensitivities of the two methods weresimilar, around the sub-ppb level. The extremely low LOD (0.6 ng/l) of the LC-MS/MSmethod reported by Shao et al. (2005) is questionable, as they failed to detect any BPA incanned soft drink products (they should have been able to detect BPA with the claimedLOD), and, in their later publication, the LOD of the same method for egg and milk was ashigh as 0.1 ng/g (Shao et al., 2007).The other advantage of LC methods, especially LC-MS-based methods, is that free BPA andconjugated BPA in a sample extract could be separated by LC and detected simultaneously;thus, deconjugation of the sample by enzymes is not needed. This was demonstrated byVolkel, Bittner & Dekant (2005); LC-MS/MS was used to analyse BPA and BPA-glucuronide in urine extracts simultaneously, with LOQs of 3.42 µg/l and 26.3 µg/l,respectively. Two MS/MS transitions of precursor ion m/z 403 to product ions m/z 113 andm/z 227 were monitored to quantify and confirm BPA-glucuronide.2.2.2 GC-MSGC-MS is also one of the methods frequently used for the determination of BPA in both foodand biological samples because of its higher resolution and lower LOD compared with LC-MS methods, despite the tedious derivatization step required. Although derivatization of BPAis not essential for confirmation purposes (Biles, McNeal & Begley, 1997), quantitativedetermination of BPA using GC-MS without derivatization is rare (Casajuana & Lacorte,2004). Symmetrical peaks could still be obtained for underivatized BPA with new GCcolumns, especially with thick coating films, but the performance will start to deteriorateafter a few injections. Thus, derivatization of BPA is always recommended for quantitativeanalysis by GC-MS. For analysis by GC-MS in electron impact ionization mode, thederivatization chemicals used most frequently are the acetylation reagent acetic anhydride(Goodson, Summerfield & Cooper, 2002; Kawaguchi et al., 2004, 2008; Thomson & 22
  • Chemistry and Analytical MethodsGrounds, 2005; Cao et al., 2008; Cao, Corriveau & Popovic, 2009; Vinas et al., 2010) and thesilylation reagents BSTFA with or without the stimulator trimethylchlorosilane (TMCS)(Arakawa et al., 2004; Kuo & Ding, 2004; Fernandez et al., 2007; Vinas et al., 2010) and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MtBSTFA) with tert-butyldimethyl-chlorosilane (tBDMCS) (Moors et al., 2007). Pentafluoropropionic acid anhydride (Dirtu etal., 2008), PFBBr (Brock et al., 2001; Yoshimura et al., 2002; Kuklenyik et al., 2003;Tsukioka et al., 2003) or pentafluorobenzoylchloride (Geens, Neels & Covaci, 2009) wasused for the derivatization of BPA for GC-MS analysis in electron capture negativeionization or negative chemical ionization mode.The electron ionization (EI) mass spectrum of BPA derivatized with acetic anhydride (BPAdiacetyl) is similar to that of underivatized BPA, with m/z 213 being the most abundant ion(used for quantification) and other ions (m/z 228, 270, 312) used for confirmation. The mostabundant ion in the EI mass spectrum of BPA derivatized with BSTFA is m/z 357 (used forquantification), and ion m/z 372 is used for confirmation. The molecular ion m/z 616 is themost abundant for BPA derivatized with pentafluorobenzoylchloride in its electron capturenegative ionization mass spectrum, with m/z 406 [M-C6F5COCH3]− being the confirmationion. The most abundant ion for BPA derivatized with PFBBr is m/z 407, which is due to theloss of a pentafluorobenzyl group from the pentafluorobenzyl diether of BPA duringchemical ionization (Brock et al., 2001; Kuklenyik et al., 2003; Tsukioka et al., 2003).Although Yoshimura et al. (2002) claimed that only one of the two hydroxyl groups in BPAwas derivatized by PFBBr, and thus m/z 407 is the molecular ion, Brock et al. (2001)confirmed the identity of the pentafluorobenzyl diether of BPA by its EI mass spectrum inwhich both the molecular ion m/z 588 [M]+ and another ion m/z 573 [M-CH3]+ (the mostabundant ion) were observed.Isotope-labelled BPA has been used in almost all GC-MS analyses of food and biologicalsamples for BPA. Although some of the early GC-MS methods showed relatively high LODs(Goodson, Summerfield & Cooper, 2002; Thomson & Grounds, 2005), the majority of theGC-MS methods for BPA showed good sensitivity, with LODs at sub-ppb levels.A GC-MS/MS method is also reported for the determination of BPA in urine (Arakawa et al.,2004). MS/MS transitions of precursor ion m/z 357 to product ions m/z 191, 267, 341 weremonitored for BPA derivatized with BSTFA. However, this method had no obvious benefit interms of LOD (0.38 ng/ml) compared with the GC-MS methods.2.2.3 Enzyme-linked immunosorbent assayEfforts were made in the early 2000s to develop ELISA methods for BPA (Ohkuma et al.,2002). Commercial ELISA kits for BPA are now available (IBL International; JapanEnviroChemicals Ltd) and have been used for the determination of BPA in biologicalsamples (Ikezuki et al., 2002; Yamada et al., 2002; Fukata et al., 2006; Kuruto-Niwa et al.,2007). Although the ELISA method for BPA is convenient and popular among non-analyticalchemists, it should be used with care.Cross-reactivity is one of the issues with the ELISA method. The ELISA method cannotdistinguish between free BPA and conjugated BPA, as both can generate responses with thekit. Cross-reactivity of the ELISA kit for BPA from IBL International is as high as 85% forBPA-glucuronide and 68% for BPA-sulfate. Cross-reactivities of chemicals with structuressimilar to BPA are also relatively high: 15.6% for bisphenol B and 6.0% for bisphenol E for 23
  • Toxicological and Health Aspects of Bisphenol Athe ELISA kit for BPA from Japan EnviroChemicals Ltd. Thus, ELISA results must beconfirmed by GC-MS or LC-MS for peak identity.The ELISA method should be validated for the matrices to be applied, and results should becompared with those obtained with well-established methods at different levels for accuracy.As the ELISA method for BPA is not accurate at levels around its LODs (sub-ppb), it is notsuitable for the determination of BPA at low levels in any matrices.Direct analysis for BPA without sample preparation using the ELISA method is possible onlyfor a simple matrix such as water. For food and biological samples, sample preparation andtreatment (solvent extraction followed by SPE, etc.) are still required to generate cleanextracts for analysis by ELISA (Kuruto-Niwa et al., 2007). It is thus logical to predict thatELISA methods are unlikely to be applied widely for the determination of BPA in food andbiological samples, even for qualitative screening purposes. ELISA can be a good fastscreening method for BPA, but, again, only for samples with a simple matrix such as water.2.3 Method validationThe published methods used for the determination of BPA in food and biological sampleshave been validated for free BPA to a certain extent. Certified reference materials for BPAare not available; thus, in-house reference materials have been used to check accuracy insingle-laboratory validations. Method performance parameters, summarized in Tables 2 and3, were acceptable in general. For biological samples, however, there is almost no evidenceof the methods being validated for conjugated BPA. The only study in which the method wasvalidated for conjugated BPA does not involve the deconjugation step with enzymes toconvert conjugated BPA to free BPA, as the conjugated BPA was analysed directly with LC-MS/MS together with free BPA (Volkel, Bittner & Dekant, 2005). This could be partly due tothe unavailability of conjugated BPA standards from reliable sources. Considering the factthat results from biomonitoring have been used for BPA exposure assessments and themajority of the BPA in biological samples is in the conjugated form, validation of methodsfor conjugated BPA will be essential to ensure the validity of the results. Information onvalidation of ELISA methods is very limited. No method performance parameters wereprovided at all for the method used to determine BPA levels in human placenta samples(Schönfelder et al., 2002); thus, the validity of those results is uncertain.Proficiency test programmes for BPA, such as the Food Analysis Performance AssessmentScheme (FAPAS) programme, are available, and some laboratories have participated in thesetests regularly or occasionally. Although most laboratories performed well with the analysis,there are still some (about 10%) that reported unacceptable results, with z-scores greater than2.0 in the 2009 and 2010 FAPAS proficiency tests for BPA. The samples used in proficiencytests are usually simple matrices, such as alcohol or oil commonly used as food simulants inmigration studies. Thus, proficiency tests are limited in testing the method robustness, andinterlaboratory studies should be conducted using real food or biological samples.3. CONCLUSIONS AND RECOMMENDATIONSSensitive and reliable analytical methods are available for the determination of BPA in bothfood and biological samples. Solvent extraction and SPE are the most commonly used andmost effective methods for the extraction of BPA in food and biological samples. Although 24
  • Chemistry and Analytical Methodsisotope dilution methods based on MS and MS/MS are the most reliable for the detection ofBPA, many of the results of BPA determination in both food and biological samples havebeen generated by non-MS-based methods.The majority of methods used to measure free and total BPA in food and biological sampleshave been validated for certain performance parameters, such as accuracy, precision,recovery and LOD. Most methods fulfil the requirements of single-laboratory validation. Forbiological samples, however, validation of methods for conjugated BPA is very limited; onlyone study validated its method for conjugated BPA for some parameters. Proficiency testingprogrammes for measuring BPA are available, and some laboratories have participatedregularly or occasionally, but validation of methods for BPA through interlaboratorycollaborative studies has not yet been conducted. It is difficult to rule out cross-contaminationwith trace levels of free BPA during sample collection, storage and analysis because of theubiquitous presence of BPA in the environment.The Expert Meeting recommends that:• Analytical methods should be validated according to published guidelines for single- laboratory validation, such as the IUPAC guidelines (Thompson, Ellison & Wood, 2002), to include at least the following method performance parameters: LOD, LOQ, repeatability, recovery, linearity and range of calibration curve.• MS- or MS/MS-based isotope dilution methods should be used for the determination of BPA whenever possible. Results from non-MS-based methods should be confirmed by MS methods, especially for food and biological samples.• The ELISA method could be used for screening purposes, but it is not adequate for the quantitative determination of BPA in food and biological samples.• Efforts should be made to produce commercially available, high-purity conjugated BPA standards for method validation purposes for biological samples.• Efforts should be made to avoid cross-contamination during sample preparation and analysis, particularly when measuring unconjugated BPA concentrations, and method blanks and certified reference materials (if available) should be included in the analysis.• Laboratories are encouraged to participate in current proficiency testing programmes to assess the reliability of the data they are producing.• Interlaboratory studies should be conducted to validate methods for different types of food and biological samples.REFERENCESAckerman LK et al. (2010). Determination of bisphenol A in U.S. infant formulas: updated methods and concentrations. Journal of Agricultural and Food Chemistry, 58:2307–2313.Arakawa C et al. (2004). Daily urinary excretion of bisphenol A. Environmental Health and Preventive Medicine, 9:22–26.Bendito MD et al. (2009). Determination of bisphenol A in canned fatty foods by coacervative microextraction, liquid chromatography and fluorimetry. Food Additives & Contaminants. Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment, 26(2):265–274.Biles JE, McNeal TP, Begley TH (1997). Determination of bisphenol A migrating from epoxy can coatings to infant formula liquid concentrates. Journal of Agricultural and Food Chemistry, 45:4697–4700.Braunrath R, Cichna M (2005). Sample preparation including sol-gel immunoaffinity chromatography for determination of bisphenol A in canned beverages, fruits and vegetables. Journal of Chromatography A, 1062(2):189–198. 25
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