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1、<p><b>  附 錄</b></p><p>  Determination of some endocrine disrupter chemicals in urban wastewater samples using liquid chromatography–mass spectrometry</p><p>  Abstract:An ana

2、lytical method for the simultaneous determination of trace amounts of fourteen endocrine disrupter chemicals (EDCs) in urban wastewater samples has been developed. The studied compounds were: bisphenol A and its chlorina

3、ted derivatives (monochloro, dichloro, trichloro and tretrachlorobisphenol A), three alkylphenols (4-n-nonyl, 4-n-octyl and 4-(tert-octyl) phenol) and six well known phthalate esters (dimethyl, diethyl, di-n-butyl, butyl

4、benzyl, bis(2-ethylhexyl) and di-n-octyl phth</p><p>  Keywords: Alkylphenols; Bisphenol A; Bisphenol A chlorinated derivatives; Phthalate esters; Liquid chromatography–mass spectrometry (LC–MS); Wastewater

5、analysis</p><p>  1. Introduction</p><p>  In recent years, a specific category of human made chemicals, called commonly endocrine disrupter chemicals (EDCs), has attracted a great deal of publi

6、c and scientific attention because of their suspected carcinogenic and estrogenic properties. In 1996, the European Union (EU) defined this class of compounds as “exogenous substances that cause adverse health effects in

7、 an intact organism, or its progeny, consequent to changes in endocrine function” [1]. </p><p>  Emerging evidence from wildlife and laboratory studies indicates that some synthetic chemicals may interfere w

8、ith the endocrine system in a broad variety of ways, including the initiation of hormones, blocking their action or accelerating their breakdown [2].</p><p>  Aware of the problem, both, the EU and the US En

9、vironmental Protection Agency (EPA) have authored a“priority” list of substances for further evaluation of their role in endocrine disruption [3,4] indicating the need to assess the levels and effects of EDCs. A report f

10、rom the EU [5] proposed a candidate list of 533 substances for further evaluation of their role in endocrine disruption. Moreover, incremental growth in dysfunctions related to male reproductive health has been demonstra

11、ted [6, 7]. </p><p>  Most EDCs are introduced into the environment by way of anthropogenic inputs and are therefore ubiquitous in aquatic environments receiving wastewater effluents. Although it is possible

12、 that a large amount of EDCs remain yet unidentified, it is clear that there are some identified families of compounds showing potential estrogenic activity. Plastic industry intermediates as bisphenol A or phthalate est

13、ers, detergent degradation compounds, as 4-n-nonylphenol, 4-n-octylphenol and 4-(tert-octyl)phe</p><p>  Bisphenol A is mainly used as a monomer in the preparation of epoxide resins, polycarbonate plastic an

14、d as an antioxidant in polyvinylchloride [10]. Levels of BPA have been found in different matrices such as body fluids, canned food and environmental samples as wastewaters. The use of chlorine for disinfecting wastewate

15、rs or drinking water has become widespread over the years. BPA present in waters may react with residual chlorine originally used as a disinfectant, producing chlorinated BPA der</p><p>  Alkylphenols such a

16、s 4-n-nonylphenol, 4-n-octylphenol and 4-(tert-octyl) phenol are widely used as intermediates to produce surfactants (anionic and non-ion surfactants) and as stabilizers of ethyl cellulose resin, oil-soluble phenol resin

17、 and esters [14, 15].</p><p>  Phthalate esters are one of the most abundant man-made environmental pollutants. They are widely used as additives in the manufacture of plastics to make them flexible. Because

18、 of their properties as plasticizers in plastic production and because its use has increased significantly in recent years, they are often found in environmental matrices such as waters and soils [16]. Six of the most co

19、mmonly used phthalates are dimethyl, diethyl, di-n-butyl, butylbenzyl, bis(2-ethylhexyl) and di-n-octyl </p><p>  Gas chromatography (GC) and liquid chromatography (LC), after previous concentration and clea

20、n-up, have been used for determining these compounds in water samples. Commonly, their preconcentration and clean-up involves liquid–liquid extraction (LLE) with cyclohexane [20] or dichloromethane [21, 22], solid-phase

21、extraction (SPE) [16–19, 23–25] or solid-phase microextraction (SPME) [26]. After an extensive bibliographic revision, SPE seems to be the most used procedure for the isolation, preconcen</p><p>  In this pa

22、per a new liquid chromatographic–mass spectro-metric (LC?MS) method has been developed for determination of EDCs in wastewaters. It involves a SPE procedure for the extraction of studied compounds from the samples. The e

23、xperimental conditions for SPE were optimized and the method was validated under the best conditions using urban wastewater samples from Granada City. The method is simple, reducing the potential for analyte loss during

24、the extraction, avoiding derivatization steps, min</p><p>  2. Experimental</p><p>  2.1. Reagents</p><p>  All reagents were of analytical grade unless specified otherwise. Water (

25、18 MΩ/cm) was purified with a Milli-Q plus system (Millipore, Bedford, MA, USA).</p><p>  4-n-Nonylphenol (4-NP), 4-n-octylphenol (4-OP), 4-(tert-octyl) phenol (4-t-OP), bisphenol F, bisphenol A and tretrach

26、lorobisphenol A (Cl4-BPA) were supplied by Sigma-Aldrich (Madrid, Spain). Monochloro, dichloro and trichloro bisphenol A (Cl-BPA, Cl2-BPA, Cl3-BPA) were synthesized in our laboratory [27]. Phthalate esters dimethyl(DMP),

27、 diethyl(DEP), di-n-butyl (DBP), butylbenzyl (BBP), bis(2-ethylhexyl) (BEHP) and di-n-octylphthalate (DOP) were supplied by Fluka (Buchs, Switzerland).</p><p>  Stock standard solutions (100 mg L? 1) of each

28、 EDC were prepared in acetonitrile and stored in dark bottles at 4 °C until use, remaining stable for at least three months.</p><p>  Acetonitrile (HPLC-gradient grade), methanol, diethyl ether, dichlor

29、omethane, hexane, o-phosphoric acid, acetic acid and sodium hydroxide were purchased from Panreac (Barcelona,Spain). Formic acid and trifluoroacetic acid were supplied by Sigma-Aldrich. All solvents and reagents were che

30、cked to ensure they were free of contamination from phthalate esters.</p><p>  Solid-phase extraction sorbents used were silica-based bonded C18 cartridges LiChrolut RP-18 (500 mg, 3mL) from Merck (Darmstadt

31、, Germany).</p><p>  All solvents and solutions prepared for LC were filtered through 0.22μm cellulose acetate disk filters (Millipore) before use.</p><p>  2.2. Instrumentation and software<

32、/p><p>  Analyses were performed using a Waters Alliance 2695 (Mildford, MA, USA) high-performance liquid chromatograph equipped with a quaternary pump, a vacuum membrane degasser, a thermostated column compart

33、ment, an automatic autosampler, an automatic injector and connected “on-line” to a Waters 2996 photodiode array detector (DAD) and a Waters Micromass ZQ 2000 quadrupole mass spectrometer system that can use either atmosp

34、heric pressure chemical ionization (APCI) or electrospray ionization (ESI) inte</p><p>  AGeminiC18 (150 mm×4.6 mm i.d., 5μm particle size) from Phenomenex (Torrance, CA, USA), a Chromolith RP-18e (100

35、mm×4.6 mm i.d.) from Merck and a Zorbax Eclipse XDB-C8 (150 mm×4.6 mm i.d., 5μm particle size) from Agilent Technologies (Palo Alto, CA, USA) liquid chromatographic columns were tested. </p><p>  S

36、PE was carried out on a Supelco (Madrid, Spain) vacuum manifold for 12 columns connected to a Supelco vacuum tank and to a vacuum pump.</p><p>  Statgraphics software package [28] was used for the statistica

37、l analysis of data.</p><p>  2.3. Sample preparation</p><p>  Urban wastewater samples were collected from different points in the city of Granada. They were placed in glass bottles previously c

38、leaned with nitric acid (1:1; v/v) and the usual precautions were taken to avoid contamination. Samples were centrifuged at 4500 rpm (2400 ×g) for 10 min, filtered through a 0.22 μm cellulose acetate disk filter (Mi

39、llipore) and stored in the dark at 4 °C, until treatment was performed, which occurred within 48 h of sample collection in all cases. The analysis was pe</p><p>  2.4. Solid-phase extraction procedure&l

40、t;/p><p>  Prior to extraction, the wastewater samples were spiked with bisphenol F as a surrogate at a concentration of 1 μgL-1. The LiChrolut RP-18 solid-phase extraction cartridges were conditioned with 5mL

41、of diethyl ether, 5mL methanol and 5mL of deionized water on a SPE manifold at a rate of 1–2mL min? 1 500mL of wastewater was passed through the SPE cartridges at a flow rate of 2–3mL min? 1. When the extraction was comp

42、leted the cartridges were cleaned with 3mL of 10% (v/v) methanol in water and dri</p><p>  Fig.1. LC-UV chromatograms using different columns: (A) Zorbax Eclipse XDB-C8 (0.8mL min?1); (B) Chromolith RP-18e (

43、0.6mL min?1); and (C) Gemini C18 (0.8mL min?1). Peak identifications: (1) BPF (S); (2) DMP; (3) BPA; (4) Cl-BPA; (5): DEP; (6) Cl2-BPA; (7) Cl3-BPA; (8) Cl4-BPA; (9) 4-t-OP; (10) BBP; (11) DBP;(12) 4-OP; (13) 4-NP; (14)

44、DOP; (15) BEHP.</p><p>  2.5. Chromatographic conditions</p><p>  Chromatographic separation of compounds was performed on a Gemini C18 column (150 mm×4.6 mm i.d., 5μm particle size) from P

45、henomenex (Torrance, CA, USA). The standards and samples were separated using a gradient mobile phase consisting of 1.0% (v/v) acetic acid aqueous solution (solvent A) and acetonitrile (solvent B). The gradient condition

46、s were: 0–30 min,45–80% B; 30–31 min, 80–100% B; and then washing column with 100% B for 7 min and return to 45% B in 2 min; finally, reconditioning the colu</p><p>  Fig.2. Mass spectra for studied EDCs (AP

47、CI + and APCI ?).</p><p>  2.6. Mass spectrometric conditions</p><p>  The APCI interface in positive mode, for phthalate esters and alkylphenols, and negative mode, for bisphenol A and its chlo

48、rinated derivatives, was chosen for the identification and quantification of the compounds. The optimization of the APCI–MS conditions was made by direct injection of standard solutions of each compound (1 mg L? 1) and t

49、he selected parameters were as follows: source temperature, 120 °C; cone temperature, 20 °C; desolvation temperature, 350 °C; discharge voltage, 4.0 kV in po</p><p>  3. Results and discussion

50、</p><p>  3.1. Preconcentration procedure</p><p>  Due to the complex nature of wastewater, a SPE was selected as appropriate to extract analytes from the samples previously filtered.</p>

51、<p>  Solid-phase extraction procedure, described in Section 2.4, was optimized by the authors in a previous work [29].</p><p>  Fig.3. LC–MS, SIM mode, chromatograms for EDCs (APCI + and APCI-).</p

52、><p>  3.2. Liquid chromatographic separation</p><p>  Previous to the coupling with the mass spectrometer, an optimization of the liquid chromatographic separation was carried out using a UV-DAD.&

53、lt;/p><p>  A Gemini C18 (150 mm×4.6 mm i.d., 5μm particle size), a Chromolith RP-18e (100mm×4.6 mm i.d.) and a Zorbax Eclipse XDB-C8 (150mm×4.6mm i.d., 5μm particle size) columns were tested.<

54、;/p><p>  The three tested columns gave good resolution for all studied analytes (Fig. 1) but the Gemini C18 column presents the advantage of providing a good resolution for the critical pair bisphenol F (peak

55、1) and bisphenol A (peak 2). Consequently, this column was selected for our purpose because it gives the best resolution at shorter times.</p><p>  Different mobile phases were studied in order to optimize t

56、he separation and peak shapes of all compounds. Two organic solvents (methanol and acetonitrile) commonly used in reversed phase liquid chromatography were evaluated. Acetonitrile gave better results than methanol. Moreo

57、ver, acidification of the LC eluent was necessary to suppress the ionicmobility of the analytes [30]. Formic acid, acetic acid and trifluoroacetic acid were tested as additives. The best separation and ionization of comp

58、o</p><p>  Lastly, in order to improve the detection limits of the method, a study was performed to evaluate the possibility of increasing the injection volume. A range from 10 to 100μL was studied and no ex

59、tra broadening of the peaks was observed, even at maximum value. Accordingly, 100μL was chosen as the injection volume.</p><p>  3.3. Mass spectrometric analysis</p><p>  APCI and ESI interfaces

60、 were evaluated for the compounds. APCI interface in positive mode (for phthalate esters and alkylphenols) and negative mode (for bisphenol A and its chlorinated derivatives) was selected because it presents, in general,

61、 higher sensitivity for studied compounds. SIM mode was used for quantitative analysis.</p><p>  BPA shows the base peak at 227 m/z corresponding to the molecular ion in negative mode [M?1]ˉ and it was used

62、 as target ion and the peak at 212 m/z corresponding to the loss of a benzylic methyl group [M?15] ˉ was used as the qualifier ion. The molecular ion peak of its chlorinated derivatives appears at 261, 295/297, 329/331 a

63、nd 363/365/367 m/z, for mono, di, tri and tretrachlorobisphenol A, respectively. For alkylphenols the base peak was 107 m/z corresponding to methylphenolic group [Ph?CH2]</p><p>  3.4. Analytical performance

64、</p><p>  Calibration graphs for wastewater samples treated according to the procedure described above were made using SIM mode. Bisphenol F (at 1μgL? 1 level) was used as surrogate. Fig. 3 shows a represent

65、ative chromatogram in SIM mode for studied EDCs.</p><p>  For calibration purposes, six concentration levels were prepared and subsequently the extraction procedure previously explained was applied (each lev

66、el was prepared by duplicate, and the central calibration sample was analyzed three times). Calibration curves were constructed using analyte/surrogate peak area ratio versus concentration of analyte. Linearity of the ca

67、libration graphs was tested using the lack-of-fit test, according to the Analytical Methods Committee [31].</p><p>  Limits of quantification (LOQs) were calculated in order to determine if analytes were pre

68、sent in real samples. Criteria for method performance have been proposed that include the decision limit, CCα, the detection capability, CCβ and limit of quantification [32]. The decision limit is the limit from which it

69、 can be inferred that a sample is contaminated with an error probability α. </p><p>  The detection capability is the smallest content of analyte that may be detected, identified and/or quantified in a sampl

70、e with an error probability β. Decision limit and detection capacity which are better adjusted to a statistical evaluation are implemented. Thus, LOQs, CCα, were calculated.</p><p>  The analytical and stati

71、stical parameters for each EDC are summarized in Table1. </p><p>  To assess intra-day accuracy and precision of the assay, samples at three concentration levels each one were spiked, extracted and analyzed

72、by duplicate. The procedure was repeated on five consecutive days to determine inter-day- variability. Each day, stock solutions of the analytes were prepared. Results obtained are shown in Table 2.</p><p>&

73、lt;b>  Table 2</b></p><p>  Results obtained from the recovery assay made with the wastewater sample number 6</p><p>  3.5. Application and validation of the method</p><p>

74、  The proposed method was applied to urban wastewater samples picked up from different points of the city of Granada. The obtained results, summarized in Table 3, show that some of the selected samples contain different

75、amounts of DEP, 4-t-OP, 4-OP, 4-NP, DOP and BEHP.</p><p>  Of the six assayed samples, in only one sample (sample 6, collected near to an industrial slaughterhouse equipped with a pre-depuration system) stud

76、ied EDCs were not detected. This sample was used for a subsequent recovery study.</p><p>  The presence of4-t-OP (4 samples), 4-OP (2 samples) and 4-NP (2 samples) could be attributed to the use of alkyl phe

77、nol ethoxylates (APEOs) as household laundry detergents, because these compounds are their principal degradation products. Also, the presence of some phthalate esters: DEP (5 samples), DOP (2 samples) and BEHP (2 samples

78、), could be explained by the degradation of the plastic residues present in the wastewater samples.</p><p>  On the other hand, bisphenol A and its chlorinated derivatives were not detected in any studied sa

79、mples, according to the previously obtained results by the authors [22].</p><p>  Due to the absence of certified materials, in order to validate the method, a recovery assay was performed. Prior to our deli

80、berate spiking with the fourteen EDCs, at three concentration levels, samples free of contamination within the low limit detected by our method, were selected to carry out the validation assay (Table 3, sample 6). Sample

81、s were filtered just after spiking and analyzed using the proposed method. The concentration of each compound was determined by interpolation from the stan</p><p>  As the P-values calculated in all cases ar

82、e greater than 0.05 (5%), the null hypothesis appears to be valid, i.e., recoveries are close to 100% (Table 2).</p><p><b>  Table 3</b></p><p>  Determination of studied EDCs in urb

83、an wastewater samples</p><p>  4. Conclusions</p><p>  The determination and quantification of a series of EDCs by liquid chromatography with APCI–MS detection in wastewater was successfully per

84、formed on a Gemini C18 column, with a linear gradient composed of acetonitrile and 1% acetic acid aqueous solution. A solid-phase extraction procedure was used for preconcentration of analytes and removal of interference

85、s. The analytical performance of the proposed method was validated and the method has been successfully used for the determination of these c</p><p>  采用液相色譜-質(zhì)譜法測定城市污水樣品中的一些內(nèi)分泌干擾物</p><p>  摘要:現(xiàn)已

86、研究出一種同時測定城市污水樣品中十四種微量內(nèi)分泌干擾物(EDCs)的分析方法。研究的化學物質(zhì)有: 雙酚A以及它的氯化衍生物(一氯、二氯、三氯、四氯雙酚A),三種烷基酚(4-壬基酚、4-辛基酚、4-叔辛酚)以及六種眾所周知的鄰苯二甲酸酯(鄰苯二甲酸二甲酯、鄰苯二甲酸二乙酯、鄰苯二甲酸二丁酯、鄰苯二甲酸丁基苯甲基二酯、鄰苯二甲酸二辛酯)。該方法包括了使用固相萃取(SPE)程序和隨后的液相色譜分離LC-MS光譜探測儀對樣品進行提取以及分析物的

87、預(yù)濃縮。雙酚F作為一種代替物。量化限在含12 ngL-1 的鄰苯二甲酸二乙酯與含69 ngL-1的4-辛基酚之間波動。該方法在對西班牙格拉納達城市的污水樣品中所含化學物質(zhì)的測定得到了圓滿的應(yīng)用,并使用標樣對該方法的回收率進行了驗證。</p><p>  關(guān)鍵詞: 烷基酚;雙酚A;雙酚A氯化衍生物;鄰苯二甲酸酯類;液相色譜-質(zhì)譜聯(lián)用(質(zhì));污水分析</p><p><b>  1

88、前 言</b></p><p>  近年來, 一種被稱為一般內(nèi)分泌干擾物(EDCs)的人類創(chuàng)造的特殊化學物質(zhì)已經(jīng)吸引了大量公眾和科學界的注意,在于他們的致癌性和雌激素性令人懷疑。1996年,歐盟(EU)將這類化合物定義為“導致在一個完整的生物內(nèi)或者對其后代復(fù)合物的內(nèi)分泌功能產(chǎn)生不良健康影響的外源性物質(zhì)”[1]。</p><p>  來自野生動物和實驗室研究的新興證據(jù)表明一些合成

89、化學品可能干擾以激素的啟動的方式廣泛存在的內(nèi)分泌系統(tǒng),從而阻止他們的行動或加速他們的故障[2]。</p><p>  意識到這個問題后,歐盟和美國環(huán)境保護署(EPA)兩者都已經(jīng)任命了一個“優(yōu)先”物質(zhì)清單,這些物質(zhì)是為了進一步評估表明需要估計EDCs的水平和效果的內(nèi)分泌干擾物的作用[3、4] 。歐盟的一份報告[5]提出了一個候選名單上的533種物質(zhì),這些物質(zhì)是為了進一步評價它們在內(nèi)分泌干擾物中所起的作用。此外,已經(jīng)

90、證實有關(guān)男性生殖健康在系統(tǒng)功能失調(diào)方面的遞增[6、7]。人類和野生動物兩者都暴露在這些化合物的復(fù)雜混合物中。所累積的影響可能只會出現(xiàn)在后代。同時產(chǎn)生的效應(yīng)可能是不可逆的以及威脅到人類的可持續(xù)發(fā)展。</p><p>  大多數(shù)的EDCs是通過人為輸入的方式引入環(huán)境的,因此普遍存在于接收廢水排出物的水環(huán)境中。雖然可能大量的EDCs仍然還未知,但有一些確定的顯示潛在雌激素活性的家族化合物已經(jīng)明朗化了。塑料行業(yè)的中間體,

91、如:雙酚A、鄰苯二甲酸酯或4-壬基酚、4-辛基酚、4-叔辛酚類的洗滌劑降解化合物在科學文獻中得到了充分的描述。現(xiàn)已知,鄰苯二甲酸酯、烷基苯和雙酚A(BPA)是潛在的內(nèi)分泌荼毒生靈化學物質(zhì)[8、9]。讓我們來看看到現(xiàn)在為止他們的一些使用。</p><p>  雙酚A主要用作環(huán)氧樹脂、聚碳酸酯塑料單體的制備和作為一種薄膜中的抗氧化劑[10]。在不同的基質(zhì)中,例如:體液、罐頭食品和污水環(huán)境樣品中,已經(jīng)發(fā)現(xiàn)了雙酚A的標準

92、。多年來,氯消毒廢水或飲水已得到了廣泛的應(yīng)用。目前水域中雙酚A可能與最初用作消毒劑的余氯反應(yīng),從而產(chǎn)生氯化雙酚A的衍生[11]。最近已經(jīng)確定了聚氯雙酚A,同時用活性污泥法做的生物降解試驗也顯示生物降解并不容易[12]以及氯化雙酚A比雙酚A更具有細胞毒[13]。</p><p>  如作為中間體的4-壬基酚、4-辛基酚和4-叔辛酚等烷基酚被廣泛應(yīng)用于生產(chǎn)表面活性劑(陰離子和非離子表面活性劑)和作為含乙基纖維素樹脂、

93、油溶性酚醛樹脂和酯[14、15]的穩(wěn)定劑。</p><p>  鄰苯二甲酸酯類化合物是最豐富的、人造的環(huán)境污染物之一。它們被廣泛應(yīng)用于使塑料更加柔韌的制造塑料的添加劑中。近年來因為塑料產(chǎn)品的特性如增塑性以及它的使用已經(jīng)大幅度增加,因此它們經(jīng)常在例如水和土壤等環(huán)境基質(zhì)中發(fā)現(xiàn)。最常用的六種鄰苯二甲酸酯類是鄰苯二甲酸二甲酯、鄰苯二甲酸二乙酯、鄰苯二甲酸二丁酯、鄰苯二甲酸丁基苯甲基二酯、和鄰苯二甲酸二辛酯。其中鄰苯二甲酸

94、二乙基己基酯是應(yīng)用最廣泛的,產(chǎn)量可多達總生產(chǎn)增塑劑的四分之一[17]。廢水成了引入這些污染物進入環(huán)境中最常見的方式[16 - 19],結(jié)合上述污染物以及將相對大量的這些化合物釋放到環(huán)境中是一項極高的工業(yè)活動。</p><p>  在預(yù)濃縮和凈化后, 氣相色譜(GC)和液相色譜(LC)已被用于測定水樣中的這些化合物。通常,它們的預(yù)濃縮和凈化涉及環(huán)己烷[20]或二氯甲烷[21、22]的液-液萃取(LLE)、固相萃取(

95、SPE)[16 - 19,23 - 25]或固相微萃取(SPME)[26]過程。經(jīng)過書目的廣泛修改后,固相萃取似乎成了最常用的環(huán)境基質(zhì)中EDCs的分離、預(yù)濃縮和凈化過程的處理方法。</p><p>  本文提出了一種測定廢水中EDCs的新型的液相色譜-質(zhì)譜法(LC-MC)。它涉及到一個提取已知混合物樣品的固相萃取過程。對固相萃取技術(shù)的試驗條件進行了優(yōu)化,并在最佳的條件下使用從格拉納達城市的污水樣品驗證了該方法。該

96、方法簡單,在提取過程中可減少潛在的分析物損失,避免衍生化步驟的產(chǎn)生且使溶劑的用量最小化,從而減少對環(huán)境的污染。</p><p><b>  2 實驗部分</b></p><p><b>  2.1 實驗試劑</b></p><p>  除非有明確的說明,否則所有的試劑都是分析級別的。水(18兆歐 /厘米)是通過加入的微Q系

97、統(tǒng)凈化的。</p><p>  4-壬基酚(4-NP)、4-辛基酚(4-OP)、雙酚F(BPF)、雙酚A(BPA) 、14-氯化雙酚A是由西格瑪奧德里奇(馬德里,西班牙)提供的。一氯雙酚A、二氯雙酚A、三氯雙酚A(Cl-BPA, Cl2-BPA, Cl3-BPA)是由實驗室合成 的。鄰苯二甲酸二甲酯(DMP)、鄰苯二甲酸二乙酯(DEP)、鄰苯二甲酸二丁酯(DBP)、鄰苯二甲酸丁基苯甲基二酯(BBP)、鄰苯二甲酸二

98、乙基己基酯(BEHP)、鄰苯二甲酸二辛酯(DOP)是由弗蘭克 (瑞士)所提供的。</p><p>  每個EDC的標準溶液(100毫克毎升)都是在乙腈作為溶劑的條件下制備的,儲存在4°C的黑暗的瓶子中并靜置至少三個月后使用。</p><p>  乙腈(高效液相色譜梯度級)、甲醇、二乙基醚、一氯甲烷、己烷, 鄰磷酸、乙酸、氫氧化鈉是從Panreac(巴塞羅那,西班牙)購買的。甲酸和

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