Article pubs.acs.org/JAFC
Development of a Highly Specific Fluorescence Immunoassay for Detection of Diisobutyl Phthalate in Edible Oil Samples Xiping Cui,† Panpan Wu,† Dan Lai,† Shengwu Zheng,† Yingshan Chen,† Sergei A. Eremin,‡ Wei Peng,§ and Suqing Zhao*,† †
Department of Pharmaceutical Engineering, Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, Guangdong 510006, People’s Republic of China ‡ Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov State University, Moscow 119992, Russia § School of Public Health, Guangzhou Medical University, Guangzhou, Guangdong 510006, People’s Republic of China ABSTRACT: The diisobutyl phthalate (DiBP) hapten containing an amino group was synthesized successfully, and the polyclonal antibody against 4-amino phthalate−bovine serum albumin (BSA) was developed. On the basis of the polyclonal antibody, a rapid and sensitive indirect competitive fluorescence immunoassay (icFIA) has been established to detect DiBP in edible oil samples for the first time. Under the optimized conditions, the quantitative working range of the icFIA was from 10.47 to 357.06 ng/mL (R2 = 0.991), exhibiting a detection limit of 5.82 ng/mL. In this assay, the specific results showed that other similar phthalates did not significantly interfere with the analysis, with the cross-reactivity less than 1.5%, except for that of DiBAP. Thereafter, DiBP contamination in edible oil samples was detected by icFIA, with the recovery being from 79 to 103%. Furthermore, the reliability of icFIA was validated by gas chromatography−mass spectrometry (GC−MS). Therefore, the developed icFIA is suitable for monitoring DiBP in some edible oil samples. KEYWORDS: fluorescence immunoassay, phthalate esters, diisobutyl phthalate, edible oil
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INTRODUCTION Phthalate esters (PEs), which have been used in large scale since the 1950s,1 comprise a large number of compounds and now are frequently used in medical application, food packing, personal care production, etc. However, these compounds are not chemically bonded to the polymeric matrix; they will migrate from their original containers to the surrounding environment. Therefore, people are likely to be exposed to these contaminants through ingestion, inhalation, or dermal exposure in their lifetime,2 even including intrauterine development.3 In recent years, PEs have captured great public concern for their adverse effects on human health. Certain PEs, such as diisobutyl phthalate (DiBP), have been reported to disrupt endocrine function and induce developmental toxicity. The conclusion derived from male mice experiments has shown that DiBP reduces anogenital distance and testosterone production during their development,4 and the finding in the human population is consistent with the result.5 It has also been demonstrated that exposure to PEs in some certain extents could cause several human diseases, such as disorders of the male reproductive tract and cancer.6 In addition, Meeker et al. has concluded that DiBP exposure during gestation could be associated with preterm birth.7 Thus far, because PEs have already affected human health seriously, many kinds of them are limited in use. In Europe, DiBP is classified as reproductive toxicants (see the European Union Cosmetics Directive at http://ec.europa.eu/consumers/ sectors/cosmetics/documents/directive/) and is banned in cosmetics. However, DiBP remains a component in the manufacture of other products, which means that exposure to © 2015 American Chemical Society
DiBP is quite ubiquitous. While we have learned that DiBP is detected in all oil samples from a U.S. retail market,8 we should be alert that DiBP is likely to be released into the environment through its leaching from the coating of plastic products or other sources. In addition, the presence of the monoester metabolites of DiBP in the general population has demonstrated that people have the possibility to be exposed to these phthalates.2,9 Consequently, it is quite important and necessary to quickly find out a rapid, reliable, simple, and sensitive method for the determination of DiBP. Up to now, the major analytical methods applied to detect DiBP are high-performance liquid chromatography (HPLC) and gas chromatography (GC), generally combined with mass spectrometry (MS).10−12 Although these analytical techniques provide reliable and accurate results, they are time-consuming, expensive, and in need of skilled personnel, which means that they are unsuitable for the analysis of a large number of samples. Moreover, owing to extensive sample treatment methods, the analytical cost is hardly affordable. Therefore, it urges us to find a more suitable method to detect DiBP. On the contrary, many analytical methods developed from immunoassay technology have been used for the determination of PEs for their reliability, low cost, speediness, ease of use, selectivity, time efficiency, and portability of the procedure.13−16 By now, there are some commonly used labels, such as enzymes, biotins, radioactive nuclei, fluorescein, etc., Received: Revised: Accepted: Published: 9372
August 13, October 5, October 8, October 8,
2015 2015 2015 2015 DOI: 10.1021/acs.jafc.5b03922 J. Agric. Food Chem. 2015, 63, 9372−9378
Article
Journal of Agricultural and Food Chemistry
against PBS (0.01 M, pH 7.4) at 4 °C to remove unconjugated DiBAP. The antigen was further purified by passing through a Sephadex G-50 column at 4 °C. Production of Antibody against DiBP. According to the immunization protocol reported previously,22 three male New Zealand white rabbits with initial body weight of 3000 ± 500 g were selected and immunized with BSA−DiBAP. After 7 days of boost for each, the blood samples were drawn and the serum was purified by precipitation with saturated ammonium sulfate solution. After that, the purified serum was aliquoted into vials and stored at −20 °C until use. After 4 months, a high titer (1:12, 800) of antibody in serum was detected by the indirect enzyme-linked immunosorbent assay (ELISA) with OVA−DiBAP as the coating antigen based on the procedure described by Cao et al.23 Conjugation of Fluorescein Isothiocyanate with Goat Antirabbit immunoglobulin G (IgG). The fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC), are usually used as labels because of their large extinction coefficients, high quantum yields, and stability.18,24 The conjugation of FITC with goat anti-rabbit IgG (1:10 goat anti-rabbit IgG/FITC) was prepared according to the method by Marshall et al.19 Briefly, the appropriate amount of goat anti-rabbit IgG was brought into PBS with an equal volume (0.01 M, pH 7.1), while FITC was dissolved in sodium bicarbonate buffer (0.5 M, pH 9.5). Then, they were mixed in the protein solution. The reaction proceeded with stirring for 4 h at 20 °C. Thereafter, the conjugates were dialyzed against PBS (pH 7.1) for 4 h and treated with Sephadex G-50 for filtration of the remaining free dye. Pretreatment of Edible Oil Samples. Each sample (1.0 mL) was extracted with methanol (5 mL). Each extraction was carried out with mixing for 10 min in a 10 mL centrifuge tube at room temperature and was followed by ultrasonic vibration for 5 min. The extraction solution was centrifuged for 10 min at 10 000 rpm to remove the oil residue. The oil residue was then extracted twice with the same procedure. The two methanol extracts were pooled and evaporated, and the residue was redissolved in n-hexane to 1 mL. The extraction solution was further purified on a Florisil silica column (10 × 50 mm). The Florisil silica column was first activated by passing n-hexane (20 mL) over the column. Then, the extracted solution was loaded into the column, which had been eluted with 20 mL of n-hexane and 20 mL of 5% acetone−n-hexane solution successively. The 5% acetone−n-hexane solution was collected. After evaporation of the solvent, the purified sample was redissolved in n-hexane or 15% ethanol−PBS to obtain a constant volume of 1 mL for GC−MS and icFIA analyses, respectively. Fluorescence Immunoassay Procedure. The icFIA was performed in Corning 96-well microtiter immunoassay plates (flat bottom black polystyrene). DiBAP−OVA solution (100 μL, 30 μg/mL in 0.1 M sodium carbonate buffer at pH 9.6) was coated on microtiter plates at 37 °C for 1 h and incubated at 4 °C overnight. The unbound coating antigen was washed with PBS-T 3 times, and non-specific binding sites were blocked with 200 μL of blocking solution (1% OVA in 10 mM sodium phosphate) at 37 °C for 1 h. The plates were washed 3 times again and stored at 4 °C for a short time until use. The binding procedure between antibody and analyte (v/v, 1:1) was performed with mixing for 10 min in a small glass tube, and then 100 μL of reaction solution was added to each microwell. After incubation for 1 h, the well was washed 3 times and 100 μL of goat anti-rabbit IgG−FITC (diluted 1:300) was added. The reaction was carried out at 37 °C for 2 h. To obtain a stable fluorescence signal, 100 μL of a 30% (v/v) glycerin solution in 0.05 M carbonate/bicarbonate buffer at pH 9.2 was added and the wells were incubated for 3 min at room temperature. The glycerin solution was then discarded, and the emptied wells were incubated for 15 min at 37 °C. After that, the wells were washed for the fourth time. Fluorescence intensity was measured under the settings: λex = 485 nm, and λem = 538 nm. The fluorescence intensity difference (ΔF) between F and F0 was calculated, in which F0 was the fluorescence intensity in the absence of labeled antibody. Standard curves were obtained by plotting the (F − F0)/(Fmax − F0) values against the DiBP concentration, where F and Fmax were the fluorescence intensities in the presence and absence of analyte,
but fluorescein is a more relatively nontoxic, reliable, and sensitive dye.17,18 In addition, fluorescein could be easily combined with the antibody and then be formed as a complex that is readily detected by ultraviolet (UV) spectra,19 which has been widely used in virology, cytology, immunoassay, etc. Therefore, it is really possible to develop a highly specific fluorescence immunoassay for detection of DiBP in edible oil samples, which has not been done before. In this work, we have successfully synthesized the hapten and produced the polyclonal antibody against DiBP. On the basis of the polyclonal antibody against DiBP and fluorescein, a simple, rapid, and sensitive indirect competitive fluorescence immunoassay (icFIA) is established and used for the detection of DiBP in edible oil samples for the first time. Under the optimal conditions, the result has demonstrated that icFIA is reliable for the detection of DiBP compared to GC−MS.
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MATERIALS AND METHODS
Apparatus. Nuclear magnetic resonance (NMR) spectra were obtained with a DRX-400 NMR spectrometer (Bruker, Germany). The fluorescence intensity was measured with a multimode plate reader (Infinite 200, Tecan, Mannedorf, Switzerland). Ultraviolet (UV) spectra were carried out on an UV-3010 spectrophotometer (Hitachi, Tokyo, Japan). Solution Preparation. Phosphate-buffered saline (PBS; 0.2 g of NaH2PO4·2H2O, 2.9 g of Na2HPO4·12H2O, and 8.0 g of NaCl in 1 L of distilled water at pH 7.4) was stored at 4 °C. PBS-T [10 mM 0.05% (v/v) Tween 20 in PBS at pH 7.4]. Carbonate buffer (CB; 0.75 g of Na2CO3 and 1.466 g of NaHCO3 in 0.5 L of distilled water at pH 9.6) was stored at 4 °C. Blocking solution was 1% ovalbumin (OVA) in 10 mM sodium phosphate. Reagents. All reagents were of analytical grade or better. Bovine serum albumin (BSA), OVA, Freund’s complete adjuvant, and Freund’s incomplete adjuvant were obtained from Sigma (St. Louis, MO). Other reagents were purchased from Guangzhou Huaxin Technology Co., Ltd. (Guangzhou, China). Palladium on carbon (Pd/ C) and DiBP were obtained from Aladdin Co., Ltd. (Shanghai, China). Synthesis of Hapten. To synthesize an effective hapten, we chose to retain the ester bond of DiBP and to introduce an amino group via a two-step chemical reaction: esterification and reduction. The esterification procedures were performed essentially as described by Zhang and Sheng.20 Briefly, the reduction was carried out by a clean and safe catalyt in the hydrogenation reaction. Specifically, 0.4 g of Pd/ C (10%) was added slowly to a solution of diisobutyl 4-nitrophthalate (2 g, 0.0095 mol) in 15 mL of methanol at room temperature. The mixture was transferred to a flask, and the air in the flask was replaced with nitrogen. Hydrogen was then added to the flask until the reaction system was filled with purified hydrogen. The reaction was carried out with stirring at 50 °C for 3 h. The liquid residue was obtained by separating Pd/C through filtration. After evaporation of the solvent, a pale yellow crude solid was obtained and purified by silica gel chromatography. The purified product was confirmed by 1H NMR and MS. 1H NMR (400 MHz, DMSO) δ: 7.58 (d, J = 8.5 Hz, 1H), 6.68− 6.59 (m, 2H), 6.16 (s, 2H), 3.94 (dd, J = 21.4 and 6.6 Hz, 4H), 2.00− 1.86 (m, 2H), 0.92 (d, J = 6.7 Hz, 12H). MS [electron ionization (EI)] m/z calcd for C16H23NO4 [M], 293.16; found, 293.1. Thin-layer chromatography (TLC) Rf = 0.5 (CHCl3/MeOH = 4:1). Preparation of Hapten−Protein Conjugates. For preparation of immunogen and coating antigen, we conjugated diisobutyl 4aminophthalate (DiBAP) to BSA and OVA, respectively, through diazotization.21 Briefly, DiBAP (0.2 mmol) being dissolved in 0.2 mL of dioxane was added to a stirred solution containing 2 mL of 1 M HCl and 1.8 mL of dioxane. The mixture was stirred in an ice bath as 0.2 mL of 1 M sodium nitrite was added dropwise and reacted for 30 min. Then, the diazo salt solution was added dropwise to 10 mL of 0.2 M borate buffer (pH 9.0) containing 60 mg of BSA or OVA. The mixture was stirred at 4 °C for 3 h. The orange conjugate solution was centrifuged at 10000g for 5 min, and the supernatant was dialyzed 9373
DOI: 10.1021/acs.jafc.5b03922 J. Agric. Food Chem. 2015, 63, 9372−9378
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Journal of Agricultural and Food Chemistry respectively. The IC50 and the limit of detection (LOD)25 were determined as the analyte concentration was giving 50 and 90% of the maximal quenching, respectively. The dynamic (linear) range corresponded to 20−80%. GC−MS Analysis of DiBP in Edible Oil Samples. The final clean extract of the edible oil sample for the detection of DiBP employed GC−MS (Agilent 7890A, Santa Clara, CA). GC conditions were as follows: 1 μL of the final clean extract of DiBP was injected in splitless mode at 260 °C with the following oven program: 60 °C for 1 min, an increase at a rate of 20 °C/min up to 220 °C, holding for 1 min, then another increase at a rate of 5 °C/min up to 290 °C, and holding at 290 °C for 2 min. The GC−MS analysis was performed in the positive-ion impact ionization technique. Ionization voltage was set at 70 eV. The transfer line and source temperatures were both maintained at 200 °C. The carrier gas, He, was injected at a flow rate of 0.7 mL/min, and the mass spectrometer was tuned in the normal mode program (emission current of 150 μA) to obtain the correct relative abundance of the following fragments: m/z 149, 223, 205, and 167.
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DiBAP at 286 nm and OVA at 280 nm. The results above were consistent with another report,21 indicating that the complete immunogens were synthesized successfully. Concentration of Coating Antigen and Antibody. With goat anti-rabbit IgG−FITC dilution at 1:300, the optimal working concentration of coating antigen was determined by checkerboard titration. The coating antigens were dispensed in rows at six concentrations (5−50 μg/mL), while various dilutions of antibodies being diluted with 15% ethanol−PBS were dispensed in columns with the dilution ratio starting at 1:1000. Figure 2 indicates that the fluorescence intensity was
RESULTS AND DISCUSSION
Synthesis of Hapten−Protein Conjugates. The initial and critical step in the development of effective immunoassays for DiBP lies in the design of an appropriate hapten for DiBP. To obtain a complete immunogen and coating antigen for DiBP, we first modified the DiBP structure to introduce an amino group by a two-step chemical reaction by use of 4nitrophthalate acid. The total yield of the process was more than 95%. Diisobutyl 4-aminophthalate was synthesized successfully according to 1H NMR data. As known, small compounds with a low molecular weight are not immunogenic and do not elicit an immune response. Therefore, they must be conjugated to carrier proteins to obtain an antigen. DiBAP was conjugated to BSA or OVA, being used as an immunogen and a coating antigen, respectively. The final conjugates were purified by Sephadex G-50, and the protein content of the hapten− protein conjugates was calculated to be 7−10 mg/mL. Their structures were measured with UV−vis spectroscopy. As shown in Figure 1, the DiBAP−BSA conjugate had the absorption peak at 337 nm, which was different from DiBAP at 286 nm and BSA at 280 nm. Similarly, the DiBAP−OVA conjugate had the absorption peak at 333 nm, which was different from
Figure 2. Influence of the antibody and coating antigen concentrations on the competition step of icFIA with second antibody solution at the diluted ratio of 1:300. Each point corresponds to the mean value of three replicates. The error bars represent standard deviation (SD).
attenuated significantly, while the concentration of antibody was decreased, especially when the concentration of coating antigen ranged from 20 to 50 μg/mL. For the purpose of achieving high sensitivity and reducing the cost, the antibody dilution at the ratio of 1:2000 was selected in this assay. For a further evaluation of the performance of coating antigen in the assay exactly, the concentrations of coating antigen, ranging from 20 to 50 μg/mL, were applied to plot inhibitory curves. As shown in Figure 3, the sensitivity of the icFIA was slightly higher when the concentration of coating antigen was 30 μg/mL, which means that, under such conditions, the interaction between antibody and analyte was most favored. The results of Figures 2 and 3 led to the conclusion that the optimal concentration of coating antigen was 30 μg/mL. Fluorescence Immunoassay Optimization. To monitor the low amount of DiBP in some edible oil samples, it is essential to develop a highly sensitive icFIA by optimizing assay conditions. For this purpose, not only the concentrations of coating antigen and antibody but other parameters, such as the dilution of goat anti-rabbit IgG−FITC, pH, and ionic strength, which could affect icFIA, were in need of investigation and optimization to achieve high detection efficiency. The optimal working dilution of goat anti-rabbit was determined from the competition experiments carried out under their various dilutions of IgG−FITC, starting at 1:100 and being serially diluted by a factor of 2 in PBS buffer. The result in Figure 4A shows that the rate of fluorescence intensity (estimated from maximum intensity, Fmax) decreased rapidly
Figure 1. UV absorption spectra of DiBAP, BSA, DiBAP−BSA conjugate, OVA, and DiBAP−OVA conjugate dissolved in 10% methanol/PBS (v/v). 9374
DOI: 10.1021/acs.jafc.5b03922 J. Agric. Food Chem. 2015, 63, 9372−9378
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Journal of Agricultural and Food Chemistry
rabbit IgG−FITC diluted 300 times was chosen as the labeled antibody for further studies. Similarly, to evaluate the effect of pH on the assay, the assay was performed in the medium with its pH ranging from 5.7 to 8.5. As shown in Figure 4B, the medium with pH at about 7.4 could obtain the maximum value of the fluorescence intensity. Regardless of higher or lower pH values, they could all inhibit the fluorescence intensity. Therefore, in this assay, pH 7.4 was selected to be the best choice for obtaining the optimal sensitivity. Moreover, the interaction between coated antigen and antibody characterized by weak intermolecular bonds could be affected by the changes in the ionic strength. The effect of the ionic strength needs be investigated by employing different concentrations of PBS ranging from 0.005 to 0.05 M. Fluorescence intensity had significantly changed under different concentrations of the buffer. The result shown in Figure 4C suggests that the optimum concentration of PBS, reflecting the highest fluorescence intensity, was 0.01 M for this assay. In addition, as mentioned in a previous publication, fluorescence self-quenching, which may affect the sensitivity of fluorescence immunoassay,26 is sometimes observed when a relatively large number of fluorescent compounds are introduced in the recognition molecule, such as antibodies. In this part, according the method developed by Petrou et al.,27,28 the fluorescence self-quenching was evaluated by comparing the fluorescence intensity of this assay treated or not with 30%
Figure 3. Influence of the coating antigen concentration on the inhibitory step with second antibody solution at the diluted ratio of 1:300 and the antibody solution at the diluted ratio of 1:2000 in PBS. Each point corresponds to the mean value of three replicates. The error bars represent SD.
with the increasing dilution ratio of IgG−FITC. The immunoassays for DiBP were more sensitive with goat anti-rabbit IgG diluted 100−400 times than other dilutions. Hence, goat anti-
Figure 4. Influence of the (A) goat anti-rabbit IgG−FITC concentration, (B) pH, (C) ionic strength, and (D) 30% glycerin treatment on the competition step of DiBP icFIA. Each point corresponds to the mean value of three replicates. The error bars represent SD. 9375
DOI: 10.1021/acs.jafc.5b03922 J. Agric. Food Chem. 2015, 63, 9372−9378
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Journal of Agricultural and Food Chemistry
Figure 5. Indirect fluorescence immunoassay calibration curve and the linear range of the calibration curve. Each point corresponds to the mean value of three replicates. The error bars represent SD.
glycerin. As shown in Figure 4D, fluorescence self-quenching was suppressed effectively after treatment with 30% glycerin when antibody dilution was from 500 to 4000 but the fluorescence intensity was nearly the same under a low antibody concentration. These results suggested that fluorescence self-quenching could be eliminated by a simple treatment with 30% glycerin. Calibration Curve. In the optimal conditions, the competitive inhibition curve of the assay for detecting DiBP has been established. Figure 5 shows that the IC50 value of the assay was 61.2 ng/mL, with a detection limit of 5.82 ng/mL (10% inhibition). The linear working range of the icFIA was between 10.47 and 357.06 ng/mL. It is obvious that the method could be applied to the same detection. Specificity. The binding between antibody and analyte is easily affected by slight structural differences. Therefore, the cross-reactivity (CR) of the icFIA method was assessed by comparing the IC50 of the analogue with that of DiBP. The CR was calculated with the following formula: CR (%) = IC50 (DiBP)/IC50 (cross reactant) × 100% Kuang et al.29 argued that the antibody could identify the length of ester linkage in the benzene ring. In this study, seven structurally similar analogues related to DiBP (Table 1) were detected with the icFIA method to evaluate the selectivity of the antibody. Table 1 lists that the icFIA method had high selectivity for DiBP, with the CR less than 1.5%, as compared to other phthalate esters, except for that of DiBAP. The result was consistent with the study by Kuang et al.30 The CR value of DiBAP, theoretically, should be higher than that of DiBP, because the antibody was raised against DiBAP− BSA conjugated in our work. However, we found that the CR value of DiBAP was just only 12.07%, which was much lower than that of DiBP (100%). The sensitivity and CR values of antibody are commonly dependent upon several factors, such as the structure of haptens and the physicochemical properties of chemicals. The strength of binding and CR, as is known, might be determined by the electrostatic and hydrophobic interaction through different electron distributions among haptens.21 When DiBAP was conjugated with BSA via diazotization, the
Table 1. CR of DiBP Structurally Related Compounds
electron density on the benzene ring was dispersed because of aromatic resonance with the nitrogen−nitrogen double bond. Thus, the antibody was much easier to recognize DiBP, which had a relatively low electron density on the benzene ring 9376
DOI: 10.1021/acs.jafc.5b03922 J. Agric. Food Chem. 2015, 63, 9372−9378
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Journal of Agricultural and Food Chemistry
accurate results, GC−MC is not only time-consuming but in need of expensive equipment and skilled personnel, while the icFIA method just takes a few hours and is cost-effective and easy to use. Therefore, icFIA is a much more practical method for rapidly and sensitively detecting DiBP in edible oil. In summary, a rapid and sensitive analytical method, icFIA, based on the polyclonal antibody was developed. After optimization of the conditions, the quantitative working range of the icFIA was from 10.47 to 357.06 ng/mL (R2 = 0.991), exhibiting the detection limit of 5.82 ng/mL. In addition, icFIA demonstrating its CR with other similar phthalates was less than 1.5%. Thereafter, the DiBP contamination in edible oil samples was detected by icFIA, with the recovery being from 79 to 103%. Furthermore, the reliability of icFIA was validated by GC−MS. Overall, all of the results above indicate that the developed icFIA is an promising method for rapid detection of DiBP in edible oil samples, being used to address a global issue.
compared to that of DiBAP. Moreover, DiBAP was not present in environment samples, which was just used as hapten in this work. Therefore, the data proved that the antibody used in the developed icFIA could identify DiBP well. Analysis of Spiked Samples. To demonstrate the practicability of the optimized icFIA developed above, it was applied to the determination of DiBP in edible oil samples. To evaluate the recovery of icFIA for DiBP, several edible oil samples were spiked with DiBP and determined by icFIA. Three concentrations of DiBP standard, ranging from 10 to 100 μg/L, were added in the edible oil samples to evaluate this icFIA system. The recoveries are illustrated in Table 2, ranging from 79 to 103%. Table 2. Recovery of DiBP from Spiked Edible Oil Samples Measured by the Optimized icFIA edible oil samples
DiBP levels (μg/L, n = 3)
added (μg/L)
total found (μg/L)
SD (n = 3)
recovery (%)
sample 1
40.32
sample 2
75.83
10 50 100 10 50 100 10 50 100 10 50 100 10 50 100
48.88 90.03 140.45 86.06 124.82 177.34 150.28 190.20 241.04 64.18 105.42 155.29 125.37 163.34 220.12
1.47 0.42 1.08 0.43 1.34 0.67 0.96 0.87 2.24 0.92 0.98 0.22 1.24 0.53 1.4
86 99 103 102 97 102 83 96 99 79 98 99 87 93 103
sample 3
141.95
sample 4
56.23
sample 5
116.68
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-15820258676. Fax: +86-020-61017546. Email:
[email protected]. Funding
This study was supported by the National Natural Science Foundation of China (41271340) and the Guangdong Natural Science Foundation (S2013030013338 and S2013040013021). The authors express their deep thanks for financial support. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Shirley Gee of University of California, Davis and Dr. Ying Zhang of the School of Foreign Languages at Guangdong University of Technology for assisting with the language editing.
To validate the quality of icFIA, five edible oil samples were analyzed by both icFIA and GC−MS methods after the simple pretreatment. The result shown in Figure 6 suggests that the results obtained from icFIA compared to those obtained with GC−MS methods were linear, with the correlation coefficients higher than 0.997, further indicating the reliability of icFIA and supporting the possibility for the detection of DiBP. Although these two analytical techniques could both provide reliable and
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REFERENCES
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Figure 6. Correlation analysis of real samples by GC−MS and icFIA. 9377
DOI: 10.1021/acs.jafc.5b03922 J. Agric. Food Chem. 2015, 63, 9372−9378
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DOI: 10.1021/acs.jafc.5b03922 J. Agric. Food Chem. 2015, 63, 9372−9378
