107034pe

http://www.chemistrymag.org/cji/2008/107034pe.htm	Jul. 1, 2008 Vol.10 No.7 P.34 Copyright

Determination of phenolic environmental estrogens in pickled quail eggs by matrix solid phase dispersion -liquid chromatography

Zhou Jianke, Zhao Ruifeng, Wang Lishuang, Yang Lifeng
(Research Center of Physics and Chemistry Analysis, Hebei University; Hebei Province Key Laboratory of Analytical Science and Technology, Baoding, 071002, China)

Abstract The method of Matrix solid phase dispersion (MSPD) was applied to extract three phenolic environmental estrogens in sample (Florisil used as dispersant, dichloromethane elute the target compounds). The phenolic compounds were determined by HPLC. A Diamonsil C18 column (5 mm, 250mm×4.6mm) was used for separation. Mobile phase were composed of methanol and water (the ratio of volume was 85:15), detecting wavelength was 227nm. The response of detector for Bisphenol A, Diethylstibestrol and Nonylphenol were linear in range of 0.5-50 mg/mL and correlation coefficients were greater than 0.9998. the method precision and accuracy were satisfactory with recovery from 90.20% to 93.62%and relative standard deviations from 0.83% to 2.65%.
Keywords Matrix solid phase dispersion; Liquid chromatography; Pickled quail eggs; phenolic environmental Estrogens

1. INTRODUCTION
Bisphenol A，Diethylstibestrol and nonylphenol belong to the phenolic environmental estrogens, have estrogen activity and antagonizing androgen effect. These compounds can enter human body through food chain, cumulate for a long time in clay, eject difficultly even do not eject. Teratogenic, carcinogenic and toxic properties of these compounds have been reported in the literature. Therefore, the monitoring and detecting their residues in food can be a significant route to human healthy. HPLC is an analytical method with the advantage of direct detection and simple operation for phenolic environmental estrogens ^[1], Liu Haiwen et al. detected BPA, NP and other five environmental estrogens by RP-HPLC in plastic toys^[2] and Liu Hong et al. used RP-HPLC for determining DES in milk ^[3]. But, the determination of trace contaminants in complex matrices, such as food, often requires multifarious sample extraction and preparation process prior to instrumental analysis. Matrix solid-phase dispersion (MSPD) is an effective sample preparation technique, first reported in 1989, by American professor Baker ^[4]. MSPD has found particular application as an analytical process for the preparation, extraction and fractionation of solid, semi-solid and/or highly viscous biological samples ^[5]. For example, Pensado et al. described the performance of MSPD for the extraction of polycyclic aromatic hydrocarbons (PAHs) in fish tissue ^[6]; Kunihiro Kishida et al. used MSPD to extract residual sulfonamides in chicken^[7]; and Zheng Ping et al. applied MSPD to extract aflatoxins in hot chilli products^[8]. Compare with classical extraction methods, MSPD combines sample homogenization, cell broken, extraction and clean-up in a single step, which can avoid the general drawbacks, such as the use of a large amount of solvent and glassware the laborious extraction procedure and the occurrence of troublesome emulsions. In the experiment, we applied MSPD to treat pickled quail eggs, detected the three phenolic environmental estrogens simultaneity by HPLC, the result was satisfactory.

2. EXPERIMENTAL SECTION
2.1 Apparatus and reagents
The chromatographic system consisted of LC-10AT liquid delivery pump, 7725i model manual injector and SPD-10A UV/VIS detector from Shimadzu (Japan). The N-2000 Double-Channel Chromatograph Date Operation System was purchased from Intelligent Information Technology Graduate School of Zhejiang University. The UV-265 Ultraviolet-visible Spectrophotometer was from Shimadzu (Japan). The SZ-93 automatic double water distilling apparatus was from Shanghai Yarong Biochemistry Instrument Company.
    Bisphenol A (BPA), Diethylstibestrol (DES) and nonylphenol (NP) were purchased from Sigma (St. Louis, MO, USA). Acetonitrile, methanol, n-hexane and dichloromethane, all HPLC grade, were supplied by kermel (Tianjin, China). Ethanol, petroleum ether (fraction 60-90) and other reagents were analytical reagent. The Florisil (150-250mm) was obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).
2.2 Chromatographic conditions
The analytical column was a reversed-phase Diamonsil C18 column (5mm，250mm×4.6mm i.d.). The proposed mobile phase was methanol- water (85:15, v/v). The flow rate was 1.0ml/min. The temperature of the column was kept at room temperature. The optimal detected wavelength was 227nm. The injection volume was 5.0mL.
2.3 Preparation of standard solutions
The standard stock solution (1.0mg/mL) was prepared by dissolving 10.0mg BPA, 10.0mg DES and 10.0mg NP in a 10mL volumetric flask and make up to volume with methanol. A series of standard solutions were prepared by diluting the standard stock solution with methanol.
2.4 Sample pretreatment
An aliquot of the yolk sample (0.5 g) was gently blended with 1.0 g of Florisil (1.5 g Florisil when treat albumen sample) into a glass mortar (50mL capacity) using a glass pestle, until a homogeneous mixture was obtained. This homogenized sample was transferred and packed into a glass syringe (10mL) with a piece of filter paper on the bottom, put another filter paper on the top of mixture and compressed using the syringe plunger. Interfering compounds such as lipin were washed with 2ml petroleum ether (This process was for the yolk sample merely). The target component was eluted with 6mL of dichloromethane by gravity (using 8mL of dichloromethane eluted for treat albumen sample), allowing the eluate to drip slowly into a 10mL volumetric flask, diluting with dichloromethane to volume. Transferred 2mL solution from volumetric flask through 0.45mm microvoid filter film into a graduated tube, concentrated under a nitrogen stream to dry, added 0.2mL mobile phase into the tube, injected 5mL into the HPLC system.

3. RESULTS AND DISCUSSION
3.1 Multi-factorial optimizations of MSPD extraction conditions
3.1.1 The selection of dispersant　
In our work, we researched the efficacy of XAD-2 resin, Chromosorb, neutral activated alumina, acidic activated alumina and Florisil that acts as the dispersant. It has been demonstrated that XAD-2 resin, Chromosorb, neutral activated alumina and acidic activated alumina as the dispersant blended with sample, after treatment and chromatographic analysis, the recovery is lower than 40% because impurity interfere. Comparatively, Florisil as the dispersant, the target compounds had better elution efficacy, there is no impurity interference basically, and the recovery is more than 80%.We has been investigated the impact of different ratio of sample and Florisil. Figure 1 show that 1:2 is the best ratio for yolk sample treatment and explains that 1:3 is the best ratio for albumen sample treatment.
3.1.2 The choice of purge and elution solvents
Lipids may be the main interference in the analysis of Trace compounds in biological sample. In Chromatographic Analysis, the Lipids can affect the active surface of the stationary phase and degrade the resolving power of the column, so we must be avoided or reduced the presence of lipids in the extracts. Both n-hexane and petroleum ether can remove lipids, the experimental result show that petroleum ether is better than n-hexane. We had has been investigated the impact of different eluent, such as methanol, Ethanol, Acetonitrile and dichloromethane. By compared the recovery, applied dichloromethane as eluent, the recovery is maximal. Thus, we selected petroleum ether to wash off lipids and other interference, dichloromethane as the best eluent. To evaluate the elution volume of dichloromethane, we used 4mL, 6mL, 8mL, and 10ml dichloromethane to perform elution, Figure 2 show the result that 6mL dichloromethane can fulfill better elution for yolk sample preparation and 8mL dichloromethane can fulfill better elution for albumen sample preparation. Because of many unknown interfering compounds in the yolk sample, they could be eluted when increasing the volume of dichloromethane. When the nonylphenol was detected, there would appear a peak of impurity near the peak of nonylphenol, if the volume of dichloromethane goes beyond 6mL. The interferer led that the integral of peak area was influenced, so the recovery of nonylphenol reduced obviously.

Figure 1 Influence of sample and Florisil's proportion

Figure 2 Influence of eluting volume

3.2 Optimization of Chromatographic Conditions
Using Ultraviolet-visible Spectrophotometer to scan standard solution from 200-320nm. The measurement of absorption spectra was conducted at 227 nm which gave an average maximum absorbance for all target compounds. We have tested the single methanol and the single acetonitrile as Mobile Phase. The peaks of BPA and DES could not be separated. Therefore, we tested methanol-water system with different Volume ratio. The best chromatogram with complete separation of all target compounds and interfering peaks with clear / short retention time was obtained with the mobile phase of methanol-water (85:15, v/v). The three phenolic environmental estrogens were detected under the optimum conditions. The chromatograms of three phenolic environmental estrogens were shown in Figure 3. Because nonylphenol consist of 11 isomeride, have low molar absorption coefficient, the peak was severe stretching under the proposed chromatographic conditions.

Figure 3 Chromatogram of standard substance
1- Bisphenol A, 2- Diethylstibestrol, 3- nonylphenol

3.3 Linearity and limits of detection
A series of working solutions were prepared by diluting the mixed standard solution containing three phenolic environmental estrogens. The working solutions were detected under the optimized chromatographic conditions. The calibration graphs of the standard compounds were found to be linear over the concentration range studied. Linear equations, correlation coefficients and detection limits are listed in Table 1. On the basis of three times the noise level, the limit of detection (LOD) was obtained. The background interferer brought on the result that the detection limit of yolk sample was higher than the albumen sample's in the experiment.

Table.1 Linearity and the detection limits

Components	Detecting range (mg/mL)	Linear equation	Correlation coefficient (n=5)	Detection limit of yolk sample (mg/g )	Detection limit of albumen sample (mg/g )
Bisphenol A	0.5-50	Y=8988.4X +1826.7	0.9999	0.032	0.005
Diethylstibestrol	0.5-50	Y=9397.7X +1878.7	0.9998	0.19	0.05
nonylphenol	0.5-50	Y=4616.1X +861.4	0.9999	0.06	0.01

Y-peak area; X-the concentration of the detected compound

3.4 Sample analysis and recoveries
Under the optimum experimental conditions, we determined the yolk and albumen of pickled quail eggs, which purchased from supermarket. Positive samples had occurrence of BPA in the albumen, the content is 0.034mg/g, but neither DES nor NP was detected in the analyzed samples. Table 2 summarizes the average recoveries from the yolk and albumen at the spiked level of 2.0mg/g obtained under the proposed experimental conditions. Average recoveries were all above 89% with RSD of 0.83-2.65% (n=5). The chromatograms of spiked sample are shown in Figure 4. So the established method of MSPD extraction followed by HPLC/UV can be used for rapid and precise analysis of the three phenolic environmental estrogens.

Table 2 Recoveries and precision

sample	Components	Sample contents /mg/g	Added contents /mg/g	Determined contents /mg/g	Recoveries /%	RSD /% (n=5)
yolk	Bisphenol A	n.d.	2.0	1.809	90.45	2.65
	Diethylstibestrol	n.d.	2.0	1.872	93.62	2.09
	nonylphenol	n.d.	2.0	1.832	91.60	0.83
albumen	Bisphenol A	0.034	2.0	1.861	91.35	1.40
	Diethylstibestrol	n.d.	2.0	1.804	90.20	1.56
	nonylphenol	n.d.	2.0	1.831	91.55	1.64

n.d. =not detected (below the detection limit).

Figure 4 Chromatograms of spiked yolk sample (A) and albumen sample (B)
1- Bisphenol A, 2- Diethylstibestrol, 3- nonylphenol

ACKNOWLEDGEMENT This work was supported by the National Account Measure Standard (Chemistry Section) Resource Sharing Terrace Capital Construction Foundation of China.（No:2005DKA10800）

REFERENCES
[1] Liu X Y, Zhang H X, Liu M C. Journal of Instrumental Analysis (Fenxi Jiance Xuebao), 2007,26 (2): 288～294.
[2] Liu H W, Lin L, Liu Q. Chinese Journal of Public Health (Zhongguo Gonggong Weisheng), 2006, 22 (8): 1003～1004.
[3] Liu H, Xie C N. Applied Prevent Medicine (Yingyong Yufang Yixue), 2006, 12 (6): 374～375.
[4] Barker S A, Long A R, Short C R, Journal of Chromatography A, 1989, 475 (2): 353～361.
[5] Bogialli S, Corcia A D. J. Biochem. Biophys. Methods, 2007, 70: 163～179.
[6] Pensado L, Casais M C, Mejuto M C et al. Journal of Chromatography A, 2005, 1077 : 103～109.
[7] Kunihiro K, Naoto F. Journal of Chromatography A, 2001, 937: 39～55.
[8] Zheng P, Sheng X, Yu X F et al. Chinese Journal of Chromatography (Se Pu), 2006, 24 (1): 62～64.

基质固相分散-液相色谱法测定腌制鹌鹑蛋中酚类环境雌激素
周建科，赵瑞峰，王立双,杨立凤
(河北大学理化分析中心, 河北省分析科学技术重点实验室河北保定 071002)
摘要硅酸镁（Florisil）作固相分散剂与样品混合，二氯甲烷洗提3种酚类环境雌激素，液相色谱法测定。色谱条件为：Diamonsil C18色谱柱（5 mm，250mm ×4.6mm），甲醇-水(85:15,V/V)为流动相，紫外检测波长227nm。双酚A、己烯雌酚、壬基酚在0.5-50 mg/mL检测范围内呈良好线性关系，相关系数大于0.9998，回收率90.20％-93.62％，相对标准偏差（RSD）0.83％-2.65％。
关键词 基质固相分散；液相色谱；腌制鹌鹑蛋；酚类环境雌激素

[ Back ] [ Home ] [ Up ] [ Next ]