Food Chemistry 172 (2015) 385–390

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Analytical Methods

Vortex-assisted hollow fibre liquid-phase microextraction technique combined with high performance liquid chromatography-diode array detection for the determination of oestrogens in milk samples Peijin Wang a, Yu Xiao b, Wenjun Liu c, Juan Wang a, Yaling Yang a,⇑ a b c

Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China Kunming Nanjiang Pharmaceutical Corporation of Limited, Yunnan Kunming 650215, China Sichuan Kelun Pharmaceutical Co., Ltd., Chengdu 610071, China

a r t i c l e

i n f o

Article history: Received 8 October 2013 Received in revised form 21 August 2014 Accepted 10 September 2014 Available online 28 September 2014 Keywords: Vortex-assisted hollow fibre liquid-phase microextraction Milk Oestrogens High performance liquid chromatography Nonanoic acid

a b s t r a c t A rapid, simple, sensitive and environmentally friendly method has been developed for the determination of three oestrogens (17b-estradiol (17b-E2), estrone (E1), and diethylstilbestrol (DES)) in milk samples by using vortex-assisted hollow fibre liquid-phase microextraction (VA-HF-LPME) and high performance liquid chromatography. Method is based on the microextraction of oestrogens from sample solution into 15 lL of nonanoic acid as extracting agent, which is placed inside the hollow fibre followed by vortexmixing. Vortex provided effective and mild mixing of sample solution and increased the contact between analytes and boundary layers of the hollow fibre, thereby enhancing mass transfer rate and leading to high recovery of target analytes. The extraction equilibrium is achieved within 2 min. Parameters influencing the recovery were investigated and optimized. The proposed technique provided good linearity (>0.9984), repeatability (RSD = 2.56–4.38), low limits of detection (0.06–0.17 ng mL1), and high enrichment factor (330). Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Steroid oestrogens are a large group of lipophilic, low-molecular weight, high oestrogenic active compounds, which could bring out the feminine characteristics, control reproductive cycles and pregnancy, influence bone, skin, the cardiovascular system, and immunity (Zhao, Huai, Sun, Suo, & Chen, 2009). They are roughly classified as natural and synthetic oestrogens. Naturally occurring oestrogens include estradiol (E2) and its most common metabolites or precursors: estrone (E1) and estriol (E3). These compounds have been suspected of having adverse effects on the endocrine system in wildlife (Monteleone et al., 2001) and humans (Hochberg et al., 1996). Several reports have shown some dramatic increase of oestrogen-dependent diseases, such as testicular, breast, prostate, ovarian, and corpus uteri cancers. Milk which is considered to be a rich source of steroids oestrogens is an important source of protein for people’s daily life. It has been reported that approximately 60–80% of oestrogens come from milk and ⇑ Corresponding author at: Faculty of Life Science and Technology, Kunming University of Science and Technology, Yunnan Province 650500, China. Tel.: +86 13888316388; fax: +86 0871 63801956. E-mail address: [email protected] (Y. Yang). http://dx.doi.org/10.1016/j.foodchem.2014.09.092 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

other dairy products in Western diets. bE2 of mammary biosynthesis has been shown in cow, goat, and sheep (Maule, Davis, & Fleet, 1983). DES and E1 were reported can affect milk protein content (Ann, Troy, Robert, & Ronald, 2012; Laura, Gabriela, Verónica, Enrique, & Mónica, 2012). These hormones can pass from the bloodstream and can be finally excreted in milk by the mammary gland which synthesizes milk proteins and lactose during late pregnancy and lactation (George & Georgios, 2013). Very recently, a strong epidemiologic correlation between female cancer incidence rate and milk and dairy products consumption, was revealed (Ganmaa & Sato, 2005). Therefore, developing a selective, accurate and sensitive analytical method for detecting oestrogens in milk samples is important for the investigation of potential use of oestrogens and for protecting the health of humans. Various chromatographic analytical methods have been reported for the determination of oestrogens, including micellar electrokinetic chromatography (MEKC) (Wen, Li, & Liu, 2013), high performance liquid chromatography (HPLC) using diode-array, fluorescence and electrochemical detections (Du, Wu, & Zhou, 2013), gas chromatography (GC), gas chromatography with mass spectrometry (GC–MS) (Hansen, Naja, & Nielsen, 2011; Natalia, Caban, Stepnowski, & Kumirska, 2012), liquid chromatography with mass spectrometry (LC–MS) (Chen, Shi, & Wu, 2012; Guo,

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Liu, & Qu, 2013). As for preconcentration and clean-up techniques for oestrogens, hollow fibre liquid-phase microextraction (HF-LPME) is an excellent pretreatment method that is highly compatible with HPLC and overcomes many of the disadvantages of traditional extraction techniques, such as solid-phase extraction (SPE) (Sadowski & Gadzala-Kopciuch, 2013), solid-phase microextraction (SPME), microsolid-phase extraction (l-SPE) (VakiliNezhaad, Mohsen-Nia, Taghikhani, Behpoor, & Aghahosseini, 2004), liquid–liquid extraction (LLE) (Gunter et al., 2008) and dispersive liquid–liquid microextraction (DLLME) (Wu, Chen, & Feng, 2012). Hollow fibre liquid-phase microextraction (HF-LPME) technique, originally proposed by Pedersen-Bjergaard and Rasmussen (1999) has gained considerable interest in the analytical area and has been widely applied to a variety of environmental and biological samples. In HF-LPME procedure, the analytes are extracted from an aqueous sample (donor phase) into an organic acceptor phase supported by a porous-walled polypropylene hollow fibre (Ebrahimzadeh, Shekari, Saharkhiz, & Asgharinezhad, 2012; Hansson & Nilsson, 2009; Wu & Hu, 2009). The volume of sample in HF-LPME can range between 5 mL and greater than 1 L, however, the volume of the extraction solvent (acceptor phase) is, in most cases, in the range 2–30 lL (Pedersen-Bjergaard & Rasmussen, 2008), thus, the very high analyte enrichment factor can be obtained. Although classical HF-LPME procedure take long time (more than 30 min), and the repeatability is not good, the major advantages of HF-LPME are simplicity, negligible volume of solvents used, high enrichment factor, large pH tolerance range, excellent sample clean-up and low cost (Hadjmohammadi, Karimiyan, & Sharifi, 2013). In general, most HF-LPME reports performed using stirring to help mass transfer were very slow (Hadi, Makahleh, & Saad, 2012; San, Alonso, Bartolome, & Alonso, 2012; Xu, Liang, Shi, Zhao, & Wang, 2013). Ultrasound-assisted hollow fibre liquid–liquid–liquid microextraction was reported by Yu-Ying Chao (Chao, Tu, Jian, Wang, & Huang, 2013) for the determination of chlorophenols in water samples. However, to the best of our knowledge, vortex-mixing has not yet been applied for determination oestrogens using hollow-fibre liquid phase microextraction. In this paper, we reported the vortex-assisted hollow fibre liquid-phase microextraction (VA-HF-LPME) in a U-shaped configuration. Vortex-mixing was utilized as an assisted approach to accelerate the mass transfer and minimize fluid loss. The extraction equilibrium is achieved within 2 min. Three oestrogens were employed as model compounds to evaluate the potential of the proposed method for milk naturally contain these oestrogens, and they can cause adverse health effects. HF-LPME approach for the analyses of oestrogens in milk has been reported by Bárbara Socas-Rodríguez. In that study, oestrogens were extracted into organic solvent (1-octanol) under stirring conditions with an addition of salt to improve the extraction efficiency through a saltingout effect (Socas, Asensio, Hernandez, & Rodriguez, 2013). In the proposed method, nonanoic acid which has a lower volatility and an appropriate viscosity was used as extraction solvent and no salt was added. Several parameters affecting the performance of the system were optimized including the selection of organic extraction solvent and eluent, donor phase pH, vortex-mix time, the fibre length, and salt addition.

solutions were stored at 4 °C (stable for at least 2 month). Working solutions were prepared daily by an appropriate dilution of the stock solutions. Spiked whole milk and skimmed milk samples were prepared by adding the standards at 1, 10 and 100 ng/mL, respectively. 1-octanol, nonanoic acid, caprylic acid, capric acid and 1-undecanol of analytical standard were obtained from Aladdin Chemistry (Shanghai, China). Acetonitrile (HPLC grade) was obtained from Merck (Darmstadt, Germany). 2.2. Instrumentation and materials Chromatographic separation and evaluation were performed on an HPLC system (containing a vacuum degasser, an auto sampler, a quatpump, and a diode-array detector; Agilent 1200 Series, Agilent Technologies, Calif., U.S.A.) equipped with a reversed phase C18 analytical column of 150  4.6 mm (Agilent TC-C18). Empower software was used for spectra recording of the studied oestrogens and spectra confirmations of peaks in the studied samples. The detection wavelength was set at 280 nm. Acetonitrile and water were used as mobile phase with the gradient program (Zou et al., 2012) as follows: 0–4.5 min, 35:65; 6.0–16 min, 55:45; acetonitrile:water, v/v (Table 3). Next, the system was allowed to stabilize for 2 min under the initial conditions. The flow rate was set at 1.0 mL min1. The column temperature was maintained at 25 °C and the injection volume was 20 lL. A vortex agitator (Kylin-Bell Lab Instruments Co. Ltd., Jiangsu, China) was used for vortex-mixing. A centrifuge with centrifugal vials (Shanghai surgical instrument factory, 80-2, Shanghai, China) was used for phase separation. An ultrasonic cleaner (Kunshan ultra-sonic instrument plant, Jiangsu, China) was used in the procedure. The porous hollow fibre used to support the organic phase was Q3/2 polypropylene (Wuppertal, Germany) with 600 lm inner diameter, 200 lm of wall thickness and pores of 0.2 lm. A 1.0 mL microsyringe (model 702SNR) with a sharp needle tip was used for the injection of the extraction solvent into the hollow fibre lumen. 2.3. Preparation of hollow fibre and VA-HF-LPME extraction procedure The hollow fibre was cut into 5 cm length pieces. Before use them, each piece was sonicated for 5 min in acetone in order to remove any contamination in the fibre and then, dried in air. The fibre was soaked in nonanoic acid for 15 s to impregnate the pores, and the lumen of the prepared fibre piece was filled with 15 lL nonanoic acid using a microsyringe carefully. Both open ends of the fibre were sealed by a piece of aluminium foil. Then the hollow fibre was bent to a U-shape and immersed in the 5 mL sample solution (pH 4.0, adjusted with Britton–Robinson buffer solution) containing 100 ng mL1 of each oestrogen. The sample was vortex-mixed for 2 min at room temperature (25 °C ± 3). Then, the fibre was removed from the sample solution, and its closed end was cut. The 200 lL methanol was injected into one of its end by a microsyringe. Finally the extract was filtered by a 0.45 lm membrane and injected into the HPLC system for analyses. Procedure of the VA-HF-LPME was shown in Fig. 1. A fresh hollow fibre was used for each extraction to decrease the memory effect. 2.4. The stirring hollow fibre liquid-phase microextraction (ST-HFLPME) procedure

2. Experimental 2.1. Chemicals and reagents 17b-estradiol (17b-E2), estrone (E1), and diethylstilbestrol (DES) of analytical standard were all purchased from Sigma (Sigma, USA). Standard stock solutions of 17b-E2, E1 and DES were prepared in methanol at a concentration of 500 lg mL1. The stock

For comparison with VA-HF-LPME, the conventional ST-HFLPME method was applied to the spiked milk. The hollow fibre was cut into 8.0 cm length pieces. Before use them, each piece was sonicated for 5 min in acetone in order to remove any contamination in the fibre and then, dried in air. The fibre was soaked in commonly used solvents 1-octanol and 1-undecanol for 15 s to impregnate the pores, and the lumen of the prepared fibre piece

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HPLC

Milk Sample Vortex-mix Fig. 1. Procedure of the VA-HF-LPME.

was filled with 15 lL extraction solvent using a microsyringe carefully. Both open ends of the fibre were sealed by a piece of aluminium foil. Then the hollow fibre was bent to a U-shape and immersed in the 5 mL sample solution containing 100 ng mL1 of each oestrogen and a 10  6 mm magnetic stirrer bar and stirring for 2 and 40 min, respectively. Then, the fibre was removed from the sample solution, and its closed end was cut. Then 200 lL methanol was injected into one of its end by a microsyringe. Finally the extract was filtered by a 0.45 lm membrane and injected into the HPLC system for analyses. 2.5. Sample preparation Two types of homogenized samples (whole milk and skimmed milk) were purchased from retail markets in Kunming, China. These samples were frozen at 20 °C until analysis. 0.1 g NaAc, 0.2 g MgSO4 and 2.0 mL acetonitrile were added into a 10 mL centrifuge vial with 5 mL milk sample for protein precipitation, followed by centrifuging at 4500 rpm for 10 min. The supernatant was collected to a new vial, and treated again according to the above method. Then, the supernatant was diluted to 5 mL with Britton–Robinson buffer solution (pH 4.0) for VA-HF-LPME procedure. 2.6. Calculations The enrichment factor (EF) was calculated by the following equations:

EF ¼ C RP; final =C SP; initial

ð1Þ

Recovery ¼ ðC found  C real Þ=C spiked

ð2Þ

3.1.1. Selection of organic extraction solvent and eluent The type of organic solvent used in HF-LPME is an essential consideration for an efficient extraction of target analyte from aqueous solution. Firstly, it should provide high solubility for the target analytes, and should have a low solubility in water. Addition, the ideal organic extraction solvent should have a low volatility and an appropriate viscosity to prevent diffusion and volatile loss. Compared with the other extractant, long chain alcohols and acids have those particular properties, which have special extraction efficiency for the analytes. Thus, five organic solvents (1-octanol, nonanoic acid, caprylic acid, capric acid, 1-undecanol) with different viscosities, volatilities and partition coefficients were evaluated in this work. As shown in Fig. 2, nonanoic acid was the most suitable extraction solvent as it resulted in the highest response. Nonanoic acid has larger viscosity, less volatility and better compatibility in polarity with oestrogens, those characteristics attributed to lower solvent loss during extraction and higher recovery. The eluent which was used for the elution of the extraction solvent after VA-HF-LPME extraction can affect chromatographic behaviour of the analytes. The studied eluents include methanol, ethanol and acetonitrile, obtaining more reproducible results (RSD 1.9%) and higher recovery by using methanol. 3.1.2. Effect of fibre length In general, a short fibre length will provide a high enrichment factor for the concentration of trace analytes in HF-LPME procedure. However, too short fibre membranes cannot provide sufficient extractant to promote analytes transport to extraction solvent. Thus, the fibre lengths of 2, 4, 5, 6, 8, 10 cm were examined. The results in Fig. 3a indicated that 5 cm was sufficient, and no significant effect was found when the fibre length ranged from 6 to 10 cm. Accordingly, an HF length of 5 cm which containing approximately 15 lL extraction solvent was selected for the subsequent experiments. 3.1.3. Donor phase pH The pH value of the donor phase is a critical parameter during the extraction. It can change the partition coefficient of analytes between the sample solution and extraction solvent. Therefore, the effect of the pH on the recovery was studied in the range 1.0–7.0 by adjusting with Britton–Robinson buffer solutions. The results in Fig. 3b show that the recoveries are the highest when the pH value is 4.0. The results can be explained by the principle that the lower pH value can inhibited the ionization of the oestrogens. All analytes will be in the neutral form at low pH value, which facilitates the extraction from sample solution. Thus, pH 4.0 was subsequently utilized in the remaining studies.

where CRP, final and CSP, initial are the final and initial concentrations of the oestrogens in the acceptor and the donor phases, respectively. Cfound is concentration of analyte after addition of a known amount of standard into the real sample, Creal is concentration of analyte in real sample and Cspiked is concentration of a known amount of standard, which was spiked into the real sample.

3.1. Optimization of the VA-HF-LPME procedure Several different experimental parameters that can influence the extraction efficiency were investigated in our experiments using the spiked samples of oestrogens at concentration of 100 ng mL1 in aqueous solutions. Total of 5 replicates were performed to obtain a mean value. The optimization procedure was done as described in VA-HF-LPME procedure.

80

Recovery / %

3. Results and discussion

estradiol estrone diethylstilbestrol

100

60

40

20

0

nonanoic acid caprylic acidcapric acid 1 - octanol 1-undecanol

Extraction solvent

Fig. 2. Effect of extraction solvent on the recovery.

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Recovery / %

Recovery / %

b

90

90

80

70

60

100

100

a

estradiol estrone diethylstilbestrol

2

4

6

8

10

80 70 estradiol estrone diethylstilbestrol

60 50

1

c

90

Recovery / %

100

2

3

4

5

6

80

70 estradiol estrone diethylstilbestrol

60

7

50 0

1

pH

Fiber length (cm)

2

3

4

5

Time/min

Fig. 3. (a) Effect of fibre length, (b) donor phase pH and (c) vortex-mix time on the recovery.

Vortex-assisted

membrane solvent (Tahmasebi, Yamini, & Saleh, 2009). Thus no salt was added in the subsequent experiments.

Ultrasound-assisted

Fig. 4. Comparison of VA-HF-LPME and UA-HF-LPME in terms of extraction condition at 2 min.

3.1.4. Effect of vortex-mixing time Vortex-mix time is one of the main factors in VA-HF-LPME. The extraction can be accelerated by vortex-mix of the sample solution. Vortex-mix facilitates analyte diffusion from donor phase, through the organic solvent, into the acceptor phase. For the present study, the effect of the vortex-mix time was studied over the time range of 0.5–5 min (Fig. 3c). It can be observed that the recoveries increased with the increase of vortex-mix time from 0.5 to 2 min; beyond 2 min, the recoveries remained constant and then a slight decrease. This may be due to the fact that the analytes were thrown out from fibre membrane because of the high shear forces. Therefore, a vortex-mix time of 2 min at 2000 rpm was selected for the procedure. 3.1.5. Effect of salt addition The effect of salt addition on recoveries was examined by adding sodium chloride to aqueous samples at the concentration levels of 0–15% (w/v). The results showed that the salt addition produced a little decrease in the recoveries. This effect may be due to increasing interactions between the analytes and salt in sample solution with increasing salt concentration. Such interactions would tend to restrict movement of the analytes from the donor phase to the

3.1.6. Comparison with UA-HF-LPME and ST-HF-LPME A comparison was made of VA-HF-LPME and UA-HF-LPME for ultrasound is also a helpful means to assist the mass transfer. The VA-HF-LPME was done at optimal conditions as discussed in Section 3.1. The recoveries of analytes with VA-HF-LPME at 2 min are higher (88–94%) than UA-HF-LPME (76–82%). Fig. 4 describes the extraction condition of VA-HF-LPME and UA-HF-LPME at 2 min. It indicates that the sample solution was emulsified when using ultrasound-assisted approach. This may be due to that some extractant overflowed from fibre membrane in powerful ultrasonic radiation, resulting in difficulty in mass transfer to the hollow fibre pores and decreased recovery. A comparison was also made of VAHF-LPME and mostly reported ST-HF-LPME at 2 and 40 min, respectively. The recoveries of ST-HF-LPME at 2 and 40 min are 6–10% and 86–95%, respectively. These results showed that the proposed novel method provides high recoveries in a shorter period of time due to the application of vortex-mixing as driving force which facilitates mass transfer kinetics. Taking all of the above into account, vortex-mixing was proven to be a good approach in HFLPME procedure. 3.1.7. Comparison of the proposed method with other reported methods Table 1 provides a comparison between the proposed method and other reported pretreatment techniques coupled with HPLC from the viewpoint of sample preparation, recovery, extraction time, solvent amount. As listed in Table 1, the analytical performance of this novel extraction is certain advantages and feasibility. 3.2. Method evaluation To evaluate the application of the proposed VA-HF-LPME method for the quantitative determination of oestrogens, calibration plots over the concentration ranges were determined and summarized. For quantitative purposes, the linear dynamic range

Table 1 Comparisons of the proposed method with other sample preparation techniques for the determination of oestrogens. Analytes

Sample preparation

Recovery (%)

Extraction time (min)

Solvent amount

References

bE2, E1, DES E1, E2, E3, DS, DES, EE2

UASEME MSPE

89.8–104.4 83.4–108.5

5 3

>500 lL 5 mg

E2, EE, E1, DES, DHDS E1, 17a-E2, 17b-E2, E3, EE2, DES, DS, HEX, 2OHE2 17b-E2, E1, DES

DLLSME HF-LPME

81.9–99.8 87–118

30 60

200 lL 10 lL

Zou et al. (2012) Gao, Luo, Bai, Chen and Feng (2011) Zhong, Hu, Hu, and Li (2012) Socas et al. (2013)

VA-HF-LPME

87.6–94.2

2

15 lL

This work

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P. Wang et al. / Food Chemistry 172 (2015) 385–390 Table 2 Analytical results for milk samples over 2 days after VA-HF-LPME-HPLC/DAD method. Analyte

Sample

Spiked (ng/ml)

Recovery (%) Day 1

RSD (%) (n = 5)

Recovery (%) Day 2

RSD (%) (n = 5)

17b-Estradiol

Whole milk

10 100 500 10 100 500 10 100 500 10 100 500 10 100 500 10 100 500

87.74 92.24 91.33 89.17 93.03 91.16 93.02 91.79 90.37 89.94 94.25 93.77 88.25 90.13 87.94 92.35 89.72 88.98

4.29 3.16 2.38 3.67 2.85 1.17 3.95 3.02 1.09 3.58 2.27 0.94 4.36 3.27 2.33 3.89 4.21 3.06

90.24 88.93 92.18 89.94 91.25 90.73 90.24 93.57 91.25 90.44 88.69 91.25 90.66 88.43 87.68 89.45 90.27 87.93

3.97 4.25 2.19 4.03 2.78 1.45 3.74 3.25 2.47 2.89 3.45 1.49 3.77 2.83 1.66 4.21 3.29 2.14

Skimmed milk

Estrone

Whole milk

Skimmed milk

Diethylstilbestrol

Whole milk

Skimmed milk

The chromatographic overall run time is 16 min.

were 0.9984, 0.9991, 0.9989 for 17b-E2, E1, and DES, respectively. The regression equation for 17b-E2, E1, and DES were y = 865.42x + 71.09, y = 1020.33x + 42.16, y = 900.27x + 76.14, respectively. The sensitivity was also evaluated in terms of LOD as concentration giving the signal-to-noise ratio of 3 (S/N = 3). The LOD was 0.08 ng mL1 for 17b-E2, 0.17 ng mL1 for E1, and 0.06 ng mL1 for DES, respectively. The LOQ was 0.32 ng mL1 for 17b-E2, 0.58 ng mL1 for E1, and 0.25 ng mL1 for DES, respectively. The relative standard deviation (RSD%) in quintuple of 100 ng mL1 for 17b-E2, E1, and DES were 2.56, 2.28, 4.38, respectively. All the performance characteristics of the proposed method were shown in Table 2. All the data were obtained under the optimized conditions.

Table 3 The gradient elution conditions HPLC. Time (min)

Acetonitrile (%)

Water (%)

0 4.5 6 12 16

35 35 55 55 35

65 65 45 45 65

Injection volume, 20 lL; flow rate, 1.0 mL min1; wavelength, 280 nm.

was assessed by plotting the peak area against the concentration of the respective compounds. A linear behaviour in the range of 5– 1000 ng mL1 was observed. The correlation coefficients (r) values

DES

mAU 1000

a

800 600 17β-E2

400

E1

nonanoic acid

200 0 mAU 350

2

4

6

10

8

12

14

min

b

30 0 250 200 150 100 50

17β-E2

0

2

4

6

8

DES

E1

10

Fig. 5. HPLC–DAD chromatograms: (a) sample milk spiked with oestrogens by VA-HF-LPME (100 ng mL

12 1

14

min

) and (b) standard oestrogens without VA-HF-LPME (100 ng mL1).

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3.3. Analysis of real milk samples To evaluate the effectiveness of the established method in larger range of milk samples, it was applied to the analysis of whole and skimmed spiked milk samples. No oestrogen was found by detecting milk samples directly, so we spiked oestrogens at three different levels over 2 days (Table 3). The recoveries are in the range of 86.24–94.25%, with RSD% (n = 5) of 0.94–4.42. Fig. 3 shows typical HPLC chromatograms of extracted and preconcentrated oestrogens. Fig. 5a is the chromatogram of oestrogens after extraction from milk spiked with VA-HF-LPME, and Fig. 5b is the chromatogram of standard oestrogens without VA-HF-LPME. The results show that the proposed method is effective for the determination of trace amounts of oestrogens in the real milk sample. 4. Conclusions A simultaneous vortex-assisted hollow fibre liquid-phase microextraction (VA-HF-LPME) method has been developed for determination of three oestrogens in milk samples. It was demonstrated that the proposed procedure is comparable to, or even better than, most works that are available for monitoring of oestrogens in mixtures or alone. Moreover, the problem of prolonged extraction time of traditional ST-HF-LPME procedure was solved. The target analytes were enriched very quickly into the acceptor phase with continuous vortex-mixing instead of stirring. Coupled with high performance liquid chromatography diode-array detector, the method has been proven to be simple, rapid, and reliable for oestrogens assay in milk samples. In a wider perspective, handling milk samples with the aid of VA-HF-LPME is a potential field of investigation. Acknowledgement This study was supported by Analysis Test Research Center of Kunming Univ. of Science and Technology, Yunnan Province, China. References Ann, L. M., Troy, L. O., Robert, F. R., & Ronald, S. K. (2012). Estrone and estrone sulfate concentrations in milk and milk fractions. Journal of the Academy of Nutrition and Dietetics, 112, 1088–1093. Chao, Y. Y., Tu, Y. M., Jian, Z. X., Wang, H. W., & Huang, Y. L. (2013). Direct determination of chlorophenols in water samples through ultrasound-assisted hollow fiber liquid–liquid–liquid microextraction on-line coupled with highperformance liquid chromatography. Journal of Chromatography A, 1271, 41–49. Chen, Q. C., Shi, J. H., & Wu, W. (2012). A new pretreatment and improved method for determination of selected estrogens in high matrix solid sewage samples by liquid chromatography mass spectrometry. Microchemical Journal, 104, 49–55. Du, X. J., Wu, Y., & Zhou, H. (2013). Development of a HPLC-ECD method for the simultaneous determination of three synthetic estrogens in milk. Analytical Methods, 5, 2822–2826. Ebrahimzadeh, H., Shekari, N., Saharkhiz, Z., & Asgharinezhad, A. A. (2012). Simultaneous determination of chloropheniramine maleate and dextromethorphan hydrobromide in plasma sample by hollow fiber liquid phase microextraction and high performance liquid chromatography with the aid of chemometrics. Talanta, 94, 77–83. Ganmaa, D., & Sato, A. (2005). The possible role of female sex hormones in milk from pregnant cows in the development of breast, ovarian and corpus uteri cancers. Medical Hypotheses, 65, 1028–1037. George, K., & Georgios, T. (2013). Rapid multi-method for the determination of growth promoters in bovine milk by liquid chromatography–tandem mass spectrometry. Journal of Chromatography B, 930, 22–29. Gunter, G. C. K., Caterina, D. A., Sue, M. A., Shirley, A. R., Angela, A. M., & Sheila, A. B. (2008). Phytoestrogen content of beverages, nuts, seeds, and oils. Journal of Agricultural and Food Chemistry, 56, 7311–7315. Guo, F., Liu, Q., & Qu, G. B. (2013). Simultaneous determination of five estrogens and four androgens in water samples by online solid-phase extraction coupled with high-performance liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 1281, 9–18. Hadi, H., Makahleh, A., & Saad, B. (2012). Hollow fiber liquid-phase microextraction combined with high performance liquid chromatography for the determination

of trace mitiglinide in biological fluids. Journal of Chromatography B, 895, 131–136. Hadjmohammadi, M., Karimiyan, H., & Sharifi, V. (2013). Hollow fibre-based liquid phase microextraction combined with high-performance liquid chromatography for the analysis of flavonoids in Echinophora platyloba DC. and Mentha piperita. Food Chemistry, 141, 731–735. Hansen, M., Naja, J. W., & Nielsen, F. K. (2011). Determination of steroid hormones in blood by GC–MS/MS. Analytical and Bioanalytical Chemistry, 400, 3409–3417. Hansson, H., & Nilsson, U. (2009). Assessment of a dynamic hollow-fibre liquid phase microextraction system for human blood plasma samples. Talanta, 77, 1309–1314. Hochberg, Z., Hochberg, R., Chayen, N., Reiss, Z., Falik, A., Makler, M., et al. (1996). Clinical, biochemical, and genetic findings in a large pedigree of male and female patients with 5 alpha-reductase 2 deficiency. Journal of Clinical Endocrinology and Metabolism, 81, 2821–2827. Laura, K., Gabriela, A. A., Verónica, L. B., Enrique, H. L., & Mónica, M. (2012). Perinatal exposure to xenoestrogens impairs mammary gland differentiation and modifies milk composition in Wistar rats. Reproductive Toxicology, 33, 390–400. Maule, W. F. M., Davis, A. J., & Fleet, I. R. (1983). Endocrine activity of the mammary gland: Oestrogen and prostaglandin secretion by the cow and sheep mammary glands during lactogenesis. British Veterinary Journal, 139, 171–177. Monteleone, P., Luisi, M., Colurcio, B., Casarosa, E., Monteleone, P., Ioime, R., et al. (2001). Plasma levels of neuroactive steroids are increased in untreated women with anorexia nervosa or bulimia nervosa. Psychosomatic Medicine, 63, 62–68. Natalia, M., Caban, M., Stepnowski, P., & Kumirska, J. (2012). Simultaneous analysis of non-steroidal anti-inflammatory drugs and estrogenic hormones in water and wastewater samples using gas chromatography–mass spectrometry and gas chromatography with electron capture detection. Science of the Total Environment, 441, 77–88. Pedersen-Bjergaard, S., & Rasmussen, K. E. (1999). Liquid–liquid–liquid microextraction for sample preparation of biological fluids prior to capillary electrophoresis. Analytical Chemistry, 71, 2650–2656. Pedersen-Bjergaard, S., & Rasmussen, K. E. (2008). Liquid-phase microextraction with porous hollow fibers, a miniaturized and highly flexible format for liquid– liquid extraction. Journal of Chromatography A, 1184, 132–142. Sadowski, R., & Gadzala-Kopciuch, R. (2013). Isolation and determination of estrogens in water samples by solid-phase extraction using molecularly imprinted polymers and HPLC. Journal of Separation Science, 36, 2299–2305. San, R. I., Alonso, M. L., Bartolome, L., & Alonso, R. M. (2012). Hollow fiber-based liquid-phase microextraction technique combined with gas chromatography– mass spectrometry for the determination of pyrethroid insecticides in water samples. Talanta, 100, 246–253. Socas, R. B., Asensio, R. M., Hernandez, B. J., & Rodriguez, D. M. A. (2013). Hollowfiber liquid-phase microextraction for the determination of natural and synthetic estrogens in milk samples. Journal of Chromatography A, 1313, 175–184. Tahmasebi, E., Yamini, Y., & Saleh, A. (2009). Extraction of trace amounts of pioglitazone as an anti-diabetic drug with hollow fiber liquid phase microextraction and determination by high-performance liquid chromatography-ultraviolet detection in biological fluids. Journal of Chromatography B, 877, 1923–1929. Vakili-Nezhaad, G. R., Mohsen-Nia Taghikhani, V., Behpoor, M., & Aghahosseini, M. (2004). Salting-out effect of NaCl and KCl on the ternary LLE data for the systems of (water + propionic acid + isopropyl methyl ketone) and of (water + propionic acid + isobutyl methyl ketone). Journal of Chemometrics, 36, 341–348. Wen, Y. Y., Li, J. H., & Liu, J. S. (2013). Dual cloud point extraction coupled with hydrodynamic-electrokinetic two-step injection followed by micellar electrokinetic chromatography for simultaneous determination of trace phenolic estrogens in water samples. Analytical and Bioanalytical Chemistry, 405, 5843–5852. Wu, C. Q., Chen, D. Y., & Feng, Y. S. (2012). Determination of estrogens in water samples by ionic liquid-based dispersive liquid–liquid microextraction combined with high performance liquid chromatography. Analytical Letters, 45, 1995–2005. Wu, Y. L., & Hu, B. (2009). Simultaneous determination of several phytohormones in natural coconut juice by hollow fiber-based liquid–liquid–liquid microextraction-high performance liquid chromatography. Journal of Chromatography A, 1216, 7657–7663. Xu, X., Liang, F. H., Shi, J. Y., Zhao, X., & Wang, Z. M. (2013). Determination of hormones in milk by hollow fiber-based stirring extraction bar liquid–liquid microextraction gas chromatography mass spectrometry. Analytica Chimica Acta, 790, 39–46. Zhao, Y., Huai, X., Sun, Z. W., Suo, Y. R., & Chen, G. C. (2009). 10-Ethyl-acridine-2sulfonyl chloride: A new derivatization agent for enhancement of atmospheric pressure chemical ionization of estrogens in urine. Chromatographia, 70, 45–55. Zhong, Q. S., Hu, Y. F., Hu, Y. L., & Li, G. K. (2012). Dynamic liquid–liquid–solid microextraction based on molecularly imprinted polymer filaments on-line coupling to high performance liquid chromatography for direct analysis of estrogens in complex samples. Journal of Chromatography A, 1241, 13–20. Zou, Y., Li, Y. H., Jin, H., Zou, D. Q., Liu, M. S., & Yang, Y. L. (2012). Ultrasound-assisted surfactant-enhanced emulsification microextraction combined with HPLC for the determination of estrogens in water. Journal of the Brazilian Chemical Society, 23, 694–701.