Determination of Estrogens in Milk by HPLC using Salting-Out Extraction.
Summary: A simple salting-out assisted liquid-liquid extraction (SALLE) method with acetonitrile combined with high-performance liquid-chromatography was developed and applied for the determination four kinds of estrogens (estradiol (bE2), diethylstilbestrol (DES), estrone (E1) and progesterone (PES)) in milk. The influences of effective parameters such as salt (type and amount), water-miscible organic solvent, phase volume, equilibration time, centrifugation time and pH of sample solution were all studied and optimized.
Under optimized experimental conditions, calibration plots were found to be linear in the range of 0.01-5 mg/l for E1, DES and bE2, and 0.02-8 mg/l for PES, with coefficient of determinations more than 0.996, preconcentration factors of 85-89 for those estrogens in milk. The relative recoveries ranged over 95.1-102.5%, the relative standard deviations (RSD) were 0.3-4.4% (n =5). And the limits of quantification achieved with this method were 0.4x10 -3 mg/l for bE2, 0.7x10 -3 mg/l for E1, 0.65x10 -3 mg/l for DES and 2.4x10 -3 mg/l for PES, respectively.
Keywords: Estrogens, Salting-out extraction, High Performance Liquid Chromatography, Milk, Preconcentration, Acetonitrile.
Estrogens are a large group of lowmolecular, lipophilic, high estrogenic active compounds, which include natural and synthetic estrogens. Natural estrogens include estrone, estradiol and estriol. These hormones have been suspected of having adverse effects on the endocrine system in wildlife  and humans .
Several animal studies have shown that perinatal exposure to estrogens can lead to adverse effects on female and male reproductive development .
In recent years, a great number of substances, both synthetic and natural, have been applied in stock farming to improve and speed up animal growth, and (also) to decrease feed costs .
Hormones found in milk originate from the blood flow and are secreted in milk through an active transport within the mammary gland. What's more, some hormones can be synthesized by the mammary gland and excreted to milk . Because of their potential adverse effects in human health, the use of estrogens in animal fattening is prohibited in the European Community .
A selective, accurate and sensitive analytical method for detecting estrogens in milk samples is important(in vital). In this context, the final purpose of the present study is to come up with the new method to determine the estrogens in milk.
A number of methods have been reported for determining estrogens in milk by biological assays and chromatographic technique. Biological assays mainly include enzyme immunoassay (EIA) and radioimmunoassay (RIA).
These methods are often affordability, easy maneuverability and high throughput. However, they are limited merely for cross-reactivity and poor inter-laboratory reproducibility. And different chromatographic techniques such as colorimetry, spectrofluorometry , gas chromatography (GC), high performance liquid chromatography (HPLC) [7, 8], and gas chromatography with mass spectrometry (GC-MS) detection [9, 10] and LC-MS .
Although GC-MS and LC-MS have sufficient selectivity and sensitivity for simultaneous determination of estrogens, the sample preparation often requites extensive cleaning steps, and the instruments are expensive. To enhance sensitivity and decrease interference of matrix, Liquid-liquid extraction (LLE) , supercritical fluid extraction (SFE) , solid-phase extraction (SPE) , solid-phase microextraction (SPME)  and microsolid-phase extraction (u-SPE)  are applied to extract(extracting) the analyte from the matrix. LLE and SPE are simple and effective but somewhat tedious or unfriendly due to the use of nonaqueous solvent.
In this present work, Salting-out extraction with water miscible organic solvent acetonitrile has shown its distinctive advantages in bioanalytical research [16, 17]. Because salting-out assisted liquid- liquid extraction (SALLE) is compatible with reversed phase HPLC, and extraction solvent evaporation is no longer required.
The extract phase can be injected into an HPLC system immediately after extraction. To our knowledge, this method of investigation of estrogens in milk by high performance liquid chromatography separation using salting-out extraction has never been reported anywhere. In this article, a simple, fast sample preparation method using salting-out extraction with thoroughly controlled conditions is presented. The analytical method was fully validated.
Results and Discussion
The purity of estrogens was confirmed by HPLC and salting-out extraction. Moreover, the HPLC chromatography of every estrogen showed one single peak. The purity of estrogens was confirmed by peak area. The influences of effective parameters such as salt (type and amount), water-miscible organic solvent, phase volume, equilibration time, centrifugation time and pH of sample solution were under studied and optimized.
Effect of Extraction Solvent
The selection of an appropriate extraction solvent is essential for the SALLE method. The extraction solvent has to meet certain requirements such as miscibility with aqueous phase and extraction capability of analytes. Based on these considerations acetonitrile, cyclohexane, n-butyl alcohol, MIBC, and acetone were tested (Fig. 1). The results show that acetonitrile exhibited the highest extraction efficiency when compared with the other solvents. Therefore, acetonitrile was selected as the extraction solvents for subsequent experiments.
Effect of Salt Type and Amount
The effect of ionic strength was extensively evaluated in traditional liquid-liquid extraction; because addition of a salt is often used to decrease the solubility of hydrophilic compounds in the aqueous phase through a salting-out and consequently increase the partition of analytes into the organic phase. In order to obtain a better phase separation and the optimum extraction efficiency, several salts, including NaCl, KCl, Na 2 SO 4, and Na 2 HPO 4 were tested (Fig. 2).
The results demonstrated that NaCl provided higher extraction efficiency than other salts. What's more, the different amount of NaCl was tested (Fig. 3). Therefore, 2g NaCl was selected for further experiments.
Effect of the Volume of Acetonitrile
It has been well known that the adjustment of solvent amounts used is an important operating variable industrially. For successful recovery of estrogens, it is desirable to use a minimum amount of acetonitrile for maximum extraction of estrogens. In order to obtain the effect of concentration of acetonitrile, different initial concentrations were tested (Fig. 4). And it could obtain a good recovery at 3ml. For all subsequent works, 3ml acetonitrile was selected in all later studies.
Effect of pH
pH plays an important role on subsequent extraction . In this study, the pH was varied in the range of 3-7. These pH values stand for acidic and neutral conditions respectively. Fig. 5 shows the variation of recovery of estrogens with the increase of the pH value of the aqueous phase in solvent extraction. Based on the experiment results, the pH value of 5 was selected for aqueous sample solution.
Effect of Equilibration Time
The equilibration time is also an important operating variable industrially. For the sake of discussing the effect of time on extraction of steroids hormone, different time was tested (Fig. 6). It has been observed that 4 min was beneficially chosen for all subsequent experiments, thus 4 min was selected in all later studies.
Effect of Centrifugation Time and Rates
It was found that the increase of centrifuge rate has no considerable effect upon the extraction efficiency and analytical signal. The effect of centrifugation time on phase separation of milk was studied in the range 2-20 min at 4500 rpm. The results show that 10 min was enough to get a complete phase separation. So a centrifugation time of 10 min was selected as optimum.
Comparison with other Methods
In order to compare the proposed method with the previously reported methods in HPLC recovery of estrogens in water, the application of the proposed SALLE method and the conventional LLE were showed in Table-1. And the equation of recovery (R) of each estrogen was as follows: where C ext and C 0 were the concentration of analyte in the extraction phase and initial concentration of analyte in sample solution, respectively.
where V ext and V aq were the volume of the extraction phase and volume of sample solution, respectively. As shown in Table-1, the SALLE method is superior to LLE.
Table-1: The recovery of the proposed method and LLE.
Analyte###CH 2 Cl 2###CCl 4###SALLE
The conventional PPT methods were also compared with in Fig. 7. According to the effect of impurity peak and peak area, acidification acetonitrile, ammonium acetate and magnesium sulfate were chosen to eliminate the interference of protein.
So as to validate the accuracy and precision of the proposed method under the selected conditions, spiked samples had been tested. Chromatograms of milk samples spiked at 0.2 mg/l are shown in Fig. 8 using the proposed method. And chromatograms of milk samples spiked at 0.2 mg/l are shown in Fig. 9 without enrichment. The results were satisfied, showing no obvious interferences.
Experimental Reagents and Chemicals
Beta E2, DES, E1 and PES (shown in Table-2) were all purchased from Sigma (Sigma, USA). All reagents used were HPLC grade, and purified water from a Milli Q system was used throughout the experiments. Standard stock solutions containing these compounds were prepared in methanol at a concentration of 500ug/ml. Working solutions were prepared daily by an appropriate dilution of the stock solutions. In addition, NaCl, KCl, Na 2 SO 4 , ZnSO 4 , MgSO 4 , ZnCl 2 , ammonium acetate, Na 2 HPO 4 , n-butyl alcohol, MIBC, CH 2 Cl 2, CCl 4 , cyclohexane and acetone were prepared immediately before each experiment.
Britton-Robinson buffer solution includes 0.04mol/L boric acid, 0.04mol/L phosphoric acid and 0.04mol/L acetic acid. The vessels used for trace analysis were washed with methanol and purified water before usage. Milk was purchased from local market.
Table-2: The structures of four estrogens.
Procedure For the extraction and preconcentration of estrogens, 3 ml acetonitrile , 2 g NaCl was added to 8 ml purified water spiked with each estrogens. The pH of the samples was adjusted to pH 5 by adding 1 mol/L hydrochloric acid (HCl), and centrifuged once (4,000rpm for 5min). After phase separation, 20uL the supernatant was directly injected in the HPLC for subsequent analysis.
Milk sample was complex, composed of proteins and lipids. The removal of all proteins and lipids in milk samples was carried out for using acidification acetonitrile. So acidification acetonitrile 6 ml, ammonium acetate 0.6 g and magnesium sulfate 2.4 g were added to 15 ml milk and centrifuged twice (4500 rpm for 10 min) to remove fat component and cellular debris. 1 ml of acetonitrile was added to this centrifugal tube (8 ml) , adjusted pH 5 and centrifuged once(4,000rpm for 5min). The supernatant was used for analysis.
The Liquid-Liquid Extraction (LLE) Procedure
For comparison with SALLE, the conventional LLE method was performed with the spiked water under study. In LLE procedure, the extract solvents were commonly selected as organic solvents such as CH 2 Cl 2 and CCl 4. 10 ml water mixed with 0.5 ml extract solvents (e.g. CH 2 Cl 2 and CCl 4 ) was placed in an ultrasonic water bath at 35 kHz of ultrasonication frequency for 10 min. Following centrifugation, the organic phase was diluted to 1.0 ml with acetonitrile and 20.0 ul was injected into the HPLC system.
The Protein Precipitation (PPT) Procedure
For comparison with SALLE, the conventional PPT method was also applied to prepare the spiked milk under study. Usually, inorganic salt such as ZnSO 4 , MgSO 4 and ZnCl 2 was applied for protein precipitation.10 ml milk mixed with 12.6 mg precipitants (e.g. ZnSO 4 , MgSO 4 and ZnCl 2 ) was centrifuged at 4000 rpm for 15 min. The supernatant was removed to a screw cap glass centrifuge tube with conical bottom by a syringe, following the SALLE procedure.
The HPLC system used includes an Agilent 1200 series binary pump, an Agilent 1200 series DAD detector and a Rheodyne 7225i injector. The separations were performed on an Agilent TC-C18 column (150 mmx4.6 mm, particle size, 5um). The mobile phase was acetonitrile: water (55:45, v/v) at a flow-rate of 1.0 ml/min. The injection volume was 20 uL, and the DAD detector was chosen at 200 nm. The column temperature was 25.
In summary, the salting-out extraction was applied as an effective method for the extraction of four kinds of estrogens in aqueous samples. Table-3 showed estrogens of the proposed method. Through the study pH=5, 2 g NaCl, 3 ml acetonitrile and extracting 4min were the best choices. Under optimized experimental conditions, calibration plots were found to be linear in the range of 0.01-5 mg/l for E1, DES and bE2, and 0.02-8 mg/l for PES, respectively, with coefficient of determinations more than 0.996, preconcentration factors of 85-89 for those estrogens.
The relative recoveries ranged over 95.1-102.5%, the relative standard deviations (RSD) were 0.3-4.4% (n =5). The high recoveries and precision showed the optimal experimental conditions were satisfied. So the proposed method is a simple, rapid, and effective method for the simultaneous determination of four kinds of estrogens with their very low concentration in milk. What's more, this study is the first investigation of estrogens in milk by high performance liquid chromatography separation using salting-out extraction.
Table-3: The performance characteristics of the proposed method.
###x (mg/l); y(peak###mg/l###mg/l
Estradiol###y = 1729.3x - 108###R 2 = 0.9976###0.01-5.0###0.4x10 -3
Estrone###y = 1123.2x - 7.3069 R 2 = 0.9992###0.01-5.0###0.7x10 -3
Diethylstibestrol y = 1102.9x - 34.351 R 2 = 0.9969###0.01-5.0###0.65x10 -3
Progesterone###y = 578.48x - 49.14###R 2 = 0.9962###0.02-8.0###2.4x10 -3
The work was greatly supported by the Medical Neurobiology Key Laboratory of Kunming University of Science and Technology, Basic and Applied Research Project in Yunnan province (2008ZC082M) and the Analysis and Testing Foundation of Kunming University of Science and Technology (No. 2011295).
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Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China.
Kunming Medical University, Kunming 650500, China.
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|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Aug 31, 2013|
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