A cheap, reliable and practical high-performance liquid chromatography—tandem mass spectrometric method was developed for the simultaneous determination of seven anabolic steroids in eggs, including trenbolone, boldenone, nandrolone, stanozolol, methandienone, testosterone and methyl testosterone. The analytes were extracted from the egg samples using methanol.
The extracts were subjected to the removal of fat by freezing-lipid filtration and then further purified by liquid—liquid extraction using tert -butyl methyl ether. The analytes were separated on a Luna C18 column by a gradient elution program with 0. This method was validated over 1. The correlation coefficients r for each calibration curve are higher than 0. The decision limits of the steroids in eggs ranged from 0. The average recoveries were between The between-day and within-day relative standard deviations were in the range of 2.
High matrix suppression effects were observed for all compounds of interest. Anabolic androgenic steroids AASs refer to substances that can be used to promote muscle growth. In animal husbandry, AASs are used in increasing the weight of meat-producing animals, enhancing nitrogen retention and building up proteins, which in turn results in an improvement of muscle growth and higher carcass quality 1 , 2 ; AASs are banned from use. Evidence of the illegal use of AASs for growth-promoting purposes has been presented.
Residues of these compounds in edible animal products are a potential risk for the consumers. Therefore, monitoring of AAS residues in animal-derived food is very important. Over the years, many analytical methods have been developed to determine AAS in biological samples.
Gas chromatography—mass spectrometry GC—MS and tandem MS methods have been used to determine and confirm AAS residues 3—7 , and currently, liquid chromatography—mass spectrometry LC—MS and tandem MS methods are preferred because they provide easier sample preparation without a derivatization step, and because the detection of substances i. For example, Van Poucke et al. Currently, only a few papers have been reported to analyze steroid hormones in eggs.
In addition, there are quantities of endogenous progesterone in eggs, so the method is unfit for the determination of progesterone. The aim of this work is to develop and validate a practical, cheap and reliable LC—MS-MS method for the simultaneous screening and quantification of seven anabolic androgenic steroid compounds including trenbolone, boldenone, nandrolone, stanozolol, methandienone, testosterone and methyl testosterone in eggs by removing fat with a freezing technique and liquid—liquid extraction protocol.
The method was successfully applied to the determination of seven AASs in eggs from local supermarkets. Ehrenstorfer GmbH Augsburg, Germany. Hexane, sodium acetate and sodium carbonate all analytical reagents were purchased from Guangzhou Chemical Reagent Factory Guangzhou, China.
Working standards at concentrations of 0. The chromatographic system was composed of an Agilent series high-performance liquid chromatography system, including quaternary pump and autosampler Milford, MA. The mass system included an Applied Biosystems API triple quadrupole mass spectrometer with electrospray ionization interface and Analyst 1. The mobile phase consisted of a gradient of 0. The linear elution gradient profile was as follows: The mass analyses were performed using an electrospray source in positive ionization mode.
The ionspray voltage was 5. Curtain gas and ion source gas 1 and gas 2 were set at 20, 55 and 50 psi, respectively. Multiple reaction monitoring MRM experiments were conducted. The optimization of the primary mass spectrometry parameters was performed by flow injection analysis for each compound.
Whole eggs were homogenized in a blender. A 2 g portion of the sample was weighed into a 50 mL polypropylene centrifuge tube with a screw cap.
Two milliliters of sodium acetate buffer 0. The mixture was sonicated for 1 min in an ultrasonic bath, and the solution was decanted to a new 50 mL centrifuge tube. The upper ether layer was collected and transferred to another centrifuge tube and re-extracted once under aqueous layer with 5 mL of TBME. The solutions were filtered through a 0.
According to the criteria, the performance characteristics of a conventional method include calibration curves, specificity, recovery, repeatability, reproducibility, decision limit, detection capability and stability. Egg was used for full validation purposes and validation data were obtained for egg matrix.
Negative eggs obtained from local layer farms were used as blank matrix samples. Blank egg was prepared as described previously. The extract residues were used to prepare concentrations of 1. The matrix-matched calibration standard curves were made by plotting the response of the respective AAS versus its concentration in the matrix solution. Five analyses were performed and repeated on three days.
The acceptance criterion was that the coefficient of correlation r must be more than 0. An aliquot of 0. Five replicates for each concentration level were performed and repeated on three consecutive days. The spiked samples were allowed to stand for 30 min before extraction and then prepared and analyzed as described previously.
The extraction recoveries of AAS at the spiked samples were determined by measuring the peak area response from samples spiked with a particular standard solution of AAS before extraction with those from blank egg samples extracted and spiked with same concentration of analytes after extraction.
The precision was expressed as relative standard deviation RSD. The analysis of more than egg samples collected from different local markets was performed to check the selectivity of the proposed method, and then a spike with a concentration of 1. The results were evaluated by the presence of interfering substances around the AAS retention time.
Stability must be taken into account during the validation of residue analysis. The measured values were compared to those of freshly prepared standard solutions. All calculations and data analysis were made with Origin 6. The simple water—acetonitrile mobile phase has a pH of approximately 7, which should dissociate any residual silanols to weakly anionic species that can strongly retain the basic SNZ.
Therefore, these compounds are expected to be separated well, and the pH value of mobile phase should be adjusted to be acidic. Based on these trials, a water—acetonitrile mobile phase containing 0. A Luna C18 column was enough to separate the compounds of interest. MS parameters for the steroids were optimized in positive electrospray ionization full scan mode. Stanozolol has no 3-oneene functional group, but has a conjugated imine nitrogen group, which is also easily protonated Because the anabolic steroids are insoluble in water, methanol and acetonitrile are used to precipitate protein and extract these compounds from complex matrices.
Egg matrices are rich in protein and lipid components, which are known to cause significant ion suppression effects in positive ion electrospray mode However, the sample solution became very complex after hydrolysis, and poor recoveries were obtained. Therefore, a fast and practical method was established for the determination of unconjugated steroid fractions in eggs. A higher recovery was obtained when extracting the target analytes from egg samples with methanol than with acetonitrile when the freezing-lipid filtration step was performed.
This defatted method could save organic solvent and achieve higher recoveries than the conventional defatted protocol with n -hexane liquid—liquid distribution. For further purification and liquid—liquid extraction with TBME was conducted according to the previous study The developed sample preparation procedure not only obtained high recoveries, but was also cheap. In comparison with the authors' previous study 13 , the current sample preparation procedure avoided emulsification in the final centrifuge.
Moreover, the cost of sample preparation using SPE cleanup is more expensive, so the developed method is practical. The proposed sample preparation procedure is also satisfactory for extracting progesterone from egg, however, progesterone is difficult to quantify due to its high endogenous amounts in eggs Compared with the background noise in egg matrix, the results demonstrated that no interfering peaks could be detected within the 2.
The chromatographic resolution and peak performance were good. Therefore, the specificity of method was satisfying for the compounds under investigation. Typical MRM chromatograms of spiked blank egg sample, 1. For the confirmation of seven AAS residues in egg and on the basis of European Union EU guidelines, the concept of identification points IPs was applied for confirmatory purposes Confirmation of trace residues in complex matrices adopted in the EU guidelines also requires the determination of relative abundance criteria.
In the samples of egg under analysis, the relative intensities of the MRM transitions met those of the standards in solvent mobile phase. In all cases for the 1. That is, the ion ratios of the qualifier ion to the corresponding quantitation ion for each compound investigated were within the acceptability criteria set by EU guidelines.
The matrix-matched calibration standard curves for detection of the steroid residues were obtained by performing a linear regression analysis in the range of 1. Good linearity was obtained for each analyte. The correlation coefficients r for the calibration curves are higher than 0. The data of the pure solvent calibration standard curves are omitted. Recoveries for the AASs were estimated with the matrix-matched standard solutions.
At the three spiked levels of 1. The intra-day repeatability was between 2. The recovery rate and the precision of the developed method were acceptable for the residue analysis. Therefore, the prepared sample solutions should be injected within two days for good quantification.
The areas of the seven compounds in solvent free matrix were compared with those obtained from the corresponding blank extracts spiked with the same amount of the steroids after extraction The experiment showed that the area ratios matrix to solvent for seven AAS drugs were between 0.
High matrix suppression effects were observed. Therefore, for evaluation of the proposed method and analysis of real samples, the matrix-matched standard solutions are suitable and scientific.
The method developed was used to determine the seven AAS drug residues in over egg samples collected from local wholesale markets, farmer's markets and supermarkets. All of the studied compounds were less than 1.