Stilbestrol is a synthetic non-steroidal estrogen substance, which can produce all pharmacological and therapeutic effects as the same natural estradiol does. It is mainly used for functional bleeding and amenorrhoea caused by estrogen low disease and hormone imbalance. It can also be used for stillbirth before induction of labor, in order to improve the muscular layer of uterine sensitivity to oxytocin. Pregnant women taking the drug, the female offspring in adolescence of cervical and vaginal adenosis and cancer incidence will rise and the male offspring reproductive tract abnormalities and abnormal sperm rate also will increase. Diethylstilbestrol can cause nausea, vomiting, loss of appetite, headache and other symptoms and long term using can make endometrial hyperplasia that may result in uterine bleeding and hypertrophy. The international standard provides stilbestrol residues detection limit is 1 μ g/kg in liver and muscle tissue in cattle and pigs.
Weighed 5.00 g (accurate to 0.01 g) homogenized sample, then put it into 50.0 mL centrifuge tube, and 15.0 mL t-butylmethyl ether was added, vortex for 1 min and oscillation for another 10 min. Centrifuged the solution for 5 min at 6000 r/min speed at -4 oC and transfered the supernate to the condensed bottle. Extracted the residue with another15.00 mL t-butyl methyl ether then combined the twice extracts. The solution was evaporated in the water bath at 45 oC. Then added 80 % acetonitrile solution 2 mL to the concentrated bottle, making the residue dissolved, and transferred it to the 10 mL centrifuge tube. The concentrated bottle was washed with 5 mL n-hexane and the n-hexane was transferred to the 10mL centrifuge tube. Then centrifuged the mixed solution for 10 min at 6000 r/min speed at 15 oC and the supernate was removed. The lower layer would be used finally.
Activate the Cleanert PEP-2 (500mg / 6mL) column using 3.0 mL t-butyl methyl ether, 3.0 mL methanol, 3.0 mL water in sequence. The lower layer solution flowed through the column at the speed of every 3~4 seconds per drop. Using 3.0 mL solution ( methanol:wate = 3:7,V:V) to wash the column and added another 3.0 mL water to balance the system. Next with methanol: 2 % ammonia solution (1:9, v/v) 3 mL flushing, discarding the lotion, and drained. Elute the sample using 1 % formic acid / methanol: methyl t-butyl ether (1:9, v/v) 7 mL into a pipe, then dried it in nitrogen. Then the residues were dissolved by 1 mL 80 % acetonitrile water solution. Filter the samples (0.22 μm) for detection.
Column: Unisol C18, 2.1 × 50 mm, 2.5 μm, 100 Å;
Mobile phase: A: water, B: acetonitrile;
Column Temperature: 35 °C;
Injection Volume: 5 μL;
Ion source: ESI
Scan mode: negative
Electrospray voltage: -5500 V
Atomizer pressure: 55 psi
Curtain Gas pressure: 12 psi
Aux Gas Pressure: 50 psi
Ion source temperature: 550 °C
Detection mode: MRM
Table 1 Gradient elution conditions of HPLC chromatography
Table 2 Mass spectrum parameters of Diethylstilbestrol
Results and Discussion
The standards of Diethylstilbestrol were added in samples at 2.5 μg/kg and 25.0 μg/kg followed by detection using SPE HPLC-MS/MS. As showed in table 3, the spiked recoveries were in the range of 80 %-96 %, and the RSDs in the range of 2.26 % - 8.95 % showed good reproducibility. As showed in fig.1 and fig.2, the peak shape of different drugs was satisfactory and its retention time was stable after purification and separation by Cleanert PEP-2 SPE column and Unisol C18 column.
Table 3 Recoveries and retention time for Diethylstilbestrol (n = 3)
Adding standard (μg/kg)
This study developed a LC-MS/MS method for the detection of Diethylstilbestrol. Combined with solid phase extraction, it also achieved quantification of Diethylstilbestrol residues in pork. With this method, 2.5μg/kg and 25 μg/kg spiked samples could be directly analyzed, and the spiked recoveries were in the range of 80%-90%. Solid phase extraction method showed good stability and the columns showed excellent reproducibility, which pointed out that this method could be used for the quantification of Diethylstilbestrol residues in animal derived food.