| Anabolic hormones were obtained by reformed the structure of natural testosterone, which the activity of sex hormone was reduced and the activity of protein assimilation was improved. Anabolic hormones were used in food-producing animals since the 50 s of last century due to their ability to decrease fatty accumulation, increase lean meat percentage and then improve the feed conversion. The abuse of anabolic hormones in food-producing animals causes their accumulation in animal tissues, which may be eaten by humans, thereby leading to health risks in humans. Therefore, the use of such hormonal growth promoters in food-producing animals has been banned in the European Union since 1988, and the 235 announcement of Chinese Ministry of Agriculture has claimed the use of 17α-methyltestosterone and trenbolone was prohibited in all animals and the residues of 17α-methyltestosterone and trenbolone must not be found in all edible tissues.A rapid and sensitive liquid chromatography-mass spectrometry method for the simultaneous determination of eight anabolic hormones(testosterone, 17α-methyltestosterone, trenbolone, trenbolone-acetate, boldenone, nandrolone phenylpropionate, nandrolone and norethindrone) in pig hair samples was described. The digestion solvents and time, type of extraction solvent, shaking time on the rocker, and other pretreatment methods were all optimized in this study. The difference of accumulation of 17α-methyltestosterone between different approaches in pig hairs was observed.This method involves subjecting 100 mg of pulverized hair to a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride, subsequent extraction of the anabolic hormones with methyl tert-butyl ether, purification with C18 solid phase extraction column and redissolved with the mobile phase at last. The separation of the target analytes was performed on an Agilent Zorbax SB-Aq column(150 ×2.1 mm i.d., 3.5 μm) with acetonitrile and 0.1% aqueous formic acid. Quantitative analysis of eight anabolic hormones was performed in positive electrospray ionization and multiple reaction monitoring mode in this study.The gradient elution program was as follows: initial time, 30% B; 1.0–3.0 min, 55% B; 6–12 min, 98% B; 12.5–18 min, 30% B. The injection volume was 5 μL. The limits of detection of eight anabolic hormones ranged from 0.3 ng/g to 3 ng/g and the limits of quantification ranged from 0.75 ng/g to 6 ng/g. The correlation coefficients for all of the compounds were greater than 0.99 over a dynamic range of 10–1000 ng/g. The mean recoveries of target compounds obtained for low, middle, high spiking levels range from 75.2% to 112.7%. The intra and inter-day relative standard deviations ranged from 1.4% to 8.2% and 2.47% to 8.58%, respectively. The results indicate a higher sensitive and robust method was obtained in this study, which meet requirement of multi-residue determination in pig hairs.The difference of accumulation of 17α-methyltestosterone between different approaches was studied. Six pigs(20.0 ± 3.5 kg) were randomly assigned to two groups, three pigs were fed with a mixture containing feed and 17α-methyltestosterone(4 mg/kg BW per day) for thirty consecutive days, another three pigs were given a single intramuscular injection with 17α-methyltestosterone. The pig hairs were collected over a period of 61 days. The hair samples were conducted and then analyzed by liquid chromatography tandem mass spectrometer system. The accumulation was analyzed by time as X-axis and concentration of 17α-methyltestosterone in pig hair as Y-axis. The results showed that the peak of concentration appeared on day 10 and following a period of decline in the multiple dose oral 17α-methyltestosterone group, and then a leveling-off was observed on about day 16. The peak of concentration appeared on day 10 and then rapidly declined in the injection 17α-methyltestosterone group. And 17α-methyltestosterone was still detected on day 61 in two groups, the significant diversity of the accumulation between different method of administration was observed.The significance of residue of 17α-methyltestosterone in pig hair was also investigated in this study. The results indicated that 17α-methyltestosterone was detected during the treatment(30 days) and withdraw period(31 days) in oral administration group, and also be found within 61 days after single dose administration of 17α-methyltestosterone in pig hair. Compared with using serum, urine, animal tissues as the matrixes, which have a shorter detection time, a wide surveillance window can be obtained by using hair as the matrix. The time of misuse of 17α-methyltestosterone can be inferred by analyzing the accumulation of 17α-methyltestosterone in pig hair. And this proposed method provided more powerful evidence to control the illicit use of anabolic hormones in pig. |
