| The study included three parts. The part I established a method with isotope internal standard to detect Clenbuterol Hydrochloride (CL) in ovine body fluid (plasma and urine), tissues (liver, lung, kidney, heart, spleen, muscle and hair) using liquid chromatography tandem mass spectrometry. The part II studied the elimination and residue of CL in ovine body fluid (plasma, urine and saliva), tissues (eye, liver, lung, kidney, heart, spleen, muscle and hair). The part Ⅲ studied residual prediction model of CL between plasma and organs (liver, lung, kidney, heart, spleen and muscle) and inter-organ.Part I. The experiment used liquid chromatography tandem mass spectrometry to establish the method with isotope internal standard to detect CL in ovine body fluid (plasma and urine), tissues (liver, lung, kidney, heart, spleen, muscle and hair). All samples which added D9-CL internal standard, extracted, purified and applied matrix compensation to eliminate the effect of matrix, were detected by using LC-MS/MS. After optimizied, the CL linearity was0.05-200ng/mL in plasma,0.15-200ng/mL in urine,0.15～100ng/g in liver, heart, lung, kidney, spleen, muscle,0.5～200ng/g in hair and the correlation coefficient was between0.9941and0.9999. The limits of detection were0.01ng/mL for plasma,0.04ng/mL for urine,0.05ng/mL for tissues and0.17ng/mL for hair. The limits of quantification were0.03ng/mL for plasma,0.13ng/mL for urine,0.15ng/mL for tissues and0.5ng/mL for hair. The recovery rates were109.42%～112.34%for plasma,94.65%～104.37%for urine,95.29%～132.03%for tissues and100.71%～106.68%for hair. The relative standard deviation was lower than13.90%. The optimized LC-MS/MS could accurately detect CL residual concentrations of ovine body fluid (plasma and urine) and tissues (liver, lung, kidney, heart, spleen, muscle and hair) based on the above mentioned data analysis.Part Ⅱ.The experiment studied the elimination and residue of CL in ovine body fluid (plasma, urine, saliva and bile) and tissues (eye, liver, lung, kidney, heart, spleen, muscle and hair).24sheep weighted30±5kg were orally given CL at a dosage of15μg/kg once a day for21days successively and21days for withdrawal. Body fluid and tissue samples were collected at specific time after treatment and withdrawal. As the results showed, CL was rapidly absorbed in sheep and detected in plasma in1h after the first CL delivery. During the administration period, two apparent absorption peaks appeared in plasma at the7th day and20th day, which were6.35±3.28ng/mL and4.61±0.79ng/mL, respectively. The elimination of CL in plasma was biphasic, CL concentration in plasma decreased rapidly after8-24hours of withdrawal and its half-life was13.72h. In24～505hours of withdrawal, CL concentration in plasma decreased slowly, the half-life at this period was108.28h. Urine was the main approach to excrete CL, CL concentration in urine was significantly higher than plasma. CL in urine could be detected in2h after the first delivery and its concentration was80.19±60.33ng/ml. The elimination pattern of CL in urine was biphasic, its half-life was12.23h in0-3rd day of withdrawal and145.64h in3rd-21st day of withdrawal. CL concentration of saliva decreased in O-48h after withdrawal, but affected by several factors. The elimination regression equation and the elimination half-life of CL in saliva could not be determined. CL had different residual concentrations in different tissues (eye, liver, lung, kidney, heart, spleen and muscle). The highest concentration was detected in eyes. Besides, the elimination rate was also different in different tissues. It was much slower in liver, spleen, heart, whereas in lung, kidney, muscle, it eliminated more rapidly. Accumulation of CL in hair was larger and it eliminated slowly, it kept rising or constant after withdrawal. The18th day of withdrawal, the CL residual concentration in hair was9.29±6.34ng/g. The CL residual concentrations in retina and liver were51.24ng/g(without SD owing to the number of eyes) and6.46±1.72ng/g at21st day of withdrawal, respectively. So eye, liver and hair could be used as ideal target to detect the illegal CL using.Part Ⅲ. The expriment studied residual prediction model of CL (R2>0.9, P<0.01) and discussed the CL concentration relationships between plasma and organs (liver, lung, kidney, heart, spleen and muscle) and inter-organ. This model could predict CL residual concentrations of some tissues based on plasma and another tissues data, avoided the misjudgment relying on the plasma data. The inter-organ prediction model of CL could have a great importance of tracing food security. |
PDF Full Text Request