피페라진은 회충, 요충, 사상충, 십이지장충, 맹장충, 장내 기생충 등 기생충 구충에 사용되는데, 적절한 투여량 및 휴약기간에 대한 자료가 부족하다. 따라서 본 연구의 목적은 육계 조직(근육, 간, 신장, 지방)에서 피페라진의 잔류 소실 데이터를 확인하고 휴약기간을 추정하는 것이다. 신뢰성을 확보하기 위한 LC-MS/MS의 방법은 파라미터(직선성, 정확성, 정밀성, 회수율, 검출한계, 정량한계 등)을 이용하여 검증하였다. 4주령의 육계를 사용하였다. 제품 A(18.7 g/kg)를 사료에 혼합하고 275 mg/bird (Fx)의 단일 용량으로 경구 투여하였다. 제품B(1.1 mL/5 mL)를 물에 희석하고 위관 영양법을 통해 275 mg/kg의 단일용량으로 경구 투여하였다. 육계 조직(근육, 간, 신장, 지방)은 투여 종류 후 6시간, 1일, 3일, 5일, 7일에 채취하여 조직들을 전처리 후 LC-MS/MS로 분석하였다.
그 결과, 제품A의 함량은 공칭농도 대비 평균 516.6±10.8 g/kg, 제품B의 함량은 공칭농도 242.5±2.7 g/kg으로 나타났다. Fx와 Wx의 함량은 평균 10.3±1.8 mg/mL 및 62.4±5.3 mg/mL로 검출되었으며, 공칭 농도와 비교하여 110.3±1.8% 및 113.5±5.3%였다. 제품 A와 제품 B의 조제에는 이상이 없음을 확인하였으며, 제품 A와 B를 피페라진의 잔류 소실 데이터를 확인하고 휴약기간을 추정하기 위해 성별에 관계없이 무작위로 Fx 30마리, Wx 30마리의 육계에 Fx, Wx를 투여하였다.
육계 조직 내 잔류된 피페라진을 정량화 하기 위해 샘플을 전처리 후 LC-MS/MS로 분석하였다. 근육의 일내 정확성은 95.5-106.4%, 정밀성 6.3-10.2%, 그리고 일간 정확성 87.4-100.0%, 정밀성 2.4-8.0%, 회수율 68.3-72.6%, 결정계수(R2) 0.9801, 검출한계 10 μg/kg, 정량한계 25 μg/kg. 간의 일내 정확성은 91.6-107.5%, 정밀성 4.6-11.7%, 일간 정확성 97.3-103.2%, 정밀성 3.7-8.4%, 회수율 77.6-88.9%, 결정계수(R2) 0.9927, 검출한계 10 μg/kg, 정량한계 25 μg/kg. 신장의 일내 정확성은 89.7-99.1%, 정밀성 8.6-11.0%, 일간 정확성 94.5-104.0%, 정밀성 3.4-9.9%, 회수율 83.5-101.3%, 결정계수(R2) 0.9866, 검출한계 10 μg/kg, 정량한계 25 μg/kg. 지방의 일내 정확성은 94.3-98.3%, 정밀성 4.2-11.1%, 일간 정확성 87.6-97.2%, 정밀성 2.5-10.0%, 회수율 60.6-68.3%, 결정계수(R2) 0.9866, 검출한계 10 μg/kg, 정량한계 25 μg/kg이다.
휴약기간은 투여 종료 후 6시간,1,3,5,7일의 분석 값을 이용하여 선형회귀곡선을 수행하고 95% 신뢰구간 중 상위 99% 값을 적용하여 설정하였다. 제품 A, 제품 B의 Fx, Wx에서 데이터를 도출한 결과, 조직 중에서는 투여 종료 6시간 후 잔류 농도가 가장 높았으며, 조직 별 잔류량은 신장, 간, 근육, 지방 순으로 신장이 가장 높았다. 사료첨가군에서는 근육, 간, 신장에서는 투여 종료 후 1일까지 잔류되었고, 3일째에는 정량한계 이하로 소실되었다. 따라서 휴약기간은 투여 종료 후 3일로 설정하는 것이 합리적이라고 판단되었다. 지방의 경우 사료첨가군은 투여 종료 후 3일 후 검출되었으며 휴약기간의 판단이 가능하였다. 그 결과로 지방이 잔류허용기준 이하로 떨어진 날은 약4.7일이었다. 음수첨가군에서는 근육과 신장에서 투여종료 후 1일까지 잔류되었고, 3일째에는 정량한계 이하로 소실되었다. 따라서 휴약기간은 투여 종료 후 3일로 설정하는 것이 합리적이라고 판단되었다. 지방과 간의 경우 투여종료 3일 후 검출되었으며 휴약기간의 판단이 가능하였다. 그결과 지방에서 잔류허용기준 이하로 떨어진 날은 약 4.8일, 간에서 잔류허용기준 이하로 떨어진 날은 약 3.4일로 휴약기간의 판단이 가능하였다.
결론적으로 육계의 각 조직에 대한 피페라진의 휴약기간 (사료첨가군)은 근육 3일, 간 3일, 신장 3일, 지방 5일로 결정하였고, 음수첨가군은 근육 3일, 간 4일, 신장 3일, 지방 5일로 결정하였다.

The Residue Study for Setting Withdrawal Period of Piperazine in the Broiler Tissues using Liquid Chromatography-Tandem Mass Spectrometry Lee Hong-Hee Department of Veterinary Medicine Graduate School of Konkuk University Piperazine is used to deworm parasites such as roundworms, pinworms, filarial worms, hookworms, cecum worms, and intestinal worms, there was a lack of data on the appropriate dosage and withdrawal period. Therefore, the objectives of this study were to determine the residue depletion data of Piperazine in broiler tissues (muscle, liver, kidney, and Fat) and estimate a withdrawal interval. To ensure the reliability of the reported results, the LC- MS/MS method was validated using the following performance parameters (Linearity, Accuracy, Precision, Recovery, LOD, LOQ). 4-week-old broiler chickens were used in the experiment. Product A (18.7 g/kg) was mixed into the feed and administered orally at a single dose of 275 mg/bird (Fx). Product B (1.1 mL/5 mL) was diluted in water and administered orally at a single dose of 275 mg/kg via gavage. The broiler tissues (muscles, liver, kidneys, and fat) were collected on the 6 hours, 1st, 3rd, 5th and 7th days after the end of the administration, and tissues were prepared and analyzed by LC-MS/MS. The content of product A was found to be 516.6±10.8 g/kg on average and product B was found to be 242.5±2.7 g/kg on average compared to the nominal concentration. The contents of the Fx and Wx were detected as 10.3±1.8 g/kg and 62.4±5.3 mg/mL on average and compared to the nominal concentration, 110.3±1.8%, and 113.5±5.3%. It was confirmed that there was no abnormality in the preparation of product A, product B, Fx, and Wx were administered. To quantify the Piperazine residue in broiler tissues, samples were prepared and analyzed by LC-MS/MS. The intra-day accuracy of the muscle was 90.5- 106.4%, and precision was 6.3-10.2%, and inter-day accuracy was 87.4- 100.0%, and precision was 2.4-8.0%, and recovery was 68.3-72.6%, and coefficient of determination ( ) was 0.9801, and limit of detection (LOD) was 10 μg/kg, and limit of quantification was 25 μg/kg. The intra-day accuracy of the liver was 91.6-107.5%, and precision was 4.6-11.7%, and inter-day accuracy was 97.3-103.2%, and precision was 3.7-8.4%, and recovery was 77.6-88.9%, and coefficient of determination ( ) was 0.9927, and limit of detection (LOD) was 10 μg/kg, and limit of quantification was 25 μg/kg. The intra-day accuracy of the kidney was 89.7-99.1%, and precision was 8.6- 11.0%, and inter-day accuracy was 94.5-104.0%, and precision was 3.4-9.9%, and recovery was 83.5-101.3%, and coefficient of determination ( ) was 0.9897, and limit of detection (LOD) was 10 μg/kg, and limit of quantification was 25 μg/kg. The intra-day accuracy of fat was 94.3-98.3%, and precision was 4.2-11.1%, and inter-day accuracy was 87.6-97.2%, and precision was 2.5-10.0%, and recovery was 60.6-68.3%, and coefficient of determination ( ) was 0.9866, and limit of detection (LOD) was 10 μg/kg, and limit of quantification was 25 μg/kg. The withdrawal period was set by performing linear regression fit using the analysis values of 6 hours, 1st, 3rd, 5th, and 7th days after the end of the administration and applying the top 99% value of the 95% confidence interval. As a result of deriving data from Fx, and Wx of product A, product B, Among the tissues, the highest residual concentration was observed on 6 hours after the end of the administration, for the residual amount by tissues, the kidney was the highest in the order of kidney, liver, muscle, and fat. In the Fx group, residues remained in the muscles, liver, and kidneys until 1 day after the end of administration and decreased below the LOQ on the 3rd day. Therefore, it is judged appropriate to set the medication withdrawal period to 3 days after the end of administration. In the case of fat, it was detected 3 days after the end of administration, and a withdrawal period was established. As a result, the number of days when fat fell below the MRL standard was approximately 4.7 days. In the Wx group, residues remained in the muscles and kidneys until 1 day after the end of administration and decreased below the LOQ on the 3rd day. Therefore, it is judged appropriate to set the medication withdrawal period to 3 days after the end of administration. In the case of fat and liver, it was detected 3 days after the end of administration, and a withdrawal period was established. As a result, the days when fat fell below the MRL standard were about 4.8 days, and the days when liver fell below the MRL standard were about 3.4 days. Finally, the Fx groups of the withdrawal period of Piperazine for each tissue of the broiler were set to 3 days for muscle, 3 days for liver, 3 days for kidney, and 5 days for fat, and Wx group set to 3 days for muscle, 4 days for liver, and 3 days for kidney, and 5 days for fat. Keyword: Piperazine; Withdrawal period; MRL; Accuracy; Precision; Recovery; LC-MS/MS