미꾸라지에서의 Nitrofuran계 대사물질인3-amino-2-oxazolidone (AOZ), 5-morpholinomethyl-3-amino-2-oxazolidinone (AMOZ), 1-ammino-hydantoin (AHD)와 semicarbazide (SEM)의 잔류량을 검사하기 위해HPLC-MS/MS를 이용한 신속한 정량법이 개발되었다. 2-nitrobenzaldehyde (2-NBA)를 이용해 50℃에서 1시간 동안 산 가수분해와 유도체화 과정을 거친 뒤에, 액-액 분배로 정제와 추출을 하였다. 회수율은 음성시료에 3가지 농도 0.5, 1.0, 2.0 μg/kg의 표준액을 첨가하여 평가하였고 평균 회수율은 75.1-108.1% 이었다. 정밀성(%RSD)은 일내 8.7% 이하, 일간 8.5% 이하였다. 직선성은 NBAOZ는 0.2-20 μg/Kg, NBAMOZ는 0.8-20 μg/Kg, NBAHD는 0.2-20 μg/Kg, NBSEM 는 0.1-20 μg/Kg 범위에서 모두 상관계수 0.99이상이었다. 검출한계(LOD)는 NBAOZ 0.06 μg/Kg, NBAMOZ 0.24 μg/Kg, NBAHD 0.06 μg/Kg, NBSEM 0.03 μg/Kg이었고, 정량한계(LOQ)는 NBAOZ 0.2 μg/Kg, NBAMOZ 0.8 μg/Kg, NBAHD 0.2 μg/Kg, NBSEM 0.1 μg/Kg 이었다. 가수분해 및 유도체화 소요시간을 1시간으로 줄여 만든 신속 간편한 이 시험법이미꾸라지 중 nitrofuran metabolites잔류량 분석에 적합함을 확인할 수 있었다.

A rapid method using HPLC-MS/MS has been developed for quantitative determination of the metabolites of nitrofurans, namely 3-amino-2-oxazolidone (AOZ), 5-morpholinomethyl-3-amino-2-oxazolidinone (AMOZ), 1-ammino-hydantoin (AHD) and semicarbazide (SEM) in loach. The extraction procedure was founded on simultaneous acidic hydrolysis and derivatization using 2-nitrobenzaldehyde (2-NBA) for 1 hour at 50℃, followed by purification with liquid-liquid extraction. Recovery was evaluated by spiking standards into blank samples at three levels (0.5, 1.0 and 2.0 μg/kg), and the mean recovery was 75.1-108.1%. Precision values expressed as the relative standard deviation (%RSD) were ≤ 8.7% and ≤ 8.5% for intra-day and inter-day precision, respectively. Linearity was studied in the range of 0.2-20 μg/Kg for NBAOZ, 0.8-20 μg/Kg for NBAMOZ, 0.2-20 μg/Kg for NBAHD, and 0.1- 20 μg/Kg for NBSEM, and the obtained coefficient correlations (r) were ≥ 0.99 for all compounds. Limits of detection (LODs) for the derivatized nitrofuran metabolites were established at 0.06 μg/Kg for NBAOZ, 0.24 μg/Kg for NBAMOZ, 0.06 μg/Kg for NBAHD, and 0.03 μg/Kg for NBSEM. Limits of quantification (LOQs) were established at 0.2 μg/Kg for NBAOZ, 0.8 μg/Kg for NBAMOZ, 0.2 μg/Kg for NBAHD, and 0.1 μg/Kg for NBSEM. This simplified rapid method for reducing the derivatization and hydrolysis times can be applied to the determination of nitrofuran residues in loach.

Materials and Methods
  Reagents, Chemicals and Samples
  Stock and Working Standard Solution
  Extraction procedure
  Chromatographic Separation Parameters
  Instrumentation and Apparatus
  Method Validation
  Selectivity and Specificity
  Linearity
  Sensitivity (LOD, LOQ, CCα and CCβ)
  Precision, Accuracy and Recovery
 Results
  Extraction Optimization
  Optimization of LC-MS/MS Conditions
  Method Validation
  Selectivity and Specificity
  Linearity
  Sensitivity (LOD, LOQ, CCα and CCβ)
  Precision, Accuracy and Recovery
 Discussion
 Acknowledgemen
 국문 요약
 References

• Eun Chae Ryu(Gyeongin Regional Food & Drug Administration, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University)
• Yun-jeong Han(Gyeongin Regional Food & Drug Administration)
• Seong-soo Park(Gyeongin Regional Food & Drug Administration)
• Chul-joo Lim(Gyeongin Regional Food & Drug Administration)
• Sunok Choi(Gyeongin Regional Food & Drug Administration) Correspondence to
• Se Chang Park(College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University)