GC-ECD를 이용한 한약재 길경(Platycodi Radix) 중 살균제 Prochloraz의 분석

오경석 ( Gyeong-seok Oh ),윤명섭 ( Myung-sub Yoon ),양승현 ( Seung-hyun Yang ),최훈 ( Hoon Choi ) 한국환경농학회 2021 한국환경농학회지 Vol.40 No.4

본 연구는 한약재 길경 중 Imidazole계 살균제 prochloraz 및 그 대사체 2,4,6-T의 잔류분석법을 확립하였다. 한약재 중 길경을 대표 시료로 선정하고 GC-ECD를 이용한 prochloraz 정량 시험법을 개발하였다. 한약재 길경 중 prochloraz 잔류물을 acetone로 추출하고, dichloromethane로 분배하고 pyridine hydoxyde를 이용하여 분해한 뒤, 이온억압 분배과정 후, NH₂ cartridge로 정제하였다. 한약재 길경 중 prochloraz의 경우 정량한계는 0.04 mg/kg으로 결정되었으며, MLOQ 수준의 회수율은 89.7%, MLOQ 10배 수준에서는 82.5%의 우수한 회수율을 보였으며, 분석오차는 최대 2.8%로 재현성 역시 양호하였다. 2,4,6-T의 경우 정량한계는 0.02 mg/kg으로 결정되었으며, MLOQ 수준의 회수율은 83.0%, MLOQ 10배 수준에서는 82.1%의 우수한 회수율을 보였으며, 분석오차는 최대 2.8%로 재현성 역시 양호하였다. 본 연구에서 확립한 prochloraz 및 그 대사체 2,4,6-T의 잔류분석법은 국내·외 한약재의 잔류농약 검사 및 분석에 적용 가능할 것으로 기대된다. BACKGROUND: Prochloraz has been widely used as an imidazole fungicide on fruits and vegetables in Korea. Analytical approaches to evaluate prochloraz residues in herbal medicine are required for their safety management. In this study, we developed a GC-ECD method for quantitative determination of prochloraz in Platycodi Radix. The metabolite 2,4,6-trichlorophenol (2,4,6-T) was used as a target compound to evaluate total prochloraz residues as it is categorized to a representative residue definition of prochloraz. All residues containing 2,4,6-T were converted to 2,4,6-T and subjected to GC-ECD. METHODS AND RESULTS: In order to verify the applicability, the method was optimized for determining prochloraz and it metabolite 2,4,6-T in Platycodi Radix. Prochloraz and its metabolite 2,4,6-T residuals were extracted using acetone. The extract was diluted with and partitioned directly into dichloromethane to remove polar co-extractives in the aqueous phase. The extract was decomposed to 2,4,6-T, and then the partitioned ion-associate was finally purified by optimized aminopropyl solid-phase extraction (SPE). The limits of quantitation of the method (MLOQs) were 0.04 mg/kg and 0.02 mg/kg, respectively for prochloraz and 2,4,6-T, considering the maximum residue level (MRL) of prochloraz as 0.05 mg/kg in Platycodi Radix. Recovery tests were carried out at two levels of concentration (MLOQ, 10 MLOQ) and resulted in good recoveries (82.1-89.7%). Good reproducibilities were obtained (coefficient of variation < 2.8%), and the linearities of calibration curves were reasonable (r² > 0.9986) in the range of 0.005-0.5 μg/mL. CONCLUSION(S): The method developed in this study was successfully validated to meet the guidelines required for quantitative determination of pesticides in herbal medicine. Thus, the method could be useful to monitor prochloraz institutionally in herbal medicine.

가수분해 후 GC-ECD를 이용한 축산물 중 살균제 Prochloraz 및 그의 대사물의 동시분석

박지수 ( Ji-su Park ),최훈 ( Hoon Choi ) 한국응용생명화학회(구 한국농화학회) 2020 Journal of Applied Biological Chemistry (J. Appl. Vol.63 No.2

축산물 중 살균제 prochloraz 및 그의 대사체의 잔류량을 함께 정량분석할 수 있는 단성분 분석법을 개발하고자 하였다. 축산물 중 prochloraz의 잔류분의 정의가 ‘prochloraz와 2,4,6-trichlorophenol기를 포함하고 있는 대사산물의 합을 prochloraz로 함’이기 때문에 prochloraz 및 대사산물을 모두 2,4,6-trichlorophenol (2,4,6-TCP)로 분해시킨 후 GC/ECD로 정량하는 정밀 분석법을 확립하였다. 대표 축산물인 소고기, 돼지고기, 닭고기, 우유 및 계란 중 prochloraz 잔류분을 acetone으로 추출하고 2,4,6-TCP로 분해한 후 dichloromethane으로 분배하고 aminopropyl SPE로 정제한 다음 2,4,6-TCP를 정량하였다. 본 분석법을 통한 기기정량한계와 분석법정량한계는 prochloraz의 경우 각각 0.01 μg/mL과 0.02 mg/kg이었으며 2,4,6-TCP는 0.005 μg/mL과 0.01 mg/kg이었다. 2,4,6-TCP를 이용한 표준검량선은 0.005-0.2 mg/L 범위에서 결정계수(R²) ≥0.995으로 직선성을 확인하였다. 분석법의 회수율은 분석정량한계 및 정량한계의 10배 처리수준에서 실시하여 모두 90% 이상이었고 변이계수는 10% 이하이었다. 따라서, 본 연구에서 확립된 분석법은 축산물 중 prochloraz 및 대사체를 정량분석하는데 우수한 정확성 및 정밀도를 보였으며, 축산물 중 prochloraz 잔류분석을 위한 정밀시험법으로써 분석개발기준을 충족하였다. The analytical method was established for simultaneous determination of fungicide prochloraz and its metabolites in several animal commodities using gas chromatography (GC) coupled with electron capture detector (ECD). Samples including beef meat, pork meat, chicken meat, milk, and egg were hydrolyzed with pyridine hydrochloride which converts prochloraz and its metabolites to 2,4,6-trichlorophenol (2,4,6-TCP) because residue definition for prochloraz was ‘sum of prochloraz and its metabolites containing the 2,4,6-trichlorophenol moiety, expressed as prochloraz’, for compliance with MRLs from animal commodities. Therefore, residual prochloraz was extracted with acetone, decomposed to 2,4,6-TCP, partitioned with dichloromethane, purified with aminopropyl SPE and quantified as 2,4,6-TCP with GC-ECD. The instrumental limit of quantitation and method LOQ (MLOQ) was 0.01 μg/mL and 0.02 mg/kg for prochloraz and 0.005 μg/mL and 0.01 mg/kg for 2,4,6-TCP, respectively. The linearity of the calibration curve was good with R²>0.995 in the range of 0.005- 0.2 μg/mL. Fortification levels of prochloraz were 0.02 mg/kg (MLOQ) and 0.2 mg/kg (10MLOQ) for recovery tests. Overall recoveries of prochloraz were >90% with <10% of coefficient variation (C.V.). This established analytical method was fully validated and could be useful for quantification of prochloraz and its metabolites in animal commodities as official analytical method.

생약 길경(Platycodon Grandiflorum) 중 살균제 Prochloraz 분석법 개발

오경석 ( Gyeong-seok Oh ),양승현 ( Seung-hyun Yang ),윤명섭 ( Myung-sub Yun ),강동현 ( Dong-hyun Kang ),김민 ( Min Kim ),김재영 ( Jae-young Kim ),남근우 ( Gnu Nam ),최훈 ( Hoon Choi ) 한국환경농학회 2021 한국환경농학회 학술대회집 Vol.2021 No.-

Prochloraz 단성분 분석법은 이미다졸계 살균제인 prochloraz 및 그의 2,4,6-trichlorophenol moiety를 함유하는 대사산물을 분석대상으로 하였다. 대상 농약들은 환경이나 식물체내에서 구조가 변환되어 일반적으로 3개의 독성 대사물을 생산하는데, N-formyl-N-propyl-N-[2-(2,4,6- trichlorophenoxy) ethyl]urea와 N-propyl-N-[2-(2,4,6- trichlorophenoxy)ethyl]urea로 전환되고 이것은 다시 2,4,6-trichlorophenol로 분해된다. 현재 농산물 중 prochloraz의 잔류분 정의는 prochloraz와 2,4,6-trichlorphenol 기를 포함하고 있는 대사산물의 합을 prochloraz로 함으로써, 본 분석법의 모든 분석대상 화합물을 2,4,6-trichlorophenol으로 분해한 후 2,4,6-trichlorophenol로써 정량한 후 모화합물인 prochloraz로써 정량하도록 하였다. 해당 농약성분의 잔류시험법 확립을 위한 생약으로는 길경(Platycodon grandiflorum)을 사용하였다. 분석법의 경우 검체 5g에 습윤화 과정 이후 acetone을 첨가하여 분석 대상을 추출하였고, 추출액은 농축과정 없이 과량의 물과 혼합한 후 dichloromethane로 분배 회수한 후 prochloraz 대사체들을 분해하기 위하여 pyridine hydrochloride를 첨가하여 고온에서 분해하였다. 분해액을 이온억압분배법에 따라 분배하고 NH2 cartridge로 정제한 다음 GC/ECD로 분석하였다. Prochloraz 분석법 정량한계는 0.05 mg/kg을 목표로 설정하였으며, 이를 위해 prochloraz의 분해산물인 2,4,6-trichlorophenol의 ILOQ(Instrumental Limit of Quantitation, 기기상의 정량한계)는 S/N≥10을 만족하는 0.01 μg/mL이었으며 희석배수 2를 고려하여 분석법 정량한계(Method Limit of Quantification, MLOQ)는 2,4,6-trichlorophenol 0.02 mg/kg이다. 2,4,6-trichlorophenol의 ILOQ와 MLOQ를 prochloraz로써 환산할 경우 prochloraz의 ILOQ는 0.02 mg/kg이고 MLOQ는 0.04 mg/kg으로 분석법 개발기준을 만족하였다.

Degradation of Prochloraz by Rice Bakanae Disease Pathogen Fusarium fujikuroi with Differing Sensitivity a Possible Explanation for Resistance Mechanism

( Seon Hwa Kim ),( Myung Ryeol Park ),( Young Cheol Kim ),( Se Weon Lee ),( Byung Ryeol Choi ),( Si Woo Lee ),( In Seon Kim ) 한국응용생명화학회 2010 Applied Biological Chemistry (Appl Biol Chem) Vol.53 No.4

The fungicide prochloraz was subjected to degradation by the pathogen causing rice Bakanae disease, Fusarium fujikuroi, in order to gain an insight into the mechanisms of sensitivity and resistance to the fungicide. Growth-inhibiting assays of pathogens conducted on potato dextrose agar (PDA) plates by a paper-disc agar-diffusion method. Significant growth inhibition of the sensitive strain CF106 was observed at the recommended treatment level of prochloraz, whereas negligible growth inhibition of the resistant strain CF245 was observed at the same treatment level. The strain CF245 was shown to be able to grow on PDA with 500 mg/L of the fungicide, which is significantly higher than its recommended treatment level. Growth-inhibiting assays of pathogens were also conducted in potato dextrose broth (PDB) medium supplemented with prochloraz at different concentrations, measuring their biomass weights over the incubation period. Significant growth inhibition was observed in the strain CF106 at a level of 0.5 mg/L, but negligible growth inhibition was observed in the strain CF245 at the same treatment level with the strain CF106. The strain CF245 could grow in PDB supplemented with 1.0 mg/L of prochloraz. The degradation of prochloraz by the two strains was evaluated by gas liquid chromatography and mass spectrometry analyses. The strain CF245 completely degraded 1.0 mg/L of prochloraz in 5 days after incubation, whereas no degradation of prochloraz was observed by the strain CF106 at the same treatment level. Liquid chromatography Q-TOF MS detected N-(2-(2,4,6-trichlorophenoxy)ethyl)propan-1-amine as a major degradation product of prochloraz by the strain CF245. These results indicated that the degradation of prochloraz may account for the reduced sensitivity of the strain CF245 to prochloraz.

Degradation of Prochloraz by Rice Bakanae Disease Pathogen Fusarium fujikuroi with Differing Sensitivity: a Possible Explanation for Resistance Mechanism

Kim, Seon-Hwa,Park, Myung-Ryeol,Kim, Young-Cheol,Lee, Se-Weon,Choi, Byung-Ryeol,Lee, Si-Woo,Kim, In-Seon The Korean Society for Applied Biological Chemistr 2010 Applied Biological Chemistry (Appl Biol Chem) Vol.53 No.4

The fungicide prochloraz was subjected to degradation by the pathogen causing rice Bakanae disease, Fusarium fujikuroi, in order to gain an insight into the mechanisms of sensitivity and resistance to the fungicide. Growth-inhibiting assays of pathogens conducted on potato dextrose agar (PDA) plates by a paper-disc agar-diffusion method. Significant growth inhibition of the sensitive strain CF106 was observed at the recommended treatment level of prochloraz, whereas negligible growth inhibition of the resistant strain CF245 was observed at the same treatment level. The strain CF245 was shown to be able to grow on PDA with 500 mg/L of the fungicide, which is significantly higher than its recommended treatment level. Growth-inhibiting assays of pathogens were also conducted in potato dextrose broth (PDB) medium supplemented with prochloraz at different concentrations, measuring their biomass weights over the incubation period. Significant growth inhibition was observed in the strain CF106 at a level of 0.5 mg/L, but negligible growth inhibition was observed in the strain CF245 at the same treatment level with the strain CF106. The strain CF245 could grow in PDB supplemented with 1.0 mg/L of prochloraz. The degradation of prochloraz by the two strains was evaluated by gas liquid chromatography and mass spectrometry analyses. The strain CF245 completely degraded 1.0 mg/L of prochloraz in 5 days after incubation, whereas no degradation of prochloraz was observed by the strain CF106 at the same treatment level. Liquid chromatography Q-TOF MS detected N-(2-(2,4,6-trichlorophenoxy)ethyl)propan-1-amine as a major degradation product of prochloraz by the strain CF245. These results indicated that the degradation of prochloraz may account for the reduced sensitivity of the strain CF245 to prochloraz.

Prochloraz와 fludioxonil 혼용침지소독에 의한 벼 키다리병 방제

박우식,최효원,한성숙,신동범,심형권,정은선,이세원,임춘근,이용환 한국식물병리학회 2009 식물병연구 Vol.15 No.2

These experiments were conducted to improve the effect of seed disinfection on rice seed severely infected Bakanae disease by seed soaking into mixed solution of prochloraz EC and fludioxonil FS. We investigated the effects of various concentrations of two fungicides mixed solution on spore germination and mycelial growth of Fusarium fujikuroi. Mycelial growth was inhibited 100% at 10 μg/ml of prochloraz and 33.3% at 80 μg/ml of fludioxonil. Spore germination was inhibited 81.4% at 40 μg/ml of prochloraz. Interestingly, mixed solution of 5 μg/ml or 10 μg/ml of each fungicide inhibitied 100% of mycelial growoth and 99.2% of spore germination, respectively. Severely infected rice seeds soaked into mixed solution composed of 125 μl/ml of prochloraz and 50 μl/ml of fludioxonil showed 2.1% of disease symptoms compared to 20.4% of prochloraz 125 μl/ml, but higher concentrations of prochloraz decreased the seedling stand rate. When the seed soaking time was longer and temperature was higher, control effect on Bakanae disease was improved, but seedling stand was lower about 80% over 35℃. These experiments were conducted to improve the effect of seed disinfection on rice seed severely infected Bakanae disease by seed soaking into mixed solution of prochloraz EC and fludioxonil FS. We investigated the effects of various concentrations of two fungicides mixed solution on spore germination and mycelial growth of Fusarium fujikuroi. Mycelial growth was inhibited 100% at 10 μg/ml of prochloraz and 33.3% at 80 μg/ml of fludioxonil. Spore germination was inhibited 81.4% at 40 μg/ml of prochloraz. Interestingly, mixed solution of 5 μg/ml or 10 μg/ml of each fungicide inhibitied 100% of mycelial growoth and 99.2% of spore germination, respectively. Severely infected rice seeds soaked into mixed solution composed of 125 μl/ml of prochloraz and 50 μl/ml of fludioxonil showed 2.1% of disease symptoms compared to 20.4% of prochloraz 125 μl/ml, but higher concentrations of prochloraz decreased the seedling stand rate. When the seed soaking time was longer and temperature was higher, control effect on Bakanae disease was improved, but seedling stand was lower about 80% over 35℃.

세포벽의 형태학적 변화와 ABC Transporter에 기초한 벼키다리병원균 Fusarium fujikuroi CF337의 살균제 prochloraz에 대한 저항성 반응

양유리 ( You Ri Yang ),이시우 ( Si Woo Lee ),이세원 ( Se Won Lee ),김인선 ( In Seon Kim ) 한국환경농학회 2012 한국환경농학회지 Vol.31 No.1

BACKGROUND: The resistance of rice bakanae disease pathogens against the fungicide prochloraz has been reported. Understanding the resistance mechanisms is an important for better control of the pathogens. In the present study, we investigated the resistance mechanisms of Fusarium fujikuroi CF337 (CF337) against prochloraz. METHODS AND RESULTS: Morphological changes in the cell wall of CF337 grown in potato dextrose broth (PDB) with or without prochloraz was investigated by transmission electron microscopy. Growth inhibition of CF337 was examined in PDB containing prochloraz or an ABC transporter inhibitor or both of them. Cell wall thickness of CF337 grown in PDB with prochloraz was significantly increased from 80.73±1.99 nm to 193.11±7.07 nm. Significant inhibition in the growth of CF337 was observed in the presence of both prochloraz and the inhibitor, but no growth inhibition was observed in the presence of the inhibitor or prochloraz. Sequence analysis of ATP-binding cassette transporter (ABC) gene of CF337 showed 70 to 80% similarities to the genes of the pathogens resistant to other fungicides. CONCLUSION: Efflux transporter system and changes in cell wall thickness were suggested as resistance mechanisms of CF337 against prochloraz.

국내 벼키다리병균의 Prochloraz 약제에 대한 저항성 변화

최효원,이용환,홍성기,이영기,남영주,이재금,한송희,Choi, Hyo-Won,Lee, Yong Hwan,Hong, Sung Kee,Lee, Young Kee,Nam, Young Ju,Lee, Jae Guem,Han, Song Hee 한국균학회 2015 韓國菌學會誌 Vol.43 No.2

국내에 분포하는 벼키다리병균의 prochloraz에 대한 저항성 정도를 조사하기 위하여 연도별로 균주를 수집하고, 병원성을 검정하였으며, 한천희석법을 사용하여 각 균주의 prochloraz에 대한 minimum inhibitory concentration (MIC) 값과 effective concentration of 50% ($EC_{50}$) 값을 조사하였다. 그 결과 2006년부터 2007년 분리균의 MIC 값은 $3.125{\sim}6.25{\mu}g/mL$인 균주가 가장 높은 빈도로 분포하였고, 2013년부터 2014년 분리균의 MIC 값은 $6.25{\sim}12.5{\mu}g/mL$의 농도에서 분포 비율이 가장 높았다. 또한 2006년부터 2007년 분리 균주의 평균 $EC_{50}$값은 $0.3142{\mu}g/mL$이었으며, 2013년부터 2014년 분리 균주는 $0.8124{\mu}g/mL$이었다. 2006년부터 2007년 분리 균주의 prochloraz에 대한 저항성 기준 $EC_{50}$값은 $0.6{\mu}g/mL$로 하는 것이 타당한 것으로 생각된다. 이 기준에 의한 저항성 균주의 비율은 2006년부터 2007년 분리 균주는 약 6.5%였고, 2013년부터 2014년 분리 균주에서는 41.6%로 저항성 균주의 빈도가 크게 증가한 것으로 나타났다. To investigate the changes of the resistance to prochloraz of Fusarium species causing bakanae disease, Fusarium isolates were collected from various regions in Korea, and pathogenicity tests were performed using rice seeds. Minimum inhibitory concentration (MIC) and effective concentration of 50% ($EC_{50}$) values of isolates were determined using the agar dilution method. High frequency distribution of MIC values of prochloraz against isolates collected in 2006~2007 and 2013~2014 years were $3.125{\sim}6.25{\mu}g/mL$ and $6.25{\sim}12.5{\mu}g/mL$, respectively. The mean $EC_{50}$ value of isolates increased from $0.3142{\mu}g/mL$ in 2006~2007 to $0.8124{\mu}g/mL$ in 2013~2014. Based on the $EC_{50}$ value of isolates collected in 2006~2007, the resistant baseline of prochloraz was determined as $0.6{\mu}g/mL$. Compared with the ratio of resistant isolates in 2006~2007, the ratio of resistant isolates in 2013~2014 increased from 6.5% to 41.6%.

GC-ECD를 이용한 배추, 사과, 감귤, 고추, 현미 중 살균제 Prochloraz의 분석법 확립

이은미 ( Eun Mi Lee ),이혜리 ( Hye Ri Lee ),류명주 ( Myoung Joo Riu ),박희원 ( Hee Won Park ),나예림 ( Ye Rim Na ),송혁환 ( Hyuk Hwan Song ),금영수 ( Young Soo Keum ),김정한 ( Jeong Han Kim ),Young Zhe Zhu 韓國環境農學會 2009 한국환경농학회지 Vol.28 No.4

Prochloraz를 2,4,6-Trichlorophenol (2,4,6-TCP)로 전환시켜 분석하는 방법을 확립하였으며, 배추, 사과, 고추. 감귤, 현미를 대사 농작물로 선정하였다. 농산물 시료를 마쇄, acetonitrile로 추출하여 dichloromethane으로 분배하고 농축액에 pyridine hydrochloride를 넣고 1시간 가열 분해를 한다. 반응액을 다시 dichloromethane으로 분배한 후 GC-ECD로 분석하였다. Prochloraz의 정량한계는 0.01 mg/kg 이었다. 회수율은 0.1 mg/kg 수준에서 105-113%, 0.5 mg/kg 수준에서 82-87% 이었고, 반복 간 분석오차는 10% 미만이었다. 이러한 분석법 개선 과정에서 기존의 soxhlet 추출과 환류장치를 쓰지 않고 용매추출법과 작은 vial을 성공적으로 적용하였고, 간편, 신속, 경제적이며 정밀한 분석법을 확립하였다. Analytical method for prochloraz in cabbage, apple, pepper, mandarin, and hulled rice was established by conversion it to 2,4,6-trichlorophenol (TCP). Crop samples were extracted with acetonitrile, and partitioned with dichloromethane. The sample extracts were hydrolyzed with pyridine hydrochloride in a vial by heating for 1 hour, and analyzed with GC-ECD after partitioning with dichloromethane. Method quantification limit (MQL) of prochloraz was 0.01 mg/kg. Recoveries at 0.1 mg/kg level was 105-113% while at 0.5 mg/kg level was 82-87%. In both of the cases CV was less than 10%. Through this procedure soxhlet extraction and refluxing apparatus of conventional method were discarded and simple solvent extraction and small vial were successfully employed, resulting in simple, rapid, economic and more precise method.

Effect of Prochloraz on Electrolytic Leakage and Spore Germination of Puccinia recondita Causing Wheat Leaf Rust

Kim, Heung-Tae,Jang, Kyung-Soo,Park, Gyung-Ja,Lee, Sun-Woo,Cho, Kwang-Yun The Korean Society of Plant Pathology 2003 Plant Pathology Journal Vol.19 No.4

The effects of prochloraz on membrane permeability and germination of uredospores of Puccinia recondita were investigated to determine its potential mode of action on wheat leaf rust control activity. Disease control activity of ergosterol biosynthesis inhibitors (EBIs) and their activities on uredospore membrane permeability and germination were examined with wheat leaf rust pathogen, both in vitro and in vivo. While wheat leaf rust was not controlled by prochloraz, electrolytic leakage and spore germination of P. recondita uredospore was the highest with the use of prochloraz among the eight fungicides tested. Prochloraz stimulated uredospore of P. recondita to germinate at a higher ratio. Although certain EBIs, such as hexaconazole, showed excellent control activity, their effects on uredospore membrane permeability and germination was much inferior to prochloraz. Therefore, results of this study suggest that effects of EBIs on membrane permeability and germination of uredospore are not always correlated with their disease control activity.