Korean Journal of Environmental Agriculture Korean J Environ Agric. 2015;34(4):318-327. Korean Online ISSN: 2233-4173 Published online 2015 December 18. http://dx.doi.org/10.5338/kjea.2015.34.4.48 Print ISSN: 1225-3537 Research Article Open Access LC-MS/MS를이용한농산물중식물생장조절제 Trinexapac-ethyl과대사산물 Trinexapac의동시분석법개발 장진, 김희정, 고아영, 이은향, 주윤지, 장문익 *, 이규식, 서세정 식품의약품안전처식품의약품안전평가원식품위해평가부잔류물질과 Development of a Simultaneous Analytical Method for Determination of Trinexapac-ethyl and Trinexapac in Agricultural Products Using LC-MS/MS Jin Jang, Heejung Kim, Ah-Young Ko, Eun-Hyang Lee, Yunji Ju, Moon-Ik Chang *, Gyu-Seek Rhee and Saejung Suh (Pesticide and Verterinary Drug Residues Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Chungju 28159, Korea) Received: 17 August 2015 / Revised: 27 October 2015 / Accepted: 12 November 2015 Copyright c 2015 The Korean Society of Environmental Agriculture This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract BACKGROUND: Trinexapac-ethyl is a plant growth regulator (PGR) that inhibits the biosynthesis of plant growth hormone (gibberellin). It is used for the prevention of lodging, increasing yields of cereals, and reducing mowing of turf. The experiment was conducted to establish a determination method for trinexapac-ethyl and its metabolites trinexapac in agricultural products using LC-MS/MS. METHODS AND RESULTS: Trinexapac-ethyl and trinexapac were extracted from agricultural products with methanol/ distilled water and the extract was partitioned with dichloromethane and then detected by LC-MS/MS. Limit of detection(lod) was 0.003 mg/kg and limit of quantification(loq) was 0.01 mg/kg, respectively. Matrix matched calibration curves were linear over the calibration ranges (0.01-1.0 mg/l) for all the analytes into blank extract with r 2 > 0.997. For validation purposes, recovery studies were carried out at three different concentration levels *Corresponding author: Moon-Ik Chang Phone: +82-43-719-4204; Fax: +82-43-719-4200; E-mail: 1004@korea.kr (LOQ, 10LOQ, 50LOQ, n=5). Recoveries of trinexapacethyl and trinexapac were within the range of 73.6-106.9%, 72.7-99.2%, respectively. The relative standard deviations (RSDs) were less than 9.0%. All values were consistent with the criteria ranges requested in the CODEX guideline(cac/gl 40, 2003). CONCLUSION: The proposed analytical method was accurate, effective and sensitive for trinexapac-ethyl and trinexapac determination and it can be used to as an official method in Korea. Key words: LC-MS/MS, Maximum residue limits, Plant growth regulators(pgr), Trinexapac, Trinexapac-ethyl 서론 식물생장조절제 (PGR, Plant growth regulator) 는식물의생육을촉진시키거나억제하여생물생육을인위적으로조절하는물질로농업생산에서경제적인비용절감을위해사용되는화학물질이다 (Lee et al., 1993; Jeong et al., 2004). Trinexapac-ethyl은 1995년에 Syngenta사에서개발된 acylcyclohexanedione 유도체계통의생장조정제로신장촉진호르몬 gibberellin의합성을막아작물생장을억제하는작용을하여곡류나잔디에처리했을때도복방지, 관 318
Analytical Method Development of Trinexapac-ethyl and Trinexapac in Agricultural Products 319 Trinexapac-ethyl Trinexapac Fig. 1. Proposed metabolic pathway of trinexapac-ethyl to trinexapac (acid) in wheat sample. Table 1. Physicochemical characteristics of trinexapac-ethyl and trinexapac Property Trinexapac-ethyl Trinexapac IUPAC name ethyl (RS) 4-cyclopropyl(hydroxy)methylene-3,5-dioxocyclohexanecarboxylate 4-cyclopropyl(hydroxy)methylene-3,5-dioxo cyclohexanecarboxylic acid CAS No. 95266-40-3 104273-73-6 Classification Plant growth regulator - Molecular formula C 13H 16O 5 C 11H 12O 5 Molecular weight 252.3 224.2 Melting point 36.3 114.4 Density 1.22 g/cm 3 1.41 g/cm 3 Log Pow 1.5 (ph 5), -0.3 (ph 6.9), -2.1 (ph 8.9), (all 25 ) 1.8 (ph 2) pka 4.57 pka1 5.32, pka 2 3.93 Vapor pressure 2.2 mpa (25 ) 2.3 10-3 mpa (25 ) Solubility In water 1.1 (ph 3.5), 2.8 (ph 4.9), 10.2 (ph 5.5), 21.1 (ph 8.2) (all in g/l, 25 ). In ethanol, acetone, toluene, n-octanol 100%, n-hexane 5% (25 ) In water 13 (ph 5.0), 200 (ph 6.8), 260 (ph 8.4) (all in g/l, 25 ). In acetone 95, ethyl acetate 37, methanol 84, octhanol 17 (all in g/l, 25 ) 수량절감, 비료효과지속, 살균제기능강화등의효과를기대할수있다 (Park, 2006; Hong et al., 2009; Tae et al., 2010; Gonzales-Curbelo, 2012; MFDS, 2013) Trinexapac-ethyl 은개발이래꾸준히사용되어왔으며이에따라분석법또한다수보고된바있다. 하지만밀과같은일부농산물에서는약제살포후 48시간이내에 trinexapacethyl이 trinexapac으로대사 (Fig. 1) 되는것이확인 (Syhre et al., 1997) 되어 trinexapac-ethyl의잔류량을정확히파악하기위해서는모화합물뿐아니라대사산물에대한분석이필요하게되었다. 또한그간보고된분석법은모화합물만을분석하거나 (Ciba-Geigy, 1991; Syhre et al., 1997; Huang et al., 2014, MHLW, 2010) 대사산물 trinexapac만을분석 (Hiemstra et al., 2003) 하였으며, 대사산물분석법의경우에도밀에만적용이가능하다는한계가있다. 현재우리나라는 trinexapac-ethyl의잔류허용기준을쌀에만 0.5 mg/kg(mfds, 2013) 으로설정하였으나, 밀에대한잔류허용기준요청에따라잔류시험결과등의검토후밀에대해서도잔류허용기준을설정할예정이다. 특히밀에서는대사산물인 trinexapac으로잔류하여인체에대한노출가능성이높으므로 잔류물의정의 로설정하여잔류허용기준에 적용하여관리할예정이며, 본연구에서는 trinexapac-ethyl 과대사산물 trinexapac을동시에분석할수있는분석법을개발하고자하였다. 또한, 밀뿐아니라대사산물의잔류량이높은다른농산물에대해서도적용이가능하도록각대표농산물군에대한시험을수행하였다. 재료및방법 시약및재료 Trinexapac-ethyl(98.6%, Fig. 1, Table 1) 표준품은 Dr. Ehrenstorfer GmbH에서제공받아사용하였고대사산물인 trinexapac(98.7%, Fig. 1, Table 1) 표준품은 Chem service 에서구입하여사용하였다. Methanol, dichloromethane, acetonitrile, n-hexane 등은 HPLC 등급으로 Merck(Darmstadt, Germany) 에서구입하여사용하였다. Hydrochloric acid와 sodium chloride는 Wako(Osaka, Japan) 에서여과보조제 (celite 545) 및 anhydrous sodium sulfate는 Merck (Darmstadt, Germany) 에서 formic acid는 Sigma Aldrich (Buchs, Switzerland) 에서구입하여사용하였다. 검체는잔류허용기준설정대상인밀뿐아니라다른농산
320 Jang et al. Table 2. Analytical conditions for the determination of trinexapac-ethyl and trinexapac Instrument LC-MS/MS (Xevo TQ-S, Waters, MS, USA) HPLC conditions Column MGII C 18 (2.0 mm i.d. 100 mm, 3 μm, Shiseido) Column temperature 40 Flow rate 0.2 ml/min Injection volume 5 μl Mobile phase A: 0.1% formic acid in distilled water B: 0.1% formic acid in methanol Gradient table Time (min) Mobile phase A (%) B (%) 0 95 5 3 95 5 10 5 95 13 95 5 15 95 5 MS/MS conditions Ion mode ESI positive mode Capillary voltage 3.0 kv Source temperature 150 Desolvation temperature 600 Desolvation gas flow rate 1000 L/hr Cone gas flow rate 150 L/hr 물에도적용이가능하도록곡류, 콩류, 과일류, 과채류, 서류의종류별로생산량이많고간섭물질이많아정제가어려울것으로예상되는대표농산물을선정하여분석법의개발에사용하였다. 각각의대표농산물은현미, 대두, 감귤, 고추, 감자이며, 모두무농약농산물을구입하여균질화한후밀봉된용기에담아 -50 에보관하고실험에사용하였다. 표준용액의조제 Trinexapac-ethyl 및 trinexapac 표준품을 methanol에용해하여 1,000 μg/ml의혼합표준원액을조제하였다. 이를무처리추출물로희석하여 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 및 1 μg/ml의혼합표준용액을만든후모두갈색병에담아 4 에보관하여실험에사용하였다. 분석법의검증본연구에서는 CODEX 가이드라인 (CAC/GL 40, 2003) 에따라분석법의직선성 (linearity), 검출한계 (LOD, limit of detection), 정량한계 (LOQ, limit of quantification), 회수율시험을통한정확성 (accuracy), 정밀성 (precision), 재현성 (repeatability) 을평가하였다. 직선성확인을위해 trinexapac-ethyl 및 trinexapac 표준용액 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 및 1 μg/ml에대한각각의 peak 면적을이용하여검량선을작성하였고검량선의결정계수 (coefficient of determination, r 2 ) 를구하였다. 검출한계 (LOD) 및정량 한계 (LOQ) 는크로마토그램상에서신호대잡음비 (S/N ratio) 가각각 3, 10 이상으로하였다. 곡류, 콩류, 과일류, 채소류, 서류의각대표농산물인현미, 대두, 감귤, 고추, 감자의무처리시료에 trinexapac-ethyl 및 trinexapac 표준용액을첨가한후분석하여회수율실험을진행하였다. 회수율실험의처리농도는정량한계 (LOQ), 정량한계의 10배 (10LOQ), 정량한계의 50배 (50LOQ) 해당하는농도로수행하였으며, 각각의농도및시료에대하여 5반복으로수행하여평균및상대표준편차 (RSD, relative standard deviation) 를계산하여분석법의정확성과정밀성및재현성을평가하였다. 결과및고찰 최적기기분석조건확립현행식품공전분석법 (4.1.4.91) 에서는 trinexapac-ethyl 이 280 nm 파장의빛을흡수하는성질을이용하여 HPLC- UVD로분석하였다. 대사산물인 trinexapac 또한 trinexapacethyl 과유사한구조이므로 HPLC-UVD 의적용이가능할것이라판단하였고, 실제로 trinexapac 표준물질의분리및검출에는문제가없었다. 하지만시료에적용하였을때에는시료에서추출된극성간섭물질의영향으로분리능이떨어져분석법의선택성확보가어려웠다. 이에본분석법에서는 trinexapacethyl 과 trinexapac 의동시분석을위해액체크로마토그래프- 질량분석기 (liquid chromatograph-tandem mass spectrometer,
Analytical Method Development of Trinexapac-ethyl and Trinexapac in Agricultural Products 321 Table 3. Selected-ion of LC-MS/MS for trinexapac-ethyl and trinexapac Compound Retention time (min) Molecular weight Precursor ion (m/z) Fragment monitored (m/z) CE* Trinexapac-ethyl 10.0 252.2 253.2 Trinexapac 8.9 224.2 225.2 * Collision energy (ev) ** Quantification ion 69** 21 185 11 207 11 69** 15 111 19 165 17 Fig. 2. Efficiency of methanol/distilled water extraction (A) and dichloromethane partition (B) of trinexapac-ethyl and trinexapac in agricultural products. LC-MS/MS) 를분석기기로선정하였다. 분석용칼럼은분리범위가넓으며 trinexapac-ethyl과 trinexapac을분리할수있는 C 18 칼럼을선택하였고, 용리방식은 0.1% formic acid in distilled water와 0.1% formic acid in methanol을이동상으로사용하는기울기용리방식을선택하였다. 이동상에는 formic acid를 proton source로서첨가하여분석시이온화를용이하게하였다. 또한 LC-MS/MS는잔류분석법개발후재확인을위한분석기기로사용되므로 trinexapac-ethyl 및 trinexapac에대한잔류물의정확성및신뢰성을동시에확보할수있었다. Trinexapac-ethyl 과 trinexapac 을이온화하기위해 electrospray ionization (ESI) 의 positive-ion mode를사용하였고 Table 2에나타낸분석조건을바탕으로 total ion chromatogram (TIC) 과 mass spectrum을통해 selectedion monitoring (SIM) 분석을위한최적특성이온을선정하였다. 질량이 252.2인 trinexapac-ethyl 표준용액 (1 μg/ ml) 을일정한속도 (10 μl/min) 로질량검출기에직접주입한결과질량이 [M+H] + 형태인 253.2 mass 값을확인하였고, 질량이 224.2인 trinexapac은 225.2 mass 값을확인하였다. 이때 cone voltage 의변화 (10~70 V) 에따른최적화과정을통해 trinexapac-ethyl 의경우 28 V에서, trinexapac의경우 40 V에서최대의 peak 강도가나타남을확인하였다. 최적화된 cone voltage상태에서분석의선택성과검출강도 를향상시키기위하여 MS/MS 분석시 MRM(multiple reaction monitoring) mode로분석하였다. 이때 collision cell에서 collision energy를조절하여최적의 precursor/ product ion pair를선정하였다. 또한가장좋은감도를보이는 product ion을정량이온 (quantification ion) 으로설정하였고, 다음으로크게검출되는 product ion을정성이온 (qualification ion) 으로설정하여확인하였다. 이때최적기기분석조건은 Table 2에나타내었고, 분석조건에서선정된특성이온과머무름시간은 Table 3에나타내었다. 추출및정제조건의확립 Trinexapac-ethyl의현행분석법에서는균질검체에 methanol/phosphate buffer(ph 7)/distilled water 혼합액을가해 2시간진탕추출하여흡인여과한뒤 ph를낮추어비이온성의 trinexapac-ethyl을 dichloromethane으로분배하여아민카트리지로정제해 HPLC-UVD 로분석하였다. 추출및정제조건은현행식품공전의분석법을바탕으로 trinexapac을동시에분석할수있도록개선하였다. 추출과정에서 trinexapac-ethyl의극성도를높여극성의 methanol/distilled water 혼합액에대한추출효율을높이기위해사용한 phosphate buffer의경우, buffer를사용하지않아도중성의 ph가유지되어 trinexapac-ethyl과 trinexapac이일관되게높은추출효율 (Fig. 2 (A)) 을나타내
322 Jang et al. Fig. 3. Calibration curve of matrix matched standard of 1, trinexapac-ethyl ; 2, trinexapac in A, hulled rice; B, soybean; C, mandarin; D, pepper; and E, potato. 므로 phosphate buffer를생략하였다. 또한, 동일한분배용매 dichloromethane을사용하였을때에도 methanol/ distilled water 추출물중 trinexapac-ethyl 및 trinexapac 에대한분배효율이만족스러운수준이었다 (Fig. 2 (B)). 다만, 현미에대한추출및분배효율이다소낮았으나이는각추출및분배과정의상대적인효율을비교하기위해각단계 만단회수행한것으로, 모든과정을연속하여수행하고반복실험을통하여실험에의한오차가줄어들경우정확도가높아질것으로예상하여정제및분석방법을확정하였다. Trinexapac-ethyl은 pka가 4.57, trinexapac은 pka1 5.32, pka2 3.93이므로 1N HCl을가하여 ph를 2.5 이하로만들어두물질의이온화를막은뒤비극성용매인
Analytical Method Development of Trinexapac-ethyl and Trinexapac in Agricultural Products 323 Fig. 4. Representative MRM (quantification ion) chromatograms of trinexapac-ethyl corresponding to: matrix matched standard (0.5 mg/kg), control, recovery (0.5 mg/kg) in A, hulled rice; B, soybean; C, mandarin; D, pepper; and E, potato. dichloromethane으로액액분배를하여극성의간섭물질을제거하였다. 이는현행식품공전분석법및그간보고된 trinexapac-ethyl 및 trinexapac 분석법에서 phosphorus acid(syhre et al., 1997) 및 phosphate buffer(ciba- Geiby, 1991; MHLW, 2010; Hiemstra et al., 2003) 로이온화를억압하여추출및분배효율을높인것과같은맥락이다. 현행식품공전분석법에서는 trinexapac-ethyl 분석시추출물내효과적인약산성화합물제거를위해아민카트리지를사용했으나대사산물 trinexapac은 trinexapac-ethyl의 ethyl carboxylate기가 carboxylic acid로전환된물질로 carboxylic acid기가카트리지의고정상과강하게결합하여회수가어려웠다 (MFDS, 2013). 또한, 추출및액액분배만으로도시료중간섭물질이충분히제거되는것을확인하였기때문에흡착크로마토그래피를통한손실량을최소화하고분석에소요되는시간을줄이기위해카트리지정제는생략하였다. 추출액중유지성분을함유한지방성시료대두및현미의경우, 분배추출물에잔여물이남아회수율에영향을미쳤기때문에유지및비극성성분의정제를위해추가로 n-hexane/
324 Jang et al. Fig. 5. Representative MRM (quantification ion) chromatograms of trinexapac corresponding to: matrix matched standard (0.5 mg/kg), control, recovery (0.5 mg/kg) in A, hulled rice; B, soybean; C, mandarin; D, pepper; and E, potato. acetonitrile 을이용한분배법을적용하였다. 분석법의검증 선택성및직선성개발된분석법의직선성 (linearity) 을확인하기위하여 trinexapac-ethyl 및 trinexapac 혼합표준원액을농산물무처리추출물로희석 ( 표준원액 / 무처리시료, 10/90, v/v) 하여 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 및 1 μg/ml 5 μl를 LC-MS/MS에주입하여분석한결과모든농산물시료표준용액에서결정계수 (r 2 ) 0.999 이상으로높은직선성을보여주었다 (Fig. 3). Trinexapac-ethyl 및 trinexapac의선택성 (selectivity) 은표준용액, 무처리시료, 회수율시료의크로마토그램을서로비교함으로써평가할수있었으며, 무처리시료와회수율시료는확립된시험방법에따라시험용액을준비한후 LC-MS/ MS로분석하였다. 농산물시료중 trinexapac-ethyl 및 trinexapac의머무름시간에검출되는어떠한방해물질도검
Analytical Method Development of Trinexapac-ethyl and Trinexapac in Agricultural Products 325 Table 4. Validation results of analytical method for the determination of trinexapac-ethyl and trinexapac residues in samples Sample Hulled rice Soybean Mandarin Pepper Potato Fortification (mg/kg) Trinexapac-ethyl Average ± RSD (%) Trinexapac 0.01 106.9 ± 6.9 74.6 ± 3.6 0.1 91.6 ± 4.0 86.1 ± 2.5 0.5 87.0 ± 7.1 84.3 ± 4.1 0.01 89.0 ± 7.8 76.7 ± 8.0 0.1 100.5 ± 7.8 76.5 ± 3.3 0.5 87.8 ± 4.5 82.2 ± 3.1 0.01 73.6 ± 2.4 79.5 ± 3.2 0.1 87.7 ± 3.7 85.8 ± 9.0 0.5 86.4 ± 2.0 80.4 ± 2.2 0.01 88.6 ± 6.8 82.1 ± 8.5 0.1 101.0 ± 8.1 95.0 ± 4.9 0.5 98.5 ± 3.1 92.2 ± 2.7 0.01 96.1 ± 8.2 72.7 ± 2.7 0.1 104.6 ± 3.6 99.2 ± 2.3 0.5 91.6 ± 8.3 93.3 ± 4.0 LOQ (mg/kg) 0.01 출되지않아검체중 trinexapac-ethyl 및 trinexapac을분석하기위해개발된분석법의높은분리능과선택성을확인할수있었다 (Fig. 4, 5). 검출한계와정량한계본연구에서확립한시험용액조제및기기분석법을이용하여검체중 trinexapac-ethyl 및 trinexapac의검출한계및정량한계를구하였다. 검출한계는최소검출량이 0.0015 ng (S/N>3) 이었고아래의계산식에따라 0.003 mg/kg으로나타났다. 정량한계는최소검출량이 0.05 ng (S/N>10) 으로아래의계산식에따라 0.005 mg/kg으로나타났다. 1 최종희석부피 (ml) 검출한계 (mg/kg) = 최소검출량 (ng) 시료량 (g) 시료주입량 (μl) = 0.015 (ng) 1 5 (ml) = 0.003 5 (g) 5 (μl) 1 최종희석부피 (ml) 정량한계 (mg/kg) = 최소검출량 (ng) 시료량 (g) 시료주입량 (μl) 분석법의회수율 = 0.05 (ng) 1 5 (ml) = 0.01 5 (g) 5 (μl) 분석법의정확성과정밀성및재현성을평가하기위하여 LOQ, LOQ 10배, LOQ 50배수준인 0.01, 0.1와 0.5 mg/ kg의처리농도로 trinexapac-ethyl 및 trinexapac의회수율실험을 5회반복하여수행하였다. 각농도에서 trinexapacethyl의평균회수율은 73.6-106.9% 이었고, trinexapac의평균회수율은 72.7-99.2% 이었다. 이때상대표준편차 (RSD) 는 모두 9.0% 미만으로확인되어잔류물분석에관한 CODEX 가이드라인 (CAC/GL 40, 2003) 의잔류농약검증기준에적합함을확인할수있었다 (Table 4). 아울러 LC-MS/MS 를이용하여분석한농산물중 trinexapac- ethyl 및대사산물 trinexapac 의회수율크로마토그램은 Fig. 4, 5에제시하였다. 개발된분석법은밀과유채에만적용가능했던 Syhre 등 (1997) 의단성분분석법과달리곡류, 과일류, 과채류, 서류, 콩류에모두적용이가능하며, 정량한계가 10배낮은 0.01 mg/kg으로감도가높다는장점이있다. 또한, 각대표작물에대한회수율결과는밀의 trinexapac에대해최적화된분석법 (Hiemstra et al., 2003) 의회수율결과 (71-94%) 와동등한수준임을확인하여개발된분석법의정확성도확인할수있었다. 이전의두연구 (Shyre et al., 1997; Hiemstra et al., 2003) 와본연구에서공통적으로 trinexapc-ethyl 에비해 trinexapac 의회수율이다소낮게나타났는데, 이는 trinexapac- ethyl 이 trinexapac으로대사되면서 ethyl carboxylate기가 carboxylic acid로전환되며분자의극성이강해지고이에따라유기용매를사용한추출및분배의효율이낮아졌기때문으로판단된다. 결론 본연구에서는농산물중 trinexapac-ethyl 의잔류검사를위하여현행식품공전잔류농약분석법을모화합물 trinexapacethyl과대사산물 trinexapac의동시분석이가능하도록개선하였으며최종확립된추출 정제방법및분석조건은다음과같다 (Fig. 6).
326 Jang et al. Sample 5 g - Add 20 ml of methanol/distilled water (70/30, v/v) - Homogenize for 5 min Filtration - Wash with methanol (20 ml) - 30 ml distilled water - Adjust ph to 2.5 by 1N hydrochloric acid Liquid-liquid extraction - Add 5 g of sodium chloride - Add dichloromethane 30 ml 2 times - Collect dichloromethane layer * Hulled rice and soybean sample - Dissolve with 30 ml of saturated n-hexane in acetonitrile - Partition with 30 ml of saturated acetonitrile in n-hexane 2 times - Collect saturated acetonitrile in n-hexane layer Concentration - Dissolve with methanol 5 ml - Filter through syringe filter (nylon, 0.22 μm) LC-MS/MS Fig. 6. Flow chart for trinexapac-ethyl and trinexapac analysis. 추출 농산물검체를분쇄하여균질화한후 5 g( 곡류및콩류는약 1 kg을혼합하여표준체 420 μm를통과하도록분쇄한후그 5 g, 과일류 채소류 서류는약 1 kg를잘게갈아혼합한그 5 g) 을정밀히달아균질기용기에넣고 methanol/ distilled water 혼합액 (70/30, v/v) 20 ml을가하여진탕기에서 5분간진탕하였다. 여과지가깔려있는부흐너깔때기로여과보조제 (celite 545) 5 g을이용해흡인여과한뒤 methanol 20 ml로잔사및용기를씻어내려앞의여액과합하였다. 합친여액에 distilled water 30 ml를더하여 ph 미터기를이용해 1N hydrochloric acid(hcl) 를첨가하여 ph 2.5로조정했다. 액액분배이를 250 ml 분액깔때기에옮기고 sodium chloride 5 g 및 dichloromethane 30 ml를차례로가한후심하게흔들어층이완전히분리될때까지정치시킨후 dichloromethane 층을 anhydrous sodium sulfate에통과시켜감압농축플라스크에받는과정을 2회반복했다. 이를 40 이하의수욕상에서감압하여용매를모두날려버린후, 잔류물에 methanol 을가하여최종부피 5 ml가되게한뒤 syringe filter(nylon, 0.22 μm) 로여과하여시험용액으로사용했다. 지방성검체인현미와대두의경우, 추가로유지성분의제거를위해미리 acetonitrile로포화시킨 n-hexane 30 ml를건고물에가하여재용해후 250 ml 용량의분액여두에옮기고미리 n- hexane으로포화시킨 acetonitrile 30 ml씩으로 2회분배 추출하였다. 이를감압농축한뒤 methanol을가하여최종부피 5 ml가되게한뒤 syringe filter(nylon, 0.22 μm) 로여과하여시험용액으로사용하였다. LC-MS/MS 분석조건 Trinexapac-ethyl 및 trinexapac은액체크로마토그래프 - 질량분석기 (LC-MS/MS, Acquity UPLC Xevo TQ-S, Waters, MS, USA) 를사용하였으며기기분석조건은 Table 2, 3에나타내었다. 분석법의검출한계 (LOD) 는 0.003 mg/kg이었고, 정량한계 (LOQ) 는 0.01 mg/kg으로확인되었으며분석법의재현성, 정확성, 정밀성등을판단하기위해각농산물의대표시료 ( 현미, 대두, 감귤, 고추, 감자 ) 에대해회수율시험을진행하였다. LOQ, 10LOQ, 50LOQ 수준에서의회수율결과는모든농산물에서 trinexapac-ethyl 및 trinexapac이각각 73.6-106.9%, 72.7-99.2%(n=5) 로나타나농산물시료및처리농도에관계없이모두 CODEX 가이드라인 (CAC/GL 40, 2003) 의조건을만족하는수준이었다. 따라서본연구에서개발된분석법은국내유통되는농산물중 trinexapac- ethyl 및 trinexapac의안전관리를위한공정분석법으로사용가능함을확인하였다. 요약 Trinexapac-ethyl 은식물체중생장조절세포의신장촉진호르몬인 gibberellin의합성을막아곡류및잔디의도복방
Analytical Method Development of Trinexapac-ethyl and Trinexapac in Agricultural Products 327 지나초장억제용으로사용되는생장조절제이다. 본연구에서는일부농산물에서 trinexpac-ethyl 이대사산물인 trinexapac 으로잔류함에따라현행식품공전 (4.1.4.91) trinexpacethyl 모화합물분석법을 trinexapac-ethyl 및 trinexapac의동시분석법으로개선하였다. 대표농산물시료 ( 현미, 대두, 감귤, 고추, 감자 ) 를 methanol/distilled water 혼합액으로추출하고 HCl으로 ph를 2.5 이하로낮춘뒤 dichloromethane 으로액액분배하여간섭물질을제거하였다. 분석기기로는모화합물과대사산물을동시분석하기에적합한 LC-MS/MS를사용하였고, 이에따른분석법의검출한계 (LOD) 및정량한계 (LOQ) 는각각 0.003, 0.01 mg/kg 으로확인되었다. 모든농산물및처리농도와관계없이 trinexapac-ethyl 및 trinexapac이각각 73.6-106.9%, 72.7-99.2% 의평균회수율을보였으며상대표준편차또한 9.0% 이하로나타나잔류물분석에관한 CODEX 가이드라인 (CAC/GL 40, 2003) 을만족하는수준으로확인되어개발된분석법의재현성, 정확성, 정밀성등을검증하였다. 본연구에서개발된분석법은농산물중 trinexapacethyl의안전관리를위한공정분석법으로사용되기에적합할것으로판단된다. Acknowledgement This study was carried out with the support of "Safety Evaluation and Analysis Method on Pesticide Residues in Foods-2015(15161MFDS042)" from Ministry of Food and Drug Safety, Republic of Korea in 2015. References González-Curbelo, M. Á., Herrera-Herrera, A. V., Ravelo- Pérez, L. M., & ernández-borges, J. (2012). Samplepreparation methods for pesticide-residue analysis in cereals and derivatives. Trends in Analytical Chemistry. 38, 32-51. Hiemstra, M., & De Kok, A. (2003). Determination of trinexapac in wheat by liquid chromatographyelectrospray ionization tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 51(20), 5855-5860. Hong, B. S., & Tae, H. S. (2009). Heavy rough management of golf course by using of trinexapacethyl. Korean Journal of Golf Studies, 3(1), 99-103. Huang, H., Zhang, J., Xu, D., Zhou, Y., Luo, J., Li, M., Chen, S., & Wang, L. (2014). [Determination of 21 plant growth regulator residues in fruits by QuEChERS-high performance liquid chromatographytandem mass spectrometry]. Se pu= Chinese journal of chromatography/zhongguo hua xue hui, 32(7), 707-716. Jeong, Y. H., Kim, J. E., Kim, J-H, Lee, Y-D, Lim, C. H., & Hur, J-H. (2004). The lastest pesticide science (Revised), p. 251, Sigma Press, Seoul, Korea. Ministry of Food and Drug Safety (MFDS). (2013). Analytical methods of pesticide residues in food (fourth edition), pp. 104-105, 689-692, MFDS, Seoul, Korea. Park, J. Y. (2006). Effect of trinexapac-ethyl treatment on growth and quality turfgrass species. Dankook University Master s thesis, 1-3. Syhre, M., Hanschmann, G., & Heber, R. (1997). Problems in analyzing trinexapac-ehtyl a new plant growth regulator. Journal of Agricultural and Food Chemistry, 45(1), 178-179. Tae, H. S., Hong, B. S., Cho, Y. S., & Oh, S. H. (2010). Trinexapac-ethyl treatment for kentucky bluegrass of golf course during summer. Asian Journal of Turfgrass Science, 24(2), 156-160.