用于农药手性分离的高灵敏度综合检测

本应用中结论：我们开发了用CD和MS检测器进行SFC手性农药分离的方法。通过TIC、CD和UV的检测，可以使用手性分离清楚地识别目标手性异构体基团和其他污染物。对于每种溴康唑对映体都获得了宽的动态范围和高灵敏度，特别是在SIM色谱图中。该系统适用于许多处理手性化合物的行业Application Note Highly Sensitive and Comprehensive Detection forChiral Separation of Pesticide using SFC-CD-MS2/5Application Note Highly Sensitive and Comprehensive Detection fo r Chiral Separation of Pesticide using SFC-CD-MS Introductio n About 30% of pesticides c u rrently used wor l dwide are c h iral i somers with one or more chiral centers. It is well known that the enantiomers of chiral pesticides h ave different biological activities， tox i city on non-ta r geted organisms， r ate of metabol i sm， and b i odegradat i o n i n the environme n t. Altho u gh it is desirable to u se the more effect i ve and safe r ena n tiomer s ， most c h i r al pesticides are marketed and used as racemates due to t h e co m pl i cated process and high cost of manufacturing a sing l e enan ti ome r m olecule. I n general ， chiral sepa r at i o n by H PLC， GC， and SFC h as been o n e of the most i mportant ways fo r analyzing the syn t hetic approach to ind i vidual enantiomers.Althoug h HPLC still dominates chromatographic chiral analysis because of its ease of use， SFC has advantages such as shorter analysis time and significant reductio n in solvent consumption and cost. I n th i s study， we developed a chiral separation met h od f or pest i cides us i ng SFC with ci r cular dichro i sm detector (CD)and mass spectrometer (MS). The CD detecto r simu lt a n eously provides circular dichro i sm and UV absorbance d a ta，and enables selective iden t i fica t ion of the CD polarity of each enantio m er. MS spectra are used to distingu i sh between chiral isomer groups wit h the same m/z f rom other components including impurities. Highly sensitive and comprehensive detection with UV， CD， and MS contributes to the widely applicable and highly accurate ana l ysis for chiral separation i n the ef f icient development of a single enantiomer pesticide. The system and c h i r al sepa r ation method described here is appl i cable to many indust r i es that deal with chiral compounds. In t h is presentat i on， we will present the application of SFC to bromuconazole， a type of triazole fungicide (Figu r e 1). Figure 1. Str u cture of Bromuconazole Exper i m e ntal Apparatus Figure 2 details the JASCO SFC-CD-MS system used in this expe r ime n t ， toget h er wit h Figure 3 th at shows the schematic di a gra m of t h e system. Usi n g a splitter with a PEEK-coated capil l a r y tube， the flow f r om a c h ir a l co lu mn is d i vided i n to two cha nn els， one each to t h e CD a n d MS detec t o r s. T he CD detector provide the CD a nd UV signals s im ultaneously. Th i s system enables au t omated met h od scout i ng an a lysis to be performed on mobile p hases a n d columns. Figure 2. The JASCO SFC-CD-MS system EHIREMA 2 Figure 3. Sche m atic diagra m of t he SFC-CD-MS Analysis Conditions for Method Scouting Chiral Columns 3： CHIRALCEL OD-H H 4： CHIRALCEL OJ-H 5： CHIRALPAK IA 6：CHIRALPAK IB 7； CHIRALPAK IC 8： CHIRALPAK ID 9： CHIRALPAK IE 10： CHIRALPAK IF Column Size 4.6 mml.D. x150 mmL，5 um Mobile Phase CO，/ Modifier (90 /10) Modifier A； Methanol (MeOH) H) B； Ethanol (EtOH) Make-up Solvent Ethanol / Water (95 /5， with 0.1 % formic acid) Flow Rate Mobile phase ； 3.0 mL/min， Make up solvent ； 0.2 mL/min Column Temperature 40°C CD Detection 225nm MS Detection lonization mode： ESl-positive Measurement mode ： Scan Mass range； 100-600m/z Capillary temperature； 250°C Capillary voltage； 160V Source gas temperature； 350°C ESI voltage ； 3500V Back Pressure 15 MPa Sample 1000 pig/mL racemic bromuconazole in ethanol Injection Volume： 10uL Split Ratio： CD ： MS= ca. 20：1 JASCO INC. 28600 Mary's Cour t ， E a s to n ， M D 21601US A T el ： (800) 333-5272， Fa x ： (410) 822-7526 A ppli c a tio n Lib r ary： htt p：//www.jas co in c .c o m/a pp l icatio n s Racemic bromuconazole with 2 c h i r al centers can be separated into 4 isomer peaks using a chiral co l umn. Fig u re 4 shows the selected r esults of method scout in g analysis f or mobile phases and columns. The best sepa r atio n was observed with the combi n ation of etha n ol and CHIRALPAK AS-H (h i ghl i ghted i n yellow). This separation cond i t i on was used i n the following experiments. Figure 4. C h ro m atogra m s of b r omuconazole stand a rd obtained by method scouti n g analysis，(A) CD c h ro m atogra m s， (B) UV chromatograms. Rows show m o b i l e p h ases and columns show c h iral co l umns. Comprehensive Detection by CD and MS Detectors Figure 5 shows the r esu l ts of a bro m uco n azole standard spiked wi t h two additional pest i c i des， coumaphos and naproanilide， as contam in ants detected by CD and MS detectors. Fig ur e 5A s h ows the total ion chromatogram (T IC)from 100 to 600 m/z， extracted i on chromatogram (XIC) of m/z 378.0， CD a n d UV ch r omatograms. Figure 5B shows the MS spectra of each peak described in Figu r e 5A. As shown i n this fi gure， we can clear l y discriminate the bromuconazole isomers and other contaminants. Bromuco n azole isome r s (peaks 2， 4， 6，and 7) have simi l ar i ties in thei r spectra， and a protonated molecular ion ([M+H]+) was o b served at m/z 377.8 as a base peak in the mass spectr u m of each isomer peak.Sodiu m f ormate ad d uc t ions ([M+HCOONa]+) of coumaphos a n d n aproanilide are a l so o b served at m/z 430.9 (peak 1) and m/z 360.0 (peaks 3 and 5). Figure 6 shows the stopped -flow scanned spectra of CD a n d UV for brom u conazole standard peaks. I n the s p ec t ral m easu r ements， a 2-posi ti o n 6-port switc h ing valve is u sed i n order to d i ver t t h e f low and ‘park' each peak in the f l ow cell for stopped f low measuremen t . The spectra of brom u cona z o l e -1， 2， 3， a n d 4 cor r espond to p eaks 2，4， 6， and 7 i n Figu r e 5A， res p ect i ve l y. Accordi n g to the CD in f ormat i on i n Figu r e 5A and F i gu r e 6， peaks 2 and 6 of the bromuco n azole isomers are estimated to be an e n a n tiomeric pai r from their CD p olarity a n d m aximum wavelength. Peak 4 and 7 are also t he sam e . In t h is way， comprehensive detection by CD and MS detectors i s useful to ide n tify the target chiral isomers group and other conta m inants with chi r a l sepa r at i on. JASCO INC. USGO Bromuconazole-1一 -Bromuconazole-22一 -Bromuconazole-3 -Bromuconazole-4 Figure 6. Stop p ed-f low scanned CD and U V spectra of b r o m uconazole standard peaks (racem a te of 1000 pg/mL).The sp ect ra of b romuco n a z o l e-1， 2， 3， and 4 co r res p on d to t he pe a k 2， 4， 6， an d 7 i n F ig . 5A， res p ect i ve l y Linearity， Reproducibility and Sensitivity Figure 7 shows t h e overlaid chromatograms of S I M (Single Ion Monitoring) at m/z 378.0， CD， and UV of bromuconazole sta n dards (SIM： 0.025， 0.125， a n d 0.25 ug/mL， CD： 62.5， 250， a n d 625 u g/m L ， UV： 25，125， and 250 pg/mL of enantiome r i c conc e n t ration). Table 1 shows the lin e ari t y， r e prod u cibility of r e te n t i on t ime and peak area， and detection lim i t of the bromuconazole e na n tiome r s (t h e conce n tra ti ons i ndicate ena n tio m ers). As shown i n these results， wid e ly linear dynamic range and highly sensit i ve detections were obtained， especially in the SIM c h romatogram. JASCO INC. 28600 Mary's Cour t ， E a s to n ， M D 21601US A A ppli c a tio n Lib r ary： htt p：//www.jas co in c .c o m/a pp l icatio n s Figure 7. SIM ， CD， a n d UV c h ro m atogra m s of bromuconazole standard s (ena n t i o m er i c co n centrat i on ). Table 1. Linear it y， rep r oducibility of r e t ention tim e and peak area， and d et ectio n l im i t of b romuco n azole en a ntiomers CorrelationCoefficent %RSD (n=10) Detection Limit(S/N=3) tR Peak Area MS-SIM (m/z 378.0) 0.9994~0.9997 (0.0125~1.25 ug/mL) 0.23~0.44 1.97~2.56 0.0010~0.0023 ug/mL3 CD 0.9995~0.9998 (25~625ug/mL) 0.31~0.492 2.00~2.342 5.67~ 21.3 ug/mL4 UV 0.9997~09999(6.25~625 pg/mL) 0.32~0.452 1.55~2.122 1.29~2.41 pg/mL4 1Cal cul at e d f ro m the r e su lts o f 0.125 ug/m L. 2 Ca l c ul at e d f rom th e r e s u lt s o f 125 pag /mL . C a lcul at ed f r om th e r es ults of 0.025 ug /mL 4 Calcula t ed f r om the re s ults o f 62.5 u g/m L. Conclusion We deve l oped t h e c h iral separat i on method for pest i c i des by SFC w i th CD and MS d e tectors. Detect i on by TIC， CD， and UV enable the clear identi f ication of target ch i ral iso m er groups a n d other contaminants using ch ir al separation. Wide dynamic r a n ges and h i g h sensitivity fo r each bromuco n azole enantiomer were obtained， especial l y in the SIM ch r omatograms. This system is applicable to many i ndustr i es that deal with chiral compounds. Ref e r e nces B . S. Sekhon， J. Pest i c. Sci， 2009，34(1)，1-12. V. Pérez -Fernande z ， M. A. Garciá， M. L. Marina， J. C h romatogr. A， 2011， 1218， 6561-6582. JASCO INC. UASGO A ppli c a tio n Lib r ary： htt p：//www.jas co in c .c o m/a pp l icatio n s