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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2015 Waters Corporation INTRODUCTION Research associated with the development of new crop protection chemicals centers around the design of products that provide highly effective and specific action towards the target organism with reduced application rates. It is estimated that 25-30% of the pesticides on the market today have optical isomers. The desired activities often result from a single enantiomer in the optical isomer mixtures. It is therefore important to assess the enantiomeric purity of the chiral active ingredients in the formulation. In addition, the detection, characterisation and quantitation of the other components in the formulation are necessary to support product registration. 1 There has been an increasing adoption of supercritical fluid chromatography (SFC) on chiral stationary phases for chiral separations. The properties of a supercritical fluid, its high diffusivity and low viscosity in particular, enable high efficiency chiral separations with shorter run times. UltraPerformance Convergence Chromatography (UPC 2 ) applies the performance advantages of UPLC® to SFC, combining the use of supercritical CO 2 with sub-2- μm particle columns. UPC 2 is an orthogonal analytical technique to reversed-phase LC and can be used to solve complex separations challenges. In this study the achiral screen of a formulated pesticide product using UPC 2 showed that the minor components detected using UV and mass detection had similar structural characteristics to the active ingredient (AI), propiconazole, a triazole fungicide with two chiral centers (Figure 2). The minor components had the same m/z and shared the same isotopic pattern as the AI. Subsequent chiral resolution of the propiconazole in the formulation in combination with simultaneous UV and mass detection provided valuable spectral information which allowed the minor components to be characterised as probable stereoisomers. 1 CHIRAL AND ACHIRAL PROFILING OF A PESTICIDE FORMULATION USING ULTRAPERFORMANCE CONVERGENCE CHROMATOGRAPHY (UPC2) WITH PDA AND MASS DETECTION Marian Twohig, 1 Michael O Leary, 1 Peter G. Alden 1 1 Waters Corporation, Milford, MA, USA METHODS Achiral Gradient Conditions: 0 min 3% B, 4 min 30% B, 6 min 30% B, return to initial conditions. Chiral Gradient Conditions: 0 min 7% B, 3 min 11% B, 6 min 17% B, 7 min 40% B, return to initial conditions. Mass Detector Conditions MS system: ACQUITY QDa Detector Ionization mode: ESI + ; Capillary voltage: 1.0 kV Desolvation temp.: 600 °C ; Source temp.: 150 °C Cone voltage: 10 V; Sampling rate: 5 Hz MS scan range: 100 to 600 m/z Empower 3 FR2 Software was used for data acquisition and chromatographic data processing. REFERENCES 1. Twohig, M. O’Leary, M. McCauley, J.P. Chen, R. Chiral and Achiral Profiling of a Pesticide Formulation Using the ACQUITY UPC2 System and the ACQUITY QDa Detector. Waters application library. APNT134790013. 2. Glaser, R. Adin, I. Ovadia, D. Mendler, E. Drouin, M. (1995) Solid-state structure determination and solution-state NMR characterization of the (2R,4R)/2S,4S)- and (2R,4S)/(2S,4R)- diastereomers of the agricultural fungicide propiconazole, th(2R,4S)/(2S,4R)-symmetrical triazole constituational isomer, and a ditriazole analogue. Structural Chemistry, Vol 6, No 3. 145- 156. 3. McCauley, J.P. Twohig, M. Chen, Rui. Isolating Trace Impurities for Structural Elucidation in a Commercial Fungicide Formulation Using Preparative SFC. Chirality 2014 Prague, Czech Republic. July 27th to 30th 2014. Waters application library PSTR134807862. CONCLUSION The addition of mass detection as a complementary analytical detection technique enhances confidence in compound detection and identification. The ACQUITY UPC 2 System has column switching capabilities so that both chiral and achiral columns can be used with a choice of four co-solvents that are compatible with MS analysis. The chiral and achiral method development and analysis can be performed on the same system. The ACQUITY UPC 2 System allows high efficiency separations that can increase sample throughput compared to traditional normal-phase separations. The diasteromeric resolution of propiconazole using UPC 2 took place in less than 3 minutes, which is at least 10 times faster than normal phase methods reviewed in the literature. 1 PDA QDa Figure 1. Waters ® ACQUITY QDa Detector is a novel mass detector that can be integrated into existing liquid chromatography configurations in order to increase sensitivity and complement the results obtained using UV detectors. Achiral Chiral Separation mode Gradient Gradient Column ACQUITY UPC 2 BEH, 3.0 x 100 mm, 1.7-μm ACQUITY UPC 2 Trefoil AMY1 3.0 x 150 mm, 2.5-μm Co-solvent (B) Methanol 50:50 2-propanol/ethanol ABPR 1990 psi/137 bar 3000 psi/207 bar Flow rate 1.5 mL/min 1.5 mL/min UV detection 220 nm 220 nm Column temp. 35 ° C 20 ° C Injection volume 0.5 μL 0.5 μL Make-up solvent 98:2 methanol:water with 0.1% ammonium hydroxide at 0.3 mL/min Table 1. Summary of the analysis conditions. RESULTS AND DISCUSSION Achiral Separation ACQUITY UPC 2 chromatograms (Figure 2) of the propiconazole standard (lower trace) and the formulation sample (top trace) obtained using an ACQUITY UPC 2 BEH column. The retention times of peaks 1 and 2 (propiconazole diastereomers) in the formulation sample matched those of the propiconazole standard. Two minor peaks (peaks 3 and 4) were observed in the formulation sample with retention times (t R ) of 2.22 min and t R 2.26 min, respectively. 342 344 0 6 7 7 342 344 0 6 7 342 344 0 6 6 6 342 344 0 6 6 m/z 20.00 325.00 330.00 335.00 340.00 345.00 350.00 355.00 360.00 268.8 0 0 0 0 0 268.8 0 0 0 0 0 313.9 343.3 0 0 0 268.8 327.0 339.9 0 5 0 5 nm 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 AU 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Minutes 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 1 2 1 2 Formulation sample Propiconazole standard 3 4 1 2 3 4 1 2 3 4 34 (2R,4R)-propiconazole (2R,4S)-propiconazole (2S,4S)-propiconazole (2S,4R)-propiconazole UV Spectra PDA 220 nm MS Spectra Figure 2. ACQUITY UPC 2 UV achiral separation of the propiconazole present in the formulation sample and propiconazole standard at 220 nm using the ACQUITY UPC 2 BEH Column. The UV and MS spectra for peaks 1-4 are also shown. Chiral Separation The ACQUITY UPC 2 System has multi-column switching capabilities and a choice of up to four co-solvents which allows both achiral and chiral method development and sample analysis to be performed on the same system. Using gradient separation on an ACQUITY UPC 2 Trefoil AMY1 chiral column (Figure 3). The two diastereomer peaks of propiconazole observed in Figure 2 were baseline resolved into four individual peaks (1-4). Interestingly, the two minor isomer peaks in Figure 2 were also resolved into four peaks (5-8) in a comparable manner, indicating a similar chirality for propiconazole and the minor components. 342 344 342 344 342 344 342 344 m/z 0.00 325.00 330.00 335.00 340.00 345.00 350.00 355.00 360.00 256.9 269.9 307.9 341.0 271.1 337.6 219.2 269.9 307.9 327.0 338.8 266.4 319.8 352.7 nm 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 342 344 342 344 342 344 342 344 m/z 0.00 325.00 330.00 335.00 340.00 345.00 350.00 355.00 360.00 268.8 268.8 268.8 268.8 nm 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 Minutes 1 2 3 4 1 2 3 4 5 6 7 5 6 7 8 8 AU 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Intensity 0.0 2.0x10 8 4.0x10 8 6.0x10 8 8.0x10 8 1.0x10 9 1.2x10 9 1.4x10 9 Minutes 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 1 2 3 4 1 2 3 4 5 6 7 8 7 0 7 7 7 7 8 8 8 8 8 8 8 4 2 0 2 4 6 0 2 4 6 8 7 7 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 PDA 220 nm 56 7 8 XIC m/z 342 5 6 7 8 PDA 220 nm QDa XIC m/z 342 Rs 1,2 = 2.42 Rs 2,3 = 2.28 Rs 3,4 = 2.40 Figure 3. ACQUITY UPC 2 UV chromatogram at 220 nm showing the chiral resolution of the propiconazole stereoisomers and unknown chiral components in the formulation sample using the ACQUITY UPC 2 Trefoil AMY1 column. The XIC of m/z 342 and the UV and MS spectra for each peak are also shown. Matrix components visible in the UV chromatogram and the MS total ion chroma- togram (TIC) of the formulation sample are clearly differentiated from the isomeric peaks of interest using an extracted ion chromatogram (XIC) (inset Figure 3). The detection sensitivity and selectivity of the method are improved when using mass detection in combination with UV detection which is used in order to measure the enantiomeric purity of the chiral pesticide in the formulation. Based on the observations, it is postulated that the minor component is a regioisomer of propiconazole. A regioisomer of propiconazole originating from one of the nitrogens on the triazole ring was characterised by Glaser et al. 2 Reproducibility Empower 3 can be used to assess the peak attributes with its system suitability function. Chromatographic parameters can be determined and statistical measurements can be made on them. In this example the RSDs for 10 determinations of the retention time, area, area % height and USP resolution were <0.61% for all of the propiconazole stereoisomers (Table 2). %RSD (n=10) Propiconazole Stereoisomers t R Area Area % Height USP Resolution Peak 1 0.37 0.44 0.09 0.61 Peak 2 0.33 0.39 0.05 0.52 0.43 Peak 3 0.27 0.37 0.05 0.57 0.29 Peak 4 0.22 0.37 0.04 0.61 0.21 Table 2. RSD for ten replicate injections of the propiconazole stereoisomers in the formulation sample. The UV spectra of peaks 3 and 4 resemble those for peaks 1 and 2 (Figure 2), indicating their structural similarity. In addition, the four peaks resulted in identical mass spectra with base peaks at m/z 342 and an isotopic pattern characteristic of dichlorinated compounds. The m/z matched the protonated propiconazole. Isolation and NMR Further experiments to characterise and isolate this compound for positive identification were carried out. Methods were developed to scale separations to SFC preparative chromatography, each minor stereoisomer was isolated followed by characterisation using 1H, 13C, 2D and NOE NMR spectroscopy. These experiments revealed that the minor isomer peaks differed from propiconazole itself by the the triazole nitrogen attachment point to the methylene group that is bridging to the dioxolane ring. The assigned structures for the all of the isolated components were in agreement with the information in the prior literature by Glaser et al. 2,3
