Isolating Trace Impurities for Structural Elucidation in a Commercial Fungicide Formulation Using Preparative SFC

Importance of the Topic

Reliable identification and structural elucidation of trace impurities in commercial formulations are critical for ensuring product safety, regulatory compliance, and environmental protection. Impurities at subpercent levels can impact efficacy, toxicity, and regulatory acceptance in industries such as agrochemicals, pharmaceuticals, food, and consumer goods. Preparative supercritical fluid chromatography (SFC) offers a rapid, efficient, and environmentally friendly workflow to isolate these minor components for detailed characterization.

Objectives and Study Overview

This study aimed to develop and demonstrate a preparative SFC–based workflow to isolate and structurally elucidate trace impurities present in a commercial propiconazole fungicide formulation. Key objectives included:

• Profiling both achiral and chiral impurity patterns using analytical SFC.
• Scaling up to preparative SFC to collect impurities at >98% purity.
• Performing NMR and high-resolution mass spectrometry (HRMS) for full structural assignment.
• Separating and confirming individual enantiomers of cis and trans isomeric impurities.

Methodology and Instrumentation

Sample Preparation:

• Liquid–liquid extraction of a 150 mL commercial formulation (1.55% propiconazole) using dichloromethane and 5% sodium bicarbonate.
• Drying over sodium sulfate and concentration to yield 4.5 g crude extract containing 2.3 g active ingredient plus impurities.

Analytical and Preparative SFC Conditions:

• Analytical achiral mode: ACQUITY UPC₂ BEH column, 6% methanol/CO₂, 120 bar, 1.5 mL/min, 40 °C, UV at 222 nm.
• Analytical chiral mode: ACQUITY UPC₂ Trefoil AMY1 column, 10% 2-propanol/ethanol in CO₂, 137 bar, 2.0 mL/min, 45 °C.
• Preparative achiral isolation: Viridis SFC 80q System with 19 × 150 mm BEH column, 5% methanol/CO₂, 70 mL/min, 120 bar.
• Preparative chiral isolation: Prep 100q SFC System with Chiralpak IC (cis isomers) or AD‐H (trans isomers) columns, methanol/CO₂ modifiers.

NMR and MS:

• High‐resolution MS: Waters SQ Detector 2 in ESI mode, m/z 200–700 scan.
• NMR: 1D, 2D COSY, HSQC, NOESY, and 1D NOE at 400 MHz on a Bruker Avance III spectrometer.

Results and Discussion

Analytical SFC revealed two minor impurity peaks (<1% of formulation) in both achiral and chiral modes. Preparative SFC enabled isolation of each impurity at >98% purity. NMR and HRMS data showed that both impurities share the propiconazole backbone but differ by the attachment point of the triazole ring to the dioxolane moiety, yielding symmetrical versus asymmetrical triazole substitution. NOE correlations distinguished cis (strong NOE between carbons 11, 14, and 6) from trans isomers. Subsequent preparative chiral separations on IC and AD‐H columns yielded all four stereoisomers (cis 2R,4S and 2S,4R; trans 2R,4R and 2S,4S) with >98% enantiomeric excess, allowing unambiguous stereochemical assignments.

Benefits and Practical Applications

This SFC‐based workflow provides:

• High speed and resolution for isolating trace impurities.
• Environmentally sustainable operation with reduced organic solvent usage.
• Rapid dry‐down and quick turnaround for purity confirmation.
• Access to sufficient quantities of pure isomers for HRMS and comprehensive NMR studies.
• Capability to characterize stereochemical variants to support efficacy and safety assessments.

Future Trends and Possibilities

Advancements in preparative SFC can expand its application across broader agrochemical and pharmaceutical impurity profiling, offering even faster separations, automated scaling, and integration with high‐throughput detection. Emerging chiral stationary phases and SFC–MS interfaces will further enhance the identification of structurally diverse trace components, driving innovation in quality control and regulatory science.

Conclusion

Preparative SFC coupled with HRMS and multidimensional NMR provides a robust, efficient, and sustainable approach for isolating and structurally elucidating trace impurities in commercial formulations. The workflow successfully isolated and characterized four stereoisomeric impurities of propiconazole, demonstrating its value for regulatory compliance, safety evaluation, and product development.

Reference

• Von Reet G, Heeres J, Janssen LW. Propiconazole workflow demonstration. US Patent 2:551–560, 1976.
• Glaser R, Adin I, Ovadia D, Mendler E, Drouin M. Solid‐state structure determination and solution‐state NMR characterization of propiconazole diastereomers and analogues. Structural Chemistry. 1995;6(3):145–156.
• Chen R. Mass‐directed preparative SFC: An orthogonal tool with reduced liquid solvent usage for high‐throughput purification. Waters Application Note 720003772EN, October 2010.