Assay of the Drug SubstanceAnthralin Using the ACQUITY UPC2 System

Significance of the Topic

Anthralin is a pharmacologically active compound used in dermatological treatments. The United States Pharmacopeia (USP) prescribes a normal phase HPLC assay for its quantification, which relies on high volumes of non-polar organic solvents. Transitioning to supercritical fluid chromatography (SFC) can significantly reduce solvent consumption, costs, and environmental impact while maintaining or improving analytical performance.

Objectives and Study Overview

This study aimed to convert the USP compendial normal phase HPLC assay for anthralin into a supercritical fluid chromatography method using the Waters ACQUITY UPC2 system. Specific goals included:

• Achieve equivalent or superior chromatographic quality compared to the USP method
• Reduce analysis time and solvent usage
• Maintain key performance metrics such as resolution, reproducibility, and peak symmetry

Methodology and Instrumentation

A single anthralin sample was analyzed by both the USP HPLC method and the UPC2 method. Comparative performance data were collected under the following conditions:

• Column: Viridis™ Silica 2-Ethylpyridine, 4.6 x 150 mm, 5 μm
• Temperature: 40 °C
• Mobile Phase: 95% CO₂ / 5% methanol with 0.25% glacial acetic acid
• Flow Rate: 3.5 mL/min
• Back Pressure: 150 bar
• Detection: UV/PDA at 351 nm

Applied Instrumentation

The conversion utilized the Waters ACQUITY UPC2 system configured for UltraPerformance Convergence Chromatography. The system’s high-pressure CO₂ delivery, precise back-pressure regulator, and UV/PDA detector enabled rapid, reproducible separations under supercritical conditions.

Main Results and Discussion

Key suitability parameters met or exceeded USP requirements:

• Retention times decreased from 2.10–5.04 min (HPLC) to 1.64–2.17 min (UPC2)
• Resolution improved significantly (e.g., from ≥3.0 to 8.9 for suitability solution)
• Peak tailing factors stayed well below USP limits (≤1.05)
• Reproducibility of retention times and area counts showed relative standard deviations below 0.8%

Solvent consumption saw a drastic reduction: each HPLC run consumed 16.4 mL of hexane and 1.2 mL of dichloromethane, while UPC2 used only 1.05 mL of methanol. At current market prices, per-run solvent costs dropped from $0.90 to $0.05. The UPC2 method was 1.6 times faster, boosting throughput.

Benefits and Practical Applications of the Method

The UPC2 assay offers multiple advantages:

• Lower environmental footprint and waste disposal requirements
• Reduced exposure to hazardous solvents, improving lab safety
• Decreased operational costs per analysis
• Higher sample throughput supporting quality control and research workflows

Future Trends and Potential Uses

Expanding the adoption of supercritical fluid chromatography aligns with green analytical chemistry initiatives. Future developments may include:

• Broader replacement of normal phase HPLC assays for other active pharmaceutical ingredients
• Integration with mass spectrometry for enhanced detection sensitivity
• Automation and high-throughput screening using 96-well plate formats
• Method standardization across pharmacopoeias

Conclusion

The conversion of the USP anthralin assay from normal phase HPLC to UPC2 on the ACQUITY UPC2 system successfully delivered equal or superior analytical performance in a shorter time frame and at a significantly reduced solvent cost. The method supports laboratory efficiency, sustainability, and regulatory compliance.

Reference

Waters Corporation. February 2012. “Assay of the Drug Substance Anthralin Using the ACQUITY UPC2 System.” 720004236EN LL-PDF.