Determination of Log P for Compounds of Different Polarity Using the Agilent 1200 Infinity Series HDR‑DAD Impurity Analyzer System

Importance of the Topic

The partition coefficient (log P) provides a key measure of a compound’s lipophilicity, influencing absorption, distribution, metabolism and excretion in drug development. Reliable measurement of log P is critical for pharmaceutical research, environmental analysis and industrial quality control, as it informs formulation strategies and predicts bioavailability and toxicity.

Objectives and Study Overview

This application note demonstrates the determination of log P at physiological pH using the Agilent 1200 Infinity Series High Dynamic Range Diode Array Detector (HDR-DAD) Impurity Analyzer System. Four model drugs of varying polarity—testosterone, ibuprofen, paracetamol and pindolol—were analyzed to evaluate the system’s capability for quantifying compounds across widely differing concentrations in water and octanol phases.

Methodology and Instrumentation

Sample Preparation:

• Aqueous phases were buffered to pH 7.4 (physiological pH) and saturated with octanol; octanol phases were pre-saturated with water.
• Drug solutions were prepared at stock concentrations in both phases, followed by serial dilutions to cover high and low concentration ranges.
• After 24-hour equilibration, equal volumes of each phase were sampled for analysis.

Chromatographic Conditions:

• Column: Agilent ZORBAX RRHD Eclipse Plus C18, 4.6 × 50 mm, 3.5 μm
• Mobile phase: water (A), acetonitrile (B), 1 % TFA (C) with a gradient from 5 % to 90 % B over 8 min
• Flow rate: 1 mL/min; temperature: 30 °C; detection at 230 nm
• Injection volume: 1 µL

Instrumentation

• Agilent 1200 Infinity Series HDR-DAD Impurity Analyzer System
• Agilent 1290 Infinity DAD with 60 mm and 3.7 mm flow cells
• Agilent 1290 Infinity Quaternary Pump, Autosampler, Thermostat Column Compartment and Thermostat
• Agilent OpenLAB CDS ChemStation for data acquisition and evaluation

Results and Discussion

Calibration and Linearity:

• The HDR-DAD provided a linear dynamic range up to 6000 mAU, enabling quantitation from sub‐mAU levels (LOD around 0.41 mAU) to high absorbances (~8000 mAU).
• Calibration curves for all four compounds exhibited excellent linearity (correlation coefficients > 0.999).

Phase Distribution Analysis:

• Testosterone exhibited strong partitioning into octanol, with peak heights of ~700 mAU in octanol vs. < 0.5 mAU in water.
• Ibuprofen showed predominant octanol solubility (high mAU peaks), while pindolol favored the aqueous phase.
• Paracetamol distributed nearly equally between water and octanol.

log P Determination:
Calculated log P values at pH 7.4 closely matched literature data (e.g., testosterone ~ 3.28 vs. 3.29; ibuprofen ~ 1.27 vs. 1.37), demonstrating the method’s accuracy.

Practical Applications and Benefits

• Simultaneous quantitation of high and low concentration peaks without changing injection volumes
• Enhanced dynamic range reduces need for multiple dilutions or detector configurations
• Improved sensitivity supports accurate LOD measurement in trace analysis
• Applicable to pharmaceutical R&D, QA/QC, environmental monitoring and regulatory studies

Future Trends and Potential Applications

Advances in detector technology and integration with mass spectrometry are expected to further expand the dynamic range and compound coverage. Automation of log P workflows and coupling with online pH adjustment platforms may streamline high-throughput lipophilicity profiling. Additionally, adaptation to alternative solvent systems and greener HPLC methods may support sustainability goals.

Conclusion

The Agilent 1200 Infinity Series HDR-DAD Impurity Analyzer System provides a robust method for accurate log P determination over a wide concentration range. Its high dynamic range and low noise characteristics allow simultaneous quantitation of analytes in both aqueous and octanol phases without altering injection volumes, offering significant advantages for pharmaceutical and analytical laboratories.

Reference

1. Huesgen A.G. Comparison of sensitivity and linearity of the Agilent 1200 Infinity Series HDR-DAD and the conventional Agilent 1290 Infinity DAD. Agilent Technologies Application Note 5991-1890EN, 2013.
2. Kerns E.H. et al. Pharmaceutical profiling method for lipophilicity and integrity using liquid chromatography–mass spectrometry. Journal of Chromatography B, 2003, 791:381–388.
3. Wikipedia. Partition coefficient. http://en.wikipedia.org/wiki/Partition_coefficient (accessed February 10, 2014).