Evaluation of Immobilized Liquid Extraction (ILE) of Small Molecules from Whole (undiluted) Serum

This study is currently being conducted, and the final results will be presented at AAPS (San Diego, Nov, 2007), and published here upon its completion.

Session Date: Wednesday, November 14
Session Time: 08:00 am - 12:00 pm
Location: Exhibit Hall, San Diego Convention Center

Purpose: To evaluate and validate Immobilized Liquid Extraction as a method for extracting small molecules directly from whole (undiluted) goat serum in order that these compounds be determined and quantified by GC/MS analysis.

ILE is a technique for preparing aqueous samples for chromatographic analysis. It is fundamentally similar to traditional liquid-liquid extraction; however, a polymer that is immobilized on the surface of an ILE device, rather than an ‘organic solvent’, is used to extract compounds from a sample. Compounds are eluted from the polymer into GC or HPLC solvent.

Methods: ILE 96-Well Plates with polydimethylsiloxane (PDMS) and polyacrylate (Acrylate) extracting phases were utilized in this method. 100?L whole (undiluted) normal goat serum samples were spiked at concentrations of 25-20,000 ng/mL with imipramine and flurazepam. Sample matrix pH adjustments were made to observe extractive charactistics of each phase over a wide range of pHs (1.68-11.0). Analytes were eluted into a 70:30 acetonitrile:methanol mixture, which was used as the injection solvent. Extracts were analyzed by an HP-5971 GC/MS in SIM mode.

Results:

Extraction efficiencies of 95.4 +/- 3.2% and 94.8 +/- 5.1% were achieved for imipramine and flurazepam, respectively using a polyacrylate plate at optimal sample pH. The PDMS phase exhibited much lower extraction efficiencies of the two polar drugs at nearly all pHs, with maximums achieved at pH 8 for both imipramine and flurazepam, which had efficiencies of 53.8 +/- 3.9% and 11.2 +/- 0.8% respectively. All sample extracts were free of matrix interferants.

Conclusion: Immobilized Liquid Extraction shows promise as a technique for extracting small molecules from biological samples. The polymer phases showed extractive characteristics similar to traditional solvents over the entire range of pHs, without losing stability at pH extremes. Differences in the polarity of each phase were reflected by the drastic discrepancies in extraction efficiencies. The high extraction efficiencies exhibited by the polyacrylate phase indicate that the extracting phase is unaffected by matrix viscosity (undiluted serum).