Investigations into the utilization of internal surface reversed-phase chromatography for the analysis of drugs in serum
Abstract
The chromatographic analysis of drugs in serum is an important part of therapeutic drug monitoring (TDM) programs. Since direct injection of serum samples onto standard HPLC columns often results in clogging, a sample preparation step designed to remove proteins is usually incorporated into the analysis. This step is frequently the most time consuming part of the assay. Recently, a new type of chromatographic column, the Internal Surface Reserved-Phase (ISRP) column, was synthesized. This material allows for the direct injection of serum samples, without prior protein removal. The packing material was fashioned with the hydrophobic bonded-phase confined to the internal surface of the silica-based support, while the external surface remains hydrophilic. Pore size is controlled so as to exclude the serum protein molecules, thus eluting them unretained. Smaller analytes, such as drugs, are free to penetrate into the packing material where they undergo separation via partitioning with the internal phase. This dissertation examines the ISRP column in terms of its performance, and presents several applications. The general operating parameters associated with direct serum injection on ISRP columns are discussed. Included is a determination of concentration ranges in which organic modifiers may be used without causing protein precipitation. The efficiency and backpressure of ISRP columns are monitored with repetitive serum injections, and advantages of using more biocompatible materials (titanium and Teflon) in place of the stainless steel inlet frits is investigated. An assay for the determination of the antiarrhythmic drugs propranolol and quinidine in serum is described, utilizing fluorescence detection methods. A set of antiepileptic drugs in serum are also analyzed on an ISRP column, and phenytoin, along with two of its metabolites, are separated in a urine matrix. The protein-binding parameters of phenytoin are determined by HPLC analysis of plasma ultrafiltrates using a 3-$\mu$m ODS column. Examination of a method to isolate free drug fractions by direct sample injection on ISRP columns is also discussed.
Degree
Ph.D.
Advisors
Pinkerton, Purdue University.
Subject Area
Analytical chemistry
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