Short communicationOn-line solid-phase extraction and determination of paclitaxel in human plasma
Introduction
The applications of clinical pharmacokinetics to oncologic studies are limited by the substantial efforts needed for the acquisition of reliable analytical data. It is well known that sample clean-up is a time consuming step, which is prone to errors when complex purification procedures are required. In addition to the inevitable loss of analyte during sample clean-up, methods without internal standard calibration may be challenging to standardize.
One possible alternative is the direct injection of plasma samples onto coupled-column liquid chromatographic systems combining on-line solid-phase extraction and analytical separation. Using a column-switch technique, the analyte is quantitatively adsorbed on a restricted access silica pre-column packing such as alkyl-diol silica (ADS), a spherical silica gel possessing two different chemical binding surfaces. The pore size of this material excludes plasma proteins, whereas the selective retention of lipophilic analytes occurs on the hydrophobic internal surface. In the next step, the analytes are transferred to the analytical column and monitored after separation. This method has been successfully applied to analyze a variety of drugs in biological fluids [1] including the enantioselective analysis of atenolol [2].
One of the first drugs analyzed by on-line solid-phase extraction were epirubicin and its metabolites by Rudolphi et al., who pioneered the use of ADS materials in clinical applications [3]. In general, the cytostatic compound epirubicin and other anthracyclines are analyzed in plasma after solid-phase extraction, solvent evaporation and injection onto a high-performance liquid chromatography (HPLC) system after reconstitution of the sample [4], [5].
The lipophilic nature of the anthracyclines is a common characteristic of several anticancer drugs and may thus be exploited for the on-line analysis from biological fluids. Since anthracyclines are easily monitored by fluorescence detection, we questioned the possibility of this approach to analyze paclitaxel. This drug is monitored in the low-UV range and has been analyzed in plasma using solid-phase extraction techniques [6], [7]. To prove the feasibility of the coupled-column liquid chromatographic analysis for paclitaxel in plasma, we present a reliable on-line technique to evaluate a drug characterized by (i) a moderate molar absorptivity, (ii) an absorption maximum in the low-UV range, i.e., 229 nm, which is (iii) administered in a low dose regimen (70 mg paclitaxel/m2) when compared with standard regimens (175 mg paclitaxel/m2 and higher), and (iv) a plasma protein binding >90%. Even under these conditions, coupled-column liquid chromatographic analysis of paclitaxel in plasma has proven to be an excellent tool for pharmacokinetic investigations.
Section snippets
Chemicals and reagents
Paclitaxel was obtained as a reference substance for chromatography (ICN Pharmaceuticals, CA, USA) and for therapeutic administrations (kindly supplied by EBEWE Pharmaceuticals, Austria). Water, acetonitrile, and methanol were of HPLC grade (LiChrosolv, Merck, Germany); ammonium acetate and glacial acetic acid were of analytical grade (Merck).
Solvent A was a mixture of methanol–water (5:95, v/v), solvent B was 20 mM ammonium acetate buffer (adjusted to pH 5.0 with glacial acetic
Sample loading
Plasma samples were loaded onto the C4-ADS clean-up column using 5% methanol in water (solvent A). Although concentrations up to 15% methanol in water did not elute paclitaxel from the C4-ADS material within 60 min, there was no difference in the elution profile of the plasma matrix when comparing 5, 10, and 15% methanol in water. To avoid the precipitation of plasma proteins, 15% methanol in water is the upper limit of organic solvent for the loading of human plasma as suggested by the
Conclusions
Coupled-column liquid chromatography allows for the on-line solid-phase extraction and analysis of paclitaxel in human plasma. Even under difficult circumstances such as low-dose regimens and unfavourable detection conditions, the merits of the described analytical method are a rapid evaluation combined with an excellent performance.
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