ABSTRACT
Tacrolimus is an immunosuppressive drug essential for preventing organ rejection after transplantation. Since tacrolimus strongly binds to erythrocytes, therapeutic monitoring requires its quantification in whole blood lyzate, representing one of the most difficult to analyze biological fluids due to its high protein load. In this communication, we report on the successful combination of whole blood hemolysis employing ionic liquids, followed by sample preparation by means of on-line solid phase extraction (SPE) using restricted access materials (RAM), which permitted the efficient removal of hemoglobin and other large biomolecules. Among six different tested RAM columns, highest hemoglobin depletion and analyte extraction efficiency was obtained with a polymer-based, glycoprotein-coated RAM stationary phase (Biotrap 500 MS) operated at an alkaline pH of 10.7. Analyte quantification was performed by high-performance liquid chromatography-selected reaction monitoring tandem mass spectrometry (HPLC-SRM-MS/MS). The ability to quantify tacrolimus in therapeutically relevant concentrations in whole blood hemolyzates was demonstrated via external calibration with lower limits of detection and quantification of 2.00 and 7.23 ng mL(-1), respectively. Moreover, the investigation of heparin-pretreated blood samples during blood sampling led to an increase in sensitivity for the analyte, while the method appeared to be more robust with ethylenediaminetetraacetic acid as anticoagulant.
Subject(s)
Erythrocytes/chemistry , Hemoglobins/chemistry , Immunosuppressive Agents/blood , Tacrolimus/blood , Adsorption , Calibration , Chromatography, High Pressure Liquid , Edetic Acid/chemistry , Hemolysis , Heparin/chemistry , Humans , Hydrogen-Ion Concentration , Ionic Liquids/chemistry , Limit of Detection , Solid Phase Extraction/methods , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Tissue Extracts/chemistryABSTRACT
OBJECTIVES: To study if functionalized ferromagnetic micro-particles used for extraction procedures prior to chromatographic analyses can be handled within an HPLC system. METHOD: We used C18-functionalized ferromagnetic micro-particles, manipulated within a high-performance liquid chromatography system for extraction of plasma samples, with a novel extractor device enabling reversible immobilization of the particles within HPLC tubing. A pilot analytical system for the quantification of itraconazole in plasma--as a representative small molecule analyte--by ultraviolet detection was configured and tested. RESULTS: The system allowed linear and reproducible quantification of the representative target analyte. CONCLUSIONS: Sample preparation of biological samples can be achieved by handling of functionalized ferromagnetic micro-particles which are handled within a high pressure liquid chromatography configuration; this offers interesting perspectives for the development of automated sample preparation devices.
Subject(s)
Chromatography, High Pressure Liquid/methods , Ferric Compounds/chemistry , Particle Size , Reproducibility of Results , Solid Phase ExtractionABSTRACT
OBJECTIVES: To study the applicability of functionalized ferromagnetic micro-particles in the sample preparation for quantification of small molecules by LC-MS/MS. DESIGN AND METHODS: A representative analyte (itraconazole) was extracted from plasma samples using small quantities of C18-modified ferromagnetic micro-particles. RESULTS: 125 microg of functionalized micro-particles was sufficient to extract the target analyte from 10 microL of plasma with a recovery rate of approximately 100%; linearity (r>0.99) and reproducibility (inter-assay CV< or =7%) of quantitative results was found. CONCLUSIONS: C18-functionalized ferromagnetic particles can be used to efficiently extract small molecule analytes from complex biological matrices for quantitative analyses using LC-MS/MS.
