Abstract:

Separation techniques such as capillary electrophoresis and liquid chromatography have found application in the analysis of biological samples. In capillary electrophoresis charged species are separated based on their mass to charge ratios under the influence of an applied electric field. In liquid chromatography, analytes are separated based on their partition between a stationary and mobile phase. Separation techniques when combined with a variety of detection techniques such as ultraviolet, laser induced fluorescence, mass spectrometry and electrochemical detection have been very successful in the quantification of analytes comprised in complex mixtures (e.g., biological samples).

Capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection was used to separate different bioconjugated CdSe/ZnS quantum dots (QDs). The QD nanocrystals studied were conjugated to the biomolecules streptavidin, biotin, and immunoglobulin G. The bioconjugated QDs showed different electrophoretic mobilities, which appear to depend upon the biomolecule that is attached to the QD and the buffer solution used. The use of a polymeric additive into the CE run buffer improved the resolution of the bioconjugates. Under CE conditions, the interaction between QD bioconjugates containing streptavidin (QDSt) and biotin (QDBi) was monitored. In a two color experiment, two different sizes of QD (i.e., 585 nm and 655 nm) were used to monitor the interaction between 655 nm QDBi and 585 nm QDSt. The use of QDs with different emission properties allows the selective monitoring of two different wavelengths while using one single excitation source. This in turn, allowed the monitoring of overlapping peaks in the electropherogram when newly formed products resulting from the interaction of the two bioconjugated QDs appeared.

Protease inhibitors (PIs) are a class of compounds that are used in the treatment of patients with the human immunodeficiency virus (HIV). These drugs inhibit the HIV-1 protease, acting on the production of viral proteins essential for the completion of the viral replication process. Monitoring free drug concentration is necessary to establish the dosage that would result in optimum free drug concentration in blood for a given patient. We developed methodology to analyze free protease inhibitor lopinavir in plasma samples. Ultrafiltration was used to isolate the free drug; then LC/MS/MS was used for sample analysis. Restrictive access media columns have been developed to simplify sample preparation processes. These columns allow the direct injection of untreated biological samples.

Restrictive access media columns exclude macromolecules such as proteins, while retaining small molecules such as drugs. Two restrictive access material columns were evaluated for the direct injection of plasma for analysis of the protease inhibitor lopinavir. In the columns studied the interaction of plasma components with the material inside the column was more than expected.

Glucose monitoring and insulin therapy are becoming a standard of care in patients in the intensive care unit. In order to control glycemic levels, frequent glucose monitoring is required. The use of tear fluid glucose concentrations was evaluated as a non-invasive sampling strategy for monitoring glucose in intensive care unit patients. Glucose in tear fluid samples from intensive care unit patients were analyzed using anion exchange chromatography with pulsed amperometric detection. The results did not show a correlation between tear fluid and blood glucose concentrations.