The analysis of biological samples using capillary electrophoresis and HPLC technologies
Dunkle, Melissa Nichole
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Capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) are two very important separation techniques. CE is a microseparation technique in which an electric field is applied to perform the separation. In CE, charged species are separated based on their charge/mass ratio, making this technique useful for separating a wide variety of components, including those found in biological samples. HPLC is the most widely used separation technique due to the ease of use and wide variety of components that can be separated. HPLC employs the use of pressure to push mobile phase through an analytical column containing a stationary phase that interacts with the solutes to effect the separation. Both CE and HPLC can use a number of different detection schemes to monitor the separated species, including ultra-violet (UV), laser-induced fluorescence (LIF), and mass spectrometry (MS). Affinity capillary electrophoresis (ACE) combines the separation power of CE with the selectivity of immunoassays. A fluorescein labeled estradiol derivative was evaluated for potential use as a competitive inhibitor in ACE. The labeling procedure rendered multiple components with the fluorescein label. Two of the components were identified as active estradiol species against the anti-estradiol antibody, examined by CE-LIF. Since only one active species had been predicted, both species were characterized using CE-MS and MS/MS, in addition to testing them in the ACE format. Nucleoside reverse transcriptase inhibitors (NRTIs) are one class of anti-HIV (human immunodeficiency virus) drugs. Because HIV is considered a pandemic, the analysis of NRTIs in human samples is very important; people all over the world are using these drugs. Currently, multiple NRTIs are given for treatment; however, there are few methods to date that separate and detect multiple NRTIs in a single run. NRTIs are metabolized in the body into their active triphosphate form. Two approaches can be followed for their separation and detection. The first approach was an indirect method, in which the active triphosphate form was dephosphorylated into the parent form of the NRTIs. HPLC with UV detection was utilized to establish an optimized separation method prior to using LC-MS/MS for the analysis of the following NRTIs in human peripheral blood monunclear cells (PBMCs): stavudine (D4T), lamivudine (3TC), the metabolized form of didanosine (ddA), zidovudine (ZDV), and the internal standard. The second approach was a direct method, in which analysis of the triphosphate form of the NRTIs was performed without dephosphorylation of the drugs. A capillary eletrophoretic method was developed that allows separation of the triphosphate NRTIs of interest: zidovudine triphosphate (ZDV-TP), stavudine triphosphate (D4T-TP), lamivudine triphosphate (3TC-TP), 2',3'-dideoxyadenoine triphosphate (ddA-TP), tenofivir diphosphate (TFV-DP), and emtricitabine triphosphate (FTC-TP). The separation was optimized using UV detection. Since UV detection does not offer appropriate detection limits, we studied the native fluorescence spectroscopic characteristics of the triphosphate NRTIs. We constructed an in-house CE-LIF system to explore LIF detection of the phosphorylated NRTIs. The system employed the use of a deep-UV laser with excitation at 224 nm. A sheath-flow cuvette was integrated into the design to improve detectability. The CE-LIF system was aligned and tested using the native fluorescence of tryptophan. The CE-LIF system was then used for the analysis of the active metabolized forms of NRTIs. With this system, we were able to separate and detect ZDV-TP, D4T-TP, 3TC-TP, ddA-TP, and the internal standard using a modification of the separation conditions previously optimized with UV detection. The detection limits obtained are not sufficiently low for the analysis of human PBMCs.