Capillary electrophoresis methodology for the analysis of biological samples
Herrmann, Jennifer Kim
MetadataShow full item record
Capillary electrophoresis (CE) is a microseparation technique used to separate a variety of bioanalytes in complex matrices. Charged solutes migrate under an applied electric field based on their charge-to-size ratios, making CE useful for separating many sizes and classes of biologically relevant molecules, including therapeutic drugs or peptides. Depending upon analyte properties and concentration, detection can be by ultraviolet absorbance (UV) or laser-induced fluorescence (LIF), which, combined with fluorescent-labeling, permits detection of biologically relevant species of interest at low nanomolar or picomolar levels. CE-UV with physiological-like buffer was used to study protein-drug interactions for HIV-therapy drugs. Interactions of three drugs, indinavir, saquinavir, and lopinavir, and two plasma proteins, human serum albumin and α-acid glycoprotein, were investigated by CE without sample preparation. These drugs and amprenavir were studied using ultrafiltration, the current clinical sample preparation for plasma sample analysis. Ultrafiltration sample preparation removes some of each free drug. Benzodiazepines are another drug class with medicinal interest. The concentrations of these drugs are low, thus sensitive analysis methods are required for their determination in biological samples. Three benzodiazepine drugs (midazolam, lorazepam, and flurazepam) were characterized for fluorescent properties with the intention of developing a new detection scheme applicable to CE methodology. Substance P (SP), an undecapeptide, is present in many biological fluids. Understanding the role of SP in biological matrices is challenging, because SP is found at low nanomolar or picomolar levels, making detection difficult. A CE-LIF method with field amplified stacking and pre-column derivatization of SP with 2,3-naphthalene dicarboxaldehyde was developed to determine SP in saliva. The method was used to analyze a saliva sample, finding a preliminary concentration of 35 nM SP. Immunoassays combined with CE-LIF methods were investigated for potential application to analyze estradiol in tears, since estradiol is an important estrogen related to fertility and disease states. Fluorescent signal amplification and competitive assays using fluorescently-labeled antigen and fluorescein-labeled estradiol, were investigated during the determination of a method compatible with low sample volumes and trace analyte levels. Different techniques are discussed and a preliminary detection limit of 75 pg/mL estradiol when using fluorescein-labeled estradiol in a competitive format.