Simultaneous determination of free concentration, total concentration, and plasma binding capacity of bioactive compounds
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Issue Date
2020-09
Authors
Cibotaru, Dorina '20
Degree
MS in Pharmaceutical Sciences
Advisor
Musteata, F. Marcel
Committee Members
Kane, Michael
Shah, Manish
Shah, Manish
Journal Title
Journal ISSN
Volume Title
Abstract
Free drug concentrations are well regarded as superior markers of pharmacological effect compared to total drug concentrations but are not used as often. Additionally, understanding drug-protein binding relationships is important for preclinical predictions of drug toxicity in humans and distribution into tissues. The current methods used for the determination of free concentration and drug-protein binding are limited to research or reference labs due to time constraints and availability of specialized machinery. A new proposed experimental method uses isotopically labeled analogues added to a sample and can be used to calculate free and total compound concentration and plasma binding capacity. Another involves multiple sequential analysis of a sample and can be used to calculate the total compound concentration and plasma binding capacity from measured free compound concentrations. Free analytes are separated using microextraction and ultrafiltration devices and measured using mass spectrometry. Testosterone and phenytoin are used as reference compounds in determining free fractions and plasma binding capacity due to their wide-spread usage as pharmacotherapy and high protein binding (\342\211\24590%). The normalization of free concentrations from patient samples using individual protein concentration and amount of compound bound to each protein can be used to help with the interpretation of biomarker concentrations in cases of variable protein binding. Resulting accuracy of analyte recovery from human serum albumin, volunteer plasma, and volunteer human serum samples support the use of SpinTip devices for microextraction and the use of Centrifree devices for ultrafiltration based on analyte recoveries between 90 and 100%, using both. Normalized compound serum concentrations were calculated, and the use of these methods may provide more insight about collected patient data. Sensitivity and uncertainty analyses reveal LCMS analysis to be the largest source of input uncertainty when calculating initial total testosterone concentrations and plasma binding capacity. Results obtained with patient samples support the use of SpinTips (R2=0.9954) and Centrifree (R2=0.9325) devices with labeled analytes for analysis of testosterone concentrations. The accuracy from using SpinTip devices was superior and the method using isotopically labeled testosterone resulted in very good accuracy for patient plasma samples, supporting the use of this method for further clinical applications.
Citation
Cibotaru D. Simultaneous determination of free concentration, total concentration, and plasma binding capacity of bioactive compounds [thesis]. Ann Arbor (MI): Proquest LLC; 2020. 67 p.
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