Quantitative analysis of multiply charged large molecules in human or rat plasma using liquid chromatography tandem mass spectrometry.

摘要

Immunoassays have traditionally been employed for the determination of plasma concentration-time profiles for pharmacokinetic studies of therapeutic proteins and peptides. These ligand binding assays have high sensitivity but require significant time for antibody generation (1 to 2 years) for assay development. Despite high sensitivity, these assays suffer from cross-reactivity that can lead to inaccurate results. As an alternative to immunoassays, this dissertation was focused on the development and validation of assays that can be used for quantitative analysis of peptides or proteins in plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS).;Two approaches were considered for measurement of proteins and peptides fortified in plasma. The first approach involved employing signature peptides as quantitative surrogates of a target protein. This approach is a multistep process that includes: computer simulated (in silico) peptide predictions, protein purification, proteolytic digestion, peptide purification, and ultimately mass spectrometry. Signature peptides were determined through in silico peptide predictions and iterative tuning processes to represent AmeviveRTM (Alefacept), a therapeutic for psoriasis, for quantification in human plasma. Horse heart myoglobin was chosen as a protein analogue internal standard to compensate for errors associated with matrix effects and to track recovery throughout the entire sample pretreatment process. Samples were prepared for analysis by selective precipitation of the target proteins with optimized pH and heat conditions followed by enzymatic digestion, dilution, and filtration. Combining selective precipitation and protein analogue internal standard lead to a method validated according to current FDA guidelines and achieved a linear range (250-10,000 ng/mL) suitable for monitoring the therapeutic levels of Alefacept (500 -6000 ng/mL) without the use of antibodies.;A second approach exploited the mass spectrometric behavior of intact polypeptides. A polypeptide can exist in multiple charge states separated by mass to charge ratio (m/z). Herein, the charge state distribution and the formation of product ions to form selected reaction monitoring (SRM) transitions for intact polypeptide quantitative analysis was evaluated in plasma. Oxyntomodulin, a 37 amino acid anorectic peptide (4449 Da), was employed as a model for analysis in rat plasma. The +7 charge state form of OXM was used to form an SRM for quantitative analysis. Two-dimensional reversed phase ion pair chromatography, a modified solid phase extraction, and a multiply charged SRM of oxyntomodulin enabled a lower limit of quantification of 1 ng/mL. Following development of the LC-MS/MS method, a validation of this approach was performed according to FDA guidelines.;Finally, to show further utility of LC-MS/MS, the validated oxyntomodulin method was used in a pharmacokinetic study with sprague-dawley rats. Rats were dosed with oxyntomodulin through intravenous or intratracheal instillation routes of administration. Plasma concentration-time profiles were determined. Using these profiles, noncompartmental parameters were determined for each dose and routes of administration.