Determination of Nanogram Levels of Peptide Drug in Rabbit and Human Plasma Using High-Performance Liquid Chromatography Coupled with Electrospray Ionization Mass Spectrometry

Abstract

High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) using an electrospray ionization (ESI) interface provides a sensitive method for the quantitative analysis of peptide drugs in complex biological matrixes. ESI HPLC-MS was applied to the analysis of a pentapeptide drug (IRI-514) in rabbit and human plasma. Prior to analysis, the plasma samples were prepared using protein precipitation followed by solid-phase extraction. The lower limit of quantitation using selected ion monitoring was determined to be 2 ng/mL, when 8 mL of human plasma spiked with 1-40 ng/mL was extracted. Rabbit plasma (1 mL) samples spiked with 10-40,000 ng of authentic drug/mL gave a linear response when a deuterated peptide analog was employed as an internal standard. A commercial ESI interface was modified to permit higher flow rates (10-20 microL/min) to enter the mass spectrometer source. The revised interface provided a 10-fold increase in sensitivities and permitted the use of standard HPLC columns (2.0-mm i.d.) and HPLC instrumentation. ESI HPLC-MS analysis was automated to provide unattended, precise, and sensitive detection of small peptides in both human and rabbit plasma. Using this methodology, a toxicokinetic study of intravenously administered IRI-514 at three dose levels indicated that the area under the curve values were dose proportional.

Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry

“…ESI response can also be affected by the percentage of aqueous or organic modifier in the mobile phase. For the quantification of the pentapeptide drug IRI-514, the post-column addition of organic modifier has been described, as sensitivity was reduced by the highly aqueous mobile phase [79]. Also co-eluting matrix components can be a reason of suppression of the ionization of the analyte in the ionization source, showing the importance of good sample preparation and chromatographic separation prior to MS detection.…”

Hyphenated chromatographic methods for biomaterials

“…They used an API ionspray source MS for the analysis of phospholipids after separation on a silicapacked column using a mixture of chloroform:methanol: water (60:55:5 v/v/v). A synthetic acid and enzymatic hydrolysis resistant pentapeptide (Ac-Arg-Pro-Asp-ProPhe-NH 2 ) developed for treatment of depression and anxiety was analyzed by HPLC-ESI-MS (Crowther et al, 1994). In addition, ESI-MS is extensively used for protein and peptide characterization.…”

Mass Spectrometry in Pharmaceutical Analysis

“…For a small peptide such as thymopentin (Arg-Lys-Asp-Val-Tyr), it can be as low as 30 s. The limited bioavailability of orally administered peptides is due in large part to enzymatic and acid hydrolysis in the gut and stomach. 7,8 Resistance to hydrolysis may be improved by modification of the amino-and carboxyl-terminii. Even so, methods that provide rapid processing of peptides in physiological fluids for analysis are needed for pharmacokinetic studies where the concentration of peptides is low at the start of extraction.…”

“…Peptides, however, generally suffer from poor pharmacokinetics after oral administration, which is generally due to the short half-life observed for many peptides. 7 This is often controlled through modification of the structure to prevent rapid digestion by proteolytic enzymes present in the gut and circulatory system. 8 MS now is also used for routine sequencing of proteins for the drug discovery process.…”