title: Method development and validation for the quantitation of the complement inhibitor Cp40 in human and cynomolgus monkey plasma by UPLC-ESI-MS.
abstract: Cp40 is a 14-amino acid cyclic analog of the peptidic complement inhibitor compstatin that binds with sub-nanomolar affinity to complement component C3 and has already shown promise in various models of complement-related diseases. The preclinical and clinical development of this compound requires a robust, accurate, and sensitive method for quantitatively monitoring Cp40 in biological samples. In this study, we describe the development and validation of an ultra-high performance liquid chromatography electrospray mass spectrometry method for the quantitation of Cp40 in human and non-human primate (NHP) plasma. Isotope-labeled Cp40 was used as an internal standard, allowing for the accurate and absolute quantitation of Cp40. Labeled and non-labeled Cp40 were extracted from plasma using reversed phase-solid phase extraction, with recovery rates exceeding 80%, indicating minor matrix effects. The triply charged states of Cp40 and isotope-labeled Cp40 were detected at m/z 596.60 and 600.34, respectively, via a Q-TOF mass spectrometer and were used for quantitation. The method was linear in the range of 0.18-3.58μg/mL (r2≥0.99), with precision values below 0.71% in NHP and 0.77% in human plasma. The accuracy of the method ranged from -2.17% to 17.99% in NHP and from -0.26% to 15.75% in human plasma. The method was successfully applied to the quantitation of Cp40 in cynomolgus monkey plasma after an initial intravenous bolus of 2mg/kg followed by repetitive subcutaneous administration at 1mg/kg. The high reproducibility, accuracy, and robustness of the method developed here render it suitable for drug monitoring of Cp40, and potentially other compstatin analogs, in both human and NHP plasma samples during pharmacokinetic and pharmacodynamic studies.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Primikyri
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: Malvina
authors.initials: M
authors.lastname: Papanastasiou
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: Yiannis
authors.initials: Y
authors.lastname: Sarigiannis
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: Sophia
authors.initials: S
authors.lastname: Koutsogiannaki
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA.
authors.forename: Edimara S
authors.initials: ES
authors.lastname: Reis
authors.affiliation: Simian Conservation Breeding and Research Center (SICONBREC), Makati City, Philippines.
authors.forename: Joel V
authors.initials: JV
authors.lastname: Tuplano
authors.affiliation: Simian Conservation Breeding and Research Center (SICONBREC), Makati City, Philippines.
authors.forename: Ranillo R G
authors.initials: RR
authors.lastname: Resuello
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Sweden.
authors.forename: Bo
authors.initials: B
authors.lastname: Nilsson
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA. Electronic address: ricklin@upenn.edu.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ricklin
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, USA. Electronic address: lambris@upenn.edu.
authors.forename: John D
authors.initials: JD
authors.lastname: Lambris
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D051056:Complement Inactivating Agents / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D005260:Female; D006801:Humans; D016014:Linear Models; D008252:Macaca fascicularis; D008297:Male; D010456:Peptides, Cyclic / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D051056:Complement Inactivating Agents; D010456:Peptides, Cyclic; C111828:compstatin
keywords: Absolute quantitation; Compstatin; Method validation; Plasma; UPLC-ESI-MS