title: LR12-peptide quantitation in whole blood by RP-HPLC and intrinsic fluorescence detection: Validation and pharmacokinetic study.
abstract: A simple, sensitive, selective and robust HPLC method based on intrinsic fluorescence detection was developed for the quantitation of a dodecapeptide (designated as LR12), inhibitor of Triggering Receptor Expressed on Myeloid cells-1, in rat whole blood. Sample treatment was optimized using protein precipitation and solid-phase extraction. Chromatographic separation was carried out in a gradient mode using a core-shell C18column (150 × 4.6 mm, 3.6 μm) with mobile phases of acetonitrile and water containing trifluoroacetic acid at 1.0 mL/min. The method was validated using methodology described by the US Food and Drug Administration guidelines for bioanalytical methods. Linearity was demonstrated within the 50-500 ng/mL range and the lower limit of quantitation was 50 ng/mL. Finally, a preliminary pharmacokinetic study after intraperitoneal injection of LR12 in rats was conducted to evaluate both LR12 monomer and its corresponding disulfide dimer, the main product of degradation. Beyond the fact that this paper describes the first fully validated method for LR12 analysis in blood samples, the approach followed here to optimize pre-analytical steps could be beneficial to develop HPLC and/or MS methods for other pharmaceutical peptides.
authors: CITHEFOR, Université de Lorraine, EA 3452, Nancy, France.
authors: M
authors: M
authors: Parent
authors: CITHEFOR, Université de Lorraine, EA 3452, Nancy, France.
authors: A
authors: A
authors: Boudier
authors: CITHEFOR, Université de Lorraine, EA 3452, Nancy, France.
authors: P
authors: P
authors: Maincent
authors: INSERM U1116, Université de Lorraine, Vandœuvre-lès-Nancy, France.
authors: S
authors: S
authors: Gibot
authors: INSERM UMR S970, PARCC, France.
authors: H
authors: H
authors: Ait-Oufella
authors: INOTREM, Vandœuvre-lès-Nancy, France.
authors: A
authors: A
authors: Boufenzer
authors: INSERM U1116, Université de Lorraine, Vandœuvre-lès-Nancy, France.|INOTREM, Vandœuvre-lès-Nancy, France.
authors: L
authors: L
authors: Jolly
authors: INOTREM, Vandœuvre-lès-Nancy, France.
authors: M
authors: M
authors: Derive
authors: Université de Lille 2, Centre Universitaire de Mesures et d'Analyses (CUMA), Lille, France.
authors: M
authors: M
authors: Kouach
authors: Université de Lille 2, Centre Universitaire de Mesures et d'Analyses (CUMA), Lille, France.
authors: J-F
authors: JF
authors: Goossens
authors: CITHEFOR, Université de Lorraine, EA 3452, Nancy, France.
authors: P
authors: P
authors: Leroy
authors: CITHEFOR, Université de Lorraine, EA 3452, Nancy, France.
authors: I
authors: http://orcid.org/0000-0001-9083-601X
authors: I
authors: Clarot
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D007274:Injections, Intraperitoneal; D057230:Limit of Detection; D008297:Male; D010455:Peptides / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics*; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides
keywords: RP-HPLC; TREM-1; bioanalytical validation; intrinsic fluorescence detection; peptide