Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg6, D-Trp7,9, mePhe8]-substance P (6-11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate.
[Arg6, D-Trp7,9 mePhe8]-substance P (6-11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met11 (M2) and G[Met11(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (microBondapak C18, 30 cm X 2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40 degrees C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G-P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1-3 and G-P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1-3. Sample clean-up by solid-phase extraction using C2-bonded 40 microm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml-100 microg/ml) normally varied by < 10%, although at the highest concentrations of M1 and M2 studied (50 microg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.
Cummings
J
J
Imperial Cancer Research Fund, Medical Oncology Unit, Western General Hospital, Edinburgh, UK.
MacLellan
A J
AJ
Mark
M
M
Jodrell
D I
DI
D000970:Antineoplastic Agents / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D010446:Peptide Fragments / Q000097:blood*; D011721:Pyrenes; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013373:Substance P / Q000031:analogs & derivatives* / Q000097:blood
D000970:Antineoplastic Agents; D010446:Peptide Fragments; D011721:Pyrenes; C046958:substance P (6-11), Arg(6)-Trp(7,9)-Me-Phe(8)-; D013373:Substance P