Electrospray LC-MS/MS quantitation, stability, and preliminary pharmacokinetics of bradykinin antagonist polypeptide B201 (NSC 710295) in the mouse.
B201 (NSC 710295), [SUIM-(Darg-Arg-Pro-Hyp-Gly-Igl-Ser-Digl-Oic-Arg)(2)], a third generation of bradykinin (BK) antagonist, has been found to possess high potency. We report the development of a highly sensitive electrospray LC-MS/MS assay method for the analysis of B201 in plasma for the first time, using an ion-trap mass spectrometer. Human or mouse plasma (0.2 ml) was spiked with B201 and the internal standard, substance P. The compounds were extracted with a preconditioned C-18 reversed-phase column and analyzed by LC-MS/MS. The analytes were separated on a 50 x 2 mm (i.d.) BetaBasic C8 column, using a gradient elution. The positive ion selected reaction monitor mode was used monitoring the transitions of ions at m/z 938.9(3+)-->816.0(2+) for B201 and 674.3(2+)-->665.7(2+) for substance P. Assay validation was performed, and the limit of quantitation (LOQ) for B201 was found to be 1 ng/ml for human plasma and 2.5 ng/ml for mouse plasma. The recovery was 78% for B201 and 88% for substance P. The assay was linear from 2.5 to 1500 ng/ml for mouse plasma monitored. Using a 0.2 ml plasma, the within-day CVs were 9.3% at 2.5 ng/ml, 6.5% at 100 ng/ml, and 3.8% at 1000 ng/ml for human plasma (n=6). For mouse plasma, the respective within-day CVs were 17.6, 9.6, and 6.2% (n=6). The between-day CVs for human plasma were 8.2, 10.9, and 2.4%, respectively, (n=3) and the respective values for mouse plasma were 11.9, 8.6 and 6.5% (n=6). Pharmacokinetics of B201 in the mouse was studied following i.v. administration at 5 mg/kg and found to conform to a two-compartment model with an initial half-life of 14 min and a terminal half-life of 44 h. Plasma B201 peak level was detected at microg/ml range and the levels were detectable for a least 24 h. Preliminary oral bioavailability was found to be about 1%. This method demonstrates that an ion trap mass spectrometer can be a powerful tool to quantify large peptides at low nanogram per milliliter with a non-isotopically labeled internal standard.
Feng
Wan Yong
WY
College of Pharmacy The Ohio State University, Columbus, OH 43210, USA. wyfeng2k@yahoo.com
Chan
Kenneth K
KK
Covey
Joseph M
JM
D000818:Animals; D001920:Bradykinin / Q000037:antagonists & inhibitors*; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D006801:Humans; D007275:Injections, Intravenous; D013058:Mass Spectrometry; D051379:Mice; D010455:Peptides / Q000032:analysis* / Q000493:pharmacokinetics*; D015203:Reproducibility of Results; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization; D013373:Substance P / Q000032:analysis
D002021:Buffers; C460096:NSC 710295; D010455:Peptides; D012996:Solutions; D013373:Substance P; D001920:Bradykinin