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.

中文翻译：

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缓激肽拮抗剂多肽B201（NSC 710295）在小鼠中的电喷雾LC-MS / MS定量，稳定性和初步药代动力学。

已发现第三代缓激肽（BK）拮抗剂B201（NSC 710295），[SUIM-（Darg-Arg-Pro-Hyp-Gly-Igl-Ser-Digl-Oic-Arg）（2）]高效力。我们首次使用离子阱质谱仪报告了用于血浆中B201分析的高灵敏度电喷雾LC-MS / MS分析方法的开发。在人或小鼠血浆（0.2 ml）中加入B201和内标物质P。将化合物用预处理的C-18反相柱萃取并通过LC-MS / MS分析。使用梯度洗脱在50 x 2 mm（id）BetaBasic C8色谱柱上分离分析物。使用正离子选择的反应监测器模式监测B201的m / z 938.9（3 +）-> 816.0（2+）和P物质674.3（2 +）-> 665.7（2+）的离子跃迁进行了验证试验，B201的定量限（LOQ）对人血浆为1 ng / ml，对小鼠血浆为2.5 ng / ml。B201的回收率为78％，P物质的回收率为88％。对于监测的小鼠血浆，该测定法的线性范围是2.5至1500 ng / ml。使用0.2 ml血浆，人体血浆（n = 6）的日内CV为2.5 ng / ml时的9.3％，100 ng / ml时的6.5％和1000 ng / ml时的3.8％。对于小鼠血浆，当日内CV分别为17.6％，9.6％和6.2％（n = 6）。人血浆的日间CV分别为8.2％，10.9％和2.4％（n = 3），小鼠血浆的日间CV值分别为11.9％，8.6％和6.5％（n = 6）。在以5 mg / kg静脉给药后研究了B201在小鼠中的药代动力学，发现其符合两室模型，初始半衰期为14分钟，终末半衰期为44 h。在microg / ml范围内检测到血浆B201峰值水平，并且该水平至少检测24小时。初步的口服生物利用度约为1％。该方法表明，离子阱质谱仪可以成为使用非同位素标记的内标物以低纳克/毫升定量大肽的强大工具。