Biotransformation Department, Bristol-Myers Squibb Research and Development, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534, USA.
Rapid Commun Mass Spectrom. 2011 Nov 15;25(21):3245-51. doi: 10.1002/rcm.5220.
An early assessment of metabolite exposure in preclinical species can provide quantitative estimation on possible active or toxic metabolites. Frequently, synthetic metabolite standards are not available at the preclinical stage, precluding the quantitation of metabolites by means of calibration curves and quality control (QC) samples. We present here an approach to determine the extent of circulating metabolites using 'metabolite standards' generated by in vitro incubations in combination with the correction for mass spectrometry response based on UV response. The study was done by coupling ultra-high-performance liquid chromatography (UHPLC) to LTQ-Orbitrap high-resolution mass spectrometry, and the quantitation was based on full scan high-resolution accurate mass analysis in combination with retention time. First, we investigated the separation capacity of a 10.5 min UHPLC method and the quantitative capability of an LTQ-Orbitrap for full scan accurate mass quantitation by spiking chemical standards of buspirone and its six metabolites in blank plasma. Then we demonstrated the use of a UV correction approach to quantitatively estimate buspirone and its metabolites in plasma samples from a rat pharmacokinetics study. We compared the concentration versus time profiles of buspirone and its six metabolites in rat plasma samples obtained using three different approaches, including using UV correction, using individual standard curves for each metabolite prepared from the synthetic standard, and using a calibration curve of the parent compound buspirone. We demonstrated the estimated metabolite exposure of buspirone using this UV correction approach resulted in rank ordering of metabolite exposure within three-fold of the value obtained with metabolite standards, in contrast to eight-fold without UV correction. The approach presented in this paper provides a practical solution to an unmet bioanalytical need for quantitative information on metabolites without standards in preclinical in vivo studies.
在临床前物种中对代谢物暴露进行早期评估，可以对可能的活性或毒性代谢物进行定量估计。通常，在临床前阶段无法获得合成代谢物标准品，这使得无法通过校准曲线和质控 (QC) 样品来定量代谢物。我们在此介绍了一种使用体外孵育生成的“代谢物标准品”来确定循环代谢物程度的方法，并结合基于紫外 (UV) 响应的质谱响应校正来进行定量。该研究通过超高效液相色谱 (UHPLC) 与 LTQ-Orbitrap 高分辨率质谱联用进行，定量基于全扫描高分辨率精确质量分析结合保留时间。首先，我们考察了 10.5 分钟 UHPLC 方法的分离能力和 LTQ-Orbitrap 对全扫描精确质量定量的定量能力，方法是在空白血浆中加入丁螺环酮及其 6 种代谢物的化学标准品进行加标。然后，我们展示了使用 UV 校正方法定量估计丁螺环酮及其在大鼠药代动力学研究中血浆样品中的代谢物。我们比较了使用三种不同方法（包括使用 UV 校正、使用从合成标准品制备的每个代谢物的单独标准曲线、以及使用母体化合物丁螺环酮的校准曲线）获得的丁螺环酮及其 6 种代谢物在大鼠血浆样品中的浓度-时间曲线。我们证明，使用这种 UV 校正方法估计丁螺环酮的代谢物暴露量，其代谢物暴露量的排序与使用代谢物标准品获得的值相差三倍，而不进行 UV 校正则相差八倍。本文介绍的方法为临床前体内研究中定量代谢物信息而无标准品的未满足的生物分析需求提供了一种实用的解决方案。
