Drug discovery calls for faster method development and high-throughput analysis in supporting drug metabolism and pharmacokinetic (PK) studies, whereas the rapid, sensitive, and accurate analysis of biological samples...Drug discovery calls for faster method development and high-throughput analysis in supporting drug metabolism and pharmacokinetic (PK) studies, whereas the rapid, sensitive, and accurate analysis of biological samples remains a significant challenge. For analysis of complex biomatrices (e.g. plasma), liquid chromatography (LC) interfaced to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) has been hampered by adverse matrix effects. For rapid assay development, it would be beneficial to improve the time-consuming method comparison and optimization steps by using generic procedures that work for a variety of compounds. However, injudiciously combining the generic procedures including protein precipitation for sample clean-up and electrospray ionization (ESI) for detection, as well as using conventional short-time isocratic or gradient LC, yield fast assay development, but often at the cost of decreased assay accuracy and increased risk of assay failure. We previously reported that the use of a mobile phase containing an extremely low concentration of ammonium formate (HCOONH4) or formic acid (HCOOH) increased analyte ESI response and controlled against matrix effects. We designated these favorable effects ‘LC-electrolyte effects’. These favorable effects can be achieved in either the positive or the negative ion ESI mode, but not for atmospheric-pressure chemical ionization (APCI). The magnitude of the LC-electrolyte effect on the analyte response depends on both the concentration of the electrolyte modifier added into the mobile phase and its identity, which is also analyte-dependent. In addition, LC is often optimized with more emphasis on improving the analytical sensitivity by concentrating the analyte on the LC column leading to a narrow and symmetric band and achieving sufficient separation between analytes and polar matrix components to avoid adverse ion suppression or enhancement of MS-MS detection. For these reasons, we proposed the so-called ‘pulse gradient system’ for conventional HPLC-based MS-MS analyses of complex biological samples, which is generic and makes method development straightforward. In order to support rapid PK studies for drug discovery, we applied the LC-electrolyte effects and the pulse gradient chromatography to the development of generic procedures that can be used to quickly generate reliable PK data for compound candidates. We herein demonstrate our approach using four model tested compounds (Compd-A,-B,-C, and-D). The analytical methods involve generic protein precipitation for sample clean-up, followed by application of fast LC gradients and the subsequent use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) for individual measurement of the tested compounds in 20 μL plasma samples. Good linearity over the concentration range of 1.6 or 8-25 000 ng/mL (r2>0.99), precision (RSD, 0.45%-13.10%), and accuracy (91%-112%) were achieved through the use of a low dose of formic acid (0.4 mmol/L or 0.015‰) in the methanol/water-based LC mobile phase. The analytical method was quite sensitive, providing a lower limit of quantification of 1.6 pg on-column except for Compd-C (8 pg), and showed negligible ion suppression caused by matrix components. Finally, the assay suitability was demonstrated in simulated discovery PK studies of the tested compounds with i.v./p.o. dosing to rats. This new assay approach has been adopted with good results in our laboratory for many recent discovery PK studies.展开更多
目的:建立一种液相色谱-质谱联用法,测定罂粟壳中吗啡、可待因、罂粟碱、蒂巴因、那可丁5种生物碱的含量。方法:采用液相色谱/质谱联用仪(三重串联四级杆质谱),色谱柱以十八烷基硅烷键合硅胶为填充剂(规格为2.1×100mm,5μm),以乙腈...目的:建立一种液相色谱-质谱联用法,测定罂粟壳中吗啡、可待因、罂粟碱、蒂巴因、那可丁5种生物碱的含量。方法:采用液相色谱/质谱联用仪(三重串联四级杆质谱),色谱柱以十八烷基硅烷键合硅胶为填充剂(规格为2.1×100mm,5μm),以乙腈-0.1%甲酸水(7:3)为流动相,流速0.4mL/min,柱温40℃。结果:5种生物碱在此条件下分离效果良好,吗啡、可待因在25~250ng/mL范围内,罂粟碱、蒂巴因、那可汀在5~50ng/mL范围内与其峰面积呈良好线性关系,相关系数r分别为0.99597、0.99686、0.99998、0.99905、0.99762。定量限(Limit of Quantitation, LOQ)分别为2.4×10-2mg/kg、2.4×10-2mg/kg、6×10-4mg/kg、6×10-4mg/kg、6×10-4mg/kg。回收率、重复性、稳定性均良好。结论:建立了一种液相色谱-质谱联用法,测定罂粟壳中吗啡、可待因、罂粟碱、蒂巴因、那可丁5种生物碱的含量,该方法高效快捷,分离效果好,定量准确稳定,为加强罂粟壳药材的质量监管提供技术支撑。展开更多
文摘Drug discovery calls for faster method development and high-throughput analysis in supporting drug metabolism and pharmacokinetic (PK) studies, whereas the rapid, sensitive, and accurate analysis of biological samples remains a significant challenge. For analysis of complex biomatrices (e.g. plasma), liquid chromatography (LC) interfaced to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) has been hampered by adverse matrix effects. For rapid assay development, it would be beneficial to improve the time-consuming method comparison and optimization steps by using generic procedures that work for a variety of compounds. However, injudiciously combining the generic procedures including protein precipitation for sample clean-up and electrospray ionization (ESI) for detection, as well as using conventional short-time isocratic or gradient LC, yield fast assay development, but often at the cost of decreased assay accuracy and increased risk of assay failure. We previously reported that the use of a mobile phase containing an extremely low concentration of ammonium formate (HCOONH4) or formic acid (HCOOH) increased analyte ESI response and controlled against matrix effects. We designated these favorable effects ‘LC-electrolyte effects’. These favorable effects can be achieved in either the positive or the negative ion ESI mode, but not for atmospheric-pressure chemical ionization (APCI). The magnitude of the LC-electrolyte effect on the analyte response depends on both the concentration of the electrolyte modifier added into the mobile phase and its identity, which is also analyte-dependent. In addition, LC is often optimized with more emphasis on improving the analytical sensitivity by concentrating the analyte on the LC column leading to a narrow and symmetric band and achieving sufficient separation between analytes and polar matrix components to avoid adverse ion suppression or enhancement of MS-MS detection. For these reasons, we proposed the so-called ‘pulse gradient system’ for conventional HPLC-based MS-MS analyses of complex biological samples, which is generic and makes method development straightforward. In order to support rapid PK studies for drug discovery, we applied the LC-electrolyte effects and the pulse gradient chromatography to the development of generic procedures that can be used to quickly generate reliable PK data for compound candidates. We herein demonstrate our approach using four model tested compounds (Compd-A,-B,-C, and-D). The analytical methods involve generic protein precipitation for sample clean-up, followed by application of fast LC gradients and the subsequent use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) for individual measurement of the tested compounds in 20 μL plasma samples. Good linearity over the concentration range of 1.6 or 8-25 000 ng/mL (r2>0.99), precision (RSD, 0.45%-13.10%), and accuracy (91%-112%) were achieved through the use of a low dose of formic acid (0.4 mmol/L or 0.015‰) in the methanol/water-based LC mobile phase. The analytical method was quite sensitive, providing a lower limit of quantification of 1.6 pg on-column except for Compd-C (8 pg), and showed negligible ion suppression caused by matrix components. Finally, the assay suitability was demonstrated in simulated discovery PK studies of the tested compounds with i.v./p.o. dosing to rats. This new assay approach has been adopted with good results in our laboratory for many recent discovery PK studies.
文摘目的:建立一种液相色谱-质谱联用法,测定罂粟壳中吗啡、可待因、罂粟碱、蒂巴因、那可丁5种生物碱的含量。方法:采用液相色谱/质谱联用仪(三重串联四级杆质谱),色谱柱以十八烷基硅烷键合硅胶为填充剂(规格为2.1×100mm,5μm),以乙腈-0.1%甲酸水(7:3)为流动相,流速0.4mL/min,柱温40℃。结果:5种生物碱在此条件下分离效果良好,吗啡、可待因在25~250ng/mL范围内,罂粟碱、蒂巴因、那可汀在5~50ng/mL范围内与其峰面积呈良好线性关系,相关系数r分别为0.99597、0.99686、0.99998、0.99905、0.99762。定量限(Limit of Quantitation, LOQ)分别为2.4×10-2mg/kg、2.4×10-2mg/kg、6×10-4mg/kg、6×10-4mg/kg、6×10-4mg/kg。回收率、重复性、稳定性均良好。结论:建立了一种液相色谱-质谱联用法,测定罂粟壳中吗啡、可待因、罂粟碱、蒂巴因、那可丁5种生物碱的含量,该方法高效快捷,分离效果好,定量准确稳定,为加强罂粟壳药材的质量监管提供技术支撑。
