采用高效液相色谱-质谱联用技术定量检测人体尿样中丙烯醛、苯、1,3-丁二烯、苯乙烯、巴豆醛的巯基尿酸类代谢物及氰化氢代谢物。将解冻后的尿样经高速离心后,取100μL上清液,加入50μL内标溶液及850μL水后上样分析。色谱分析采用XSELE...采用高效液相色谱-质谱联用技术定量检测人体尿样中丙烯醛、苯、1,3-丁二烯、苯乙烯、巴豆醛的巯基尿酸类代谢物及氰化氢代谢物。将解冻后的尿样经高速离心后,取100μL上清液,加入50μL内标溶液及850μL水后上样分析。色谱分析采用XSELECT HSS T3 C18色谱柱(150 mm×2.1 mm i.d,2.5μm),以乙腈-15 mmol醋酸铵溶液梯度洗脱,流速250μL/min,柱温40℃,在多反应监测模式(MRM)下采用负离子扫描模式进行定量分析。本方法共检测了16名吸烟者和6名非吸烟者的尿样,发现日吸烟量大于25支的吸烟者尿样中的烟气成分代谢物浓度高于非吸烟者。展开更多
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
文摘采用高效液相色谱-质谱联用技术定量检测人体尿样中丙烯醛、苯、1,3-丁二烯、苯乙烯、巴豆醛的巯基尿酸类代谢物及氰化氢代谢物。将解冻后的尿样经高速离心后,取100μL上清液,加入50μL内标溶液及850μL水后上样分析。色谱分析采用XSELECT HSS T3 C18色谱柱(150 mm×2.1 mm i.d,2.5μm),以乙腈-15 mmol醋酸铵溶液梯度洗脱,流速250μL/min,柱温40℃,在多反应监测模式(MRM)下采用负离子扫描模式进行定量分析。本方法共检测了16名吸烟者和6名非吸烟者的尿样,发现日吸烟量大于25支的吸烟者尿样中的烟气成分代谢物浓度高于非吸烟者。
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.
