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LC-electrolyte effects in combination with pulse gradient chromatography enhance overall method performance of LC-MS-MS bioanalytical assays in supporting pharmacokinetic study for drug discovery

【摘要】:正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 re- mains a significant challenge.For analysis of complex bi- omatrices(e.g.plasma),liquid chromatography(LC) interfaced to mass spectrometry(MS)or tandem mass spectrometry(MS/MS)has been hampered by adverse ma- trix effects.For rapid assay development,it would be ben- eficial 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 precip- itation for sample clean-up and electrospray ionization (ESI)for detection,as well as using conventional short- time isocratic or gradient LC,yield fast assay develop- ment,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(HCOONH_4)or formic acid(HCOOH)increased analyte ESi response and controlled against matrix effects. We designated these favorable effects‘LC-electrolyte ef- fects’.These favorable effects can be achieved in either the positive or the negative ion ESI mode,but not for at- mospheric-pressure chemical ionization(APCI).The mag- nitude of the LC-electroiyte effect on the analyte response depends on both the concentration of the electrolyte modifi- er 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 sepa- ration 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 ge- neric and makes method development straightforward. In order to support rapid PK studies for drug discov- ery,we applied the LC-electro|yte effects and the pulse gradient chromatography to the development of generic pro- cedures 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 ap- plication 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(r~2>0.99),precision(RSD,0.45% -13.10%),and accuracy(91%-112% )were achie- ved through the use of a low dose of formic acid(0.4 mmol/L or 0.015‰)in the methanol/water-based LC mo- bile phase.The analytical methed was quite sensitive, providing a lower limit of quantification of 1.6 pg on-col- umn 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.

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