| During the quantitative analysis of two classes of special drug candidates,metal chelators and hydrogen sulfide donors,poor precision,low recovery and narrow quantitative linear range always occur due to their special physicochemical properties.Furthermore,the quantification of those drugs was usually based on derivatization methods which were time consuming and unrepeatable.In this paper,simple LC-MS/MS methods with strong specificity,high sensitivity,good repeatability,high sample throughput and low analysis costs were established for the determination of a uranium chelator,catechol-3,6-bis(methyleiminodiacetic acid?CBMIDA?,and the third signaling molecule,hydrogen sulfide?H2S?,in their bulk duugs,pharmaceutical preparations and plasma samples from pharmacokinetic studies,which provides technical and data support for New Drug Application and methodological reference for the quantitative analysis of analogue compounds.1.Establishment of in vitro and in vivo LC-MS/MS analytical methods for determination of CBMIDA and their application in assay of preparation content and pharmacokinetic studyDuring the analysis of metal complexing agent by the HPLC method,deformed chromatographic peak,poor precision and accuracy,and narrow linear range always occur due to the complexation of the analyte with the ubiquitous metal ions in the analysis system.At present,the analytical methods of polyamine carboxylic acid chelating agents are based almost entirely on the determination of their metal complexes,and the formation of metal complexes is strongly influenced by the metal species used for complexation,reaction temperature,pH,reaction time and the thermodynamic stability of the complex.The analyte needed to be converted into one major species at high temperature for one or more hours to allow reliable quantitative analysis;in addition,the chromatographic separation in these methods is usually performed by ion-pairing reversed-phase liquid chromatography which is characterized by long equilibrium time and poor reproducibility.In this paper,to eatablish a fast and simple in vitro analytical method for determination of CBMIDA,an acid and water resistant Agilent Zorbax SB Aq column with good reservation for both polar and non-polar compounds was selected as the analytical column,pulse gradient chromatography was used and formic acid and EDTA were added to the mobile phase,hence problems that often occur in the analysis of metal chelators such as weak retention,poor peak shape,low precision,poor accuracy and low recovery were overcome.The addition of EDTA to the mobile phase and the sample solution shielded the interference of the metal ions in the sample matrix and the assay system.The acid in the mobile phase could increase chromatographic retention and weaken the metal coordination capacity of CBMIDA by inhibition of dissociation.As a consequence,in our method,CBMIDA was directly determined in prototype,and more obvious simplicity was obtained in comparision with the current methods.The method was applied to the determination of CBMIDA content in its materials and preparations.The lower limit of quantification was 25 ng?mL-1.The method had good linearity in the range of 252500 ng?mL-1.The RSD of precision and reproducibility test were 1.31%and 1.27%,respectively.Since CBMIDA could complex with metal ions in plasma proteins,EDTA was also required in the protein precipitant to increase the recovery of CBMIDA in plasma samples.When the in vitro assay method was directly applied to the analysis of beagle dog plasma,part of CBMIDA could combine with the plasma matrix deposited on the column,then evident peak tailing was observed and the baseline after the peak was higher than the baseline before the peak.By increasing the concentration of formic acid to further inhibit the dissociation of CBMIDA,which reduced the coordination atoms that CBMIDA provided to complex with metal ions,in cooperation with the increase of EDTA concentration,the interaction between CBMIDA and the biological matrix deposited on the column was weaken,and the problem of peak tailing and post-peak baseline drift were tackle.In consideration of the metal complexation and metabolism characteristics of polyaminocarboxylic acid chelating agents,during the method validation of plasma sample analysis,extra tests were conducted to assess the influence of anticoagulant on the plasma concentration of the test substance and the acyl glucuronide metabolites on the determination of drug prototype in animals,and the stability of the test article incubated in whole blood at 37°C.The established beagle dog and rat plasma sample analysis methods had high specificity,precision and accuracy,and good linearity in the range of 0.110?g mL-1,and had been successfully applied to the pharmacokinetic and toxicokinetic studies of CBMIDA in Beagle dogs and the pharmacokinetic study in SD rats.The method established for determination of CBMIDA in plasma samples in this paper is characterized by mild pretreatment conditions,simple procedures,high sample throughput,wide quantitative linear range and strong suitability.In addition,new contents were added to the method validation process for the special charactteristic of catechol polyaminocarboxylic acids;therefore,the method in this paper had practical reference value for method optimization of CBMIDA analogues.2.Assay of preparation content and pharmacokinetic study of H2S donor based on NBD probeH2S is the third gaseous signaling molecule discovered following NO and CO.It has been proven to be involved in the progression of many diseases,and shows potential theraputic effect on various diseases such as cardiovascular disease,digestive tract disease,acute and chronic inflammation,Parkinson's disease and Alzheimer's disease.Therefore,H2S donors have also become candidates for the development of new drugs.The widely accepted highly specific and sensitive methods for quantitative analysis of hydrogen sulfide had always used an expensive derivatization reagent,monobromodiamine;the reaction conditions were complex and demanding,and the standard calibration samples for quantitative analysis of biological samples were buffer solutions of the derivatization product?SDB?.The influence of matrix effect and extraction efficiency of the test sample on the accuracy of the analysis was not considered.The optimal reaction conditions and the measured baseline levels of H2S in the same species obtained by different research groups were inconsistent.In this study,based on the thiolysis reaction of NBD ether,the commonly used chromatographic modifier 4-Cl-NBD,and four probes NBDOMe,NBDOEt,NBDOTFE and NBDOCMR synthesized using 4-Cl-NBD as raw materials,were selected as the capture probes for the LC-MS quantification of H2S,which has strong reducibility and volatility.Firstly,LC-MS/MS detection methods were established for the probes,the reaction product and possible by-product of the probes with H2S and the hydrolysate of the probes,then the reaction conditions for the above five probes with H2S were optimized,and the stability,selectivity,reaction rate,and detection sensitivity and linearity of the probes were investigated.Thereafter,NBDOEt and 4-Cl-NBD,which showed good linearity in quantative reaction with H2S,were selected as the derivatization compounds.The lower limits of quantification were 50 nM,and good linearity was obtained in the concentration range of 50 n5?M for both compounds.The sample processing procedures were simple.The methods showed high precision,and good accuracy and reproducibility,and were successfully applied to the assay of Na2S content in its material and preparations.The sensitivity and linearity of NBDOEt and 4-Cl-NBD derivatization methods were comparable to that of the standard MBB method,while the probe cost is only one tenth of MBB.The recovery and sensitivity of H2S detected in rat plasma using NBDOEt and4-Cl-NBD probes were investigated.Given that H2S was mainly in the bound state in vivo,the recovery of H2S in the plasma of SD rat deteted using the NBDOEt probe was less than 30%,with poor precision and reproducibility,despite the fact that NBDOEt showed better specificity and faster reaction rate in vitro.The addition of antioxidant and specific reducing agents for sulfhydryl compounds in the plasma did not make any contribution to the improvement of recovery.The less specific 4-Cl-NBD probe could form the same product,NBDSH,with the sulfides in plasma as with H2S,by specific reaction at the pH of about 9.5.Due to the interference of endogenous sulfides,the linear curve constructed by Na2S spiked plasma samples could not meet the requirements of biological analysis,therefore it could not act as the calibration standard curve.In this paper,the stability of H2S in whole blood and plasma,and the linearity and recovery of H2S plasma standard calibration samples,and the linearity,precision and recovery of NBDSH in plasma were investagated.The results showed that H2S was stable in whole blood and plasma within 10 min;the absolute recovery of H2S at various concentration points of the plasma standard curve were within the range of 250%to 500%,but the recovery at the same concentration point had a good reproducibility;although the square linear correlation coefficient of the standard curve was less than 0.99,the response of the reaction product was positively correlated with the concentration Na2S spiked,and the slope of the standard curve was reproducible;The plasma standard curve of NBDSH had good linearity in the concentration range of 0.120?M,and the recovery of low,medium and high concentration plasma QC samples were 119.26%,86.75%,and 81.41%respectively,with a precision of within 15%RSD.Therefore,we can reliably quantify H2S in plasma samples by using the NBDSH plasma standard curve as the calibration standard curve.Furthermore,the inteference of endogenous sulfides was eliminated by deducting the average background value of multiple blank points collected for each subject.Finally,with the plasma preparation time controlled at 10 min,the dynamic change of H2S level in rats after tail vein injection of Na2S injection was observed using NBDSH plasma standard curve as the calibration curve.In conclution,during the development and validation of the method for determination of H2S in rat plasma in this paper,the influence of reaction recovery,matrix effect and endogenous interference on the accuracy of analysis was taken fully into account.The method is characterized by simplicity,high precision and accuracy,and good reproducibility,and provides a new technical idea for the pathophysiological mechanism and pharmacokinetic study of H2S. |
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