Study Of The Matrix For Drug Small Molecules And Their Analysis Application Based On Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS,is a new type of soft ionization biological mass spectrometry technology developed in the 1980s,which has been widely used in the detection of biological macromolecules,such as peptides,nucleic acids,oligosaccharides,proteins and synthetic polymers,with advantages of convenient sample preparation,less sample consumption,high sensitivity,fast detection speed and high throughput.It plays a very important role in the field of bio-analysis and clinical diagnosis such as proteomics,clinical microbial identification and disease biomarker screening.Similarly,because the soft ionization method can better ensure the integrity of the target molecule,producing almost no or less fragment ions,so that it also shows great advantages and potential in the structure identification and analysis of small molecule compounds.However,a large number of background signals of matrix addition molecular ion peaks,alkali metal ion addition peaks and matrix fragment peaks will be generated in the low molecular weight region（m/z<1000）when small molecular compounds analyze by using traditional organic matrices,which are a certain interference to the detection and analysis of small molecule samples,so that limits the application of MALDI-TOF MS in the analysis and detection of small molecules.Therefore,the development of new matrices such as inorganic micro and nano materials,including metal/metal oxides nanoparticle,carbon nano-materials such as carbon dots,carbon nanotubes,graphene,etc.,and metal organic frameworks（MOFs）as matrices,is an effective way to solve the limitation of MALDI-TOF MS in the analysis and application of small molecules.In addition,although the traditional matrix has the disadvantage of large background interference signals in the low molecular weight region,it has the advantages of high ionization efficiency and good data reproducibility compared with the above-mentioned inorganic micro/nano materials.For some low-molecular-weight molecules（m/z<1000）,distinguishable signals between samples and matrix through careful analysis of the spectrum,MALDI-TOF MS technology based on conventional organic matrix still occupies a place in the detection and analysis of small molecules.Furthermore,some methods,for example,increasing the molecular weight of small molecule samples by derivatization,reactive matrices formed by traditional organic matrix reacting with the sample,or structural derivatization modification of the traditional organic matrices,are also used to avoid or weaken the above-mentioned problems of traditional organic matrices in the analysis and detection of small molecules,thereby further expanding the scope of its application in the analysis of small molecules.Based on contents described above,this research is aimed to develop the method of MALDI-TOF MS for the analysis and detection of small molecule drugs,based on the understand of background signal characteristics of traditional organic matrices includingα-Cyano-4-hydroxycinnamic acid（CHCA）,2,5-dihydroxybenzoic acid（DHB）and sinapinic acid（SA）in positive and negative ion modes through experiments.In addition,based on the knowledge of the properties of MOFs and in-situ biomimetic synthesis,an attempt is made to prepare Zeolitic imidazolate frameworks（ZIF-8）coated magnetic copper-iron nanoparticles（Cu Fe₂O₄@BSA@ZIF-8）as a matrix for MALDI-TOF MS analysis.However,during the experiment,the results of the control experiment showed that the copper-iron nano-magnetic beads（Cu Fe₂O₄@BSA）coated with bovine serum albumin as the matrix exhibited lower background signal in the analysis of biologically active small molecules.Therefore,preliminary explorations to analysis biologically active small molecules were made by used Cu Fe₂O₄@BSA as the matrix.The specific research content is as follows:ⅠChromatography or chromatography-mass spectrometry are usually used to detect drug residual impurities.Compared with that,complicated sample pretreatment process is not required steps for MALDI-TOF MS,which has advantages of simple sample preparation,low consumption,fast analysis,high sensitivity and high throughput.In this part of the study,it was found that sample molecular ion peaks and matrix background signal peaks were not only distinguished,but also the molecular ion peaks of the main components and their related impurities when using CHCA as the matrix to detect rosiglitazone and its related impuritiy,reduced glutathione and oxidized glutathione（GSH/GSSG）under the positive ion mode through the screening of traditional matrices.Under the optimal experimental conditions,the rosiglitazone-related impurity was detected,and it has a linear relationship between the increase of the mass spectrum signal intensity and the impurity concentration in the range of 0.05～10.0μM,and the limit of detection was 28.6 n M.Two batches of rosiglitazone bulk drug provided by a pharmaceutical factory in Fuling,Chongqing were tested,and one batch was unqualified.The content of rosiglitazone-related impurities reached 0.31%,which was similar to the result of 0.28%measured by the classic HPLC method.In addition,for GSH/GSSG detection,there is a good linear relationship between the intensity of the mass spectrum peak and the corresponding sample GSH and GSSG in the concentration range of 0.05～10.0 m M and 0.1～10.0m M,respectively.The limits of detection are 11.6μM and 20.5μM,respectively.This method was used for the detection of GSH in glutathione tablets,and the recovery rate was in the range of 95.9～100.7%,and the relative standard deviation RSD was less than 7.0%.MALDI-TOF MS can directly analyze samples without using a separation system,providing new ideas and ways for rapid detection of drug residual impurities.ⅡClinically,drugs with large individual differences and small therapeutic indexes,often require blood-drug concentration monitoring to adjust the dosage regimen to achieve individualized administration,thereby improving the efficacy and avoiding toxic and side effects.In the previous study using CHCA as the matrix,it was found that the background signal peak of the matrix was actually weak in the range of m/z 600～1000.Therefore,using CHCA as a matrix,drugs that need to be monitor in the m/z range were be screened and analyzed,and found that the immunosuppressant cyclosporin A and the anti-tuberculosis drug rifampicin had a better signal-to-noise ratio.Cyclosporine A,an immunosuppressant,is widely used in the rejection of organ transplantation and the treatment of certain autologous diseases,which effective concentration is in the range of 83.2～374.2 n M and toxic reaction will occur above concentration of 498.9 n M.Rifampicin has a strong antibacterial effect on Mycobacterium tuberculosis.It is an effective anti-tuberculosis drug with an effective concentration of 0.6～12.2μM.Due to individual differences,blood-drug concentration monitoring is also required.In this experiment,the MALDI-TOF MS method is used to quantitatively detect cyclosporin A and rifampicin using CHCA as the matrix.Correlation between intensity of the mass signal and the concentration of cyclosporin A in the serum was found to be a linear in the range of 90.0 n M～9.0μM with a detection limit of 36.0 n M.The quantitative analysis of rifampicin with rifapentin as an internal standard has a linear relationship between 0.5～200.0μM with a detection limit of 0.2μM.When employed to assay drugs in the serum,the approach shows good precision and recovery.The results show that MALDI-TOF MS can quickly and effectively detect the drug concentration in the serum and can be used to monitor the blood drug concentration of therapeutic drugs.ⅢAs mentioned above,in this experiment,it would intend to prepare Cu Fe₂O₄@BSA@ZIF-8 as the matrix for MALDI-TOF MS analysis.However,the result showed that Cu Fe₂O₄@BSA had better response in the analysis of biologically active small molecules when be used as the matrix.Therefore,Cu Fe₂O₄@BSA was used as the matrix to detect a series of representative small molecules,including amino acids,nucleosides,peptides,oligosaccharides,fatty acids and procaine and other small drug molecules.The results showed that Cu Fe₂O₄@BSA as a matrix produced almost no matrix-related signals in the positive ion mode and has little interference in the detection of analytes.In addition,the matrix also exhibited good dispersibility and salt tolerance.Finally,the Cu Fe₂O₄@BSA matrix was used for the quantitative analysis of procaine with the linear range of 4.0～400.0μM and the detection limit of 2.1μM.It shows that Cu Fe₂O₄@BSA had the potential to be used as an effective matrix for MALDI for small molecule analysis.In summary,some preliminary explorations have been made in this research,based on MALDI-TOF MS technology,using biologically active small molecules as the analysis object,focusing on the use of traditional matrices and the development of new matrices,in drug impurity inspection,therapeutic drug blood concentration monitoring,and other active small molecule analysis.It expands the application of MALDI-TOF MS analysis technology in the field of biologically active small molecules,which is of great significance in biomedicine and clinical diagnosis.