Development Of A New Zeolite Matrix For MALDI-TOF-MS And Its Application To Quantitative Analysis Of Small Molecules

As a soft ionization mass spectrometry technique,matrix-assisted laser desorption/ionization time-of-flight mass spectrometry?MALDI-TOF-MS?is widely used in analysis of biomacromolecules and organic macromolecules.MALDI-TOF-MS has the advantages of high throughput,fast and simple sample processing.Especially the soft ionization method can ensure the integrity of the molecule.It has great advantages and potential in the identification and analysis of small molecules.Due to the interference and inhibition effects of traditional small molecules matrices in the low mass region?m/z<1000?,MALDI-TOF-MS is not the most effective in analyzing small molecules.The mesoporous zeolite material is a crystalline zeolite rich in mesopores,which not only retains the excellent surface adjustability and high thermal stability of the zeolite material,but also improves the adsorption and protection properties of small molecules because of the introduction of mesopores.The combination of traditional matrix and inorganic mesoporous materials works as a MALDI matrix,showing the advantages of inhibiting matrix fragments,simplifying the spectrum,improving sensitivity and repeatability.The traditional matrix combined with inorganic mesoporous materials as the MALDI matrix can effectively solve the above problems.In this paper,CHCA loaded on zeolite as matrix was firstly used for MALDI-TOF-MS analysis of small molecules,and the quantitative analysis method of enrofloxacin in samples by MALDI-TOF-MS was established.Firstly,we investigated the effect of CHCA loaded on zeolite as matrix for MALDI-TOF-MS analysis of small molecules such as Sub P and fluoroquinolones.Under the same condition,we compared with CHCA and investigated the effect of ZSM-5 with different silicon-aluminum ratio?SiO₂/Al₂O₃?and Beta and ZSM-5 zeolite carrier pairs with different mesopores on the detection of Sub P.The results showed that CHCA loaded on zeolite as matrix has the advantages of inhibiting alkali metal ion peaks,eliminating interference with fragment ions,simplifying and improving mass spectra,improving measurement repeatability and ionization efficiency.In contrast,it was regarded that the strong surface acidity and sufficient cavity for loading CHCA were beneficial for interference fragments suppression and peak intensity enhancement.Additionally,it was successfully applied in the detection of enrofloxacin and ciprofloxacin in complex samples.Secondly,we investigated the effect of the CHCA loaded on Beta zeolite as matrix for MALDI-TOF-MS analysis of small molecules such as enrofloxacin.In the experiments,H⁺,Na⁺and Ag⁺ion exchanged Beta zeolites were prepared,respectively.These new matrixes were examined and compared in the analysis of enrofloxacin at the same MALDI-TOF-MS conditions.The results indicated that the enrofloxacin could be selectively ionized by using metal ion exchanged zeolite.Additionally,CHCA loaded on BetaNa zeolite with the mass ratio of 4:2 was capable of suppressing fragments and promoting peak intensity of enrofloxacin.Based on previous research,it was presumed that the interaction between the activity site on zeolite surface and enrofloxacin would affect the ionization efficiency of MALDI-TOF-MS analysis.The composite matrix was successfully applied to the screening of illicit fluoroquinolone drugs in Chinese veterinary drug samples.Thirdly,CHCA loaded on the BetaNa zeolite as a novel composite matrix was further established for the quantitative analysis of enrofloxacin in complex samples by MALDI-TOF-MS.Aiming at the complex samples of milk and egg liquid,the method of quantitative analysis of enrofloxacin by MALDI-TOF-MS was established by adding isotope internal standard ENR-D₅.Through the optimization of the pretreatment method of the actual samples,the best extraction solvent was determined,the unnecessary pretreatment steps such as purification were reduced,the speed of the whole analysis process was accelerated.The exact content of enrofloxacin in the milk and egg liquid positive samples was calculated by ENR/ENR-D₅ abundance ratio to take into the standard solution curve equation,though adding the same concentration of isotope internal standard to the positive samples in the enrofloxacin standard curve solution.The linearity was good within 0.5 mg/kg⁶.0 mg/kg,and the recoveries ranged from 92%to 101%with the addition level of 0.75 mg/kg³.0 mg/kg,which test results were verified by LC-MS/MS.