| The drug analysis in the biological samples is a base for the research of PK/PD. The determination of target drugs in vivo is a challenging task, just because the target drugs generally are the lower concentration and impacted easily by inorganic salts and endogenous compounds. The combination of liquid chromatography and mass spectrometry (LC-MSn) has become widespread, and has had a significant impact on all major areas of analytical science. Along with many successful applications, a number of limitations of LC-MSn have been noted. Poor sensitivity using ESI has been observed for compounds. Some of the small and highly polar compounds are difficult to retain on the reversed-phase chromatographic columns even with high aqueous mobile phases. In the thesis, chemical derivatization was applied in the pre-processing firstly. Derivatization changes the structure of the drugs and therefore changes their physical and chemical properties, resulting in high ionization efficiency, low matrix effect and low disturbance by inorganic salts and endogenous compounds in LC-MSn. Additional, hydrophilic interaction chromatography/tandem mass spectrometry had also been studied in this thesis and applied in the determination of some typical drugs.On the foundation of functional group, including phenols, alcohol, ketones and aldehydes, carboxylic acids, amines et al., some kinds of typical drugs with different functional group were selected to research the method of derivatization. Meanwhile, it is necessary that the derivatized strategies are convenience, quick and have characteristics of high efficiency and the stabile products. According to the result of research, some derivatized reagents were selected : the detection of phenols by LC-MSn using dansyl chloride (DNS-Cl) derivatization, the detection of ketones and aldehyde by LC-MSn using toluene sulfonyl hydrazine (TSH), the detection of carboxylic acids by LC-MSn using methanol and ethanol, the detection of hydroxyl group by LC-MSn using glycidyltrimethylammonium (GTMA), the detection of amines by LC-MSn using 9-fluorenylmethyl chloroformate (FMOC-Cl). After the selection of derivatized reagents, we paid attention to derivatized conditions. The critical factors influencing the derivatization include molar ratio of reactants, catalyst, temperature, time and solvent. Compared with the direct determination, the changes on the chromatogram and mass spectrum after derivatization were observed and discussed.①Through the research of typical drugs (KL-1, paeonol, caffeic acid, chrysophanol) , the common derivatized conditions was that the acetonitrile as the reaction solvent, the reaction temperature was 60 ?C, and the reaction time was 10 - 15 min, the pH of buffer was 10 - 10.5, the aqueous phase : organic phase was about 1: 3 - 1: 5. In MS performance, the [M+1]+ ions increase of 233. In LC performance, the retention time of the compounds became longer after derivatization, which could avoid the interference of endogenous substances and decrease the matrix effect.②Through the research of typical drugs (gestodene, mifepristone and paeonol) , the common derivatized conditions was that the reaction temperature was about 40– 60 ?C , the reaction time was not more than 8 h and the concentration of the catalyst was about 0.5 - 5 %. In MS performance, the [M+1]+ ions increase of 168. Just because the derivatized product conclude the hydrazone, which result in the higher ionization efficiency than that of the drugs.③Through the research of typical drugs (L20051117, FS-3 and SH-1) , the common derivatized conditions was that the ratio of HCl : methanol or HCl : ethanol was 1 : 9, the reaction temperature was about 60 - 70 ?C and the reaction time was 20 - 40 min. In MS performance, the [M+1]+ ions increase of 14 in methylation and increase of 28 in ethylation. In general, the detection of some carboxylic acids may not sensitive in the ESI mode. This may be due to matrix effects and high background noise which can not meet detection requirements in vivo. Good peak shape and strong response of target compounds were observed with the LC-MSn system after derivatization.④Through the research of typical drugs (voglibose, D-galactose, D-lylose, glucose) , the common derivatized conditions was that the reaction temperature was about 55 ?C, the reaction time was not less than 4 h, the concentration of derivatized reagents was about 50 % and that of the catalyst was about 1 N. In MS performance, the [M+1]+ ions increase of 116. Resulting in the tertiary amines group of the derivatized product, the high ionization efficiency, good peak shape and well signal noise ratio were reached easily.⑤Through the research of typical drugs (gabapentin) , the common derivatized conditions was that the reaction temperature was about 40 ?C , the reaction time was between 20 - 40 min and the pH of the buffer was about 10. In MS performance, the [M+1]+ ions increase of 222. Through the structural modification for small amines, the apolarity of derivatized product increase sharply, the retention time became longer than underivatized drugs. But the sensitivity of products did not increase.Based on above research, we seeked one typical drugs of each chemical group, and validated the LC-MSn with derivatized methods completely. And then these methods have been applied to the PK/PD research. It is believed that these LC-MSn with derivatized methods are successfully, which lead to increasing the sensitivity, raising the retention time, decreasing the matrix effect, avoiding the interference of endogenous substances , getting good peak shape and signal noise ratio.Additionally, HILIC-MSn method was also established with the highly polar compounds, which were difficult to retain on the reversed-phase chromatographic columns even with high aqueous mobile phases. Some of the typical drugs (memantine hydrochloride, gabapentin and terazosin hydrochloride) were as the target drugs. HILIC can result in advantages compared to reversed-phase chromatography as follows: MS sensitivity improvement due to higher organic content in the mobile phase, longer retention time, and less matrix effects. Compared to the derivatization, HILIC may avoid the time-consuming preprocessing.In summary, LC-MSn with chemical derivatization is an effect way, which may solve basically the determination problems of some drugs, including the poor stability, the shorter retention time, the lower ionization efficiency and the higher matrix effects. In this thesis, the derivatized method was selected with the good reaction efficiency, sensitivity, speed, and selectivity. After the validation, the LC-MSn with the derivatization can meet the requirement of the quantification the typical drugs in biological samples. And then these methods were applied for determining those of typical drugs in plasma of animal and human.
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