降糖活性物6-苄氧基-9-(-4-氯甲苯酰基)-四氢化咔唑-3-羧酸在人工胃肠液中的稳定性研究及降解产物分析
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摘要
目的:考察降糖活性物6-苄氧基-9-(-4-氯甲苯酰基)-四氢化咔唑-3-羧酸(简称"ZgO2")在人工胃肠液中的稳定性,并分析其降解产物。方法:采用高效液相色谱法,以吲哚美辛为内标,检测ZgO2在空白人工胃液(pH 1.3,不含酶)、空白人工肠液(pH6.8,不含酶)、人工胃液(pH 1.3,含胃蛋白酶)和人工肠液(pH 6.8,含胰蛋白酶)中孵育6 h的含量变化,计算ZgO2剩余百分比;利用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF/MS)技术分析对照样品(ZgO2对照品溶液)、空白样品(未加入ZgO2的孵育溶液)、孵育样品(稳定性试验中ZgO2浓度变化最明显的孵育体系)的总离子流图,比较差异峰,通过MS图推测降解产物。结果:ZgO2在空白人工肠液和人工肠液中孵育不同时间的剩余百分比均在90%～110%范围内;ZgO2在酸性(pH 1.3)条件中不稳定,在孵育3 h时剩余百分比约为50%,继续孵育剩余百分比无明显变化,人工胃液(含酶)中的剩余百分比低于空白人工胃液(不含酶)的剩余百分比,但相差不明显。孵育样品与对照样品、空白样品的差异体现在负模式扫描下保留时间为5.23 min时,该时间点色谱峰的MS显示,低能量扫描通道存在m/z 320.127 2[M-H]-的准分子离子峰,高能量通道可见碎片离子峰230.081 5、276.138 2,推测ZgO2在酸性(pH 1.3)条件下可能发生酰胺键断裂生成6-苄氧基-四氢化咔唑-3-羧酸。结论:ZgO2在人工肠液中稳定性良好;在酸性(pH 1.3)条件下不稳定,基本上不受胃蛋白酶的影响,降解产物可能为6-苄氧基-四氢化咔唑-3-羧酸。
OBJECTIVE:To investigate the stability of hypoglycemic candidate 6-benzyloxy-9-(-4-chlorobenzoyl)-tetrahydrocarbazole-3-carboxylic acid(abbreviated "ZgO2") in artificial gastrointestinal fluid, and to analyze its catabolites.METHODS:Using indometacin as internal standard,HPLC method was used to detect the contents of ZgO2 incubated in blank artificial gastric fluid(pH 1.3,no enzyme),blank artificial intestinal fluid(pH 6.8,no enzyme),artificial gastric fluid(pH 1.3,containing pepsin) and artificial intestinal fluid(pH 6.8,containing trypsin) for 6 h. The remaining percentage was calculated.UPLC-Q-TOF was used to analyze TIC of control sample(ZgO2 control solution),blank sample(incubation solution without ZgO2)and incubated sample(incubation system with the most obvious change of ZgO2 concentration in stability test);difference peaks were compared,and catabolites were inferred by MS map. RESULTS:The remaining percentage of ZgO2 incubated in blank artificial intestinal fluid and artificial intestinal fluid for different time ranged 90%-110%. ZgO2 was unstable in acidic condition(p H 1.3). The remaining percentage was about 50% after incubated for 3 h. There was no significant change in the remaining percentage of continuous incubation. The remaining percentage of artificial gastric fluid(including enzymes)was lower than that of blank artificial gastric fluid(without enzymes),but the difference was not obvious. The difference of incubated samples,control samples and blank samples was reflected that the retention time was 5.23 min under negative mode scanning, MS of chromatographic peaks at this time point showed that quasi-molecular ion peaks of m/z 320.127 2 [M-H]-existed in low energy scanning channels and fragment ion peaks of 230.081 5 and 276.138 2 could be seen in high energy channels. It was speculated that amide bond breakage may occur in ZgO2 under acidic(p H 1.3)conditions to generate 6-benzyloxy-tetrahydrocarbazole-3-carboxylic acid. CONCLUSIONS:ZgO2 is stable in artificial intestinal fluid;ZgO2 is not stable in acidic condition(pH 1.3),and is basically not affected by pepsin,the degradation products may be 6-(benzyloxy)-tetrahydrocarbazole-3-carboxylic acid.
OBJECTIVE:To investigate the stability of hypoglycemic candidate 6-benzyloxy-9-(-4-chlorobenzoyl)-tetrahydrocarbazole-3-carboxylic acid(abbreviated "ZgO2") in artificial gastrointestinal fluid, and to analyze its catabolites.METHODS:Using indometacin as internal standard,HPLC method was used to detect the contents of ZgO2 incubated in blank artificial gastric fluid(pH 1.3,no enzyme),blank artificial intestinal fluid(pH 6.8,no enzyme),artificial gastric fluid(pH 1.3,containing pepsin) and artificial intestinal fluid(pH 6.8,containing trypsin) for 6 h. The remaining percentage was calculated.UPLC-Q-TOF was used to analyze TIC of control sample(ZgO2 control solution),blank sample(incubation solution without ZgO2)and incubated sample(incubation system with the most obvious change of ZgO2 concentration in stability test);difference peaks were compared,and catabolites were inferred by MS map. RESULTS:The remaining percentage of ZgO2 incubated in blank artificial intestinal fluid and artificial intestinal fluid for different time ranged 90%-110%. ZgO2 was unstable in acidic condition(p H 1.3). The remaining percentage was about 50% after incubated for 3 h. There was no significant change in the remaining percentage of continuous incubation. The remaining percentage of artificial gastric fluid(including enzymes)was lower than that of blank artificial gastric fluid(without enzymes),but the difference was not obvious. The difference of incubated samples,control samples and blank samples was reflected that the retention time was 5.23 min under negative mode scanning, MS of chromatographic peaks at this time point showed that quasi-molecular ion peaks of m/z 320.127 2 [M-H]-existed in low energy scanning channels and fragment ion peaks of 230.081 5 and 276.138 2 could be seen in high energy channels. It was speculated that amide bond breakage may occur in ZgO2 under acidic(p H 1.3)conditions to generate 6-benzyloxy-tetrahydrocarbazole-3-carboxylic acid. CONCLUSIONS:ZgO2 is stable in artificial intestinal fluid;ZgO2 is not stable in acidic condition(pH 1.3),and is basically not affected by pepsin,the degradation products may be 6-(benzyloxy)-tetrahydrocarbazole-3-carboxylic acid.
引文
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