Dickinson R G, King A R
Department of Medicine, University of Queensland, Royal Brisbane Hospital, Australia.
Ther Drug Monit. 1989 Nov;11(6):712-20. doi: 10.1097/00007691-198911000-00018.
Reactivity of glucuronide and sulfate conjugates was taken into account in development of a simple isocratic HPLC method for direct assay of diflunisal (DF) and its acyl glucuronide (DAG), phenolic glucuronide (DPG), and sulfate (DS) conjugates. Whereas DPG was stable over the pH range 0-9, DAG was highly labile at neutral to slightly alkaline pH, undergoing rearrangement (isomerisation via acyl migration), hydrolysis, and in the presence of methanol, transesterification to DF methyl ester. The 2-, 3-, and 4-O-acyl positional isomers of DAG appeared as three pairs of peaks. Interconversion between partners of each pair occurred even under acidic conditions inhibitory to acyl migration, implicating mutarotation. DS was stable at neural to slightly alkaline pH, but underwent hydrolysis under relatively strongly acidic conditions. However, this hydrolysis was remarkably catalyzed (e.g., by 1,000-fold) in the presence of solvents (i.e., solvolysis) such as diethyl ether and ethyl acetate. DS (an acid) could not be extracted from aqueous solution because of this acidic solvolysis. Suitable conditions for simultaneous direct analysis (nonextractive, nonconcentrative) of DF and its reactive (DAG and DS) and unreactive (DPG) conjugates were achieved by working at pH of approximately 4.5. The procedure thus developed is suitable for plasma, urine, and bile samples, and has revealed the presence of new, as yet unidentified, metabolites of DF.
在开发一种用于直接测定二氟尼柳（DF）及其酰基葡萄糖醛酸苷（DAG）、酚性葡萄糖醛酸苷（DPG）和硫酸酯（DS）结合物的简单等度高效液相色谱法时，考虑了葡萄糖醛酸苷和硫酸酯结合物的反应活性。虽然DPG在pH值0 - 9范围内稳定，但DAG在中性至微碱性pH条件下高度不稳定，会发生重排（通过酰基迁移进行异构化）、水解，并且在甲醇存在下会转酯生成DF甲酯。DAG的2 - O - 酰基、3 - O - 酰基和4 - O - 酰基位置异构体表现为三对峰。即使在抑制酰基迁移的酸性条件下，每对峰的对应异构体之间也会发生相互转化，这涉及变旋现象。DS在中性至微碱性pH条件下稳定，但在相对强酸性条件下会发生水解。然而，在二乙醚和乙酸乙酯等溶剂存在下（即溶剂解），这种水解会显著加速（例如加速1000倍）。由于这种酸性溶剂解作用，DS（一种酸）无法从水溶液中萃取出来。通过在约4.5的pH值下进行操作，实现了对DF及其活性（DAG和DS）和非活性（DPG）结合物的同时直接分析（非萃取、非浓缩）的合适条件。由此开发的方法适用于血浆、尿液和胆汁样本，并揭示了DF新的、尚未鉴定的代谢产物的存在。
