3S-Pharmacological Consultation and Research GmbH, Koenigsbergerstrasse 1, 27243, Harpstedt, Germany,
Anal Bioanal Chem. 2013 Oct;405(25):8295-310. doi: 10.1007/s00216-013-7237-y. Epub 2013 Aug 16.
Diosmin is a flavonoid often administered in the treatment of chronic venous insufficiency, hemorrhoids, and related affections. Diosmin is rapidly hydrolized in the intestine to its aglicone, diosmetin, which is further metabolized to conjugates. In this study, the development and validations of three new methods for the determination of diosmetin, free and after enzymatic deconjugation, and of its potential glucuronide metabolites, diosmetin-3-O-glucuronide, diosmetin-7-O-glucuronide, and diosmetin-3,7-O-glucuronide from human plasma and urine are presented. First, the quantification of diosmetin, free and after deconjugation, was carried out by high-performance liquid chromatography coupled with tandem mass spectrometry, on an Ascentis RP-Amide column (150 × 2.1 mm, 5 μm), in reversed-phase conditions, after enzymatic digestion. Then glucuronide metabolites from plasma were separated by micro-liquid chromatography coupled with tandem mass spectrometry on a HALO C18 (50 × 0.3 mm, 2.7 μm, 90 Å) column, after solid-phase extraction. Finally, glucuronides from urine were measured using a Discovery HSF5 (100 × 2.1 mm, 5 μm) column, after simple dilution with mobile phase. The methods were validated by assessing linearity, accuracy, precision, low limit of quantification, selectivity, extraction recovery, stability, and matrix effects; results in agreement with regulatory (Food and Drug Administration and European Medicines Agency) guidelines acceptance criteria were obtained in all cases. The methods were applied to a pharmacokinetic study with diosmin (450 mg orally administered tablets). The mean C max of diosmetin in plasma was 6,049.3 ± 5,548.6 pg/mL. A very good correlation between measured diosmetin and glucuronide metabolites concentrations was obtained. Diosmetin-3-O-glucuronide was identified as a major circulating metabolite of diosmetin in plasma and in urine, and this finding was confirmed by supplementary experiments with differential ion-mobility mass spectrometry.
地奥司明是一种黄酮类化合物，常用于治疗慢性静脉功能不全、痔疮及相关疾病。地奥司明在肠道内迅速水解为其苷元地奥司明，后者进一步代谢为轭合物。本研究建立并验证了三种新方法，用于测定人血浆和尿液中的地奥司明、游离型地奥司明（经酶去结合后）及其潜在的葡萄糖醛酸代谢物地奥司明-3-O-葡萄糖醛酸苷、地奥司明-7-O-葡萄糖醛酸苷和地奥司明-3,7-O-葡萄糖醛酸苷。首先，采用高效液相色谱-串联质谱法（HPLC-MS/MS），在 Ascentis RP-Amide 柱（150×2.1mm，5μm）上，在反相条件下，经酶解后，对游离型地奥司明（经酶去结合后）进行定量。然后，采用微液相色谱-串联质谱法（μLC-MS/MS），通过固相萃取，从血浆中分离葡萄糖醛酸代谢物。最后，采用 Discovery HSF5 柱（100×2.1mm，5μm），经简单稀释后，用流动相进行尿液中葡萄糖醛酸苷的测定。所有方法均通过线性、准确度、精密度、定量下限、选择性、提取回收率、稳定性和基质效应进行验证；在所有情况下，均获得符合监管（美国食品药品监督管理局和欧洲药品管理局）指南接受标准的结果。该方法应用于地奥司明（450mg 口服片剂）的药代动力学研究。血浆中地奥司明的 C max为 6049.3±5548.6pg/mL。地奥司明及其葡萄糖醛酸代谢物浓度之间存在很好的相关性。地奥司明-3-O-葡萄糖醛酸苷被鉴定为地奥司明在血浆和尿液中的主要循环代谢物，这一发现通过采用差分离子淌度质谱的补充实验得到了证实。
