HPLC determination
A Validated HPLC Determination of the flavone Aglycone Diosmetin in Human Plasma
Thu, 2013-12-26 14:48 — Feras I. QanazeDiosmetin, 3′,5,7-trihydroxy-4′-methoxyflavone, is the aglycone of the flavonoid glycoside diosmin that occurs naturally in foods of plant origin. Diosmin exhibits antioxidant and anti-inflammatory activities, improves venous tone and it is used for the treatment of chronic venous insufficiency. Diosmin is hydrolyzed by enzymes of intestinal microflora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the determination of diosmetin in human plasma was developed and validated. Diosmetin and the internal standard 7-ethoxycoumarin were isolated from plasma by liquid–liquid extraction and separated on a C8reversed-phase column with methanol–water–acetic acid (55:43:2, v/v/v) as the mobile phase at 43°C. Peaks were monitored at 344 nm. The method was linear in the 10–300 ng/mL concentration range (r > 0.999). Recovery for diosmetin and internal standard was greater than 89.7 and 86.8%, respectively. Intra-day and inter-day precision for diosmetin ranged from 1.6 to 4.6 and from 2.2 to 5.3%, respectively, and accuracy was better than 97.9%. Copyright © 2004 John Wiley & Sons, Ltd.
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TwitterA Validated Solid-Phase Extraction HPLC Method for the Simultaneous Determination of the Citrus Flavanone Aglycones Hesperetin and Naringenin in Urine
Thu, 2013-12-26 14:36 — Feras I. QanazeA simple, specific, precise, accurate, and robust HPLC assay for the simultaneous analysis of hesperetin and naringenin in human urine was developed and validated. Urine samples were incubated with β-glucuronidase/sulphatase and the analytes were isolated by solid-phase extraction using C18 cartridges and separated on a C8 reversed phase column using a mixture of methanol/water/acetic acid (40:58:2, v/v/v) at 45 °C. The method was found to be linear in the 50–1200 ng/ml concentration range for both hesperetin and naringenin (r > 0.999). The accuracy of the method was greater than 94.8%, while the intra- and inter-day precision for hesperetin was better than 4.9 and 8.2%, respectively and for naringenin was better than 5.3 and 7.8%, respectively. Recovery for hesperetin, naringenin and internal standard 7-ethoxycoumarin was greater than 70.9%. The method has been applied for the determination of hesperetin and naringenin in urine samples obtained from a male volunteer following a single 300 mg oral dose of each of the corresponding flavanone glycosides hesperidin and naringin. The intra- and inter-day reproducibility through enzyme hydrolysis was less than 3.9% for both total (free + conjugated) hesperetin and naringenin. Stability studies showed urine quality control samples to be stable for both hesperetin and naringenin through three freeze–thaw cycles and at room temperature for 24 h ( error ≤ 3.6%).
Simultaneous Reversed-Phase High-Performance Liquid Chromatographic Method for the Determination of Diosmin, Hesperidin and Naringin in Different Citrus Fruit Juices and Pharmaceutical Formulations
Thu, 2013-12-26 13:41 — Feras I. QanazeDiosmin, hesperidin and naringin are flavonoid glycosides that occur naturally in citrus fruits. They exert a variety of pharmacological properties such as anti-inflammatory, antioxidant and free radical scavenging and antiulcer effects and also inhibit selected cytochrome P-450 enzymes resulting in drug interactions. A reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of diosmin, hesperidin and naringin in different citrus fruit juices and pharmaceutical preparations. Diosmin, hesperidin, naringin and the internal standard rhoifolin were separated using tetrahydrofuran/water/acetic acid (21:77:2, v/v/v) as the mobile phase at 34 °C, using a C8 reversed-phase column. The method was linear in the 0.25–20.0 μg/ml concentration range for all three flavonoid glycosides (r>0.999). The method has been successfully applied to the determination of all three flavonoid glycosides in several samples of different citrus fruit juices sold in Greece and for the determination of diosmin and hesperidin in pharmaceutical preparations.
