IDENTIFICATION AND DETERMINATION OF THE MAJOR CONSTITUENTS IN AN ANTIANGIOGENESIS HERBAL FORMULA, QING-LUO-FANG, BY HPLC—DAD—ESI/MS

Abstract

A high-performance liquid chromatography—diode array UV detection—electrospray ionization tandem mass spectrometry (HPLC—DAD—ESI-MS) method was established for the simultaneous qualitative and quantitative analysis of the major bioactive constituents in Qing-Luo-Fang (QLF), an anti-angiogenesis herbal formula. The separation was performed on a Phenomenex Gemini C₁₈ column using gradient elution. The mobile phase A was ammonium acetate (10 mM) in water; and mobile phase B was methanol-acetonitrile (1:3, v/v) containing 10 mM ammonium acetate and 0.2% ammonia. Twelve compounds were identified by online ESI-MS, and eight of which were quantitated by HPLC—DAD. The eight compounds are major alkaloid compounds present in QLF, including magnoflorine, oxymatrine, oxysophocarpine, sinomenine, sophocarpine, matrine, palmatine, and berberine. The method was validated and the linearities (r² > 0.999) and recoveries (ranged from 95.24 to105.50%) were acceptable. The limits of detection (LOD) and the limits of quantitation (LOQ) of these alkaloids ranged from 0.63 to 6.70 ng/mL and from 2.01 to 20.69 ng/mL, respectively. The intra- and inter-day precision was less than 3% and the accuracy ranged from 97.4% to 102.7%. These results prove that this established method can be used to identify the structure of active constituents responsible for the pharmacological effects of QLF and to control the quality of QLF.

Keywords:

• alkaloids
• HPLC—DAD—ESI-MS
• qing-luo-fang
• simultaneous analysis

ACKNOWLEDGMENTS

This study was supported by the China FANEDD (No. 200366), NSFC (No. 30772710) and National S & T Major Project-Created Major New Drugs Project (No. 2009ZX09301-003, No. 2009ZX09103-382).

Notes

^a The number in crude drug item means which crude drug the ingredient belongs to: (1) Sophora flavescens, (2) Sinomenium acutum, and (3) Phellodendron amurense.

^a [(Mean of measured concentration-spiked concentration)/spiked concentration] × 100.

^b (S.D./mean) × 100.