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Development of a Simple, Rapid and Reproducible HPLC Assay for the Simultaneous Determination of Hypericins and Stabilized Hyperforin in Commercial St. John's Wort Preparations

Development of a Simple, Rapid and Reproducible HPLC Assay for the Simultaneous Determination of Hypericins and Stabilized Hyperforin in Commercial St. John's Wort Preparations

G C de los Reyes, R T Koda

J Pharm Biomed Anal. 2001 Dec;26(5-6):959-65.

PMID: 11600308

Abstract:

A reversed-phase HPLC method was developed and validated for the simultaneous determination of hypericins and stabilized hyperforin in St. John's wort extract. The sample solution was prepared by extraction of the finely powdered extract with methanol-water (80:20, v/v) containing 5% HP-beta-cyclodextrin, and adjusted to pH 2.5 with orthophosphoric acid. Diluted extract solutions, maintained at 0 degrees C, were injected into a C18 column. The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.3% v/v phosphoric acid (90:10, v/v) at a 1.5 ml/min flow rate with simultaneous fluorescence (315/590 nm, excitation/emission) and UV (273 nm) detection. Quantification of the marker compounds (hypericin, pseudohypericin, hyperforin) was achieved by use of standard curves generated by plotting peak heights versus concentrations. Validation studies demonstrated that this HPLC method is simple, rapid, reliable, and reproducible. The standard curves were linear over the concentration ranges, 0.5-2.5 microg/ml (hypericin), 0.35-1.6 microg/ml (pseudohypericin) and 5-50 microg/ml (hyperforin). The intra-day coefficients of variation obtained for hypericin, pseudohypericin and hyperforin were < or = 4.4%, < or = 5.4%, and < or = 2.8%, respectively; inter-day CVs were < or = 5.8%, < or = 4.9%, and < or = 2.5%, respectively. This method may be applied for the routine standardization of St. John's wort products against hyperforin and the hypericins, the putative antidepressant principles in the herbal.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP84082804	Powdered St. John′s Wort Extract		84082-80-4	Price

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