Evaluation of major active components in St. John’s Wort dietary supplements by high-performance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation

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Abstract

A RP-HPLC method with photodiode array detection and LC–electrospray ionization (ESI) MS confirmation was established for the determination of major active components in St. John’s Wort dietary supplement capsules. The samples alternatively were extracted with ethanol–acetone (2:3) using a 55°C water-bath shaker or an ambient temperature ultrasonic bath. Extracts were separated by RP-C18 chromatography using a 95-min water–methanol–acetonitrile–trifluoroacetic acid gradient. The major components were identified by photodiode array detection and then confirmed by LC–ESI-MS. The quantification of components was performed using an internal standard (luteolin). This method may serve as a valuable tool for the quality evaluation of St. John’s Wort dietary supplement products.

Introduction

The use of herbs as dietary supplements and as over-the-counter (OTC) drugs has increased dramatically in the past few years because of the sense that “natural” is better and lower cost in comparison to most western drugs. In Europe, herbal supplements are traditionally regulated as OTC drugs. They are, however, dispensed by licensed pharmacists or other health care professionals [1] and steps have been taken to ensure quality or proper use. The German government has prepared monographs defining quality standards and potency tests for over 350 single plant drugs. France also has officially recognized more than 200 medicinal plants and provided specifications governing their sale [2]. By comparison, in the USA, according to the Dietary Supplement Health and Education Act (DSHEA) issued in 1994, herbal products are generally classified as dietary supplements, a category somewhere between food and OTC drugs. Under the DSHEA law, a manufacturer may make structure–function claims for a product on its label provided that these claims are supported by scientific evidence. It is not required, however, that the manufacturers demonstrate either safety or efficacy prior to marketing, and no regulations govern product quality [3].

Dietary supplements represented a US$6.5 billion market in 1996 alone and more than half of Americans use dietary supplements according to a recent market research report [4]. Among these dietary supplements, St. John’s Wort is one of the most popular products. In 1997, it is estimated that nearly 7.5 million Americans took St. John’s Wort [5]. Sales in the USA increased 20-fold between 1995 and 1997, from US$10 million to US$200 million annually [6]. In Germany, St. John’s Wort is the most common antidepressant. It is prescribed four-times more often than fluoxetine hydrochloride [7].

St. John’s Wort (Hypericum peforatum) is a herbaceous perennial plant that is distributed worldwide. It has been used as a medicinal herb throughout history. It has become very popular because of its reported beneficial effects on the nervous system, especially as an antidepressant [8]. St. John’s Wort extracts contain at least 10 constituents or groups of components that could contribute to its pharmacological effects. These components include naphthodianthrones (hypericin, on whose content most of the available preparations are standardized), flavonoids (rutin, hyperoside, isoquercitrin, quercitrin and quercetin), phloroglucinols (hyperforin and adhyperforin), and biflavonoids (biapigenin and amenthoflavon). The mechanism of action of the postulated antidepressant effects is unclear [9].

Because of the fast growing market for St. John’s Wort in the USA increasingly more products are sold. The preparations vary widely in content. The amount of authentic plant material is limited and other species with different composition may be substituted. The method generally used for standardization of St. John’s Wort products based on the hypericin content is the spectrophotometric determination of naphthodianthrones. It is not as selective as high-performance liquid chromatography (HPLC) methods and can be manipulated easily (e.g., by adding colorants) [10]. In the present work, a method involving solvent extraction, HPLC RP-C18 column chromatography, photodiode array detection (DAD), liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) confirmation and internal standard quantitation is developed for determining the major components in St. John’s Wort dietary supplements.

Section snippets

Chemicals and samples

Rutin, quercitrin, quercetin, hypericin, luteolin and trifluoroacetic acid (TFA) were purchased from Sigma (St. Louis, MO, USA). Hyperoside and isoquercitrin were purchased from Indofine (Belle Mead, NJ, USA). Pseudohypericin, hyperforin and adhyperforin were isolated from St. John’s Wort dry plant (purchased from health food store, Little Rock, AR, USA). The purity and structural identity of each isolated compound were chemically characterized by HPLC–DAD, nuclear magnetic resonance (NMR) and

Results and discussion

Several HPLC methods have been developed for the analysis of St. John’s Wort [12], [13], [14], [15], [16], [17]. All of these methods either used three mobile phases or inorganic buffer systems. In the current method two mobile phases compatible with common LC–MS systems were used. More than 10 major components were detected (two wavelengths, 284 nm and 590 nm) using a 95-min program. A typical chromatogram is shown in Fig. 1. The LC–DAD–ESI-MS data for standard reference compounds and

Acknowledgements

F.F.L. and J.R. were supported in part by an appointment to the ORAU Research Program at the National Center for Toxicological Research, administered by the Oak Ridge Associated Universities through an interagency agreement between the US Department of Energy and the US Food and Drug Administration.

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Current address: PHAD-ARD, Novartis Pharmaceutical Corporation, East Hanover, NJ 07936, USA.

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