Analysis of goldenseal, Hydrastis canadensis L., and related alkaloids in urine using HPLC with UV detection

Abstract

A screening method was developed to extract and detect berberine and hydrastine alkaloids from goldenseal root powder and urine samples using HPLC with UV detection. The isocratic method was developed to detect alkaloids in 5 mL of urine prior to drug screening. Urine samples were spiked with the alkaloids at varying concentrations and extracted twice with 3:1 chloroform:2-propanol (CHCl₃:2-propanol). The extracts were combined, concentrated using nitrogen gas and the residue was then reconstituted with a mobile phase of acetonitrile:buffer (32:68). A 17 min isocratic run time was performed with a flow rate of 2.0 mL/min, and UV detection at 230 nm using a C₁₈ (250 mm × 4.6 mm) column at room temperature. The method showed good linearity for berberine (r² = 0.9990) and hydrastine (r² = 0.9983) over a range of 11.80 ng/mL to 17.64 μg/mL. The LOD for berberine in urine was 12.74 ng/mL and the LOD for hydrastine in urine was 54.48 ng/mL. Urine samples were spiked with goldenseal root powder and liquid extract as part of a blinded study to determine whether berberine and hydrastine alkaloids could also be extracted in vitro from goldenseal and show a presence in urine samples. Out of the 37 blinded urine samples extracted the two spiked samples were correctly identified based on the presence or absence of berberine and hydrastine. The results demonstrated that this method will enable laboratories to test for the herbal supplement in submitted urine samples prior to drug testing, avoiding false negative results.

Introduction

With the widespread use of drug testing, there have been many controlled substance users and abusers who are willing to try masking agents or herbal supplements to pass a routine urine drug screen test. Many drug users try to subvert the drug testing process by in vivo adulteration by using herbal products for “flushing” as a means to pass the testing. Today, as in the past, people are under the assumption that goldenseal is one such remedy and can be used as a detoxifying agent to cleanse the body of certain illegal substances. As a result, goldenseal is taken orally with the idea that a person using the herbal supplement can pass a routine urine drug test. There has been conflicting research published stating if ingested goldenseal or other detoxifying agents on the market, actually do mask the illegal drugs that should appear in a routine urine drug test [1], [2]. Dietary supplements are also found to contain goldenseal, which may stand alone or be paired with Echinacea [3]. The Dietary Supplement Heath and Education Act (DSHEA) does not require the contents of goldenseal alkaloids in dietary supplements to be placed on the label [3]. Due to the use of goldenseal and these other detoxifying agents; toxicology laboratories may see false negative urine samples in routine drug testing methods that are currently employed. The purpose of this project is to create a screening method for toxicology laboratories to utilize in the detection of the major alkaloids, berberine and hydrastine, from goldenseal, Hydrastis canadensis L., in urine samples using high performance liquid chromatography (HPLC) with ultra-violet (UV) detection.

Goldenseal, Hydrastis canadensis L., is a slow growing perennial herbaceous plant from the buttercup family located in North America, particularly in the areas of southeastern Canada and northeastern United States [4]. It is believed to be an herbal remedy that was traditionally used by Native American Indians. Traditionally, Native Americans used the root of the goldenseal plant as a dye for clothing and then later extended the uses to treat skin disorders, digestive problems, liver conditions, diarrhea, and eye irritations. Today, goldenseal is one of the top selling herbal remedies on the market [4]. People may take the herb to help treat wound healing, bladder infections, fungal infections, common colds and flu's, and sinus and chest congestion [4].

There are three major components in the composition of goldenseal that are assumed to cause bioactivity: berberine, hydrastine, and canadine. The two main components, which were the focus of this study, are isoquinoline alkaloids. These isoquinoline alkaloids are berberine (Fig. 1) and hydrastine (Fig. 2), which both are plant alkaloids. Hydrastis canadensis L. has been reported to contain several isoquinoline alkaloids, including 2–4% hydrastine [5] and 2–3% berberine by weight [6]. Canadine was not evaluated in this study due to the low weight percent in goldenseal samples. Canadine is also difficult to obtain in the United States of America. There are also three other minor components of goldenseal that are not widely studied which are berberastine, hydrastinine tetrahydroberberastine, and canalidine [5]. A number of alkaloid containing plants have been substituted for goldenseal, including Chinese goldenthread (Coptis chinensis), yellow root (Xanthorhiza simplicissima), and Oregon grape (Mahonia aquifolium).

The main objective of the project was to create a screening method for toxicology laboratories to detect goldenseal, and related alkaloids, in urine samples using HPLC. In this article, we describe an isocratic HPLC method with UV detection adapted from Weber et al. [7] and Chen and Chang [8] to determine the weight percent of the two major isoquinoline alkaloids found in goldenseal, berberine and hydrastine, and to identify and quantify these alkaloids in human urine samples.