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A method previously validated to determine caftaric acid, chlorogenic acid, cynarin, echinacoside, and cichoric acid in echinacea raw materials has been successfully applied to dry extract and liquid tincture products in response to North American consumer needs. Single-laboratory validation was used to assess the repeatability, accuracy, selectivity, LOD, LOQ, analyte stability (ruggedness), and linearity of the method, with emphasis on finished products. Repeatability precision for each phenolic compound was between 1.04 and 5.65% RSD, with HorRat values between 0.30 and 1.39 for raw and dry extract finished products. HorRat values for tinctures were between 0.09 and 1.10. Accuracy of the method was determined through spike recovery studies. Recovery of each compound from raw material negative control (ginseng) was between 90 and 114%, while recovery from the finished product negative control (maltodextrin and magnesium stearate) was between 97 and 103%. A study was conducted to determine if cichoric acid, a major phenolic component of Echinacea purpurea (L.) Moench and E. angustifolia DC, degrades during sample preparation (extraction) and HPLC analysis. No significant degradation was observed over an extended testing period using the validated method.
Echinacea is a genus of flowering plants endemic to North America; species used medicinally include Echinacea angustifolia DC, E. pallida (Nutt.), and E. purpurea (L.) Moench (1). These species and their extracts are most often used for the prevention and treatment of upper respiratory tract infections, such as colds and flu, and as an immune stimulant (2). Phytochemical constituents include the caffeic acid derivatives (also called phenolics), alkamides, glycoproteins, and polysaccharides (3, 4). Root and aerial plant parts may be used raw or in formulations, while homeopathic remedies may include the whole plant (5). According to a 2002 U.S. Food and Drug Administration (FDA) survey, echinacea was the most used nonvitamin/nonmineral dietary supplement (6). In a 2007 National Center for Complementary and Alternative Medicine survey, echinacea was the third most commonly used nonvitamin, nonmineral natural product among adults, and the most administered natural product in children in the United States (7). In 2009, it was still the fifth-highest-selling herbal dietary supplement (8). It is also the second most used Natural Health Product in Canada (9, 10). This popularity has resulted in production and availability of a wide variety of products for which analytical methods to support quality assessments by manufacturers and regulators are needed.
Echinacea commercial products have diversified recently to include capsules, tablets, powders, tinctures, teas, and other beverages, as well as personal care products. Many capsules and tinctures incorporate other popular immune-boosting ingredients such as goldenseal (Hydrastis canadensis L.), zinc, and vitamin C, which may interfere with extraction and analysis of echinacea components. Up to 80% of commercial echinacea products contain E. purpurea (11), and many incorporate both root and aerial parts. Cichoric acid is the most prominent phenolic component of E. purpurea root (12, 13), making it an important marker for possible standardization and QC purposes (3, 14). It, along with the other phenolic compounds, may play a critical role in stimulating the immune system; these are often of most importance during the quantification and qualification of echinacea products (2, 15–18). However, it has been reported that the cichoric acid is especially susceptible to degradation by endogenous enzymes during the processing of fresh echinacea plant material (19). There is also concern that other phenolic components may undergo similar degradation during preparation of plant materials for analysis (20, 21). Because cichoric acid and alkamides are sensitive to extraction conditions, they may be good indicators of reproducible extract production (3, 5, 17).
Although recent animal research suggests E. purpurea may be an effective immunomodulator (22, 23), clinical trials have not been able to conclusively demonstrate efficacy or nonefficacy (24–34). In an attempt to derive meaning from these trials, several meta-analyses were conducted (35–38), and flaws in study designs were noted by the authors of those analyses. These included poorly defined outcome measures, inconsistencies in clinical intent (prevention versus treatment, natural infection versus rhinovirus challenge, dose), and limited quality of evidence (39). Another major issue is a general lack of adequate chemical and botanical descriptions of test articles used in botanical clinical trials (40, 41). Manufacturers and consumers of echinacea preparations are faced with similar issues. Nonstandardized material can and does result in the production of batches of the same product that have completely different phytochemical composition (42). There have also been numerous cases of misidentification and/or adulteration involving both commercial products and research materials (2, 14).
Although alkamides, polysaccharides, glycoproteins, and phenolics have all been hypothesized as potential active ingredients of echinacea, most of the published methods for ensuring identity and potency were developed to quantify the phenolic marker compounds, cichoric acid, echinacoside, and/or total phenolics (12, 16, 17, 43–47). Until recently (48), none of these methods had been validated according to the guidelines published by AOAC INTERNATIONAL (49). To address this issue, an AOAC expert review panel convened in 2005 to identify a published echinacea phenolics method with potential to be widely implemented by industry. The Institute for Nutraceutical Advancement method (15), reviewed by Perry et al. (17) and included in the American Herbal Pharmacopeia monograph for quantifying phenolic marker compounds in raw materials and extracts (2), was chosen for further optimization and single-laboratory validation (SLV). The resulting study, published by Brown et al. (48), was conducted only on echinacea biomass and not on extract raw materials and finished products containing echinacea as well as other ingredients.
This paper describes a matrix extension SLV study of the HPLC method for the quantification of the major phenolic compounds in echinacea of Brown et al. (48), conducted according to AOAC INTERNATIONAL guidelines (49). The new matrixes include echinacea raw materials and finished products with goldenseal, vitamin C, zinc, and other possible interfering species. It also presents key details of an analyte stability study (21) designed to determine if cichoric acid degrades during extraction and HPLC analysis.
This HPLC method is used to detect and quantify five phenolic compounds commonly found in Echinacea spp. raw materials, powdered extract finished products, and tinctures. The phenolic compounds are caftaric acid, chlorogenic acid, cichoric acid, cynarin, and echinacoside.
All test materials were stored at room temperature. Whole root and aerial parts of E. angustifolia, E. purpurea, and E. pallida were harvested in 2008 under the supervision of Wendy Applequist (Missouri Botanical Gardens, St. Louis, MO) and provided by Naturex (South Hackensack, NJ). The herbarium specimens for these three species were deposited with the Missouri Botanical Garden Herbarium, Voucher Nos. 217, 218, and 216, respectively.
For caftaric acid, cichoric acid, and echinacoside, 1000 ppm stock solutions were prepared by dissolving individual reference materials in extraction solvent. Chlorogenic acid and cynarin stock solutions were also prepared at 1000 ppm, but then diluted to 200 and 100 ppm, respectively. These stock concentrations were then diluted to appropriate concentrations to establish retention time and combined at different concentration levels for external calibration.
The equations used to determine the average weight of finished products, based on 20 capsules, are as follows:
where C = weight of capsule with shell and fill content (g), and S = weight of empty shell (g).
The calculation used to determine phenolic concentration is as follows:
where A = peak area (mAu × s), B = intercept of the calibration curve, and D = slope of the calibration curve.
To quantify the individual phenolic compounds on a % (w/w) basis, the following calculation was used:
where C = concentration (µg/mL) from linear regression analysis, FV = the final volume (mL) of the sample preparation, D = the dilution factor of the sample preparation, and W = the sample weight (mg).
To quantify the individual phenolic compounds on a part per million (µg/mL) basis for tinctures, the following calculation was used:
where C = concentration (µg/mL) from linear regression analysis, FV = the final volume (mL) of the sample preparation, D = the dilution factor of the sample preparation (25), and W = the sample weight (mg).
For the validation study, the following equations were used for evaluating precision:
where SD(r) = population SD (σ/n, where σ = sum of squares and n = number of replicates).
Within-day: average and SDs of four data points within-day.
Within-laboratory: average and SDs of 12 data points over 3 days (separate batches on 3 days).
Chromatographic resolution, Rs, was calculated using the following equation:
where tR = retention time, min, W = width of peak at baseline, min, A = earlier-eluting peak, and B = later-eluting peak. Baseline resolution requires an Rs >1.5.
Refer to Table 2 for the approximate concentration of the individual phenolic compounds for each linearity determination (calibration level). All solutions not immediately used were stored at −20°C.
This method was validated according to AOAC INTERNATIONAL guidelines for conducting an SLV (49).
In a separate experiment, stability of extracted test solutions was assessed by combining 125 mg E. purpurea with 25 mL methanol–water (60 + 40), vortexing 30 s, and shaking for 20 min on a wrist-action shaker. A portion of the final extracted solution was filtered into seven separate HPLC vials kept at room temperature. From six of these vials, 30 consecutive HPLC injections were made over the course of 9 h. The seventh vial was stored at room temperature for 6 days and then analyzed. The concentration for each of the five analytes was calculated for each sample. Degradation would be indicated if the actual concentrations were less than the expected concentrations.
Identification of analytes in test materials was performed by comparing peak tR values and UV profiles to the individual reference standards diluted to within the method calibration curve concentrations. A gradient elution was used for the analysis of the five major phenolic compounds in Echinacea spp. (Table 1). The order of elution was caftaric acid (4.18–4.23 min), chlorogenic acid (4.50–4.57 min), cynarin (7.57–7.69 min), echinacoside (7.81–7.92 min), and cichoric acid (12.96–13.13 min). A representative chromatogram of a mixed calibration standard illustrating this elution order can be seen in Figure 1.
Quantification of the analytes was carried out by linear regression analysis using quadruplicate samples prepared on three separate days at seven concentration levels. The analytical range used for each phenolic is listed in the section Preparation of Calibration Solutions. Test matrixes included E. purpurea, E. angustifolia, and E. pallida in root and/or aerial raw materials; E. purpurea and E. angustifolia in powdered extract (loose, in capsule as single botanical ingredient, or in capsule with other botanical ingredients); and E. purpurea and/or E. angustifolia in tincture (ethanol or glycerite).
Baseline resolution (Rs >1.5) was achieved for each analyte within the calibration range. Rs >4.0 and Rs >3.0 was achieved between caftaric acid/chlorogenic acid and cynarin/echinacoside peaks, respectively. There was no evidence of chromatographic interference with analytes of interest by goldenseal, zinc, vitamin C, reishi, astragalus, or elderberry in formulations.
All of the calibration curves generated over the course of the study appeared linear upon visual inspection. All of the RSDs were above 99.5%. These results confirm that the curves were linear over the expected concentration range for echinacea materials.
Variance checks showed that the method used was applicable for the analytes. The MDL and LOQ for each of the analytes are reported in Table 4.
For some of the test materials, chlorogenic acid, cynarin, and echinacoside were not detected; for that reason, no response values were reported for these analytes, and no precision analysis was performed. The responses observed for all other analytes, in all test articles, were above the detection limit of the method and were thus reported as detected. For all reported materials, ANOVA indicated no significant differences for between-day precision. Average HorRat values for raw materials and dry finished products (Tables 5a and b) were 0.49 and 0.55, respectively, while average HorRat values for the tinctures (Table 5c) were lower than expected at 0.19. The low tincture HorRat values could be attributed to the use of volumetric glassware and high within-laboratory precision since they were consistently low among all analytes.
Two spike recovery studies were conducted to determine method accuracy. The first study was designed to emulate raw Echinacea spp. materials containing 1.8, 4.7, and 8.6% (w/w) total phenolics (sum of all five analytes) spiked onto P. quinquefolius L. root powder. Recovery (Table 6) over the three levels, averaged over all samples, was 99.8%. A second study used materials designed to resemble commercial Echinacea spp. extracts containing 0.9, 1.8, and 3.8% (w/w) total phenolics. The average recovery (Tables 7a–c) over these three levels was 100.0% (1.39% RSD).
Significant cichoric acid degradation was observed when E. purpurea and E. angustifolia root samples were extracted with highly aqueous extraction solvents [ethanol–water (20 + 80)]. No advantage was observed when 1 mM ascorbic acid was added to the methanolic extraction solution. The cichoric acid concentrations in liquid extracts of echinacea materials prepared by the validated method [methanol–water (60 + 40)] did not degrade over the entire 100 min test period.
Concentrations of each of the five analytes in an E. purpurea root sample extracted with methanol–water (60 + 40) were found to be stable through 30 injections over a 9 h period, based on a ≤4% difference in peak areas. A sample of this solution stored at room temperature for 6 days did not show any significant degradation of any analyte.
The reported method, previously validated for echinacea raw materials, was extended to extracts, extract in capsules, and tinctures, and subjected to an SLV study, according to AOAC guidelines. All parameters investigated were found to be in compliance with those guidelines. As such, the described method is considered suitable for the purpose of determining caftaric acid, chlorogenic acid, cynarin, echinacoside, and cichoric acid in E. purpurea, E. angustifolia, and E. pallida powdered commercial extracts alone or in combination with H. canadensis L., zinc, and ascorbic acid (Vitamin C), and extracts in ethanolic or glycerite tinctures alone or in combination with other ingredients. In the interest of establishing an Official Method of AnalysisSM for determination of phenolic compounds in Echinacea spp., a collaborative study of the described method is planned.
This work was funded in part by the Growing Forward Program, a joint venture between the British Columbia Ministry of Agriculture and Lands and Agriculture and Agri-Food Canada. We gratefully acknowledge the National Institutes of Health–Office of Dietary Supplements and the FDA for providing for the acquisition of authenticated plant material used in this study.
Paula N. Brown, British Columbia Institute of Technology, 3700 Willingdon Ave, Burnaby, BC, V5G 3H2, Canada.
Michael Chan, British Columbia Institute of Technology, 3700 Willingdon Ave, Burnaby, BC, V5G 3H2, Canada.
Lori Paley, British Columbia Institute of Technology, 3700 Willingdon Ave, Burnaby, BC, V5G 3H2, Canada.
Joseph M. Betz, National Institutes of Health, Office of Dietary Supplements, 6100 Executive Blvd, Suite 3B01, Bethesda, MD 20892.