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High‐performance liquid chromatography determination of hydrastine and berberine in dietary supplements containing goldenseal
High-Performance Liquid Chromatography Determination of Hydrastine and Berberine in Dietary Supplements Containing Goldenseal EHAB A. ABOURASHED,1 IKHLAS A. KHAN1,2 1
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677
2
Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, Mississippi 38677
Received 26 June 2000; revised 21 November 2000; accepted 5 December 2000
ABSTRACT: Goldenseal (Hydrastis canadensis L., Ranunculaceae) is an ingredient of various dietary supplements intended for enhancing general body immunity. Many goldenseal products are currently available in the United States, either alone or in combination with echinacea. In most products, the content of the main active alkaloids of goldenseal, hydrastine and berberine, is not indicated on the label. A high-performance liquid chromatography (HPLC) method has been developed for the detection and quanti®cation of hydrastine and berberine in a number of products obtained from the 1 United States market. The method uses a Phenomenex Luna C18 column, a mobile phase consisting of solvent A (100 mM sodium acetate/acetic acid, pH 4.0) and solvent B (acetonitrile/methanol; 90/10, v/v). Elution was run at a ¯ow rate of 1.0 mL/min, with a linear gradient of 80± 40% A in B over 20 min and ultraviolet detection at 290 nm. A wide range of content variation was observed for both alkaloids in the tested samples. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:817±822, 2001 Keywords: goldenseal; hydrastine; berberine; HPLC; dietary supplements
INTRODUCTION Goldenseal (Hydrastis canadensis L., Ranunculaceae) is a native American plant that has a history of folk medicine use in the treatment of many ailments, such as gastrointestinal disturbances, urinary disorders, hemorrhage, skin, mouth, and eye infections, and in¯ammation.1,2 Currently, echinacea/goldenseal dietary supplements are among the top 13 herbal products in the United States Food, Drug, and Mass market (FDM), with sales exceeding $44 million in 1999.3 The common structure/function label claim of such products is to maintain a healthy immune function during the ¯u season. Extracts of H. canadensis were Correspondence to: I. A. Khan (Telephone: 662-915-7821; Fax: 662-915-7989; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 817±822 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
found to possess antibacterial, antimalarial, and smooth muscle relaxant activities.4±7 The pharmacological and antimicrobial effects of the extracts are mainly attributed to the high content of the isoquinoline alkaloids, hydrastine and berberine (Figure 1), even though their oral bioavailability is low.2,8 The popularity of goldenseal as an ingredient in natural ¯u remedies may be justi®ed by the recent ®nding that the extract increased the primary IgM response in a group of goldenseal-treated subjects during the ®rst 2 weeks of treatment.9 On the other hand, the administration of goldenseal may result in various adverse reactions, such as digestive disorders, nausea and vomiting, CNS and respiratory depression, seizures, and heart block.10 In spite of the potential for toxic effects, under the Dietary Supplement Health and Education Act (DSHEA), alkaloid content is not required on product label. Also, in addition to possible hazards, products
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 7, JULY 2001
817
818
ABOURASHED AND KHAN
Figure 1. Chemical structures of the major goldenseal alkaloids.
with inadequate alkaloid content, and therefore poor pharmacological bene®t, can be a common problem when appropriate analytical quality control methods are lacking. A number of spectrophotometric, gravimetric, chromatographic, and electrophoretic methods have been reported for the determination of hydrastine and berberine in goldenseal extracts.11± 17 Also, two previously reported methods are based on reversed-phase high-performance liquid chromatography (HPLC).18,19 However, in both reports, method information was partially disclosed, the analytical run in one method was relatively long (40 min) with obvious peak overlap for hydrastine and tailing for berberine, and, from an application perspective, no herbal products containing goldenseal were analyzed by either method.18,19 In continuation to our investigation of various herbal products for their content uniformity and to bring attention to the necessity for establishing acceptable standards for dietary supplements marketed in the United States, we report herein the development and utilization of a fast and simple HPLC method for the determination of hydrastine and berberine in a number of selected goldenseal products purchased locally. Parameters included in method validation were linearity, accuracy, precision, speci®city, limit of detection, and limit of quantitation.
EXPERIMENTAL SECTION Materials Berberine chloride and ( ÿ )-b-hydrastine were purchased from Sigma (St. Louis, MO). Goldenseal-containing products were purchased either from local retailers or via the Internet: ``Sample 1'' (Herbal Single Goldenseal Root, Nature's Way Products, Inc., Springville, UT); ``Sample 2'' (Goldenseal Root, Thompson Nutritional ProJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 7, JULY 2001
ducts, Boca Raton, FL); ``Sample 4'' and ``Sample 5'' (Herbal Formula Echinacea with Goldenseal, Nature's Way, different batches); ``Sample 6'' (Echinacea-Goldenseal, Nature's Resource Products, Mission Hills, CA); ``Sample 7'' (EchinaceaGoldenseal, NaturPharma, American Fork, UT); ``Sample 8'' (Echinacea & Goldenseal Root, Futurebiotics, Hauppauge, NY); ``Sample 9'' (Echinacea Goldenseal, Natrol, Inc., Chatsworth, CA); ``Sample 10'' (BeeFense, Premier One Products, Inc., Park City, UT); ``Sample 11'' (Echinacea/Goldenseal, Harvest of Nature, Wilmington, DE); and ``Sample 12'' (Echinacea-Goldenseal, Planetary Formulas, Soquel, CA). Crude goldenseal powder (Sample 3) was obtained from the Australasian College of Herbal Studies (Lake Oswego, OR). HPLC grade acetonitrile and methanol were purchased from Fisher Scienti®c (Fairlawn, NJ). Deionized water was obtained from a Milli-Q Plus analytical deionization system (Bedford, MA). Sodium acetate was purchased from Sigma (St. Louis, MO). Instrumentation and Chromatographic Conditions A component HPLC system (Waters, Millford, MA) consisted of a 600E controller, a 600 pump, a 712 WISP autoinjector, and a 996 UV photodiode array detector. A pre-packed Luna, 15 cm 4.6 mm (5 mm particle size), C18 HPLC column and SecurityGuard cartridge system (Phenomenex, Torrance, CA) were operated with a mobile phase consisting of solvent A (0.10 M sodium acetate/acetic acid, pH 4.0, in water) and solvent B (acetonitrile/methanol, 90/10, v/v), continuously degassed with helium. Elution was run at a ¯ow rate of 1.0 mL/min with a linear gradient of 80±40% A in B over 20 min and ultraviolet (UV) detection at 290 nm. After each run, the column was eluted with methanol for 5 min, followed by reequilibriation with the starting solvent for 10 min. Chromatographic data were processed with Millennium 32 software, version 3.05.01 (Waters, Millford, MA) installed on a Quantex PC. Standard Solutions and Sample Preparation Hydrastine and berberine (1.0 mg each) were placed in a 10-mL volumetric ¯ask and the volume was brought to 10 mL with methanol. Solutions with known concentrations were prepared from the standard stock solution by serial dilution with methanol.
HPLC DETERMINATION OF HYDRASTINE AND BERBERINE IN GOLDENSEAL DIETARY SUPPLEMENTS
Accurately weighed powdered goldenseal from each sample (obtained by combining the contents of three capsules or by direct weighing of crude powder) was exhaustively extracted as follows: 100.0 mg of powder was sonicated in 3 mL of methanol for 15 min in a screw-capped polyethylene tube. The tube was centrifuged at 1500 rpm for 5 min, and the supernatant was transferred to a 10-mL volumetric ¯ask. The same procedure was repeated twice and the combined extract was adjusted to volume with methanol. An adequate volume (2 mL) was passed through a 13-mm, 0.45-mm nylon membrane. The ®rst 1 mL was discarded and the remaining volume was collected in an HPLC sample vial, and 10.0 mL was injected into the chromatograph. For Sample 12 (liquid extract), 1 mL was transferred to a 2-mL volumetric ¯ask and diluted to volume with methanol, and 10.0 mL was injected into the chromatograph. The remaining residue was subjected to a fourth extraction with methanol (3 mL) and a ®ltered aliquot of the extract was separately injected into the chromatograph. Method Validation Calibration curves were constructed by plotting peak areas for the external standards, hydrastine and berberine, versus a 6-fold concentration range of 100.0, 50.0, 25.0, 12.5, 6.3, and 3.1 mg/ mL. Each solution was injected three times beginning with the most dilute concentration. The slope, intercept, and correlation coef®cients were calculated by linear regression analysis on the Millennium software. For validating analytical accuracy, 100.0-mg portions of Sample 1 were spiked with three different concentrations of hydrastine and berberine (Table 1). Extraction of each portion was conducted as already described. The same extraction procedure was repeated on 100 mg of unspiked Sample 1. Extracts were injected in the chromatograph and the Table 1.
alkaloid concentrations were determined. The retention time and relative standard deviation (RSD) values for measured alkaloids in all samples were used to determine method precision. Peak identity was determined by spiking a solution of Sample 1 (800 mL) with 200 mL of standard solution of hydrastine and berberine (140.0 and 100.0 mg/mL, respectively) before injection, then peak overlap was observed for the obtained chromatogram. Peak purity was established by spectral overlap.
RESULTS AND DISCUSSION The two alkaloids were well resolved with a retention time difference of > 4 min (Figure 2). Linear regression analysis of the calibration plot of both alkaloids gave the equations y 8.32 103x 4090 (hydrastine) and y 1.68 104x 1480 (berberine), with correlation coef®cients (r2) of > 0.999 (n 3). Therefore, each alkaloid had a linear response within the speci®ed range of concentrations. The ef®ciency of the triple extraction procedure was established when neither alkaloid could be detected in the chromatograms of the fourth extract of each sample. Accuracy was validated by recovery studies in which known amounts of both alkaloids were spiked into sample 1 and subjected to the same triple extraction procedure described earlier. Acceptable recoveries were obtained for both alkaloids in the range 94.82±101.71% (Table 1). Method precision was re¯ected in the fact that the mean retention times were 10.9 min (RSD 2.67%, n 36) and 15.2 min (RSD 0.83%, n 36); and that the RSD of all measurements ranged from 0.75 to 1.82% for hydrastine and from 0.01 to 0.97% for berberine (n 3/sample). The limit of quantitation of each alkaloid was taken as 3.13 mg/mL because peaks at concentrations below this level (detection limit, 1.00 mg/mL) were
Statistical Characterization of Method Accuracy
Analyte ( ÿ )-b-Hydrastine Berberine
819
Added (mg)
Mean Recovery (%, n 3)
RSDa (%)
Overall Mean Recovery (%)
RSDa (%)
9.90 32.67 98.99 11.23 37.08 112.35
101.23 99.08 94.82 101.71 98.29 95.09
1.28 1.88 0.82 1.29 1.71 0.76
98.38
3.32
98.36
3.37
a
Relative standard deviation. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 7, JULY 2001
820
ABOURASHED AND KHAN
Figure 2. Representative chromatograms of extracts from two commercially available goldenseal products. Peaks: A hydrastine, B berberine.
detectable but not quanti®able because of considerable baseline drift. Peak purity was established by comparing the UV spectra obtained at three time points for peaks corresponding to each alkaloid in all analyzed samples. All spectra were superimposable and representative ones are shown in Figure 3 for Sample 1. Spiking a solution of Sample 1 with standard alkaloid solution resulted in pure peaks at the same retention time of hydrastine (10.87 min) and berberine (14.92 min), with peak area increases of 11 and 7%, respectively, thus validating the peak identities of both alkaloids. The method was also speci®c for the detection of the two markers because no interfering constituents were detected in any of the analyzed samples as evidenced by peak purity. The analyzed samples exhibited a wide range of alkaloid concentrations that ranged from 0.00 to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 7, JULY 2001
2.51% for hydrastine and from 0.00 to 4.35% for berberine (Table 2). All samples had a hydrastine/ berberine ratio of 1/1.5, typical of goldenseal.8 The only exception was Sample 10, whose label listed seven herbal ingredients (goldenseal, echinacea, garlic, elderberry, ginger, peppermint, and cayenne) and in which neither alkaloid could be detected. Highest concentrations were observed in single-component goldenseal products (Samples 1 & 2) and in the crude powder (Sample 3). Sample 1 was the only product to show a label claim of 5% total alkaloids, and the total value obtained in our analysis (6.86%) exceeded the label claim. For all the remaining samples, the total alkaloid content was < 5%, which is not unexpected because all were multicomponent products in which echinacea powder was included in the formulation. Therefore, the relative ratios
HPLC DETERMINATION OF HYDRASTINE AND BERBERINE IN GOLDENSEAL DIETARY SUPPLEMENTS
821
Figure 3. Peak purity validation by UV spectral overlap: (A) Overlaid spectra of hydrastine peak at 10.78, 10.88, and 10.99 min; (B) overlaid spectra of berberine peak at 16.39, 16.52, and 16.71 min.
of herbal components in mixtures should be clearly stated so that quantitative analysis may accurately determine marker compound levels in each herbal component. In this context, the labels of only three products (Samples 8, 9, and 10) showed the relative ratio of goldenseal powder to other mixture components. Based on the label claims, the alkaloid concentration in the goldenseal component of these products could be recalculated to assess the quality of the ingredient raw materials (Table 3). It is to be noted that the 5% total alkaloids (label of Sample 1) is not a standard value recommended by any major
authority (e.g., United States, British or German Pharmacopeias), but rather a ®gure that is close to the reported total alkaloid concentration estimated for goldenseal.8,20 In conclusion, even though quality appraisal is not the main focus of this work, it is clear that content uniformity is an issue that needs to be considered to achieve a minimum level of product conformity. The wide range of alkaloid concentrations estimated in this study implies that goldenseal products, among many other dietary supplements on the United States market, need to conform to common acceptable quality control
Table 2. Concentration (%) of Hydrastine and Berberine in Selected Goldenseal-Containing Products (n 3) Product 1a 2a 3a 4b 5b 6 7 8 9 10 11 12
Form
Lot#
Exp. Date
Hydrastine (RSD %)
Berberine (RSD %)
Capsule Capsule Crude Capsule Capsule Capsule Capsule Capsule Capsule Capsule Tea bag Liquid Extract
907330 16427 Ð 810423 612166 HD12070 704081 97VF514 934864 001309 Ð Ð
11/2002 6/2002 Ð 10/2003 12/2001 4/2000 4/2002 Ð 8/2001 3/2000 Ð Ð
2.51 (1.46) 2.27 (1.22) 1.35 (0.75) 0.60 (0.98) 0.58 (1.07) 0.54 (1.06) 0.22 (0.56) 0.94 (1.00) 0.11 (1.82) 0.00 (0.00) 1.91 (1.46) 1.83 (0.96)
4.35 (0.75) 3.41 (0.62) 1.79 (0.34) 1.03 (0.61) 0.86 (0.41) 0.82 (0.97) 0.34 (0.22) 1.27 (0.69) 0.17 (0.40) 0.00 (0.00) 2.83 (0.90) 1.81 (0.47)
a
Exclusively goldenseal. Different batches of the same brand.
b
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ABOURASHED AND KHAN
Table 3. Adjusted Alkaloidal Content in the Goldenseal Portion of Multicomponent Products Adjusted Concentration (%) Sample 8 9 10
Goldenseal (mg) 200 125 50
Other (mg)
Factor
Hydrastine
Berberine
300 360 400
2.50 3.88 9.00
2.35 0.43 0.00
3.18 0.66 0.00
criteria. The method described in this paper can be applied as an analytical tool for determining the levels of hydrastine and berberine in commercial products containing goldenseal, either as a single component or in combination with echinacea, to establish one of the primary criteria for quality and conformity of herbal dietary supplements.
ACKNOWLEDGMENT The authors acknowledge the United States Department of Agriculture, Agricultural Research Service Speci®c Cooperative Agreement No. 58-6408-7-012 for partial support of this work.
REFERENCES 1. Fetrow CW, Avila JR. 1999. Complementary & Alternative Medicines. Pennsylvania: Springhouse, pp. 301±305. 2. Tyler VE, Foster S. 1994. Tyler's Honest Herbal. 4th ed. New York: Haworth Herbal, pp. 195±197. 3. Blumenthal M. 1999. Herb market levels after ®ve years of boom: 1999 sales in mainstream market up only 11% in ®rst half of 1999 after 55% increase in 1998. Herbalgram 47:64±65. 4. Knight SE. 1999. Goldenseal (Hydrastis canadensis) versus penicillin: A comparison of effects on Staphylococcus aureus, Streptococcus pyogenes, and Pseudomona aeruginosa. Bios 70:3±10. 5. Vennerstrom JL, Klayman DL. 1988. Protoberberine alkaloids as antimalarials. J Med Chem 31:1084±1087. 6. Baldazzi C, Leone MG, Casini ML, Tita B. 1998. Effects of the major alkaloid of Hydrastis canadensis L., berberine, on rabbit prostate strips. Phytother Res 12:589±591. 7. Bolle P, Cometa MF, Palmery M, Tucci P. 1998. Response of rabbit detrusor muscle to total extract and major alkaloids of Hydrastis canadensis. Phytother Res 12:S86±S88. 8. DerMardersosian A. 2000. The Review of Natural Products. St. Louis: Facts and Comparisons. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 7, JULY 2001
9. Jalees R, Dillow JM, Carter SM, Chou J, Le B, Maisel AS. 1999. Increased production of antigenspeci®c immunoglobulins G and M following in vivo treatment with the medicinal plants Echinacea angustifolia and Hydrastis canadensis. Immunol Lett 68:391±395. 10. PDR for Herbal Medicine. 1998. Montval: Medical Economics Company. 11. El-Masry S, Korany MA, Abou-Donia AHA. 1980. Colorimetric and spectrophotometric determinations of hydrastis alkaloids in pharmaceutical preparations. J Pharm Sci 69:597±598. 12. Caille G, Leclerc-Chevalier D, Mockle JA. 1970. Spectrophoto¯uorometric determination of the alkaloids berberine, tetrahydroberberine, and hydrastine, and application to the dry extract and tincture of Hydrastis canadensis. Can J Pharm Sci 5:55±58. 13. Stanisals E. Gleye J, Rouf®ac R. 1971. Spectrometric determination of hydrastine content in the rhizome of hydrastis, Hydrastis canadensis. Ann Pharm Fr 29:27±32. 14. Zwaving JH, De Long-Havenga EHJ. 1972. Determination of hydrastine in Hydrastis ¯uid extract. Pharm Weekbl 107:137±146. 15. Datta D, Bose PC, Prabhas C, Ghosh D. 1971. Thin layer chromatography and UV spectrophotometry of alcoholic extracts of Hydrastis canadensis. Planta Med 19:258±263. 16. Sturm S, Stuppner H. 1998. Analysis of isoquinoline alkaloids in medicinal plants by capillary electrophoresis-mass spectrometry. Electrophoresis 19:3026±3032. 17. Unger M, Stockigt J. 1997. Improved detection of alkaloids in crude extracts applying capillary electrophoresis with ®eld ampli®ed sample injection. J Chromatogr, A 791:323±331. 18. Hermes RE, Valdez LS. 1996. HPLC method for the determination of alkaloids in extracts of goldenseal (Hydrastis canadensis). In: 211th ACS National Meeting; 1996; New Orleans, LA, 1996:217036. 19. Leone MG, Cometa MF, Palmery M, Saso I. 1996. HPLC determination of the major alkaloids extracted from Hydrastis canadensis L. Phytother Res 10:S45±S46. 20. Wagner H. 1988. Pharmazeutische Biologie. Stuttgart: Gustav Fischer Verlag.
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677
2
Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, Mississippi 38677
Received 26 June 2000; revised 21 November 2000; accepted 5 December 2000
ABSTRACT: Goldenseal (Hydrastis canadensis L., Ranunculaceae) is an ingredient of various dietary supplements intended for enhancing general body immunity. Many goldenseal products are currently available in the United States, either alone or in combination with echinacea. In most products, the content of the main active alkaloids of goldenseal, hydrastine and berberine, is not indicated on the label. A high-performance liquid chromatography (HPLC) method has been developed for the detection and quanti®cation of hydrastine and berberine in a number of products obtained from the 1 United States market. The method uses a Phenomenex Luna C18 column, a mobile phase consisting of solvent A (100 mM sodium acetate/acetic acid, pH 4.0) and solvent B (acetonitrile/methanol; 90/10, v/v). Elution was run at a ¯ow rate of 1.0 mL/min, with a linear gradient of 80± 40% A in B over 20 min and ultraviolet detection at 290 nm. A wide range of content variation was observed for both alkaloids in the tested samples. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:817±822, 2001 Keywords: goldenseal; hydrastine; berberine; HPLC; dietary supplements
INTRODUCTION Goldenseal (Hydrastis canadensis L., Ranunculaceae) is a native American plant that has a history of folk medicine use in the treatment of many ailments, such as gastrointestinal disturbances, urinary disorders, hemorrhage, skin, mouth, and eye infections, and in¯ammation.1,2 Currently, echinacea/goldenseal dietary supplements are among the top 13 herbal products in the United States Food, Drug, and Mass market (FDM), with sales exceeding $44 million in 1999.3 The common structure/function label claim of such products is to maintain a healthy immune function during the ¯u season. Extracts of H. canadensis were Correspondence to: I. A. Khan (Telephone: 662-915-7821; Fax: 662-915-7989; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 817±822 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
found to possess antibacterial, antimalarial, and smooth muscle relaxant activities.4±7 The pharmacological and antimicrobial effects of the extracts are mainly attributed to the high content of the isoquinoline alkaloids, hydrastine and berberine (Figure 1), even though their oral bioavailability is low.2,8 The popularity of goldenseal as an ingredient in natural ¯u remedies may be justi®ed by the recent ®nding that the extract increased the primary IgM response in a group of goldenseal-treated subjects during the ®rst 2 weeks of treatment.9 On the other hand, the administration of goldenseal may result in various adverse reactions, such as digestive disorders, nausea and vomiting, CNS and respiratory depression, seizures, and heart block.10 In spite of the potential for toxic effects, under the Dietary Supplement Health and Education Act (DSHEA), alkaloid content is not required on product label. Also, in addition to possible hazards, products
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 7, JULY 2001
817
818
ABOURASHED AND KHAN
Figure 1. Chemical structures of the major goldenseal alkaloids.
with inadequate alkaloid content, and therefore poor pharmacological bene®t, can be a common problem when appropriate analytical quality control methods are lacking. A number of spectrophotometric, gravimetric, chromatographic, and electrophoretic methods have been reported for the determination of hydrastine and berberine in goldenseal extracts.11± 17 Also, two previously reported methods are based on reversed-phase high-performance liquid chromatography (HPLC).18,19 However, in both reports, method information was partially disclosed, the analytical run in one method was relatively long (40 min) with obvious peak overlap for hydrastine and tailing for berberine, and, from an application perspective, no herbal products containing goldenseal were analyzed by either method.18,19 In continuation to our investigation of various herbal products for their content uniformity and to bring attention to the necessity for establishing acceptable standards for dietary supplements marketed in the United States, we report herein the development and utilization of a fast and simple HPLC method for the determination of hydrastine and berberine in a number of selected goldenseal products purchased locally. Parameters included in method validation were linearity, accuracy, precision, speci®city, limit of detection, and limit of quantitation.
EXPERIMENTAL SECTION Materials Berberine chloride and ( ÿ )-b-hydrastine were purchased from Sigma (St. Louis, MO). Goldenseal-containing products were purchased either from local retailers or via the Internet: ``Sample 1'' (Herbal Single Goldenseal Root, Nature's Way Products, Inc., Springville, UT); ``Sample 2'' (Goldenseal Root, Thompson Nutritional ProJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 7, JULY 2001
ducts, Boca Raton, FL); ``Sample 4'' and ``Sample 5'' (Herbal Formula Echinacea with Goldenseal, Nature's Way, different batches); ``Sample 6'' (Echinacea-Goldenseal, Nature's Resource Products, Mission Hills, CA); ``Sample 7'' (EchinaceaGoldenseal, NaturPharma, American Fork, UT); ``Sample 8'' (Echinacea & Goldenseal Root, Futurebiotics, Hauppauge, NY); ``Sample 9'' (Echinacea Goldenseal, Natrol, Inc., Chatsworth, CA); ``Sample 10'' (BeeFense, Premier One Products, Inc., Park City, UT); ``Sample 11'' (Echinacea/Goldenseal, Harvest of Nature, Wilmington, DE); and ``Sample 12'' (Echinacea-Goldenseal, Planetary Formulas, Soquel, CA). Crude goldenseal powder (Sample 3) was obtained from the Australasian College of Herbal Studies (Lake Oswego, OR). HPLC grade acetonitrile and methanol were purchased from Fisher Scienti®c (Fairlawn, NJ). Deionized water was obtained from a Milli-Q Plus analytical deionization system (Bedford, MA). Sodium acetate was purchased from Sigma (St. Louis, MO). Instrumentation and Chromatographic Conditions A component HPLC system (Waters, Millford, MA) consisted of a 600E controller, a 600 pump, a 712 WISP autoinjector, and a 996 UV photodiode array detector. A pre-packed Luna, 15 cm 4.6 mm (5 mm particle size), C18 HPLC column and SecurityGuard cartridge system (Phenomenex, Torrance, CA) were operated with a mobile phase consisting of solvent A (0.10 M sodium acetate/acetic acid, pH 4.0, in water) and solvent B (acetonitrile/methanol, 90/10, v/v), continuously degassed with helium. Elution was run at a ¯ow rate of 1.0 mL/min with a linear gradient of 80±40% A in B over 20 min and ultraviolet (UV) detection at 290 nm. After each run, the column was eluted with methanol for 5 min, followed by reequilibriation with the starting solvent for 10 min. Chromatographic data were processed with Millennium 32 software, version 3.05.01 (Waters, Millford, MA) installed on a Quantex PC. Standard Solutions and Sample Preparation Hydrastine and berberine (1.0 mg each) were placed in a 10-mL volumetric ¯ask and the volume was brought to 10 mL with methanol. Solutions with known concentrations were prepared from the standard stock solution by serial dilution with methanol.
HPLC DETERMINATION OF HYDRASTINE AND BERBERINE IN GOLDENSEAL DIETARY SUPPLEMENTS
Accurately weighed powdered goldenseal from each sample (obtained by combining the contents of three capsules or by direct weighing of crude powder) was exhaustively extracted as follows: 100.0 mg of powder was sonicated in 3 mL of methanol for 15 min in a screw-capped polyethylene tube. The tube was centrifuged at 1500 rpm for 5 min, and the supernatant was transferred to a 10-mL volumetric ¯ask. The same procedure was repeated twice and the combined extract was adjusted to volume with methanol. An adequate volume (2 mL) was passed through a 13-mm, 0.45-mm nylon membrane. The ®rst 1 mL was discarded and the remaining volume was collected in an HPLC sample vial, and 10.0 mL was injected into the chromatograph. For Sample 12 (liquid extract), 1 mL was transferred to a 2-mL volumetric ¯ask and diluted to volume with methanol, and 10.0 mL was injected into the chromatograph. The remaining residue was subjected to a fourth extraction with methanol (3 mL) and a ®ltered aliquot of the extract was separately injected into the chromatograph. Method Validation Calibration curves were constructed by plotting peak areas for the external standards, hydrastine and berberine, versus a 6-fold concentration range of 100.0, 50.0, 25.0, 12.5, 6.3, and 3.1 mg/ mL. Each solution was injected three times beginning with the most dilute concentration. The slope, intercept, and correlation coef®cients were calculated by linear regression analysis on the Millennium software. For validating analytical accuracy, 100.0-mg portions of Sample 1 were spiked with three different concentrations of hydrastine and berberine (Table 1). Extraction of each portion was conducted as already described. The same extraction procedure was repeated on 100 mg of unspiked Sample 1. Extracts were injected in the chromatograph and the Table 1.
alkaloid concentrations were determined. The retention time and relative standard deviation (RSD) values for measured alkaloids in all samples were used to determine method precision. Peak identity was determined by spiking a solution of Sample 1 (800 mL) with 200 mL of standard solution of hydrastine and berberine (140.0 and 100.0 mg/mL, respectively) before injection, then peak overlap was observed for the obtained chromatogram. Peak purity was established by spectral overlap.
RESULTS AND DISCUSSION The two alkaloids were well resolved with a retention time difference of > 4 min (Figure 2). Linear regression analysis of the calibration plot of both alkaloids gave the equations y 8.32 103x 4090 (hydrastine) and y 1.68 104x 1480 (berberine), with correlation coef®cients (r2) of > 0.999 (n 3). Therefore, each alkaloid had a linear response within the speci®ed range of concentrations. The ef®ciency of the triple extraction procedure was established when neither alkaloid could be detected in the chromatograms of the fourth extract of each sample. Accuracy was validated by recovery studies in which known amounts of both alkaloids were spiked into sample 1 and subjected to the same triple extraction procedure described earlier. Acceptable recoveries were obtained for both alkaloids in the range 94.82±101.71% (Table 1). Method precision was re¯ected in the fact that the mean retention times were 10.9 min (RSD 2.67%, n 36) and 15.2 min (RSD 0.83%, n 36); and that the RSD of all measurements ranged from 0.75 to 1.82% for hydrastine and from 0.01 to 0.97% for berberine (n 3/sample). The limit of quantitation of each alkaloid was taken as 3.13 mg/mL because peaks at concentrations below this level (detection limit, 1.00 mg/mL) were
Statistical Characterization of Method Accuracy
Analyte ( ÿ )-b-Hydrastine Berberine
819
Added (mg)
Mean Recovery (%, n 3)
RSDa (%)
Overall Mean Recovery (%)
RSDa (%)
9.90 32.67 98.99 11.23 37.08 112.35
101.23 99.08 94.82 101.71 98.29 95.09
1.28 1.88 0.82 1.29 1.71 0.76
98.38
3.32
98.36
3.37
a
Relative standard deviation. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 7, JULY 2001
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Figure 2. Representative chromatograms of extracts from two commercially available goldenseal products. Peaks: A hydrastine, B berberine.
detectable but not quanti®able because of considerable baseline drift. Peak purity was established by comparing the UV spectra obtained at three time points for peaks corresponding to each alkaloid in all analyzed samples. All spectra were superimposable and representative ones are shown in Figure 3 for Sample 1. Spiking a solution of Sample 1 with standard alkaloid solution resulted in pure peaks at the same retention time of hydrastine (10.87 min) and berberine (14.92 min), with peak area increases of 11 and 7%, respectively, thus validating the peak identities of both alkaloids. The method was also speci®c for the detection of the two markers because no interfering constituents were detected in any of the analyzed samples as evidenced by peak purity. The analyzed samples exhibited a wide range of alkaloid concentrations that ranged from 0.00 to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 7, JULY 2001
2.51% for hydrastine and from 0.00 to 4.35% for berberine (Table 2). All samples had a hydrastine/ berberine ratio of 1/1.5, typical of goldenseal.8 The only exception was Sample 10, whose label listed seven herbal ingredients (goldenseal, echinacea, garlic, elderberry, ginger, peppermint, and cayenne) and in which neither alkaloid could be detected. Highest concentrations were observed in single-component goldenseal products (Samples 1 & 2) and in the crude powder (Sample 3). Sample 1 was the only product to show a label claim of 5% total alkaloids, and the total value obtained in our analysis (6.86%) exceeded the label claim. For all the remaining samples, the total alkaloid content was < 5%, which is not unexpected because all were multicomponent products in which echinacea powder was included in the formulation. Therefore, the relative ratios
HPLC DETERMINATION OF HYDRASTINE AND BERBERINE IN GOLDENSEAL DIETARY SUPPLEMENTS
821
Figure 3. Peak purity validation by UV spectral overlap: (A) Overlaid spectra of hydrastine peak at 10.78, 10.88, and 10.99 min; (B) overlaid spectra of berberine peak at 16.39, 16.52, and 16.71 min.
of herbal components in mixtures should be clearly stated so that quantitative analysis may accurately determine marker compound levels in each herbal component. In this context, the labels of only three products (Samples 8, 9, and 10) showed the relative ratio of goldenseal powder to other mixture components. Based on the label claims, the alkaloid concentration in the goldenseal component of these products could be recalculated to assess the quality of the ingredient raw materials (Table 3). It is to be noted that the 5% total alkaloids (label of Sample 1) is not a standard value recommended by any major
authority (e.g., United States, British or German Pharmacopeias), but rather a ®gure that is close to the reported total alkaloid concentration estimated for goldenseal.8,20 In conclusion, even though quality appraisal is not the main focus of this work, it is clear that content uniformity is an issue that needs to be considered to achieve a minimum level of product conformity. The wide range of alkaloid concentrations estimated in this study implies that goldenseal products, among many other dietary supplements on the United States market, need to conform to common acceptable quality control
Table 2. Concentration (%) of Hydrastine and Berberine in Selected Goldenseal-Containing Products (n 3) Product 1a 2a 3a 4b 5b 6 7 8 9 10 11 12
Form
Lot#
Exp. Date
Hydrastine (RSD %)
Berberine (RSD %)
Capsule Capsule Crude Capsule Capsule Capsule Capsule Capsule Capsule Capsule Tea bag Liquid Extract
907330 16427 Ð 810423 612166 HD12070 704081 97VF514 934864 001309 Ð Ð
11/2002 6/2002 Ð 10/2003 12/2001 4/2000 4/2002 Ð 8/2001 3/2000 Ð Ð
2.51 (1.46) 2.27 (1.22) 1.35 (0.75) 0.60 (0.98) 0.58 (1.07) 0.54 (1.06) 0.22 (0.56) 0.94 (1.00) 0.11 (1.82) 0.00 (0.00) 1.91 (1.46) 1.83 (0.96)
4.35 (0.75) 3.41 (0.62) 1.79 (0.34) 1.03 (0.61) 0.86 (0.41) 0.82 (0.97) 0.34 (0.22) 1.27 (0.69) 0.17 (0.40) 0.00 (0.00) 2.83 (0.90) 1.81 (0.47)
a
Exclusively goldenseal. Different batches of the same brand.
b
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Table 3. Adjusted Alkaloidal Content in the Goldenseal Portion of Multicomponent Products Adjusted Concentration (%) Sample 8 9 10
Goldenseal (mg) 200 125 50
Other (mg)
Factor
Hydrastine
Berberine
300 360 400
2.50 3.88 9.00
2.35 0.43 0.00
3.18 0.66 0.00
criteria. The method described in this paper can be applied as an analytical tool for determining the levels of hydrastine and berberine in commercial products containing goldenseal, either as a single component or in combination with echinacea, to establish one of the primary criteria for quality and conformity of herbal dietary supplements.
ACKNOWLEDGMENT The authors acknowledge the United States Department of Agriculture, Agricultural Research Service Speci®c Cooperative Agreement No. 58-6408-7-012 for partial support of this work.
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