Ephedrine Alkaloids Research Articles

Statistical collaboration to impact policy decisions

The objective of this paper is to support the following thesis: In health services research, a statistician can, and often should, contribute beyond what is generally defined as the 'classical' statistician project role. I use projects from RAND Health, the health services research division of the RAND Corporation, to demonstrate the issues that arise for the statistician in this expanded collaborative role. These projects are the 'HIV Cost and Services Utilization Study' (HCSUS) and the 'Ephedra: Clinical Efficacy and Side Effects Project'. HCSUS collected information on a nationally representative sample of people in care for HIV infection in order to determine what services were being delivered and their cost in order to guide policy decisions on the allocation of limited health care resources. The ephedra project was a systematic review conducted by the Southern California Evidence-Based Practice Centre to provide information on the existing science so that the National Institutes of Health could guide an expanded research effort to better understand the safety of ephedrine alkaloids. In order to collaborate on more than just the data analysis of a typical project, the statistician must proactively contribute to all project phases. These phases include problem formulation, study design, data collection, analysis, and the communication of results. The statistician needs to effectively and efficiently balance methodological, substantive, practical, and sometimes even ethical demands. As a result of contributing beyond standard statistical tasks, the statistician may directly impact health policy decisions.

Molecular mechanisms of toxicity of important food-borne phytotoxins

At present, there is an increasing interest for plant ingredients and their use in drugs, for teas, or in food supplements. The present review describes the nature and mechanism of action of the phytochemicals presently receiving increased attention in the field of food toxicology. This relates to compounds including aristolochic acids, pyrrolizidine alkaloids, beta-carotene, coumarin, the alkenylbenzenes safrole, methyleugenol and estragole, ephedrine alkaloids and synephrine, kavalactones, anisatin, St. John's wort ingredients, cyanogenic glycosides, solanine and chaconine, thujone, and glycyrrhizinic acid. It can be concluded that several of these phytotoxins cause concern, because of their bioactivation to reactive alkylating intermediates that are able to react with cellular macromolecules causing cellular toxicity, and, upon their reaction with DNA, genotoxicity resulting in tumors. Another group of the phytotoxins presented is active without the requirement for bioactivation and, in most cases, these compounds appear to act as neurotoxins interacting with one of the neurotransmitter systems. Altogether, the examples presented illustrate that natural does not equal safe and that in modern society adverse health effects, upon either acute or chronic exposure to phytochemicals, can occur as a result of use of plant- or herb-based foods, teas, or other extracts.

Survey on Resources of Ephedra Plants in Xinjiang

The resources of wild Ephedra plants in the Xinjiang Uygur Autonomous Region were surveyed. Ephedra plants mainly grow on the fringes of the Taklimakan Desert and Gureban-tonggute Desert. We found six genotypes of Ephedra przewalskii growing widely in Xinjiang. Three genotypes of Ephedra intermedia were limited to the northern and eastern parts, and Ephedra regeliana scattered in the northern part of Xinjiang. These Ephedra specimens were analyzed for DNA sequences of nuclear ribosomal DNA, internal transcribed spacers 1 and 2, chloroplastic DNA, trnL intron and trnL-trnF intergenic spacer. Intraspecific variation of the nucleotide sequence in E. przewalskii was found in different habitats. Norephedrine, ephedrine, pseudoephedrine, and methylephedrine contents of the specimens were determined. Although Ephedra intermedia of all three genotypes contained ephedrine alkaloids, ephedrine alkaloids were not detected in E. regeliana and E. przewalskii.

Dietary Supplements Containing Ephedrine Alkaloids Adulterated Because They Present an Unreasonable Risk; Final Rule

The Food and Drug Administration issued a final regulation declaring dietary supplements containing ephedrine alkaloids adulterated under the Federal Food, Drug, and Cosmetic Act (the act) because they present an unreasonable risk of illness or injury under the conditions of use recommended or suggested in labeling, or if no conditions of use are suggested or recommended in labeling, under ordinary conditions of use. The agency took this action based upon the wellknown pharmacology of ephedrine alkaloids, the peer-reviewed scientific literature on the effects of ephedrine alkaloids, and the adverse events reported to have occurred in individuals following consumption of dietary supplements containing ephedrine alkaloids. The final Rule was published in the Federal Register, Volume 69, Number 28, Pages 6787-6854 on February 11, 2004. This rule that became effective on April 12, 2004 is now included in the Code of Federal Regulations (21CFR Part 119). This report includes the Table of Contents of the full Federal Register publication and selected sections and tables from that publication.

Banned, but Not Forgotten

Banned, but Not Forgotten

FDA to Ban Sales of Dietary Supplements Containing Ephedra

On December 30, 2003, the FDA announced that it will publish a rule banning sales of ephedra - a dietary supplement often utilized for weight loss, increased energy, and enhanced athletic performance - because it poses an unreasonable health risk.The ban will be issued under the auspices of the Federal Food, Drug, and Cosmetic Act and the Dietary Supplement Health and Education Act of 1994 (DSHEA), in response to a process that began in June of 1997, when the FDA first proposed a mandatory warning statement on dietary supplements containing ephedra. FDAs analysis of the scientific evidence has led it to conclude that dietary supplements containing ephedrine alkaloids present an unreasonable risk of illness or injury and are therefore adulterated under Section 402(f)(1)(A) of the Federal Food, Drug, and Cosmetic Act?

Did Delay of Ephedra Ban Cause Unnecessary Deaths?

Back to table of contents Previous article Next article Clinical & Research NewsFull AccessDid Delay of Ephedra Ban Cause Unnecessary Deaths?Mark MoranMark MoranSearch for more papers by this authorPublished Online:6 Feb 2004https://doi.org/10.1176/pn.39.3.0024The Food and Drug Administration (FDA) is banning sale of dietary supplements containing ephedrine alkaloids (ephedra). The agency is also urging consumers to stop using ephedra products immediately.Health and Human Services Secretary Tommy Thompson issued a consumer alert on the safety of dietary supplements containing ephedra and has notified manufacturers of its intent to publish a final rule on dietary supplements containing ephedrine alkaloids. The rule will state that dietary supplements containing ephedrine alkaloids present an unreasonable risk of illness or injury.The rule would have the effect of banning the sale of dietary supplements containing ephedrine alkaloids when it becomes effective, 60 days following publication. Sixty-two firms that market dietary supplements containing ephedra and ephedrine alkaloids received the letter from the FDA alerting them of the final rule.Thompson said the final rule “will formalize its conclusions that dietary supplements containing ephedrine alkaloids present unreasonable risks to those who take them for any reason.”Ephedra, also called Ma huang, is a naturally occurring substance derived from botanicals. Its principal active ingredient is ephedrine, which when chemically synthesized is regulated as a drug. In recent years ephedra products have been extensively promoted for use to aid weight loss, enhance sports performance, and increase energy. The supplement has also been linked to a variety of adverse events, including psychiatric complications.This year the RAND Corporation’s Southern California Evidence-Based Practice Center issued the report “Ephedra and Ephedrine for Weight Loss and Athletic Performance Enhancement: Clinical Efficacy and Side Effects.” The report provided evidence that ephedra is associated with major health risks—including psychiatric effects—and found only limited evidence of health benefits resulting from its use.The RAND Corporation’s review of some 16,000 adverse-event reports revealed two deaths, four heart attacks, nine strokes, one seizure, and five psychiatric cases involving ephedra in which the records appeared thorough and no other contributing factors were identified.In addition, three cases of psychosis related to use of ephedra-containing herbal supplements were reported in the July 2003 Southern Medical Journal.Study authors Ruth Walton, M.D., and Gail H. Manos, M.D., of the department of psychiatry at Portsmouth Naval Medical Center in Portsmouth, Va., observed, “Toxicity may occur at only two or three times the maximum therapeutic dose of 150 mg/d. Most reports have focused on medical complications such as stroke, seizure, hepatic toxicity, or even death” (Psychiatric News, November 21, 2003).In 1997 the FDA first proposed a rule on dietary supplements containing ephedra including requiring a warning statement on these products. The agency modified this proposed rule in 2000, and a year ago the agency announced a series of comprehensive actions designed to protect Americans from the potentially serious risks of dietary supplements containing ephedra.At least one critic charged the FDA’s action comes too late.Sidney Wolfe, M.D., director of Public Citizen’s Health Research Group, claimed that the FDA knew of more than 155 deaths of ephedra users and said the agency had waited more than two years after Public Citizen first petitioned the agency to ban the supplement.The delay “shows the dangerous cowardice of FDA Commissioner Mark McClellan and HHS Secretary Tommy Thompson,” Wolfe said. “All the scientific evidence and legal authority to ban ephedra was in place at the time of our petition, which we filed in September 2001. One reason major manufacturers have stopped selling ephedra is that the companies have become uninsurable because of massive losses in product liability cases. When we filed our petition, there were reports of 81 ephedra-related deaths. Now, after that number has nearly doubled and very little ephedra is being manufactured, the FDA finally announces a ban.“This is an inexcusable dereliction of responsibility by an agency that has acted more like an ephedra sales extension agency than the public health agency it is supposed to be,” Wolfe said.More information on the FDA’s actions related to ephedra can be found online at www.fda.gov/ola/2003/dietarysupplements1028.html. ▪ ISSUES NewArchived

Dietary Supplements Containing Ephedrine Alkaloids Adulterated Because They Present an Unreasonable Risk; Final Rule

The Food and Drug Administration issued a final regulation declaring dietary supplements containing ephedrine alkaloids adulterated under the Federal Food, Drug, and Cosmetic Act (the act) because they present an unreasonable risk of illness or injury under the conditions of use recommended or suggested in labeling, or if no conditions of use are suggested or recommended in labeling, under ordinary conditions of use. The agency took this action based upon the well-known pharmacology of ephedrine alkaloids, the peer-reviewed scientific literature on the effects of ephedrine alkaloids, and the adverse events reported to have occurred in individuals following consumption of dietary supplements containing ephedrine alkaloids. The final Rule was published in the Federal Register, Volume 69, Number 28, Pages 6787-6854 on February 11, 2004. This rule that became effective on April 12, 2004 is now included in the Code of Federal Regulations (21 CFR Part 119). This report includes the Table of Contents of the full Federal Register publication and selected sections and tables from that publication.

Determination of Ephedrine Alkaloids in Botanicals and Dietary Supplements by HPLC-UV: Collaborative Study

An international collaborative study was conducted of a high-performance liquid chromatography (HPLC)-UV method for the determination of the major (ephedrine [EP] and pseudoephedrine [PS]) and minor (norephedrine [NE], norpseudoephedrine [NP], methylephedrine [ME], and methylpseudoephedrine [MP]) alkaloids in selected dietary supplements representative of the commercially available products. Ten collaborating laboratories determined the ephedrine-type alkaloid content in 8 blind replicate samples. Five products contained ephedra ground herb or ephedra extract. These 5 products included ground botanical raw material of Ephedra sinica, a common powdered extract of Ephedra sinica, a finished product containing only Ephedra sinica ground botanical raw material, a complex multicomponent dietary supplement containing Ma Huang, and a high-protein chocolate flavored drink mix containing Ma Huang extract. In addition, collaborating laboratories received a negative control and negative control spiked with ephedrine alkaloids at high and low levels for recovery studies. Test extracts were treated to solid-phase extraction using a strong-cation exchange column to help remove interferences. The HPLC analyses were performed on a polar-embedded phenyl column using UV detection at 210 nm. Repeatability relative standard deviations (RSDr) ranged from 0.64-3.0% for EP and 2.0-6.6% for PS, excluding the high protein drink mix. Reproducibility relative standard deviations (RSDR) ranged from 2.1-6.6% for EP and 9.0-11.4% for PS, excluding the high protein drink mix. Recoveries ranged from 84.7-87.2% for EP and 84.6-98.2% for PS. The data developed for the minor alkaloids are more variable with generally unsatisfactory HORRATS (i.e., >2). However, since these alkaloids generally add little to the total alkaloid content of the products, the method gives satisfactory results in measuring total alkaloid content (RSDr 0.85-3.13%; RSDR 2.03-10.97%, HORRAT 0.69-3.23, exclusive of the results from the high protein drink). On the basis of these results, the method is recommended for Official First Action for determination of EP and PS in dietary supplements exclusive of the high protein drinks.

Caffeine and ephedrine: physiological, metabolic and performance-enhancing effects.

Preparations containing caffeine and ephedrine have become increasingly popular among sportspersons in recent years as a means to enhance athletic performance. This is due to a slowly accumulating body of evidence suggesting that combination of the two drugs may be more efficacious than each one alone. Caffeine is a compound with documented ergogenicity in various exercise modalities, while ephedrine and related alkaloids have not been shown, as yet, to result in any significant performance improvements. Caffeine-ephedrine mixtures, however, have been reported in several instances to confer a greater ergogenic benefit than either drug by itself. Although data are limited and heterogeneous in nature to allow for reaching consensus, the increase in performance is a rather uniform finding as it has been observed during submaximal steady-state aerobic exercise, short- and long-distance running, maximal and supramaximal anaerobic cycling, as well as weight lifting. From the metabolic point of view, combined ingestion of caffeine and ephedrine has been observed to increase blood glucose and lactate concentrations during exercise, wheareas qualitatively similar effects on lipid fuels (free fatty acids and glycerol) are less pronounced. In parallel, epinephrine and dopamine concentrations are significantly increased, wheareas the effects on norepinephrine are less clear. With respect to pulmonary gas exchange during short-term intense exercise, no physiologically significant effects have been reported following ingestion of caffeine, ephedrine or their combination. Yet, during longer and/or more demanding efforts, some sporadic enhancements have indeed been shown. On the other hand, a relatively consistent cardiovascular manifestation of the latter preparation is an increase in heart rate, in addition to that caused by exercise alone. Finally, evidence to date strongly suggests that caffeine and ephedrine combined are quite effective in decreasing the rating of perceived exertion and this seems to be independent of the type of activity being performed. In general, our knowledge and understanding of the physiological, metabolic and performance-enhancing effects of caffeine-ephedrine mixtures are still in their infancy. Research in this field is probably hampered by sound ethical concerns that preclude administration of potentially hazardous substances to human volunteers. In contrast, while it is certainly true that caffeine and especially ephedrine have been associated with several acute adverse effects on health, athletes do not seem to be concerned with these, as long as they perceive that their performance will improve. In light of the fact that caffeine and ephedra alkaloids, but not ephedrine itself, have been removed from the list of banned substances, their use in sports can be expected to rise considerably in the foreseeable future. Caffeine-ephedra mixtures may thus become one of most popular ergogenic aids in the years to come and while they may indeed prove to be one of the most effective ones, and probably one of the few legal ones, whether they also turn out to be one of the most dangerous ones awaits to be witnessed.

Separation and Determination of Ephedrine Enantiomers and Synephrine by High Performance Capillary Electrophoresis in Dietary Supplements

The chiral separation of ephedrine alkaloids by high performance capillary electrophoresis is of great interest since the enantiomers exhibit quantitative and qualitative differences in pharmacological activity. The isomers of (−)-ephedrine, (+)-pseudoephedrine, (−)-N-methylephedrine, (+)-N-methylpseudoephedrine, (+)-norpseudoephedrine and (−)-norephedrine are the major bioactive components of E. sinica (Ma-Huang) which is a Chinese herb used for weight loss and as an energy booster in the US. However, the compounds stereoisomers are not present in the plant material. The electrophoretic separation was performed using a 110 cm × 50 μm I.D. (101.5 cm effective length) fused silica capillary. The samples were injected by pressure for 5 s at 50 mbar and the running voltage was 30 kV at the injector end of the capillary. Within 23 min, nine ephedrine compounds and synephrine were separated at 210 nm. The method was successively applied to the determination of the ephedrine compounds in dietary supplement products. Parameters affecting the resolution between (+) and (−)-enantiomers, such as pH, cyclodextrin concentration, temperature, organic modifier, buffer concentration and capillary dimensions were reported.

Demographic, pathologic, and toxicological profiles of 127 decedents testing positive for ephedrine alkaloids

The relative toxicity of ephedra-containing dietary supplements is disputed. In order to ascertain the magnitude of the problem, we reviewed all autopsies in our Medical Examiner’s jurisdiction, from 1994 to 2001, where ephedrine or any its isomers (E+) were detected. Toxicology testing results were tabulated and anatomic findings in E+ cases were compared to those in a control group of drug-free trauma victims. Of 127 E+ cases identified, 33 were due to trauma. Decedents were mostly male (80.3%) and mostly Caucasian (59%). Blood ephedrine concentrations were <0.49mg/l in 50% of the cases, range 0.07–11.73mg/l in trauma victims, and 0.02–12.35mg/l in non-trauma cases. Norephedrine (NE) was present in the blood of 22.8% (mean of 1.81mg/l, S.D.=3.14mg/l) and in the urine of 36.2% (mean of 15.6mg/l, S.D.=21.50mg/l). Pseudoephedrine (PE) was present in the blood of 6.3% (8/127). More than 88% (113/127) of the decedents also tested positive for other drugs, the most common being cocaine (or its metabolites) and morphine. The most frequent pathologic diagnoses were hepatic steatosis (27/127) and nephrosclerosis (22/127). Left ventricular hypertrophy was common, and coronary artery disease (CAD) detected in nearly one third of the cases. The most common findings in E+ deaths are those generally associated with chronic stimulant abuse, and abuse of other drugs was common in those with CAD. There were no cases of heat stroke or rhabdomyolysis. In most cases, norephedrine was not detected, suggesting it plays no role in ephedrine toxicity.

Ephedrines in over-the-counter cold medicines and urine specimens collected during sport competitions.

Ephedrine (EPH), pseudoephedrine (PEPH), phenylpropanolamine (PPA), and methylephedrine (MEPH) are ephedrine alkaloids commonly found in cold medications and are banned by the International Olympic Committee (IOC). These compounds were detected in the urinary doping tests during the national sport competitions in Taiwan. To study the sources of these compounds, 91 over-the-counter (OTC) nonprescription cold remedies, along with 1803 athletes' urine samples collected (from 1999 to 2001) in competitions were analyzed using gas chromatography-nitrogen-phosphorus detection (GC-NPD) for initial screening and GC-mass spectrometry (MS) for confirmation. We found that 80% of OTC cold medicines showed banned ephedrines in their ingredients lists, in which MEPH (52%) was the most common drug labeled. However, when these OTC cold medicines were analyzed by GC-NPD and GC-MS, EPH (35.4%) was found substantially higher than that labeled in the OTC products (1.3%). In the total urine specimens tested, approximately 2.8% contained banned ephedrines and 1.3% exceeded the IOC cutoff levels. Within the urine specimens that exceeded the IOC cutoff values, PEPH accounted for a 44% occurrence rate, followed by EPH (28%), PPA (17%), and MEPH (11%). In agreement with the other report, bodybuilders showed a high incidence rate for ephedrines misuse. Nevertheless, it is likely that the high incidence of doping violations for ephedrine-related substances was related to misuse of ephedrines present in most OTC common cold medicines and some dietary supplements for relieving cold symptoms, reducing body weight, and preserving muscle.

Determination of ephedrine alkaloids and synephrine in dietary supplements by column-switching cation exchange high-performance liquid chromatography with scanning-wavelength ultraviolet and fluorescence detection.

An HPLC method with on-line cleanup coupled to the separation column is described for determination of (-)-norephedrine, (+)-norpseudoephedrine, (-)-ephedrine, (+)-pseudoephedrine, (-)-N-methylephedrine, (+)-N-methylpseudoephedrine, and (+/-)-synephrine in finished dietary supplement products. Test portions were extracted in acidified aqueous acetone. A filtered aliquot was cleaned up on a strong cation exchange (SCX) precolumn that later was automatically coupled to the SCX analytical column. Measurement was by full-scan UV spectra for confirmation of identity by spectral matching and real-time integration of three wavelength signals for multiple quantitation. (+/-)-Synephrine was also quantitated by native fluorescence. Recovery averaged 95-100%. Determination of the major ingredients (-)-ephedrine, (+)-pseudoephedrine, and (+/-)-synephrine compared favorably to findings by an independent LC-MS analysis for a set of 25 samples. The results of a survey were reported for total ephedrine alkaloid and synephrine content and were compared to content declaration, for approximately 48 finished products.

Ephedra in perspective--a current review.

Although the traditional use of Ephedra 'ma huang' has been established for thousands of years, its resurgence in the US as a herbal dietary supplement is currently a matter of national controversy. At the heart of the debate are three important questions: (1) the identity and composition of Ephedra products with regard to ephedrine and related alkaloids; (2) the potential therapeutic utility of Ephedra supplements for weight loss or performance enhancement; and (3) potential health risks associated with such uses of Ephedra, particularly in sensitive individuals or in cases of intentional abuse for its stimulant properties. This review surveys the literature on Ephedra with regard to traditional uses, botany, chemistry, analytics, pharmacological effects and health risks. A brief discussion of the central issues in the current debate on the regulation of Ephedra in the United States is included as this is where most of the problems have occurred to date.

Determination of Ephedrine Alkaloids in Dietary Supplements and Botanicals by Liquid Chromatography/Tandem Mass Spectrometry: Collaborative Study

An interlaboratory study was conducted to evaluate the accuracy and precision of a method for ephedrine-type alkaloids [i.e., norephedrine (NE), norpseudoephedrine (NPE), ephedrine (E), pseudoephedrine (PE), methylephedrine (ME), and methylpseudoephedrine (MPE)] in dietary supplements and botanicals. The amount of ephedrine-type alkaloids present was determined using liquid chromatography with tandem mass selective detection. The samples were diluted to reflect a concentration of 0.0200 to 1.00 microg/mL for each alkaloid. An internal standard was added and the alkaloids were separated using a 5 microm phenyl LC column with an ammonium acetate, glacial acetic acid, acetonitrile, and water mobile phase. Eight blind duplicates of dietary supplements or botanicals were analyzed by 10 collaborators. Included was a negative control, ephedra nevadensis, and negative controls fortified at 2 different levels with each of the 6 ephedrine-type alkaloids. The spike levels were approximately 100 and 1000 microg/g for NE, 100 and 600 microg/g for NPE, 6500 and 65000 microg/g for E, 1000 and 10 000 microg/g for PE, 300 and 3000 microg/g for ME, and 100 and 1000 microg/g for MPE. On the basis of the accuracy and precision results for this interlaboratory study, it is recommended that this method be adopted Official First Action for the determination of 6 different individual ephedrine-type alkaloids in dietary supplements and botanicals.

Effect of a multicomponent, ephedra-containing dietary supplement (Metabolife 356) on Holter monitoring and hemostatic parameters in healthy volunteers

Herbal products containing ephedra (a natural source of ephedrine alkaloids) in combination with other ingredients exhibiting sympathomimetic properties (kola nut, guarana, and green tea extract) are widely available under names such as Xenadrine RFA-1 (Cytodyne Technologies, Lakewood, New Jersey), Metabolife 356 (Metabolife International, San Diego, California), and Ripped Fuel (Twin Laboratories, Inc., Hauppauge, New York). These products are most often taken to promote weight loss and to enhance exercise performance. Metabolife 356 has become the nation’s best-selling weight loss supplement, capturing 45% of the weight loss supplement market. Metabolife 356 contains a variety of herbs, including ma huang, guarana, ginger, ginseng, and gotu kola, as well as various other vitamins and minerals. Only a few studies have evaluated the effectiveness of ephedra-containing products on weight loss, yet the safety aspects of these products remains equivocal. Ephedracontaining dietary supplements have been associated with serious adverse effects, including myocardial infarction, cardiac arrhythmia, hypertension, hepatitis, seizure, stroke, and death. 1– 8 This study was conducted to evaluate the short-term effects of Metabolife 356 on blood pressure, heart rate, 24-hour Holter monitoring, and hemostatic parameters in healthy subjects.  Healthy men aged 20 to 40 years were included in this study. Exclusion criteria were history of consumption of any ephedra-containing supplement, any known medical condition, smoking, and current use of any prescription or nonprescription medication. Subjects were required to refrain from caffeine and exercise throughout the study period. The protocol was approved by the institutional review board. All subjects provided written informed consent before any study-related procedures were initiated. After a 7-day, caffeine-free period, subjects underwent baseline venipuncture, cuff blood pressure measurement, heart rate, 12-lead electrocardiography, and physical examination. Casual cuff blood pressures were measured using an aneroid sphygmomanometer in the dominant arm after a minimum of 10 minutes of rest in a seated position. After confi rmation of a 12hour fast, venous blood samples were collected and the serum was separated and stored frozen at 70°C. The samples were shipped frozen to the clinical laboratories and were analyzed for tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) using enzyme-linked immunosorbent assay; ephedra was assessed using liquid chromatographic/ mass spectrophotometric techniques. A baseline chemistry panel and complete blood count were also obtained to rule out any abnormalities before supplementation. Subjects with normal fi ndings at the baseline visit

The role of chemical fingerprinting: application to Ephedra

Ephedra sinica, known as Ma Huang, is one of the oldest medicinal herbs in Traditional Chinese Medicine (TCM). Preparations, namely teas, of E. sinica have been used for over 5000 years as a stimulant and as an antiasthmatic. In the West, extracts of E. sinica, E. intermedia or E. equisetina are most commonly used in dietary supplements as a stimulant and to promote weight loss. More than 50 species of Ephedra are native to both hemispheres, but the detection of ephedrine alkaloids has been limited to species in Eurasia. Currently, methods exist to quantitate the ephedrine alkaloids in extracts of plant material or dietary supplements, but the methods are not able to verify the extract is of an Ephedra species. Reverse phase high performance liquid chromatography with photodiode array detection was applied for the chemical fingerprinting of the Ephedra species. Two regions of comparison were determined in the chromatograms at 320 nm. The series of peaks between 52 and 64 min confirms an Ephedra species is being analyzed. The aforementioned peaks also could distinguish between Ephedra species from Eurasia, North America and South America. Peaks at ca. 57 and 59 min were isolated and determined to be two new compounds, 4-(2-eicosyloxycarbonyl-vinyl)-benzoic acid and 4-(2-docosyloxycarbonyl-vinyl)-benzoic acid respectively. Authentication of ground plant material as Ephedra can be achieved by this chemical fingerprinting method.

Quantitative analysis of ephedrine analogues from ephedra species using 1H-NMR.

Four ephedrine analogues such as ephedrine, pseudoephedrine, methylephedrine, and methylpseudoephedrine were determined by (1)H-NMR from Ephedra species. In the region of delta 5.0-4.0, the signals of H-1 attached to the same carbon with a hydroxyl, were well separated from each other in CDCl(3). The amount of each alkaloid was calculated by the relative ratio of the intensity of H-1 signal to the known amount of internal standard, 200 microg of anthracene. This method allows rapid determination of the quantity of four ephedrine alkaloids from Ephedra species. The amount of these alkaloids was in the range of 1.0-2.0% of dry weight depending on the plant materials.

SIMULTANEOUS SEPARATION AND DETERMINATION OF EPHEDRINE ALKALOIDS AND TETRAMETHYL-PYRAZINE IN EPHEDRA SINICA STAPF BY HPLC

A simple, sensitive, and reliable high performance liquid chromatography (HPLC) method was first developed for simultaneous determination of Ephedrine alkaloids and 2,3,5,6-tetramethylpyrazine (TMP) in Ephedra sinica Stapf. The HPLC separation was performed on a reversed phase C18 column (Nova-Pak® C18, 150 mm × 3.9 mm I.D.) with a duplicate gradient composed of methanol and phosphate buffer solution (PBS, 0.03 mol/L KH2PO4-acetic acid-triethyl amine = 400:0.25:0.1, v:v:v, pH = 6.0) to elute the solutes with a flow rate of 0.8 mL/min. The detection wavelength was set at 210 nm. Regression equations revealed linear relationships (correlation coefficients: 0.991 ∼ 0.998) between the peak area of each constituent (E, PE, NE, NPE, TMP) and its concentration. The detection limits for E, PE, NE, NPE, and TMP were 0.4, 0.1, 0.03, 0.02, 0.03 μg/mL, respectively at a signal-to-noise ratio of 3:1, and the recoveries ranged between 96.0–104.5%. The contents of E, PE, NE, NPE, and TMP in Ephedra sinica Stapf extracts were measured 0.480 g/100 g (2.5%, RSD), 0.090 g/100 g (3.0%, RSD), 0.090 g/100 g (2.4%, RSD), 0.060 g/100 g (2.2%, RSD), and 0.022 g/100 g (1.5%, RSD), respectively.