GROUP NUMBER____T2______
TITLE____ HPLC Analysis of Goldenseal Root______
DATE SUBMITTED__12-20-01__
ROLE ASSIGNMENTS
ROLE GROUP MEMBER
FACILITATOR………………………..____ Ronit Morris ____
TIME & TASK KEEPER………………___Anoop Kowshik___
SCRIBE………………………………..____ Namrata Choudhury
PRESENTER………………………….____ Howard Lopez
ABSTRACT
In this experiment, the main objectives were to develop an HPLC method to identify berberine from Goldenseal root powder and compare the concentrations of berberine in GNC Fingerprinted Goldenseal Root and Bedrock Farm Goldenseal Powder. Berberine analysis in HPLC was conducted at a wavelength of 344 nm, which produced discernible absorbance peaks for both the berberine standard and the unknown solutions. The concentration-gain parameters used for the GNC and Bedrock Goldenseal powders were 40 and 20 times greater than that of the berberine standard, respectively. However, the berberine peak could not be isolated in the unknowns, as the retention times of the main peaks of the standard and unknowns did not agree. The retention times of the peaks for the GNC and Bedrock root were 13% higher and 8.6% higher than the standard retention time, respectively. These peaks were most likely combinations of many smaller peaks representing other alkaloids or impurity compounds. The most likely variables that contributed to this broad peak are the mobile phase (which was set at 50% water, 50% MeOH), or the lack of a solid phase extraction protocol to separate non-organic impurities. The berberine was assumed to be present in the unknown because of its high solubility in water, but possibly in very low concentrations. An ultra-high concentration unknown solution was tested and revealed another small peak within the broad unknown peak, signaling the presence of at least one other compound. Further manipulation of experimental variables was needed to isolate the berberine peak, and thus, the second objective of finding and comparing the berberine concentrations of the two root powders could not be achieved.
Objectives
• To develop a method to identify berberine from Goldenseal root powder through use of isocratic, reversed phase mode HPLC.
• To determine and compare the concentrations of berberine in GNC Fingerprinted Goldenseal Root 500 mg capsules and Bedrock Farm Goldenseal Powder.
Specific Aims:
• To determine the optimal wavelength and concentration-gain conditions at which to detect berberine so as to maximize the berberine sensitivity in unknown solutions.
• To determine the optimal mobile phase and the necessity of solid phase extraction for isolating the berberine peak in unknown solutions.
• To isolate berberine from both root powder and determine concentrations.
Hypotheses:
1) The optimal wavelength for berberine detection will be approximately 235 nm in the UV spectrum
2) The optimal concentration-gain parameters for the unknowns in HPLC will be 20 to 200 times more than the optimal concentration-gain product for the standards.
3) Isolation of berberine is possible without the use of Solid Phase Extraction.
4) Percent of recovered alkaloids for both unknowns shall range between 0.5-6% of sample.
Background
Hydrastasis Canadensis (Goldenseal) is a plant whose root contains alkaloids, mainly hydrastine and berberine, which have been used for medicinal purposes as an anti-inflammatory, oxytotic stimulant, anti-bacterial agent, and mild sedative. The most important principles of goldenseal are a group of isoquinoline alkaloids consisting mainly of hydrastine (1.5 - 4%) and berberine (0.5 - 6%), lesser amounts of canadine, candaline, and related alkaliods(Willard). Note that the numbers above correspond to fractional percentages of the total alkaloids. However, another source stated that berberine is 0.5 – 6% of the total root (Polaris).
The chemical composition of berberine chloride, a salt, is C20H18NO4Cl. It is a yellow powder, which is very soluble in water or methanol. Upon dissociation, it separates into the berberinium ion and chloride ion.
Hydrastine is the principle white alkaloid of Hydrastasis, and was discovered in 1850, by Mr. Alfred B. Durand (Amer. Jour. Of Pharmacology, Vol. 23, p. 13), who described it as being insoluble in water (Felter).
In 1873, Mr. A. K. Hale (Amer. Jour. Pharmacology, 1873, p. 247) noted that Canadine (C20H21NO4), another alkaloid found in the root which resembled berberine, but being darker in color, and behaving differently toward solvents (Felter). Canadine forms an almost insoluble nitrate by means of which the alkaloid was obtained from hydrastis. The free base nitrate form of this alkaloid is insoluble in water, soluble in alcohol, whereas the sulfate form, an exception, has been found to be soluble in water (Felter).
The berberine molecule is a positive ion (cation) due to the formal charge on the nitrogen of +1. It has a polarity due to the presence of electronegative atoms on oxygen molecules, along with the formal charge on nitrogen. The Nitrogen heteoroatom places it into the group of alkaloids. The chemical structure is shown below.
Berberine is a yellow compound that occurs naturally as a berberinium salt of either chloride or sulfate, with both chloride and sulfate forms being very soluble in water (Seddon). It can also exist as berberine nitrate, although the nitrate does not naturally occur. The CRC Handbook of Physics and Chemistry rates berberine as highly soluble (a rating of 4/4, with 4 being very soluble) in diethyl ether and ethanol. Berberine is also mentioned as being soluble in water; one gram dissolves in 20ml of water (Merck). Hydrastine is given solubility ratings of 1 in water, and 3 in both acetone and benzene. Canadine is not listed as having any solubility rating within the web edition of the HBCP.
HPLC analysis is a powerful tool for the identification and quantification of active compounds of Herbal supplements, which in our case are the alkaloids of Goldenseal. Lazarowych, Etal described the use of HPLC for this purpose in their paper "Use of Fingerprinting and Marker Compounds for Identification and Standardization of Botanical Drugs: Strategies for Applying HPLC Analysis to Herbal Products. She examined several herbals such as Valerian Root and FeverFew using chemical "markers" which were basically pure standardized sources of the active compounds of the herbals. Different sources of herbals from different North American sources were analyzed for active compound concentrations, and many were found to contain little or none of these compounds (Lazarowych).
Solid Phase Extraction is a method by which aqueous samples can be processed in order to isolate and concentrate organic analytes from the sample matrix and provide a suitable sample extract for instrumental analysis. This process has a direct impact on accuracy, precision and quantitation limits and is often the rate-determining step for many analytical methods. SPE uses bonded alkyl bonded silica sorbents packed into disposable plastic or glass cartridges through which an aqueous solution of the analyte passes. Reverse phase SPE uses non-polar functional groups such as octadecyl (C18), which binds to the polar target molecule, while other compounds pass through. The solution will need to be highly non-polar to facilitate interactions of the polar analyte with the non-polar silica. The compound may then be removed fully from the tube by a washing step using a slightly more polar solvent. This procedure is highly accurate, reducing evaporation losses and exposure of analytes to organic solvents, which can be problems in traditional liquid-liquid extraction methods. (Beney, etal)
In previous research studies Goldenseal alkaloids, such as berberine and hydrastine, were run through HPLC at wavelengths corresponding to UV light. None of these studies noted using Solid Phase Extraction. In one Japanese Study, berberine standard and unknown were analyzed with a reverse phase chromatography tube at a flow rate of 0.6 mL/min, a 70 water/30 CH3CN (with 0.5 TFA) eluent, and a wavelength of 254nm (Shodex Webpage). Another study from Ansys Technologies and Metachem utilized a wavelength of 220nm with an eluent consisting of a changing mobile phase of 0.1%TFA in H2O to 0.1%TFA in MeCN (Polaris Webpage).
Materials and Apparatus:
· Reversed Phase HPLC , poly(styrenedivinylbenzene) (PSDVB) packed column
· Spectrophotometer at 344 nm
· Solid Phase extraction tube, silica gel, C18 Bonded phase
· HPLC Mobile phase: Methanol: 20mM KH2PO4. pH 2.6; 50% methanol, 50% water.
· Berberine Chloride Standard (Sigma)
· Goldenseal Powdered Root:
- Bedrock Farm Powdered Goldenseal Root
- GNC fingerprinted Goldenseal, 500 mg capsules
Methods:
A solution of 10 mg/ml berberine chloride (standard) was made in DI H2O and diluted to 1 mg/mL in 50:50 mobile phase ratio. Using the Genesis spectrophotometer, a survey scan, based against pure mobile phase, was run over a range of 200nm-500nm. Another scan was conducted in the range from 200-800 nm, which includes the visible range. No appreciable peaks were seen after 500nm in this scan. Initially the absorbance peaks went off the scale, so the solution was diluted again until a final concentration of 0.01 mg/mL was reached, which yielded absorbencies within the limitations of the spectrophotometer. The peaks in the survey scan corresponded to the wavelengths at which the detector absorbed the minimum amount of radiation that was passed through the berberine compound, corresponding to the maximum absorbance at that wavelength. A wavelength of 344 nm (UV) was chosen as the optimal wavelength and used for subsequent scans.
For the HPLC experiments, standard berberine solutions of 0.5 mg/mL were used because the detector on the built-in spectrophotometer is less sensitive at low concentrations. The sample loop only injects a volume of 20 ml in the column, which is further diluted with mobile phase. Therefore, the concentration injected must be higher in order for the detector to measure within its normal range.
As stated in the background, berberine makes up 0.5 and 6% of the total root, or of the total alkaloid content only. Thus, if berberine were 0.5 to 6% of the total root, the optimal concentration-gain product for the unknown solution would be anywhere from 20 to 200 times that of the standard (approximately). However, at that time, solubility was also a concern. At an unknown solution concentration of 100 mg/mL, the total solubility of berberine was questionable. Root powder solutions of 1mg/ml exhibited reasonable solubility, yielding golden yellow solutions, and were used in HPLC experiments. To adjust for this low concentration, the aspect of gain control was introduced. The HPLC Spectrophotometer has a gain control which amplifies the signal from solutions that contain the target compound in low concentrations. The gain control was adjusted for both the standard and unknown solutions so that HPLC experiments would result in graphs that contained the maximum possible peaks (for better sensitivity), but still lie within the detection limits of the spectrophotometer. Note that the concentration-gain parameters for the GNC root powder was 40 times that of the Berberine standard, while that of the Bedrock powder was 20 times that of the standard.
Solid Phase Extraction:
Solid phase was not used to purify goldenseal root powder solutions prior to analysis with HPLC. One literature source that used HPLC to examine different herbal compounds used solid phase extraction in experiments involving other herbals, but not on Goldenseal Root (Alltech). The root was simply pulverized in mobile phase ratio, and analyzed directly. At least 2 other sources reported isolation of berberine from goldenseal root without the use of solid phase extraction. Further evidence came from experimental data. An HPLC trial of an unknown sample of root contained one clear, visible peak with no evidence of overlap. This was thought to represent only one compound at that retention time.
Results:
Figure 1 below, is the survey scan of .01 mg/mL berberine solution. The scan was based against the 50:50 methanol-KH2PO4 mobile phase.
Figure 1: Survey Scan of .01 mg/mL Berberine Solution
Peaks in the graph occurred at 420, 344, 263 and 228nm. 344nm was chosen as the optimal wavelength because it had the broadest peak and corresponded with literature that used wavelengths in the UV spectrum.
The following graph (Figure 2) shows the HPLC spectrophotometer data collected from the LabView software for the standard at 0.5 mg/mL.
Figure 2: HPLC of berberine chloride standard at 0.5 mg/mL
The average area under the curve is 176.31 AU2. Considering a sample of 0.5 mg/mL solution in a 20 μL sample loop there is 0.01 mg of berberine. Therefore the concentration factor is 17637.13 AU2/mg. This value was used to calculate the amount of berberine in the unknown solutions. The retention times of each of the peaks agreed well, with the largest difference between times being 18.25 sec or 3.03%.
Figures 3 and 4 are the HPLC spectrophotometer graphs for the GNC and Bedrock Goldenseal Root.
Figure 3: HPLC of GNC at 1 mg/mL
Figure 4: HPLC of Bedrock at 0.5 mg/mL
Note that GNC trial 3 and Bedrock trial 1 are not aligned with the other peaks of the same unknown. These two trials were conducted in succession, and that the retention times of both trials were lower than the other trials of their respective sources (GNC, Bedrock).
The detector which converts absorbance data to electrical data on the apparatus was set at a gain of 0.01 for GNC and Bedrock roots compared to 0.2 for the standard solution. The areas obtained for the unknowns were therefore divided by 20 to agree with the rest of the HPLC results. Table 1 displays these results as well as fraction of alkaloids contained in each sample based on the concentration factor of 17637.13 AU2/mg.
Standard / GNC / BedrockAvg. Area (adjusted to .2 gain) / 176.3713 / 10.819 / 8.529
Concentration of sample (mg/mL) / 0.5 / 1.0 / 0.5
Amount berberine/alkaloid input (mg) / 0.1 / 0.000613 / 0.000484
Fraction berberine/alkaloid in sample / 3.07% / 4.84%
Table 1: Fraction Alkaloid in each Unknown
The unknowns contained alkaloid concentrations in the range given by literature values, so the data made sense. However, the data shows that the retention times of the unknowns did not match up with the standard; therefore, the calculated amounts of alkaloid are not necessarily berberine. The amounts given represent the total area under the graph of the peak, which may contain some berberine, but also other unknown compounds such as different types of alkaloids.