In the Laboratory
High-Pressure Liquid Chromatography: Quantitative Analysis of Chinese Herbal Medicine
W
W. F. Chan* and C. W. Lin Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; *
[email protected] Traditional Chinese Medicine (TCM) is extensively used in Asian countries. Nevertheless, there are growing concerns about its toxicity and lack of efficacy. Analytical methods are needed to investigate the quality of Chinese herbal preparations and the active ingredients involved. High-pressure liquid chromatography (HPLC) is one of the few analytical techniques that can be employed for the analysis of TCM preparations (1–4). The applications of HPLC in undergraduate laboratories for the analysis of food (5–9) and drugs (10, 11) can be found in several publications. However, there exists no undergraduate experiment on how to analyze TCMs. In this article, an HPLC experiment involving quantitative and qualitative analysis of TCMs has been developed for an advanced analytical laboratory course. This experiment can be completed within a four-hour time frame. Glycyrrhizae radix and cinnamomi ramulus are commonly used Chinese herbal medicines and are included in many TCM preparations. These two herbs are also used as food spices in many countries. In the present experiment, one or more of the active ingredients in these herbs are employed as markers for the analysis. The marker for glycyrrhizae radix is glycyrrhizin while cinnamic acid and cinnamaldehyde are the markers for cinnamomi ramulus (Figure 1). Two types of samples are analyzed: a decoction prepared from two dried herbs and a powder sample of a dry extract from an seven-herb preparation that includes the two herbs of interest. As the two samples contain a complicated matrix, a solid-phase extraction (SPE) technique (8) is employed before analyzing the samples.
O HC
C H
C
R
OH
OH
Experimental Chemicals Acetonitrile and phosphate buffer (pH 3) are used as the mobile phase. Phosphate buffer is prepared by mixing 25 mM phosphoric acid with 25 mM sodium dihydrogen phosphate to pH 3. Mixtures of the phosphate buffer and methanol are used for the SPE eluting process. Cinnamic acid and cinnamaldehyde were purchased from Fluka while glycyrrhizin was bought from Tokyo Kasei Kogyo Co. Ltd. Equipment The experiment requires an HPLC system with an UV–vis diode-array detector. The chromatograms are recorded at two wavelengths (254 nm for glycyrrhizin and 285 nm for cinnamic acid and cinnamaldehyde). For absorption signals greater than the set threshold in the above wavelengths, the UV spectra in the range from 190–350 nm are collected. A 25 cm C18 analytical column with a similar guard column that can successfully operate for a long period at low pH, such as the Zorbax Stable Bond, is preferred. Gradient elution with composition starting from 95% phosphate buffer∙5% acetonitrile and ending up with 10% phosphate buffer∙90% acetonitrile is employed. The mobile phase flow rate is 1 mL∙min and 10 μl of sample is injected. The column is thermostated at 20 °C. The SPE cleanup procedure uses a C18 high capacity cartridge. Procedure
cinnamic acid: R OH cinnamaldehyde: R H
COOH
O
COOH OH O
The purpose of the experiment is for undergraduate students: (a) to learn how to use markers in the analysis of TCMs, (b) to become familiar with the four steps of SPE (conditioning, adsorption, washing, and elution), (c) to understand the efficiency of gradient elution in HPLC separation as well as the merits of diode-array detection (12), and (d) to explore the difficulties in the quality control of TCMs through the analysis.
O O
glycyrrhizin O
HO
COOH OH
Figure 1. Structures of compounds used as markers for TCMs.
Before the experiment, the SPE cartridge has to be conditioned by passing through 5 mL of methanol and then filling the cartridge with deionized water. For the analysis using the HPLC system, the analytical column needs to equilibrate with the starting buffer solution (95% phosphate buffer∙5% acetonitrile) for 20 column volumes (~60 mL of volume). External calibration is employed for quantitation. Stock solutions, 1000 ppm, of the three markers are used to prepare mixed standard solutions containing concentrations of cinnamic acid (2–20 ppm), cinnamaldehyde (2–20 ppm), and glycyrrhizin (10–100 ppm). These solutions are injected into the HPLC system for analysis. The peaks in the chromatograms are identified by comparing with the UV spectra of the markers. Calibration curves for the three markers are generated from the corresponding peak areas in the chromatograms.
1982 Journal of Chemical Education • Vol. 84 No. 12 December 2007 • www.JCE.DivCHED.org
In the Laboratory
Absorbance / 10Ź3
20
A254nm
0
cinnamic acid
Cinnamic acid and cinnamaldehyde are irritating to eyes, respiratory system, and skin. Methanol is toxic by inhalation and if swallowed. It is harmful in contact with skin and can cause serious damage to eyes. Phosphoric acid is corrosive and causes burns when in contact with skin. Acetonitrile is highly flammable. It is irritating to eyes and toxic by inhalation, in contact with skin, and if swallowed. Result and Discussion Calibration curves were obtained for the three markers and the regression equations were used to determine the concentrations of markers in the decoction as well as the powder sample. The data show good linearity for concentrations of cinnamic acid, cinnamaldehyde, and glycyrrhizin up to 20, 20, and 100 ppm, respectively. The chromatograms for the decoction sample and the powder sample are shown in Figures 2 and 3. The decoction sample was prepared in a similar manner as in traditional preparation of TCMs by boiling the mixture to about one quarter of the original volume. The peaks of glycyrrhizin, cinnamic acid, and cinnamaldehyde are separated from other peaks and can easily be identified by comparing the UV spectra obtained from diode-array detector with the given UV spectra of the markers. The chromatogram at 254 nm was used for the determination of the quantity of glycyrrhizin while that at 285 nm with enhanced detection sensitivity was used for the
cinnamaldehyde
8 4
A285nm
0
4 0
5
10
Retention Time
15
20
/ min
Figure 2. Gradient separation of decoction solution obtained from dried glycyrrhizae radix and cinnamomi ramulus. Conditions: Zorbax SB-C18, 4.6 × 12.5 mm guard column + 4.6 × 250 mm analytical column, 5 μm dp; ACN: 25 mM phosphate buffer pH 3.0, 1 mL/ min; 20 °C; inj 10 μL; detect 254, 280 nm. Gradient: 5% ACN to 70% ACN at 12 minutes, 90% ACN at 15 minutes.
glycyrrhizin 4
A254nm
0 4
cinnamic acid 4
A285nm
0 4 0
5
10
Retention Time
Hazards
glycyrrhizin 40
Absorbance / 10Ź3
For the decoction sample, 4 g of glycyrrhizae radix and 4 g of cinnamomi ramulus are placed into a 250 mL beaker and 200 mL of water is added. The mixture is evaporated, via boiling, to a final volume of ~75 mL and the supernatant is transferred to a 250 mL volumetric flask. Distilled water is added up to the mark. For the powder preparation, 1 g of sample is dissolved in 50 mL warm water. The solution is allowed to cool before transferring to a 100 mL volumetric flask. Distilled water is then added up to the mark. A SPE procedure is employed to clean the two sample solutions. For each sample, 2–3 mL of solution is filtered through a 0.45 μm syringe filter to a clean and dry vial. Filtered solution, 1 mL, is pipetted to the conditioned SPE cartridge. The solution is then allowed to pass at a drop-by-drop rate by applying a pressure on top of the cartridge using a plastic syringe. The steps have to be applied in a cautious manner so that the liquid level is always above the paking bed inside the SPE cartridge. Solvent, 10 mL of 20% methanol∙80% phosphate buffer, is added to wash the cartridge. The washing is then discarded. Another 4 mL of solvent of 70% methanol∙30% phosphate buffer is added to elute the analytes. The analytes are collected in a 5 mL volumetric flask and eluted solvent is added up to the mark. The solution is injected into the HPLC system for analysis. The quantities of the three analytes in the samples are calculated from the peak areas in the chromatograms of the sample solutions. For the decoction, the quantities (mg∙g) of glycyrrhizin in glycyrrhizae radix and cinnamic acid and cinnamaldehyde in cinnamomi ramulus are reported. For the powder sample, the quantities of glycyrrhizin, cinnamic acid, and cinnamaldehyde are calculated as per gram of powder as well as per gram of herb used for making the powder.
/ min
15
20
Figure 3. Gradient separation of the seven-herb extract powder. Conditions same as in Figure 2.
cinnamic acid and cinnamaldehyde. The typical quantity of glycyrrhizin in glycyrrhizae radix was found to be in the range of 5–31 mg∙g, and the cinnamic acid and cinnamaldehyde in cinnamomi ramulus were in the range of 0.1–0.3 mg∙g and 0.05–0.24 mg∙g, respectively. It can be seen that the quantities of markers in the herbs vary greatly. This probably depends on where the plants were grown. In Figure 3, glycyrrhizin and cinnamic acid can be identified in the powder sample and the quantity of cinnamaldehyde was found to be below the limit of quantitation. The quantities of glycyrrhizin and cinnamic acid in the powder were found to be in the range of 3–10 mg∙g and 0.2–0.35 mg∙g, respectively. In the present experiment, the powder sample is a dry extract from an seven-herb preparation. According to the manufacturer information, 10 grams of powder consist of 3 g of glycyrrhizae radix and 3 g of cinnamomi ramulus. The quantities of glycyrrhizin and cinnamic acid in the herb were calculated to be in the range of 10–33 mg∙g and 0.7–1.2 mg∙g, respectively. It can be seen that the quantity of cinnamic acid extracted from cinnamomi ramulus in the powder sample is much higher than that in the decoction sample. The extraction process that is used
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In the Laboratory
to prepare the TCMs in the powder form may somehow change the level of active ingredients when compared to the TCMs prepared in the traditional decoction form. In the extreme situation, the cinnamaldehyde could not be found in the powder sample. Currently many TCM preparations are made in powder form for convenience, and it is not known how this will affect the treatment efficiency when taking the TCMs prepared in this non-traditional manner. In addition, although the quantities of markers in the powder can be determined in the present experiment, it cannot be used to correlate with the quantities of herbs used for making the powder. The reason is due to the great variation of the quantity of markers in the herbs as stated previously. Finally, apart from the above concerns the potential presence of pesticides and heavy metals in the herbs is another important safety consideration. WSupplemental
Material
Handouts for the students, notes for the instructors, and spectra are available in this issue of JCE Online.
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1984 Journal of Chemical Education • Vol. 84 No. 12 December 2007 • www.JCE.DivCHED.org
