A validated analytical method is reported for the analysis of chlorogenic and caffeic acids in Xu Duan (
There is very little published literature on the HPLC (high performance liquid chromatography) analysis of Xu Duan (
Chlorogenic and caffeic acids (structure shown in Figure
Structure of chlorogenic acid (a) and caffeic acid (b).
Both compounds are known to have antioxidant properties [
The lack of comprehensively validated HPLC methods for Xu Duan used in this study reinforced the need to develop and validate methods used for this herb. Analysis of Xu Duan had very little published literature. The validated method developed was used to quantify the analytes of interest in the raw herb sourced from different suppliers. The differences in concentration of the selected analytes in the different sourced herbs may be due to factors like growing conditions, time of harvest, postharvest treatment, storage conditions, and age of herb. This work reports a LC-PDA (liquid chromatography-photodiode array detection) method for the determination of chlorogenic and caffeic acids in the Xu Duan raw herb, with MS identity confirmation of the analyte.
LC-PDA experiments were performed on a Varian (Varian Australia, Sydney, Australia) chromatography system equipped with 2 × ProStar 210 solvent delivery modules, a ProStar 430 autosampler, a ProStar 500 column valve module, and a ProStar 335 PDA detector. This system was controlled by the Varian Star MS workstation (version 6.5). A tandem MS/MS detector with a positive electrospray ionisation ((+) ESI-MS/MS) interface was used for analyte identity confirmation. The LC system used was the same as for the LC-PDA method, except that a Varian 1200L triple quadrupole MS/MS detector replaced the PDA and only 20% of the total flow of 1 mL min−1 was diverted to the ESI source.
The herb sample used for the validation was supplied by the Win-Duc Herb Company (Sydney, NSW, Australia). This herb sample is referred to as R-I. Two other samples were purchased, one from the Chinese Ginsengs Herbal Company and the other from Pine Mountain Herbs, both suppliers are located in the Chinatown area of Sydney (NSW, Australia); these samples are referred to R-II and R-III, respectively. A sample was also supplied from the University of Western Sydney’s UniClinic (NSW, Australia), referred to as R-IV. The chlorogenic acid (95.06%) and caffeic acid (98.30%) standards were obtained from Sigma-Aldrich (CA, USA).
Methanol, ethanol, and formic acid (AR grade) used were purchased from Biolab (VIC, Australia). Mallinckrodt Baker Inc. supplied the n-hexane and ethyl acetate used for the extraction solvent optimisation. Acetonitrile (LC grade) was from J.T.Baker (NJ, USA). Ultrapure water (>18.2 MΩ/cm) was obtained from an Elga (Buckinghamshire, UK) Purelab Prima 7 water purification unit.
A mixed stock calibration solution containing an accurately known concentration of approximately 174
A mixed standard containing 8.7, 26.2, 43.6, 87.3, 130.9, and 174.5
Grind the dried Xu Duan (100 g) samples to pass through a 212
The ground raw herb sample was extracted using six different solvents (with five replicates per solvent) to determine which was most efficient. The solvents used were methanol, water, aqueous methanol (50 : 50% v/v), absolute ethanol, ethyl acetate, and hexane. Accurately weighed quantities of the sample (0.950–1.050 g) were transferred into volumetric flasks (100 mL), 95.0 mL of the solvent added, and the mixture sonicated for
The ground raw herb sample was extracted with the selected optimum solvent (which was found to be water) using three different extraction methods (with five replicates per method). The extraction methods to be used were sonication, reflux, and Soxhlet.
Using the optimal extraction method and solvent (sonication and water, resp.), the residual herb sample following the extraction described was reextracted three more times. The combined extracts were concentrated to 5.0 mL and analysed. This result gives an estimate of the extraction efficiency.
Approximately 0.5 g (0.450–0.550 g) of the raw herb sample was weighed and transferred to a 100 mL volumetric flask. 95.0 mL of water was added and the mixture was sonicated for 2 × 30 min. The sample solution was cooled to room temperature between extractions. After cooling the sample solution was made up to volume, before filtering through a 0.45
A mixed spiking solution was prepared by weighing 43.21 and 7.03 mg of chlorogenic and caffeic acids, respectively, into a 25 mL volumetric flask and was made to volume with methanol. For the 100% spike level, 250
The gradient mobile phase composition for the LC analysis of chlorogenic and caffeic acids is summarised in Table
Mobile phase composition for the HPLC analysis of Xu Duan.
Time (min) | 0.1% aqueous formic acid | % methanol |
---|---|---|
0 | 82 | 18 |
10 | 72 | 28 |
15 | 70 | 30 |
15.5 | 5 | 95 |
25 | 5 | 95 |
25.5 | 82 | 18 |
35 | 82 | 18 |
Note: the flow rate was 1 mL/min.
Column oven temperature was set to 30°C and flow rate to 1.0 mL/min. With this gradient program the column head pressure varied from 1500 to 2300 psi. The injection volume was 6
ESI-MS/MS was used for identity confirmation. The MS/MS conditions set as follows: nebulisation gas temperature 350°C, at 19 psi; needle voltage −4100 V at 15
The
Chromatograms obtained at 325 nm for chlorogenic acid and caffeic acid gave the best compromise between sensitivity and baseline noise. Figure
A HPLC-PDA chromatogram for the extract of the Xu Duan raw herb (
The results for the extraction solvent optimisation showed water to be the most efficient extraction solvent followed by 50% aqueous methanol, methanol, ethanol, hexane, and ethyl acetate. It was observed that the sonication method was the most effective at extracting both analytes. The sonication method also has the advantage of being simpler to use and is amenable to batch processing. A study was carried out to determine the analytes remaining in the herb following extraction by the selected method. The percentage residual chlorogenic and caffeic acids left over was
Sample R-I was used for the validation studies. The concentration of analytes in the unspiked sample analysed by the method was 33.2 mg/g and 1.8 mg/g for chlorogenic acid and caffeic acid, respectively. The 50, 100, and 200% spike recovery results for the Xu Duan raw herb are presented in Table
Recoveries for chlorogenic acid and caffeic acid in the Xu Duan.
Compound | %recovery ± %RSDa | |||||
---|---|---|---|---|---|---|
Chlorogenic acid | Caffeic acid | |||||
50% spike | 100% spike | 200% spike | 50% spike | 100% spike | 200% spike | |
Raw herb |
|
|
|
|
|
|
aMean recovery ± %RSD calculated from 7 replicate extractions and analyses.
For the raw herb, the average recovery across the three spiking levels for chlorogenic acid and caffeic acid is 99.88% and 87.21%, respectively. The %RSD for each recovery level varied from 2.4 to 9.6% (average 4.7%). The calibration curves obtained for each analyte showed good linearity with correlations of determination
The UV spectrum of the standard and sample peaks for chlorogenic and caffeic acids match closely as shown in Figure
Comparison of UV spectra of the standard and sample peaks for chlorogenic acid (a) and caffeic acid (b) peaks.
Identity confirmation is achieved by observing the presence of the correct
Comparison of intensities of the qualifier ion(s) for chlorogenic acid (
Analyte |
|
Relative diff. (%)b | Permitted tolerances | |
---|---|---|---|---|
Standard | Raw herb | |||
Chlorogenic acid | 85 (7) | 85 (6) | 14c | ±50% |
|
||||
Caffeic acid | 89 (61) | 89 (50) | 18c | ±20% |
135 (39) | 135 (33) | 15c | ±25% |
aAverage calculated from 5 replicates.
bRelative difference (absolute difference/relative intensity of standard) × 100.
cMaximum permitted tolerance of the European Union guidelines [
Proposed fragmentation pattern for chlorogenic acid showing the formation of the
Proposed fragmentation pattern for caffeic acid showing the formation of the
MS identity confirmation is thus achieved by observing the presence of the correct mass for the [M-H]− ion for chlorogenic acid and [M-H]+ ion for caffeic acid, the presence of the same
The chlorogenic acid and caffeic acid concentrations in the raw herbs are presented in Table
Concentration of chlorogenic acid and caffeic acid in several Xu Duan raw herbs determined by HPLC-PDA.
Sample | Mean (mg/g) ± %RSDa | Chlorogenic/caffeic ratio | |
---|---|---|---|
Chlorogenic acid | Caffeic acid | ||
R-I |
|
|
7.1 |
R-II |
|
|
6.7 |
R-III |
|
|
16.7 |
R-IV |
|
|
65.1 |
aCalculated from 5 replicates.
The concentration of chlorogenic acid in the raw herbs varies significantly, with R-I and R-IV having marginally higher levels compared to the other two samples. The caffeic acid concentrations are quite low in all the samples with the sample used for the validation having the highest concentration of caffeic acid. There also does not seem to be a consistent ratio between the concentrations of the two acids. The observed variations could be due to many factors including different growing, harvesting, and storage conditions and the age of the herb.
A HPLC-PDA method has been developed for the quantitation of chlorogenic and caffeic acids in the Xu Duan raw herb. The extraction is simple to perform and uses water for extraction. Both peaks show baseline resolution and the chromatographic analysis can be completed in 35 min. Good recoveries and precision of recoveries are obtained. Identity confirmation by ESI-MS/MS is achieved to an acceptable standard.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by the Centre for Complementary Medicine Research (University of Western Sydney).