View More View Less
  • 1 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
  • 2 State Key Laboratory Breeding Base of Dao-di Herbs, China Academy of Chinese Medical Sciences, Beijing, 100700, China
  • 3 Shenzhen University, Shenzhen, China
Open access

A reliable and rapid high-performance liquid chromatography coupled with diode array detector method (HPLC–DAD) was established and validated to determine eight gingerol simultaneously in the rhizomes of Zingiber offcinale Rosc. The separation of eight compounds (4-hydroxy-3-methoxy-benzenebutanol,3,5-dihydroxy-1-(4-hydroxy-3-methoxyphenyl) decane, 3,5-dihydroxy-1-(3,4-dimethoxyphenyl) decane, 6-gingerol, 8-gingerol, 6-shogaol, 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1,4-decadien-3-one, and 10-gingerol) were performed on an Agilent TC(2) C18 (250 mm × 4.6 mm, 5 μm) at 30 °C using acetonitrile (A) and 1% formic acid aqueous solution (B) as the mobile phase with gradient elution (0–10 min, 20%–35% A; 10–28 min, 35%–55% A; 28–35 min, 55%–60% A; 35–55 min, 60%–70% A; 55.01–60 min, 100%–100% A). The detection wavelength was set at 280 nm, and the flow rate was 0.8 mL/min. Validation of the analytical method was performed by linearity, precision, and accuracy test. All compounds were quantified with good linear calibration curves (coefficient of determination R2, >0.9999). The method showed good precision with overall coefficients of variation between 0.56% and 0.84%. The range of recovery was from 95.50% to 104.14% for the analytes. This method was successfully applied to quantify eight gingerols in Z. offcinale Rosc from different regions in China, so it can provide quality assessment for this medicine.

Abstract

A reliable and rapid high-performance liquid chromatography coupled with diode array detector method (HPLC–DAD) was established and validated to determine eight gingerol simultaneously in the rhizomes of Zingiber offcinale Rosc. The separation of eight compounds (4-hydroxy-3-methoxy-benzenebutanol,3,5-dihydroxy-1-(4-hydroxy-3-methoxyphenyl) decane, 3,5-dihydroxy-1-(3,4-dimethoxyphenyl) decane, 6-gingerol, 8-gingerol, 6-shogaol, 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1,4-decadien-3-one, and 10-gingerol) were performed on an Agilent TC(2) C18 (250 mm × 4.6 mm, 5 μm) at 30 °C using acetonitrile (A) and 1% formic acid aqueous solution (B) as the mobile phase with gradient elution (0–10 min, 20%–35% A; 10–28 min, 35%–55% A; 28–35 min, 55%–60% A; 35–55 min, 60%–70% A; 55.01–60 min, 100%–100% A). The detection wavelength was set at 280 nm, and the flow rate was 0.8 mL/min. Validation of the analytical method was performed by linearity, precision, and accuracy test. All compounds were quantified with good linear calibration curves (coefficient of determination R2, >0.9999). The method showed good precision with overall coefficients of variation between 0.56% and 0.84%. The range of recovery was from 95.50% to 104.14% for the analytes. This method was successfully applied to quantify eight gingerols in Z. offcinale Rosc from different regions in China, so it can provide quality assessment for this medicine.

Introduction

Dried ginger (Zingiber officinale Roscoe, Zingiberaceae) is a traditional Chinese medicinal herb which has been widely applied to relieve colds, inflammation, and headaches and as an antiemetic for prevention of seasickness [1]. Dried ginger includes diarylheptanoids and gingerol-related compounds, which attracts growing interest in pharmacological activity, such as antitumor, antioxidant, anti-inflammatory, and low toxicity [2]. The gingerol, which is one of the most important major bioactive component, may affect the quality and efficacy of the Zingiberis rhizome [3]. In addition, there are many differences among chemical components of gingerol from different sources. Even gingerol from the same region can differ significantly. Therefore, it is necessary to establish a qualitative and quantitative method based on the bioactive gingerol-related compounds for control of the quality of Zingiberis rhizome.

The content of gingerol directly affected the quality and efficacy of the Zingiberis rhizome. Several analytical methods were reported to determinate the main chemical gingerol components in gingeris rhizomes. These contain high-performance liquid chromatography (HPLC) [4], tandem liquid chromatography–mass spectrometry (LC–MS) [5], gas chromatography coupled with mass spectrometry (GC–MS) [6], the high-performance thin-layer chromatography (HPTLC), dual wavelength ultraviolet (UV) spectrophotometry, and capillary chromatography (CE) [7]. Among these methods, HPLC, being simple and rapid, was widely accepted for determining the contents of gingeris rhizomes. To date, only few reports are found on the quantitative determination of the eight gingerol-related compounds simultaneous in Zingiberis rhizome. Therefore, the present study was designed to develop a simple and accurate HPLC method for the simultaneous quantification of the eight major characteristic gingerols in Zingiberis rhizome, namely, 4-hydroxy-3-methoxy-benzenebutanol (B), 3,5-dihydroxy-1-(4-hydroxy-3-methoxyphenyl) decane (C), 3,5-dihydroxy-1-(3,4-dimethoxyphenyl) decane (D), 6-gingerol (E), 8-gingerol (F), 6-shogaol (G), 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1,4-decadien-3-one (H), and 10-gingerol (I) (Figure 1), which can provide the evaluation method for Zingiberis rhizome in quality standard.

Figure 1.
Figure 1.

Chemical structures of the analytes

Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00125

Materials and Methods

Chemicals and Plant Materials

Analytical standard of 6-gingerol and 6-shogaol (>98% purity) was purchased from the National Institute for Food and Drug Control (Beijing, China). 4-Hydroxy-3-methoxy-benzenebutanol,3,5-dihydroxy-1-(4-hydroxy-3-methoxyphenyl) decane,3,5-dihydroxy-1-(3,4-dimethoxyphenyl)decane,5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1,4-decadien-3-one, 8-gingerol, and 10-gingerol were extracted and identified from ginger extracts in our laboratory. The purity of all compounds was more than 98% (determined by HPLC). Acetonitrile and methanol (HPLC grade, Fisher Scientific, New Jersey, USA) as well as pure water (Wahaha Group Co., Ltd., China) were used for HPLC analysis. All other chemicals were of analytical grade. The solutions were filtered through 0.22 μm membranes before HPLC analysis.

Twelve samples of Zingiberis rhizome were collected from different sources of the herbal market or local pharmacy in the following different regions in China and identified as dried ginger, as shown in Table 1. Ginger herbs were used as samples for the test, in accordance with the “Chinese Pharmacopoeia” and “National Traditional Chinese Medicine and norms” and, under the relevant processing methods, were prepared into 40-mesh sieve analysis for the experiment. Each powdered sample (through 40 mesh) of Zingiberis rhizome was precisely weighed and extracted ultrasonically by 25 mL methanol for 20 min, cool and added methanol to compensate the weight loss of solution, and then filtered through 0.22 μm membrane prior to HPLC analysis.

Table 1.

Details of the herbal materials collected

No.Cultivation regions (provinces)Year of collections
S-1Bozhou, Anhui2013
S-2Fuzhou, Fujian2013
S-3Nanning, Guangxi2013
S-4Baise, Guangxi2013
S-5Gui yang, Guizhou2013
S-6Zhengzhou, Henan2013
S-7Linyi, Shandong2013
S-8Cheng du, Sichun2013
S-9Yaan, Sichun2013
S-10Leshan, Sichun2013
S-11Mianyang, Sichun2013
S-12Kunming, Yunnan2013

Preparation of Standard Solutions

The mixed standard stock solutions of B (3.60 μg/mL), C (11.30 μg/mL), D (7.86 μg/mL), E (280.00 μg/mL), F (118.00 μg/mL), G (26.40 μg/mL), H (2.05 μg/mL), and I (79.20 μg/mL) were prepared in methanol and stored at 4 °C. The combined solution was diluted step by step to yield a series of standard working solutions with different concentration for linear validation.

Apparatus and HPLC Analysis

HPLC was performed on a Shimadzu HPLC system (Shimadzu Corporation, Japan) equipped with an LC-20AT binary pump, an SPD-M20A diode array detector (DAD), a CBM-20A lite system controller, an SIL-20A autosampler, a DGU-20A5 degasser, and a CTO-10ASvp column oven. An Agilent TC(2) C18 column (250 × 4.6 mm, 5 μm) was maintained at 30 °C. Detection wavelength was set at 280 nm. The mobile phase consisted of acetonitrile (A) and 1.0% formic acid aqueous solution (B) at a flow rate of 0.8 mL/min; a gradient program was used as follows: 0–10 min, 20%–35% A; 10–28 min, 35%–55% A; 28–35 min, 55%–60% A; 35–55 min, 60%–70% A; 55.01–60 min, 100%–100% A. The injected volume was 10 μL. Online DAD spectra were recorded in the range of 190–800 nm. Under this condition, ginger sample with the other components of each reference was able to achieve baseline separation.

Analytical Method Validation

The linear relationship, limit of detection (LOD), limit of quantification (LOQ), recovery, precision, and relative standard deviation (RSD) are shown in Tables 2, 3, and 4 to evaluate the quality of this method. The working standard solution (mixture) that includes eight standards at seven different concentrations and was analyzed. The calibration curves were plotted in linear regression analysis of the integrated peak area (Y) versus concentrations (x). The regression equations were in the form of Y = ax + b. The working solution was further diluted to accurate concentration to study the LOD and LOQ. LOD and LOQ were described as the concentration of analyte capable of producing a peak with a signal to noise ratio was 3 and 10, respectively. The precision was determined by continuous injection of the sample solution on three consecutive days. A recovery test was used to assess the accuracy of the developed method. Three kinds of different quantities of the accurate standards were added into the samples in form of solution. The quantity of each analyte was subsequently accomplished from the corresponding calibration curve and relative standard deviation (RSD) % = (SD/mean) × 100%.

Table 2.

Linear equation and linear range of the analytes

CompoundsCalibration curveRLiner range (μg/mL)
BY = 510,123x + 197.0710.0034–0.1020
CY = 482,073x + 301.520.999950.0102–0.3060
DY = 634,312x + 178.6710.0065–0.1950
EY = 682,932x + 1318510.2800–8.4000
FY = 541,122x + 1436.610.0984–2.9520
GY = 895,112x + 404.0710.0254–0.7620
HY = 807,601x − 1801.90.999950.0181–0.5442
IY = 685,627x + 743.6610.0722–2.1660
Table 3.

Precision, repeatability, and stability of standard compounds in Zingiberis rhizome

Peak no.CompoundsPrecision (n = 6) RSD (%)Repeatability (n = 6) RSD (%)Stability (n = 6) RSD (%)
1B0.701.510.58
2C0.711.170.53
3D0.840.980.54
4E0.630.980.49
5F0.750.900.53
6G0.561.150.54
7H0.660.980.36
8I0.590.920.45
Table 4.

Recovery of the analytes in Zingiberis rhizome (n = 6)

CompoundsContained (μg)Added (μg)Found mean (μg)Recovery mean (%)RSD (%)
B29.7029.6660.38103.381.03
C108.31108.48221.28104.140.69
D70.6170.49138.3696.110.63
E2149.802146.084199.2195.500.50
F633.46626.081274.09102.330.44
G121.40121.19244.50101.570.79
H114.21114.01232.35103.631.27
I730.45729.181487.00103.750.58

Sample Analysis

The developed method was used to analyze the contents of eight compounds in twelve samples. Contents for all compounds were calculated from its corresponding calibration curve. Chromatographic peaks in the samples were identified by comparing their retention times and UV spectra with those of the reference standards.

Results and Discussion

Extraction Procedure

Extracting solvent (methanol, ethanol, and chloroform), extraction method (ultrasound extraction, reflux extraction, and cold soaking extraction), and extraction time (10 min, 20 min, 30 min, 40 min, 1 h, 2 h, 3 h, 8 h, 12 h, and 24 h) were evaluated in an effort to optimize the extraction procedures. Although chloroform gets more extractive, some contents of them are very low, and the contents of extractive are not stable every time. The results revealed that methanol extraction was better than ethanol and chloroform (Figure 2A). About the extraction method, the results are similar, so we choose the simplest method of ultrasonic extraction. The influence of the extraction time (10 min, 20 min, 30 min, and 40 min) on the efficiency of extraction was also investigated. We found that ultrasonic extraction for 20 min was found with best results, and there was no obvious difference between 20 min and 30 min (Figure 2B).

Figure 2.
Figure 2.

Total eight gingerols in the Zingiberis rhizome under different extraction conditions

Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00125

Optimization of the Chromatographic Conditions

The different mobile phases, such as water–acetonitrile and water–methanol, were tested with various gradient programs to obtain the optimal separation conditions. Water–acetonitrile had better separation and peak shape than water–methanol. One percent formic acid was added to improve the peak shape and reduce the peak tailing. Furthermore, other chromatographic variables were also optimized, including column temperatures (25 and 30 °C) and flow rates (0.8 and 1.0 mL/min). Eventually, the optimal separation was achieved on an Agilent TC (2) C18 (250 mm × 4.6 mm, 5 μm) at a column temperature of 30 °C with a flow rate of 0.8 mL/min. Under the optimal chromatographic conditions, the peaks corresponding to the different analytes were well separated within 65 min. Representative chromatograms for the eight standard analytes and samples were shown in Figure 3. The peaks of these gingerol were identified by two methods: (1) by comparing the retention times of the peaks with those of the reference compounds eluted under the same conditions and (2) by spiking the sample with stock standard solutions of analytes.

Figure 3.
Figure 3.

HPLC chromatograms of the twelve samples from different sources

Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00125

Reliability of the Method

The calibration curves were performed by measuring at least six different concentrations of standards. The LODs and LOQs of the gingerol were between 0.024 and 0.366 μg/mL and 0.073 and 1.110 μg/mL, respectively. The calibration curve formulas and relevant dates have been observed in Table 2.

Precision, Accuracy, and Stability

For the precision, the mixed standard solutions were analyzed six times under the optimized conditions within one day (n = 6). The inter-day reproducibility was validated by analyzing the aforementioned eight mixed standard solutions once per day on three consecutive days. The RSD values were summarized in Table 3. As can be seen in the table, RSDs for precisions range between 0.56% and 1.51%. The stabilities of the same solutions were analyzed at 0 h, 2 h, 4 h, 8 h, 16 h, and 24 h at room temperature. It was found that the sample solutions were stable within 24 h (RSD, ≤0.58%).

Recovery

As shown in Table 4, the recovery test was measured in triplicate in sample to evaluate the accuracy by the method of standard addition. The contents of the eight analytes in the sample were calculated from their respective calibration curves. The same volume of each analyte present in the sample was spiked into the sample six times. The fortified samples were then extracted and analyzed as described in the experimental section as mentioned above. The mean recoveries of the analytes were between 95.50% and 104.14% with RSDs less than 1.27% as shown in Table 4. The results show that the method is accurate.

Quantification of Eight Compounds in Twelve Samples from Different Sources

The developed method was applied to determine eight gingerol simultaneously in twelve samples on Zingiberis rhizome collected from different sources. Based on the analytical results (Figure 4), the concentrations of the analytes varied remarkably. The contents of the eight compounds in these samples were quantified, and the results of the mean values of three replicate injections are shown in Table 4 and Figure 4. Among the twelve batches of samples, the content variation orders of eight kinds of gingerols in Zingiberis rhizome are similar with 6-gingerol > 10-gingerol > 8-gingerol > 6-shogoal mostly, except Chengdou (S-8, Figure 4). By comparison, Z. officinale Rosc. in the Fuzhou shows the best quality, because it is the highest content among eight components besides 6-gingerol and 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1,4-decadien-3-one present in the Fuzhou. The results indicate clearly that ginger has genetic and metabolic variation in the cultivars collected from different regions. Thus, to establish a stable and efficient quality assessment method is significant.

Figure 4.
Figure 4.

The content of the gingerols in the Zingiberis rhizome from different areas

Citation: Acta Chromatographica Acta Chromatographica 30, 3; 10.1556/1326.2017.00125

Conclusions

With the growing numbers of Chinese medicine being widely used, efficient methods to evaluate and control the quality of TCM are urgently needed. However, the quality and quantity data on Chinese medicine are far from sufficient to meet the demands needed to support its widely used. The Chinese medicine contains many chemical components. Therefore, high-efficiency methods are required for qualitative and quantitative analysis of their effective compounds. Multi-components determination as markers for quality evaluation is a good choice. In the previous work, only one or two compounds have been determined in Zingiberis rhizome. This is the first report of an HPLC–DAD method to simultaneously determine eight major bioactive compounds in Zingiberis rhizome. The new method is simple, reliable, accurate, and promising to improve the quality control of Zingiberis rhizome. Results also indicated that Fuzhou and Mianyang contained the highest concentrations of gingerols. Consequently, these regions are recommended for the large scale production of ginger.

Abbreviations

HPLC–DAD high-performance liquid chromatography coupled with diode array detector

LOD limit of detection

LOQ limit of quantification

RSD relative standard deviation

LC–MS tandem liquid chromatography–mass spectrometry

GC–MS gas chromatography coupled with mass spectrometry

References

  • 1.

    Afzal, Μ.; Al-Hadidi, D.-H.; Menon, M.; Pesek, J.; Dhami, M. S. I. Drug Metab. Drug Interact. 2001, 18, 159190.

  • 2.

    (a) Jolad, S. D.; Lantz, R. C.; Solyom, A. M.; Chen, G. J.; Bates, R. B.; Timmermann, B. N. Phytochemistry 2004, 65, 19371954;

    (b) Giriraju, A.; Yunus, G. Y. Indian J. Dent. Res. 2013, 24, 397400;

    (c) Giriraju, A.; Yunus, G. Y. Indian J. Dent. Res. 2013, 24, 397;

    (d) Sabina, E. P. Food Chem. Toxicol. 2010, 48, 229235;

    (e) Abdel-Azeem, A. S.; Hegazy, A. M.; Ibrahim, K. S.; Farrag, A.-R. H.; El-Sayed, E. M. J. Diet. Suppl. 2013, 10, 195209;

    (f) Thomson, M.; Al-Qattan, K. K.; Al-Sawan, S. M.; Alnageeb, M. A.; Khan, I.; Ali, M. Prostaglandins Leukot. Essent. Fatty Acids 2002, 67, 475478;

    • Search Google Scholar
    • Export Citation

    (g) Akhani, S. P.; Vishwakarma, S. L.; Goyal, R. K. J. Pharm. Pharmacol. 2004, 56, 101105;

    (h) Altman, R. D.; Marcussen, K. C. Arthritis Rheum. 2001, 44, 25312538;

    (i) Sang, S.; Hong, J.; Wu, H.; Liu, J.; Yang, C. S.; Pan, M. H.; Badmaev, V.; Ho, C. T. J. Agric. Food Chem. 2009, 57, 1064510650.

  • 3.

    Jiang, H.; Sólyom, A. M.; Timmermann, B. N.; Gang, D. R. Rapid Commun. Mass Spectrom. 2005, 19, 29572964.

  • 4.

    Jolad, S. D.; Lantz, R. C.; Chen, G. J.; Bates, R. B.; Timmermann, B. N. Phytochemistry 2005, 66, 16141635.

  • 5.

    Deng, C.; Mao, Y.; Hu, F.; Zhang, X. J. Chromatogr. A 2007, 1152, 193198.

  • 6.

    (a) Zhou, H. Zhongguo Zhongyao Zazhi 1998, 23, 234236;

    (b) Meng, Q.; Feng, Y. F.; Guo, X. L.; Chen, G. F.; Cai, J. C. C. Zhongguo Zhongyao Zazhi 2005, 30, 750752.

  • 7.

    (a) Wang, W. H.; Juan, L. I.; Gao, H. M.; Wang, Z. M.; Zhang, L. Yaowu Fenxi Zazhi 2009, 29 12481252;

    (b) Xiang, L. I.; Wang, M.; Guo, L. H.; Yang, Y.; Lei, M. U.; Guo, Q. Med. Pharma. J. Chin. Peoples Liberation Army 2011, 23, 2021.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1.

    Afzal, Μ.; Al-Hadidi, D.-H.; Menon, M.; Pesek, J.; Dhami, M. S. I. Drug Metab. Drug Interact. 2001, 18, 159190.

  • 2.

    (a) Jolad, S. D.; Lantz, R. C.; Solyom, A. M.; Chen, G. J.; Bates, R. B.; Timmermann, B. N. Phytochemistry 2004, 65, 19371954;

    (b) Giriraju, A.; Yunus, G. Y. Indian J. Dent. Res. 2013, 24, 397400;

    (c) Giriraju, A.; Yunus, G. Y. Indian J. Dent. Res. 2013, 24, 397;

    (d) Sabina, E. P. Food Chem. Toxicol. 2010, 48, 229235;

    (e) Abdel-Azeem, A. S.; Hegazy, A. M.; Ibrahim, K. S.; Farrag, A.-R. H.; El-Sayed, E. M. J. Diet. Suppl. 2013, 10, 195209;

    (f) Thomson, M.; Al-Qattan, K. K.; Al-Sawan, S. M.; Alnageeb, M. A.; Khan, I.; Ali, M. Prostaglandins Leukot. Essent. Fatty Acids 2002, 67, 475478;

    • Search Google Scholar
    • Export Citation

    (g) Akhani, S. P.; Vishwakarma, S. L.; Goyal, R. K. J. Pharm. Pharmacol. 2004, 56, 101105;

    (h) Altman, R. D.; Marcussen, K. C. Arthritis Rheum. 2001, 44, 25312538;

    (i) Sang, S.; Hong, J.; Wu, H.; Liu, J.; Yang, C. S.; Pan, M. H.; Badmaev, V.; Ho, C. T. J. Agric. Food Chem. 2009, 57, 1064510650.

  • 3.

    Jiang, H.; Sólyom, A. M.; Timmermann, B. N.; Gang, D. R. Rapid Commun. Mass Spectrom. 2005, 19, 29572964.

  • 4.

    Jolad, S. D.; Lantz, R. C.; Chen, G. J.; Bates, R. B.; Timmermann, B. N. Phytochemistry 2005, 66, 16141635.

  • 5.

    Deng, C.; Mao, Y.; Hu, F.; Zhang, X. J. Chromatogr. A 2007, 1152, 193198.

  • 6.

    (a) Zhou, H. Zhongguo Zhongyao Zazhi 1998, 23, 234236;

    (b) Meng, Q.; Feng, Y. F.; Guo, X. L.; Chen, G. F.; Cai, J. C. C. Zhongguo Zhongyao Zazhi 2005, 30, 750752.

  • 7.

    (a) Wang, W. H.; Juan, L. I.; Gao, H. M.; Wang, Z. M.; Zhang, L. Yaowu Fenxi Zazhi 2009, 29 12481252;

    (b) Xiang, L. I.; Wang, M.; Guo, L. H.; Yang, Y.; Lei, M. U.; Guo, Q. Med. Pharma. J. Chin. Peoples Liberation Army 2011, 23, 2021.

Monthly Content Usage

Abstract Views Full Text Views PDF Downloads
Jun 2020 0 13 6
Jul 2020 0 20 18
Aug 2020 0 8 1
Sep 2020 0 12 5
Oct 2020 0 12 5
Nov 2020 0 10 0
Dec 2020 0 2 0