Abstract
Context: Fructus Corni, a traditional Chinese medicines, is derived from the dry ripe sarcocarp of Cornus officinalis Sieb. et Zucc (Cornaceae). Gallic acid, 5-hydroxymethylfurfural, morroniside, sweroside, loganin, cornin, 7-O-methyl-morroniside and cornuside are the active constituents of Fructus Corni used in many traditional Chinese medicines. This paper describes a sensitive and specific assay for the determination of the eight bioactive compounds in crude and processed Fructus Corni extracts.
Materials and methods: In this paper, the eight components were determined by high performance liquid chromatography coupled with triple quadrupole mass spectrometry (MS/MS). Quantization was based on multiple reaction monitoring using the precursor production combination for determination of the eight analytes. The analysis was performed on an Agilent Zorbax Extend C18 column (100 mm × 3.0 mm, 3.5 μm), and an electrospray ionization (ESI)-tandem interface in the positive and negative ion polarity mode was employed prior to mass spectrometric detection.
Results: With the optimized conditions, the eight bioactive compounds were detected properly within 10 min. The developed method showed good precision and reproducibility with the limits of detection ranged from 0.0042 to 12.7875 ng/mL and the average recoveries ranged from 97.08 to 103.7%.
Discussion and conclusion: This newly established method is validated as simple, reliable and accurate. It can be used as a valid analytical method for intrinsic quality control of crude and processed Fructus Corni.
Introduction
Fructus Corni, a traditional Chinese medicine, is derived from the dry ripe sarcocarp of Cornus officinalis Sieb. et Zucc (Cornaceae). Due to its biological and pharmacological activities such as anti-inflammation, antivirus, and antioxidation, Fructus Corni is increasingly given much attention as one of the most popular and cherished clinical herbal medicines in the world. It can be used as medicine, hygienic food, and cosmetic (CitationLi et al., 2008; Cao et al., Citation2009a,b; CitationLiu et al., 2009). Chemical constituents from Fructus Corni that have been studied include mainly irridoids, organic acids, triterpenes, cornustannins, and carbohydrates (CitationDing et al., 2008). The published studies on Fructus Corni have proved that loganin exhibited immune regulating activity, anti-inflammatory and antishock effects (CitationYokozawa et al., 2009). It has also been shown that the activity of the morroniside constituent contributed to effectiveness in invigorating stomach and preventing diabetic angiopathies (CitationZhou et al., 2008). 5-Hydroxymethylfurfural (5-HMF) is an endogenous product found in plants, in free or bound forms. It is found in large amounts in processed rather than crude Fructus Corni, from which it is extracted in hot aqueous infusions (CitationFu et al., 2008). Pharmacological studies on the components showed that 5-HMF had good biological activities such as anti-inflammatory activity and bacteriostatic action (CitationCao et al., 2011).
A few analytical methods have been reported for quality assessment of Fructus Corni and its processed products (CitationDu et al., 2008) . Unfortunately, due to the complex constituents in the herbal medicine, a complicated sample preparation process has usually been applied before analysis in the common high performance liquid chromatography (HPLC), which consumed large amounts of organic solvents and time. Therefore, the development of a simple and rapid method for the analysis of these constituents in Fructus Corni is of great significance. Recently, the MS/MS with multiple reaction monitoring (MRM) has led to a notable improvement in detectability and selectivity (CitationSlusarczyk et al., 2009; CitationNie et al., 2006). Due to the high selectivity of MRM mode, the sample pre-processing and optimization of chromatographic separation is greatly simplified. Furthermore, precursor and production ion monitoring can be used to increase specificity of detection and identification of known molecules.
In the present study, we focused on developing a simple and validated method using HPLC–MS/MS with MRM to simultaneously identify and determine the multiple bioactive constituents in crude Fructus Corni and its processed products. It offers a new method that can be employed for the quality control of not only crude Fructus Corni, but also its processed products.
Materials and methods
Reagents and standards
Crude Fructus Corni and its processed forms of jiu zheng pin (JZP) were collected from Henan suppliers (samples 1, 2 and 3). Gallic acid, loganin and 5-HMF were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Morroniside, cornuside, cornin, 7-O-methyl-morroniside and sweroside were obtained from Shanghai Shangyi Biotechnology Co. Ltd. (Shanghai, China). The purity for each reference compound was greater than 98% by HPLC analysis. HPLC-grade methanol (99.9% purity) was purchased from E. Merck (Darmstadt, Germany). A Milli-Q water (Millipore Co., Billerica, MA, USA) purification system was used to obtain purified water for the HPLC analysis. The structures of these eight compounds are shown in .
Figure 1. The chemical structures of the eight bioactive compounds in Fructus Corni.
HPLC and MS analysis
The chromatographic analyses were performed on an Agilent 1200 series (Agilent, Santa Clara, CA, USA) with the mobile phase consisting of (A) methanol acetic acid (0.1%, v/v) and (B) aqueous acetic acid (0.1%, v/v) using a gradient elution of 10–90% B at 0–10 min. An Agilent Zorbax Extend C18 column (100 mm × 3.0 mm, 3.5 μm) was used with a flow rate of 0.6 mL/min. The column was maintained at 40°C, and the injection volume of reference solution or sample solution was 10 μL. Determination was performed using an Agilent Technologies 6410 Triple Quad LC/MS equipped with electrospray ionization (ESI). The compounds were ionized in the positive and negative ion polarity mode. The ionization source conditions were as follows: spray voltage of 4000 V(+), 3600 V(−), source temperature of 100°C and desolvation temperature of 350°C. Nitrogen was used as nebulizer gas and pressure was set at 40 psi at a flow rate of 10 L/min. The pressure of high purity nitrogen was 0.15 MPa for collision-induced dissociation. Quantification was operated at MRM modes. The delta potential of the electron multiplier voltage was set to 400 V for data acquisition. The summary of MS/MS detection parameters is shown in . Data acquisition and processing were performed by Agilent MassHunter Workstation.
Table 1. MS/MS detection parameters for the eight bioactive compounds in Fructus Corni.
Preparation of sample solutions
The powder of crude and processed Fructus Corni samples were precisely weighed (1.000 g) and transferred into respective 25 mL dark brown calibrated flasks. They were extracted with 10 mL of 80% methanol in an ultrasonic bath for 30 min. Additional 80% methanol was added to make up the loss. Before being injected into the HPLC system, all solutions were diluted to proper concentrations and filtered through the 0.45 μm filter membranes.
Preparation of reference solutions
Reference stock solutions of gallic acid (10.58 mg) and 5-HMF (13.10 mg) were prepared in 20 mL of 80% methanol, and reference stock solutions of morroniside (5.0 mg), sweroside (2.130 mg), loganin (2.01 mg), cornin (3.72 mg), 7-O-methylmorroniside (3.41 mg) and cornuside (3.50 mg) were prepared in 10 mL of 80% methanol, respectively. Each reference stock solution after dilution with 80% methanol was mixed and then further diluted with 80% methanol to give at least six different appropriate concentrations for calibration curves. The solutions were filtered through the 0.45 μm filter membranes prior to injection.
Limits of detection and quantification
The limits of detection (LOD) and quantification (LOQ) were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively. They were performed using serial diluted reference solution of each compound under the present chromatographic conditions.
Precision, reproducibility and accuracy
Intra- and inter-day variations were chosen to determine the precision of the developed method. The intra-day variation was determined by analyzing in triplicate the same mixed reference solution for five times within a single day. For inter-day variability test, the solution was examined in triplicate for three consecutive days. Five different working solutions prepared from the Fructus Corni samples were investigated to confirm the reproducibility, and variations were expressed by relative standard deviations. A recovery test was performed with the method of standard addition. Accurate amounts of reference compounds were added to Fructus Corni samples, then extracted and analyzed as described (n = 6).
Results and discussion
Optimization of MS Conditions
Several mobile phases, including methanol-water, acetonitrile-water, methanol acetic acid (0.2%, v/v)-aqueous acetic acid (0.4%, v/v) and methanol acetic acid (0.1%, v/v)-aqueous acetic acid (0.1%, v/v), were tried to obtain chromatograms with good resolution and adjacent peaks. It was found that the combination of methanol acetic acid (0.1%, v/v)-aqueous acetic acid (0.1%, v/v) gave the best separation of the eight bioactive compounds.
To obtain better detection, the MS conditions should be optimized, such as capillary voltage, ion mode, collision energy, cone voltage and dwell times. For example, when the fragmentation energies for rosmarinic acid were optimized to be 100 V, the maximum response of the compound fragment ion peaks was achieved. In the study, it was also found that matrix effects influenced the assay results greatly. The results showed that the ESI ion mode and the assayed solution concentration were the main effective factors. In the present study, ESI in both negative and positive ion modes were tried and the results showed that ESI in negative ion mode was more sensitive for gallic acid, loganin, morroniside, cornuside, cornin, 7-O-methyl-morroniside and sweroside and ESI in positive ion mode was more sensitive for 5-HMF. In addition, the assayed solutions must be diluted to the proper concentrations.
Method validation for quantitative determination of the bioactive compounds
With the optimized conditions, the eight bioactive compounds were detected properly within 8 min (). The bioactive compounds in Fructus Corni were identified and confirmed by comparing the retention time and MS spectrum of the reference standards. The calibration curve of each compound was performed with at least six appropriate concentrations in triplicate. shows the results of linearity, LOD and LOQ for each compound. All calibration curves were linear with good correlation coefficients (R2 > 0.9922). The developed method for simultaneous identification and determination of the eight bioactive compounds showed good precision and reproducibility with the average recoveries ranged from 97.08% (sweroside) to 103.7% (morroniside).
Table 2. Summary of regression data, LOD, LOQ for the eight bioactive compounds in Fructus Corni.
Figure 2. The LC-MS/MS chromatographic profiles of Fructus Corni samples. 1: gallic acid; 2: 5-HMF; 3: morroniside; 4: sweroside; 5: cornin; 6: loganin; 7: 7-O-methyl-morroniside; 8: cornuside.
Sample analysis
The developed optimized method was used for the quantification of the eight bioactive compounds in three different crude and processed Fructus Corni. All the samples were extracted and analyzed in triplicate. The eight bioactive compounds were confirmed by mass spectral analyses (). The contents of the eight compounds in Fructus Corni samples analyzed are listed in . The results showed that the content of each compound in crude drugs and processed products varied significantly. For instance, for Fructus Corni collected from Henan suppliers (sample 1), the content of gallic acid was lower in crude drug (0.9495 mg/g), but higher in its processed product (2.1357 mg/g), 5-HMF was hardly detected in crude drug, but with higher content in its processed product (5.7855 mg/g), the contents of morroniside and cornuside in crude drug (3.6421 and 1.4351 mg/g) were higher than those in its processed product (3.4335 and 0.9803 mg/g), and the contents of sweroside, cornin and 7-O-methyl-morroniside in crude drug (0.4925, 0.1458 and 0.0604 mg/g) were lower than those in its processed product (0.5495, 0.1904 and 0.0880 mg/g). The variations might result from different processing procedures for Fructus Corni. It was reported that processing or heating could drastically increase the content of 5-HMF and tannin in Fructus Corni was hydrolyzed to generate gallic acid owing to high temperature.
Table 3. The mean contents of the eight bioactive compounds in crude and processed Fructus Corni samples (mg/g + SD, n = 5).
Figure 3. The mass spectra of the eight bioactive compounds. Each compound was collected from HPLC and analyzed by mass spectra.
It was found that there were remarkable differences between crude and processed Fructus Corni samples in terms of contents of the eight bioactive compounds. It could also be seen that the total contents of the eight compounds varied slightly in the same type of samples from Henan suppliers. Thus, it is necessary to control the main active components in Fructus Corni by good agricultural practice and the norm of Chinese medicinal materials processing.
Conclusion
For the first time, a highly sensitive and selective method for simultaneous identification and determination of the eight compounds in crude and processed Fructus Corni using HPLC–MS/MS with multiple reaction monitoring mode was established. The method is simple, precise and accurate combined with high recovery and repeatability. The simultaneous extraction and analysis of samples provided an economical method for analysis of large numbers of samples in the short duration of time. Thus, this method has potential of being applied as an analytical technique for intrinsic quality control in Fructus Corni processing industries.
Acknowledgements
The authors wish to thank Zhiwei Xu and Sucai Luo who provided suggestions to improve the English and quality of the second revision of the paper.
Declaration of interest
The authors are grateful for financial support of the Open Project of National First-Class Key Discipline for Science of Chinese Materia Medica, Nanjing University of Chinese Medicine (2011ZYX2-006), the National Natural Science Foundation of China (No.81202918, No. 81274056, No. 30873438, No.81173546), the International Science and Technology Cooperation Project of Jiangsu Province, China (No. BZ2011053), the Project of Science and Technology of Chinese Medicine of Zhejiang Province, China (No. 2009CB008), the fund of Zhejiang Modernization of Traditional Chinese Medicine ([2008]436), the Chinese Medicine Research Program of Zhejiang Province, China (2008ZA002, 2011ZB101), and the Science Foundation of Zhejiang Chinese Medical University (7211093).
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