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ORIGINAL ARTICLE
Year : 2021  |  Volume : 7  |  Issue : 1  |  Page : 86-96

Ultra-high-performance liquid chromatograph with triple-quadrupole mass spectrometer quantitation of twelve phenolic components in different parts of sarcandra glabra


1 State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, People's Republic of China
2 Fujian Key Laboratory of Integrative Medicine on Geriatric, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, People's Republic of China
3 Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Chinese Medicine and Pharmaceutical Science, Nanning, People's Republic of China

Date of Submission07-Jun-2020
Date of Acceptance15-Jul-2020
Date of Web Publication04-Feb-2021

Correspondence Address:
Dr. Wei Zhang
State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau
People's Republic of China
Dr. Zhi-Hong Jiang
State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau
People's Republic of China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_54_20

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  Abstract 


Objective: The study objective was to determine phenolic components for the quality control (QC) of cultivated Sarcandra glabra (Thunb.) Makino ( S. glabra). Materials and Methods: A sensitive, ultra-high-performance liquid chromatography-tandem mass spectrometric method for the simultaneous determination of 12 phenolic components has been developed. Six caffeoylquinic acids, two caffeoylshikimic acids, and four flavanonol glucosides were selected for the comprehensive analysis of distribution in different parts (root, stem, and leaf). Results: Twelve phenolic components were linear in the concentration range of 0.005–5.0 μg/mL ( R2 > 0.995). The relative standard deviation of intra-day and inter-day precision across three validation runs over the entire concentration range was <5%. The recovery determined was within 5% in terms of relative error. Our results showed that the 12 phenolic compounds were mainly distributed in leaves and stems far more than those in roots. Conclusions: This study provided an ultra-high-performance liquid chromatograph with triple-quadrupole mass spectrometer quantitative method of 12 phenolic components for the QC of cultivated S. glabra. It was found that the phenolic components were significantly accumulated in the aerial parts (stems and leaves) of cultivated S. glabra.

Keywords: Cultivation, quantitation, Sarcandra glabra, ultra-high-performance liquid chromatograph with triple-quadrupole mass spectrometer


How to cite this article:
Lu JG, Wang CY, Chen DX, Wang JR, Che KS, Zhong M, Zhang W, Jiang ZH. Ultra-high-performance liquid chromatograph with triple-quadrupole mass spectrometer quantitation of twelve phenolic components in different parts of sarcandra glabra. World J Tradit Chin Med 2021;7:86-96

How to cite this URL:
Lu JG, Wang CY, Chen DX, Wang JR, Che KS, Zhong M, Zhang W, Jiang ZH. Ultra-high-performance liquid chromatograph with triple-quadrupole mass spectrometer quantitation of twelve phenolic components in different parts of sarcandra glabra. World J Tradit Chin Med [serial online] 2021 [cited 2021 Apr 21];7:86-96. Available from: https://www.wjtcm.net/text.asp?2021/7/1/86/310935




  Introduction Top


Sarcandra glabra (Thunb.) Makino, belonging to the Chloranthaceae family, is an evergreen shrub that is generally distributed in the southern part of China, North Korea, Japan, India, southeastern Asia, etc.[1] The slices of dried whole plant of S. glabra (Sarcandrae Herba) are usually used in the preparation of various traditional Chinese medicinal preparations (TCMPs).[2] These TCMPs are mainly used for treating oral inflammation diseases, thrombocytopenic purpura, pneumonia, appendicitis, cellulitis, and cancer.[2] According to the studies on S. glabra,[3],[4],[5],[6],[7],[8],[9],[10],[11],[12] organic acids, sesquiterpenes, coumarins, caffeoyl derivatives, flavonoids, and polysaccharides were considered contributing to its biological activities.

Due to its significant biological properties and effects, the demand of Sarcandrae Herba has been increased in recent years. For the sustainable utilization and development of S. glabra, cultivated S. glabra have been successfully developed nowadays. At present, the quality control (QC) of Sarcandrae Herba is performed based on the Chinese Pharmacopoeia 2015 Volume I using isofraxidin and rosmarinic acid as markers. Phenolic components including caffeoyl derivatives and flavonoids are the major components in the water extracts of Sarcandrae Herba, which exert good bioactivities in antioxidation and anti-inflammation, which is consistent with the pharmacodynamic mechanisms of Sarcandrae Herba.[10],[13],[14],[15],[16],[17],[18] Several qualitative and quantitative methods have been developed for the determination and identification of S. glabra. For example, high-performance liquid chromatography (HPLC)-time-of-flight mass spectrometry was established for identifying the characteristic markers in the stems and leaves of S. glabra.[19] Li et al. reported an ultra-high-pressure liquid chromatography coupled with LTQ Orbitrap mass spectrometry method for the chemical profiling of bioactive constituents, and 17 compounds in S. glabra and their preparations were quantified by HPLC-QQQ-MS.[20],[21] However, there is still a lack of a multicomponent quantitative method of UHPLC with triple-quadrupole mass spectrometer (UHPLC-QQQ-MS) for QC of S. glabra, which could offer shorter analysis time, more specificity, and higher sensitivity. In this study, 12 phenolic components [Figure 1] including six caffeoylquinic acids, two caffeoylshikimic acids, and four flavanonol glucosides were determined in cultivated and wild S. glabra samples through UHPLC-QQQ-MS. Chemical distribution of the target compounds in different parts was also described. This study provides technical method for the authentication and QC of S. glabra and its preparations.
Figure 1: Structure of 12 target compounds

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  Materials and Methods Top


Chemicals

HPLC-grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). HPLC-grade formic acid was purchased from Fluka (Buchs, Switzerland). Water was deionized and purified by Milli-Q Plus system (Millipore, Inc., MA, USA) at 18.2 MΩ.cm. Reference standards of 5- O-caffeoylquinic acid (5-CQA), 3- O-caffeoylquinic acid (3-CQA), 4- O-caffeoylquinic acid (4-CQA), 3, 4- O-dicaffeoylquinic acid (3,4-diCQA), 3, 5- O-dicaffeoylquinic acid (3,5-diCQA), and 4, 5- O-dicaffeoylquinic acid (4,5-diCQA) were purchased from Chengdu MUST Bio-Technology Co., Ltd (Chengdu, China). Neoastilbin, astilbin, neoisoastilbin, and isoastilbin were purchased from Chengdu Alfa Biotechnology Co., Ltd (Chengdu, China).

Herbal materials

Twenty-six batches of cultivated S. glabra (9 planting from seeds and 17 planting from sprouts) were collected from different provinces in China. Two batches of wild S. glabra, as QC samples, were obtained from Youxi country, Sanming city, Fujian Province. All the herbal materials mentioned above were taxonomically identified and offered by Prof. Zhi-Hong Jiang [Table 1], and the representative herbariums of cultivated and wild S. glabra were stored in our laboratory. Each batch of sample was firstly divided into three parts, i.e., leaves, stems, and roots. Then, the samples of each part were cut into smaller pieces, grounded into powder, and passed through a 50-mesh sieve.
Table 1: Twenty-eight batches of Sarcandra glabra samples from China

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Preparation of standard and sample solutions

A mixed standard stock solution containing 5-CQA, 3-CQA, 4-CQA, 3,4-diCQA, 3,5-diCQA, 4,5-diCQA, neoastilbin, astilbin, isoastilbin, and neoisoastilbin was prepared and dissolved in 50% (v/v) methanol. The solution was stored in a refrigerator at 4°C prior to analysis.

Powdered herbal sample (0.1 g) was accurately weighed and placed into a 50-mL centrifugal tube. Then, 25 mL of 50% methanol was accurately added. The mixture was sonicated for 30 min with occasional shakings at room temperature and was centrifuged at 1800 × g for 5 min. The total weight of the tube with the sample solution was recorded, and the weight was made and kept the same before and after sonication. The supernatant was filtered through a 0.22-μm PTFE filter as sample solution.

Ultra-high-performance liquid chromatograph with triple quadrupole mass spectrometer conditions

The liquid chromatography was assembled by an Agilent 1290 infinity system (Santa Clara, CA, USA) consisting of binary pumps with an integrated vacuum degasser (G4220A), a thermostat (G1330B), a standard autosampler (model G4226A), and a thermo statted column compartment (model G1316C). The UHPLC-QQQ-MS analysis was performed on an Agilent 6460 UHPLC-QQQ-MS. Data acquisition was carried out by Agilent Mass hunter® workstation B.05.00 software (Agilent, USA).

The chromatographic separations were carried out on an Agilent Extend C18 RRHD column (1.8 μm, 100 mm × 2.1 mm I.D., Agilent). A gradient program was used with mobile phase consisting of solvent A (0.1%, v/v, formic acid in water) and solvent B (0.1%, v/v, formic acid in acetonitrile) as follows: 0–12 min, 10%–20% B; 12–13 min, 20%–40% B; 13–15 min, 40%–10% B. The injection volume was 2 μL. The flow rate was 0.3 mL/min, and the column temperature was maintained at 30°C.

Mass spectrometric data were acquired using the multiple reaction monitoring (MRM) in negative ion mode. The parameters were as follows: gas temperature, 325°C; gas flow, 9 L/min; nebulizer gas, 40 psi; and capillary voltage, 4000 V. Nitrogen served as the nebulizer and collision gas.

Method validation

Method validation was studied for linearity, precision, limit of detection (LOD), limit of quantification (LOQ), repeatability, and recovery. The linearity was prepared using the mixed standard stock solution by diluting them to a serial appropriately concentrations with 50% methanol. Intra-day precision was evaluated by performing replicate analyses ( n = 5) of the mixed standard solution at three concentration levels (0.05, 0.5, and 5.0 μg/mL), and inter-day precision was determined by repeating analyses using the same mixed standard solutions on three consecutive days. For the accuracy evaluation, repeatability and recovery was studied using the batch of S28 sample. Repeatability was obtained by analyzing the batch of S28 sample for five times, and recovery was performed by spiking the known appropriate amounts of the mixed standard solution to the batch of S28 sample with five replicate analyses. LOD and LOQ were evaluated by diluting the mixed standard solutions based on the signal-to-noise ratio (S/N) of 3:1 for LOD and S/N ratio of 10:1 for LOQ.


  Results Top


Optimization of sample solution preparation and ultra-high-performance liquid chromatograph with triple-quadrupole mass spectrometer conditions

The methanol concentration of extraction solvents (30%, 50%, 70%, and 100%) was tested for the efficiency of extraction; 50% methanol could maximize the extraction of the 12 target compounds. To optimize the extraction time, the amounts of 12 constituents extracted from the first extraction were compared with that from the second extraction. The results showed that the first extraction was enough to extract the 12 compounds completely. For accurate quantification, dilution folds of the sample solution were investigated, and the results showed that a 10-fold dilution was appropriate for the detection of the target compounds.

To obtain the optimal UHPLC-QQQ-MS conditions for detection of the target compounds, the negative mode was applied for its higher sensitivity and cleaner mass spectral background when compared to those in positive mode. The collision energy (0–40 eV) and fragmentor voltage (90–150 V) parameters were compared to obtain the highest relative abundance of the exclusive ions and production in the MRM optimized conditions. Production ion with highest abundance was used for quantitation. The finally optimized conditions for each reference standard are shown in [Table 2].
Table 2: Multiple reaction monitoring acquisition parameters for determination of phenolic compounds through ultra-high-performance liquid chromatograph with triple quadrupole mass spectrometer

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Method validation

The quantitation method validation results are summarized in [Table 3],[Table 4],[Table 5]. The linearities of target compounds showed good correlation coefficients ( R2 > 0.995) in a relatively wide concentration range. The recovery results were satisfactory with all relative standard deviations (RSDs) <5.0%, and all mean recoveries ranged from 99.3% to 107.8%. The mean values of repeatability were from 148.13 to 3750.20 μg/g, with their RSDs being <2.6%. Both the intra-day and inter-day precisions (RSDs) were <5% for all analytes in the three concentration levels. The LODs and LOQs ranged from 0.002 to 0.02 μg/mL and from 0.005 to 0.05 μg/mL, respectively. All these results mentioned above indicate that this developed method was well verified and showed excellent detection sensitivity for the target compounds.
Table 3: Linearity study of phenolic compounds for method validation

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Table 4: Precision ( n=5) relative standard deviations (%) of phenolic compounds for method validation

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Table 5: Repeatability and recovery of phenolic compounds for method validation ( n=5)

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Quantitation and distribution of twelve target compounds in different parts of Sarcandra glabra

Twenty-eight batches of S. glabra samples separated into three parts were quantitatively assessed in duplicate using the developed UHPLC-QQQ-MS method. Typical MRM chromatograms of ten reference standards and 12 target compounds in S. glabra sample (batch S28) are shown in [Figure 2]. The contents of six caffeoylquinic acids (5-CQA, 3-CQA, 4-CQA, 3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA) and four flavanonol glucosides (neoastilbin, astilbin, isoastilbin, and neoisoastilbin) were quantitated by calibration curves of reference standards. Due to the same skeletal structure, two caffeoylshikimic acids of 4-dHCQA and 5-dHCQA were calculated using calibration curves of 4-CQA and 5-CQA, respectively. The quantitative amounts of target compounds in the three parts of S. glabra samples are summarized in [Table 6][Table 7],[Table 8]. The contents of target compounds in the three parts of cultivated S. glabra samples were compared, as shown in [Figure 3]. Comparison on the contents of target compounds in the three parts from cultivated and wild S. glabra samples is presented in [Figure 4], [Figure 5], [Figure 6].
Figure 2: Typical multiple reactions monitoring chromatograms of ten reference standards and 12 target compounds in Sarcandra glabra sample (batch S28)

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Figure 3: Comparison on the contents of 12 target compounds in the three parts of cultivated Sarcandra glabra. NS: P > 0.05

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Figure 4: Comparison on the contents of the 12 target compounds in roots from different sources. *0.01 < p < 0.05; ** p < 0.01

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Figure 5: Comparison on the contents of the 12 target compounds in stems from different sources. *0.01 < p < 0.05; ** p < 0.01

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Figure 6: Comparison on the contents of the 12 target compounds in leaves from different sources. *0.01 < p < 0.05; ** p < 0.01

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Table 6: Quantitative results of 28 batches of S. glabra samples (roots, n=2)

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Table 7: Quantitative results of 28 batches of Sarcandra glabra samples (stems, n=2)

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Table 8: Quantitative results of 28 batches of Sarcandra glabra samples (leaves, n=2)

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  Discussion Top


For the cultivated S. glabra samples [Figure 3], caffeoylquinic acids (5-CQA, 3-CQA, and 4-CQA) and flavonoids (neoastilbin, astilbin, isoastilbin, and neoisoastilbin) were dominantly distributed in the leaves, and then in stems and roots. While there was a monotonic increase in concentration from the roots, leaves, and stems for other caffeoylquinic acids (3, 4-diCQA, 3, 5-diCQA, and 4, 5-diCQA) and caffeoylshikimic acids (4-dHCQA and 5-dHCQA).

These distribution results revealed that there are significant differences among roots, stems, and leaves in cultivated S. glabra samples. Leaves and stems are far more important than roots, which is almost consistent with the distribution of isofraxidin and rosmarinic acid in different parts of cultivated and wild S. glabra.[22],[23],[24],[25],[26]

Besides, [Table 6],[Table 7],[Table 8] and Figures 4-6 indicate that there were no significant differences of target compounds in cultivated S. glabra samples planting from seeds and sprouts. At the same time, some target compounds in cultivated S. glabra were entirely different with those in wild S. glabra, which need to be confirmed in the future because only two batches of wild samples were used in this study.


  Conclusions Top


The analytical method described in this article provided a sensitive and specific assay for determination of 12 phenolic components in different parts of 26 batches of cultivated S. glabra samples, with two batches of wild herb as QC samples. Our results showed that 12 phenolic components mainly accumulate in leaves and stems, and much less in roots. Besides, there are no significant differences between the herbal samples planting from seeds and sprouts. Therefore, this study may provide great help for QC and rational utilization of cultivated S. glabra.

Acknowledgments

This research was funded by the Science and Technology Development Fund, Macau SAR (File no. 0023/2019/AKP) and Guangxi Science and Technology Department Fund (File no. AD17195002).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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