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ORIGINAL ARTICLE Table of Contents  
Ahead of print publication
High-performance liquid chromatography fingerprint of marine traditional chinese medicine haliotidis


1 Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China; Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China
2 Insect Biotechnology, Justus Liebig University Giessen, Giessen, Germany

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Date of Submission14-Apr-2021
Date of Acceptance20-May-2021
Date of Web Publication21-Feb-2022
 

  Abstract 


Objective: Haliotidis, the dried muscle of abalone, has long been used as a marine traditional Chinese medicine (TCM) due to its high medicinal and nutritive values, whereas few reports contributed to its quality control. This study aimed to establish the high-performance liquid chromatography (HPLC) fingerprints of Haliotidis. Materials and Methods: The HPLC fingerprints of Haliotidis, the manufacturing products of dried muscle of abalone, were analyzed by reverse-phase HPLC, using a YMC HPLC C18 column (250 mm × 4.6 mm, 5 μm). The gradient elution mode was applied with mobile phase starting from 5% acetonitrile and 0.1% phosphoric acid aqueous solution to 100% acetonitrile in 70 min at a flow rate of 1.0 mL/min. The detection wavelength was set at 190 nm. Results: Through compared the HPLC fingerprint chromatograms of 14 batches of Haliotidis, 25 peaks were assigned as the characteristic common peaks, of which one index component L-phenylalanine was identified by comparison with its standard substance. The established HPLC fingerprint analysis method of Haliotidis was validated with good precision, repeatability, and stability. Based on the above study, a reference HPLC fingerprint chromatogram of Haliotidis was proposed. Conclusions: It is the first time to establish the HPLC fingerprint chromatogram of TCM Haliotidis, which could contribute to the establishment of its quality control system.

Keywords: Haliotidis, high-performance liquid chromatography fingerprint, marine traditional Chinese medicine, quality control


How to cite this URL:
Zhao Q, Song SY, Zhang MQ, Li X, Liu Y, Wang CY. High-performance liquid chromatography fingerprint of marine traditional chinese medicine haliotidis. World J Tradit Chin Med [Epub ahead of print] [cited 2022 Aug 8]. Available from: https://www.wjtcm.net/preprintarticle.asp?id=337873





  Introduction Top


Abalone (class Gastropoda, family Haliotidae) is a marine mollusk with a characteristic single shell and approximately 100 species of them could be found worldwide.[1],[2],[3] It has long been utilized as food and folk medicine in China, which has been recognized to have health-promoting effects through nutritious substances or bioactive components.[4] Among medicine species, Haliotis diversicolor Reeve, Haliotis discus hannai Ino, Haliotis asinina Linnaeus, and Haliotis ovina Gmelin are widely distributed and maricultured in China.[1],[5],[6],[7],[8] Organism abalone could be separated as visceral mass, shell, and muscle. The viscera mass of abalone is not considered edible and is generally discarded.[4] The shell of abalone, called Haliotidis Concha, is one traditional Chinese medicine (TCM) and has been recorded in Chinese Pharmacopoeia.[1],[6],[8],[9]

Abalone medicine, Haliotidis, is the dried muscle of abalone and has been traditionally recognized as tonic remedy in China, sold in drug stores.[1],[6],[7],[8] According to ancient records, the medicinal efficacy of Haliotidis includes nourishing yin and clearing heat, benefiting spirit and improving eyesight, nourishing blood and softening liver, regulating menstruation and promoting lactation, moistening dryness and appetizing stomach, and benefiting intestines and dredging stranguria.[1],[6],[7],[8] Moreover, Haliotidis was mainly applied to treat consumptive fever and hectic fever due to yin deficiency, cough, glaucoma and cataract, irregular menstruation, metrorrhagia and morbid leukorrhea, hypogalactia after delivery, stranguria and turbid discharge, kidney asthenia, frequent urination, and dry stool.[1],[6],[7],[8]

Even though the interest in Haliotidis was increased, there were only few reports contributing to its quality control. Haliotidis sold in drug stores varies in quality and there is no uniform quality standard, which affects its medicinal safety and efficacy. Therefore, it is imperative to establish a systematic quality standard for Haliotidis. Similar to other marine TCMs, there are usually hundreds of different constituents coexisting in a single medicinal material, making quality control extremely difficult.[10] Nowadays, fingerprint technology is a comprehensive and quantifiable identification method mainly used to evaluate the authenticity and stability of medicines,[11] which has been internationally accepted as a feasible method to evaluate the quality of TCM. Specifically, high-performance liquid chromatography (HPLC) fingerprint emerges to be the most widely used technique attributed to its convenience and efficiency.[12] In the present study, the HPLC fingerprint of Haliotidis was analyzed, for the purpose of laying foundation for the method establishment of its quality control.


  Materials and Methods Top


Apparatus and reagents

Hitachi L-2000 HPLC system (equipped with a Hitachi L-2455 photodiode array detector); YMC HPLC C18 column (250 mm × 4.6 mm, 5 μm); HPLC-grade methanol (Tianjin Siyou Fine Chemicals Co., Ltd.); HPLC-grade acetonitrile (Shanghai Xingke high purity solvent Co., Ltd.); L-phenylalanine (Shanghai Solabo Biological Technology Co., Ltd., 200 mg). All the other chemicals and reagents were of analytical grade.

Medicinal materials

The abalone medicine Haliotidis materials (batches S1 − S14) used in our research were purchased from different drug stores in Zhejiang, Shandong, and Guangdong provinces in China, with S1 and S2 from Zhoushan, Zhejiang, S3 − S8 from Qingdao, Shandong, and S9 − S14 from Qingping, Guangdong. They were all authenticated by Professor Lin Ma (from the Ocean University of China).

Preparation of sample solution

Haliotidis samples were finely ground into powder and sieved through 40-mesh screen. Pulverized sample (1.0 g) was soaked with 50 mL anhydrous methanol in round-bottom flask for 1 h, and then heated to reflux at 80°C for 30 min. The extracted solution was filtered and the residue was refluxed with 50 mL anhydrous methanol for 30 min. All of the filtrates were combined and evaporated to dryness using a rotary vacuum evaporator with water bath heated at 40°C, and then dissolved with 1 mL HPLC-grade methanol. All the solutions were stored in refrigerator at 4°C for subsequent use.

Chromatographic conditions

The HPLC analysis of Haliotidis extract was performed on a Hitachi L-2000 HPLC system. A YMC HPLC C18 column (250 mm × 4.6 mm, 5 μm) was used and kept at 30°C. The mobile phase was consisted of acetonitrile and 0.1% phosphoric acid aqueous solution with a flow rate of 1.0 mL/min. The detection wavelength was at 190 nm and the sample injection volume was 20 μL. Before injection, the sample solution was filtered through a 0.22 μm pore membrane filter for HPLC analysis.

L-phenylalanine identification and content measurement

Standard substance L-phenylalanine (1.0 mg) was dissolved in 1 mL HPLC-grade methanol and 20 μL standard solution was injected into the HPLC system under the same chromatographic condition as Haliotidis sample solution with its HPLC chromatogram recorded. The content of L-phenylalanine in Haliotidis was quantified by peak area and calculated by regression equation based on external standard method. Briefly, 0.8, 1.5, 2.6, 3.8, 4.3, 5.0, and 6.7 mg L-phenylalanine were precisely weighed and dissolved into 1 mL chromatographic methanol, respectively. Each standard solution (20 μL) was injected into the HPLC. Linear regression analysis was performed by plotting the peak area (y) against the content (x) of L-phenylalanine. According to the calibration curve and peak area, the contents of L-phenylalanine in 14 batches of Haliotidis were quantified. During the whole experiment, the injection volume and HPLC analysis condition were strictly controlled.

Method validation of high-performance liquid chromatography fingerprint analysis

The precision evaluation of the HPLC fingerprint analysis method was performed by repeatedly injecting the same sample solution for six consecutive times. The repeatability was examined by injecting six independent sample solutions prepared from the same sample. Stability was assessed by analyzing the same sample solution intraday, with sample solution injected at 0, 2, 4, 8, 12, and 24 h after preparation.[12],[13]

Statistical analysis

Data analysis was performed by a professional software named Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004A, National Pharmacopoeia Commission, Beijng, China).


  Results and Discussions Top


Optimization of extraction process

To achieve the optimum extraction efficiency, extraction solvent, and extraction method were investigated. Extraction solvent was selected by comparing the numbers of detectable peaks and the stability of the HPLC chromatograms of Haliotidis sample solution extracted with three different solvents, including anhydrous methanol, anhydrous ethanol, and their mixture solution. The results showed that among these solvents, anhydrous methanol was the most suitable extraction solvent because the HPLC chromatogram of Haliotidis extracted with anhydrous methanol was more stable and showed more detectable peaks [Figure 1]. In addition, the HPLC chromatograms of Haliotidis sample solution extracted with reflux extraction method (one common method to extract chemical ingredients from TCM) and supersonic extraction method (one relatively convenient method) were also compared. It was found that reflux extraction was superior to supersonic extraction since the HPLC chromatogram of Haliotidis extracted with reflux extraction method displayed more detectable peaks [Figure 2]. Therefore, the optimum extraction method was determined as reflux extraction and used subsequently in sample solution preparation.
Figure 1: High-performance liquid chromatography chromatograms of Haliotidis solutions extracted with different solvents. Note: From the bottom up, anhydrous methanol; anhydrous ethanol; mixed solution (anhydrous methanol: Anhydrous ethanol = 1: 1)

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Figure 2: High-performance liquid chromatography chromatograms of Haliotidis solutions extracted by reflux and supersonic extraction methods. Note: From the bottom up, 80°C reflux extraction for 30 min; supersonic extraction for 30 min; extraction solvent: Anhydrous methanol

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Optimization of high-performance liquid chromatography chromatographic conditions

To obtain better separation of ingredients and more useful chemical information in fingerprint chromatogram of Haliotidis, different mobile phase compositions, detection wavelengths, injection volumes, and gradient elution procedures were tested and compared.

On the basis of previous trials, it was found that more detectable peaks could be obtained and the baseline was improved on the HPLC chromatogram of Haliotidis when acetonitrile-0.1% phosphoric acid aqueous solution was used as mobile phase. Different sample injection volumes, 5 μL, 10 μL, and 20 μL were investigated, respectively. Among them, 20 μL injection volume was the most suitable because the HPLC chromatogram showed satisfactory resolution and peak intensity [Figure 3]. Different gradient elution retention time, 60 min and 70 min were investigated. Obviously, the HPLC chromatogram with retention time of 70 min showed better resolution [Figure 4]. Besides, when the detection wavelength was set at 190 nm, more chromatographic peaks showed strong ultraviolet absorption. Finally, a binary gradient elution system composed of acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B) was chosen for the HPLC fingerprint analysis. The elution program was set as follows: 0–5 min, 5%–5% A; 5–10 min, 5%–10% A; 10–15 min, 10%–20% A; 15–18 min, 20%–25% A; 18–20 min, 25%–25% A; 20–30 min, 25%–30% A; 30–35 min, 30%–55% A; 35–45 min, 55%–60% A; 45–50 min, 60%–80% A; 50–55 min, 80%–100% A; 55–70 min, 100%–100% A.
Figure 3: High-performance liquid chromatography chromatograms of Haliotidis solutions under different injection volumes. Note: From the bottom up, injection volume 20 μL; injection volume 10 μL; injection volume 5 μL; extraction solvent: Anhydrous methanol; extraction method: 80°C reflux extraction for 30 min

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Figure 4: High-performance liquid chromatography chromatograms of Haliotidis solutions at different retention time. Note: From the bottom up, retention time for 70 min and 60 min; extraction solvent: Anhydrous methanol; extraction method: 80°C reflux extraction for 30 min

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Blank experiment

The Haliotidis sample extract was dissolved in HPLC-grade methanol solvent before injected into HPLC for analysis. To investigate the influence of HPLC-grade methanol on HPLC chromatogram, under the above chromatographic condition, 20 μL HPLC-grade methanol solvent was injected and its HPLC chromatogram was recorded [Figure 5]. It was found that the baseline only drifted slightly at about 50–60 min. Therefore, the influence of HPLC-grade methanol on HPLC chromatogram could be negligible.
Figure 5: High-performance liquid chromatography chromatogram of high-performance liquid chromatography-grade methanol solvent

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Establishment of the high-performance liquid chromatography fingerprint of Haliotidis

The HPLC fingerprints of 14 batches of Haliotidis samples were obtained using the optimized HPLC method [Figure 6]. These HPLC fingerprint data were analyzed by the software “Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004A)” recommended by the Chinese Pharmacopoeia Committee.[10],[14] Their common mode (marked R) was automatically calculated using the software. The results indicated that the HPLC fingerprint of S11 had the highest similarity degree to the common mode R [Table 1]. Therefore, the HPLC fingerprint of S11 was selected as the reference HPLC fingerprint of Haliotidis [Figure 7].
Figure 6: High-performance liquid chromatography fingerprints of 14 batches of Haliotidis (S1 − S14) and their common mode R

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Table 1: The similarity degree among the high-performance liquid chromatography fingerprints of 14 batches of Haliotidis and their common mode R

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Figure 7: The reference high-performance liquid chromatography fingerprint of Haliotidis

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In HPLC fingerprints, common peaks with relatively high intensity and good resolutions were assigned as “characteristic common peaks” to represent the characteristics of samples.[13] By comparing retention time and absorbance of each peak, 25 peaks in the reference HPLC fingerprint of Haliotidis were selected as characteristic common peaks [Figure 7]. According to their distribution characteristics and elution order, the HPLC fingerprint could be divided into four chromatographic zones, I (0–25 min), II (25–35 min), III (35–55 min), and IV (55–70 min), involving in 9, 1, 7, and 8 characteristic peaks, respectively. From the perspective of chemical polarity, the main chemical components in Haliotidis were mainly compounds with polarity and nonpolarity in zones I and IV, respectively, while a few compounds with medium polarity in zones II and III.

Index component L-phenylalanine identification and its quantitative analysis

Organism abalone is rich in nutritious substances, such as proteins, polyunsaturated fatty acids, vitamins, and minerals, especially essential amino acids.[4] It has been reported that L-phenylalanine, as an essential amino acid, is an index component in abalone.[15] Therefore, we determined the content of L-phenylalanine in Haliotidis. Peak 5 in the reference HPLC fingerprint of Haliotidis was identified as L-phenylalanine by comparing with the standard substance L-phenylalanine with the retention time at 11.52 min [Figure 8].
Figure 8: High-performance liquid chromatography chromatograms of Haliotidis (a) and standard substance L-phenylalanine (b)

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The content of L-phenylalanine in Haliotidis was quantified by peak area and calculated by regression equation. The calibration curve of standard substance L-phenylalanine was fitted by linear regression analysis [Figure 9]. The content proportions of L-phenylalanine in 14 batches of Haliotidis were ranged from 0.12%–1.08%, with standard deviation (SD) and coefficient of variance (CV%) of 0.28% and 45.81%, respectively [Table 2]. Sample S11 had the highest L-phenylalanine content proportion of 1.08%. Samples S1, S2, S4, S6, S12, and S13 had content proportion of 0.72%−0.93%, while S3, S7, S8, S9, S10, and S14 of 0.26%−0.60%. Sample S5 had the lowest content proportion of 0.12%. The content proportion of L-phenylalanine varied in different samples might result from different original growth regions, diets, and fishing time.
Figure 9: Calibration curve of standard substance L-phenylalanine

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Table 2: Content proportion (%) of L-phenylalanine in 14 batches of Haliotidis

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Method validation of high-performance liquid chromatography fingerprint analysis

The method of HPLC fingerprint analysis for Haliotidis was validated in precision, repeatability, and stability. The relative SD (RSD) values of relative retention time (RRT) and relative peak area (RPA) were used to evaluate the quality of the fingerprint.[11] In our study, peak 5 (L-phenylalanine) was assigned as a reference peak, and the other 24 peaks were applied to calculate their own RRT and RPA. In terms of precision, the RSD value of RRT of each peak was <3.60%, and the RSD of RPA <4.90% [Table 3], which indicated that the precision of instrument was good, conforming to the technical requirement of HPLC fingerprint. In repeatability assessment, the RSD values were below 3.80% for RRT and 4.88% for RPA, which demonstrated that the HPLC analysis method was repeatable and reliable. For the stability test, the RSD values of RRT and RPA did not exceed 1.39% and 4.88%, respectively, indicating that the sample solution was stable within 24 h.
Table 3: The relative standard deviation values of relative retention time and relative peak area in precision, repeatability, and stability tests

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  Conclusions Top


In this study, the HPLC fingerprint analysis method of marine TCM Haliotidis was first established. A total of 25 characteristic common peaks were detected in the HPLC fingerprint of Haliotidis during the retention time of 70 min with a YMC HPLC C18 column (250 mm × 4.6 mm, 5 μm) and the mobile phase consisting of acetonitrile and 0.1% phosphoric acid aqueous solution. The index component L-phenylalanine was identified in the HPLC chromatograms of Haliotidis. The HPLC analysis of Haliotidis showed the established HPLC fingerprint method had good precision, repeatability, and stability. Therefore, this study provided the reference HPLC fingerprint for the quality control of Haliotidis. Whereas this study is only a preliminary experiment on the HPLC fingerprint analysis of Haliotidis, further work should be extended, such as the identification of more characteristic peaks and the determination of the contents of characteristic components.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Guan HS, Wang SG. Chinese Marine Materia Medica. Vol. 3. Shanghai, China: Shanghai Scientific and Technical Publishers; 2009. p. 80-98.  Back to cited text no. 1
    
2.
Nguyen V, Qian Z, Ryu B, Kim K, Kim D, Kim Y, et al. Matrix metalloproteinases (MMPs) inhibitory effects of an octameric oligopeptide isolated from abalone Haliotis discus hannai. Food Chem 2013;141:503-9.  Back to cited text no. 2
    
3.
Shi LF, Hao GX, Chen J, Ma SK, Weng WY. Nutritional evaluation of Japanese abalone (Haliotis discus hannai Ino) muscle: Mineral content, amino acid profile and protein digestibility. Food Res Int 2020;129:108876.  Back to cited text no. 3
    
4.
Doh HS, Park HJ. Speciation of bio-available iodine in abalone (Haliotis discus hannai) by high-performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry using an in vitro method. J Food Sci 2018;83:1579-87.  Back to cited text no. 4
    
5.
Di G, Luo X, You W, Zhao J, Kong X, Ke C. Proteomic analysis of muscle between hybrid abalone and parental lines Haliotis gigantea Reeve and Haliotis discus hannai Ino. Heredity (Edinb) 2015;114:564-74.  Back to cited text no. 5
    
6.
State Administration of Traditional Chinese Medicine. Chinese Materia Medica. Vol. 9. Shanghai, China: Shanghai Scientific and Technical Publishers; 1999. p. 36-9.  Back to cited text no. 6
    
7.
Fu XM, Zhang MQ, Shao CL, Li GQ, Bai H, Dai GL, et al. Chinese marine materia medica resources: Status and potential. Mar Drugs 2016;14:46.  Back to cited text no. 7
    
8.
Guan HS, Wang SG. Illustrated Handbook of Chinese Marine Materia Medica. Vol. 2. Shanghai, China: Shanghai Scientific and Technical Publishers; 2016. p. 42-7.  Back to cited text no. 8
    
9.
Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China. Vol. 1. Beijing, China: Chinese Medical Science and Technology Press; 2020. p. 91.  Back to cited text no. 9
    
10.
Tang DQ, Zheng XX, Chen X, Yang DZ, Du Q. Quantitative and qualitative analysis of common peaks in chemical fingerprint of Yuanhu Zhitong tablet by HPLC-DAD-MS/MS. J Pharm Anal 2014;4:96-106.  Back to cited text no. 10
    
11.
Tan J, Zheng M, Duan S, Zeng Y, Zhang Z, Cui Q, et al. Chemical profiling and screening of the marker components in the fruit of Cassia fistula by HPLC and UHPLC/LTQ-Orbitrap MSn with chemometrics. Molecules 2018;23:1501.  Back to cited text no. 11
    
12.
Liu X, Wu Z, Yang K, Ding H, Wu Y. Quantitative analysis combined with chromatographic fingerprint for comprehensive evaluation of Danhong injection using HPLC-DAD. J Pharm Biomed 2013;76:70-4.  Back to cited text no. 12
    
13.
Jiang Z, Zhao C, Gong X, Sun X, Li H, Zhao Y, et al. Quantification and efficient discovery of quality control markers for Emilia prenanthoidea DC. by fingerprint-efficacy relationship modelling. J Pharm Biomed 2018;156:36-44.  Back to cited text no. 13
    
14.
Zhu L, Fang L, Li Z, Xie X, Zhang L. A HPLC fingerprint study on Chaenomelis Fructus. BMC Chem 2019;13:7.  Back to cited text no. 14
    
15.
Jia L. The Research of Detection Method about Characteristic of Abalone Producing Area. Baoding, Hebei: Hebei University; 2010. p. 22-3.  Back to cited text no. 15
    

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Correspondence Address:
Chang-Yun Wang,
Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China; Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237
China
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/wjtcm.wjtcm_71_21



    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

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