Establishment of Variable-wavelength Fingerprint for Yixinshu Tablets

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1. Chengde Medical College, Chengde 067000, China; 2. Jingfukang Pharmaceutical Group Co., Ltd., Chengde 067000, China; 3. New Excipients of Traditional Chinese Medicine Engineering Research Center of Hebei Province, Chengde 067000, China

Abstract [Objectives] The purpose of this study was to establish the HPLC fingerprint of Yixinshu tablets, in order to provide a basis for quality control of Yixinshu tablets. [Methods] The chromatographic conditions were as follows: column, Agilent 5 TC-C18(2) (250 mm×4.6 mm, 5 μm); mobile phase, acetonitrile-0.02% phosphoric acid solution (gradient elution); column temperature, 25 ℃; detection wavelength, 280 nm (0-62 min) and 260 nm (62-125 min). Using salvianolic acid B as the reference, the fingerprints of nine batches of Yixinshu tablets were determined. The similarity between the fingerprints was evaluated according to the Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2012). [Results] A total of 25 common peaks were calibrated, 10 of which were identified. The similarity between the fingerprints of the nine batches of Yixinshu tablets and the reference fingerprint was all greater than 95%, indicating good similarity. [Conclusions] The established HPLC fingerprint can be applied to the quality control of Yixinshu tablets.

Key words Yixinshu tablet, Fingerprint, HPLC

1 Introduction

Yixinshu tablets are prescribed by Radix Ginseng, Radix Ophiopogonis, Radix Astragali, Chinese Magnoliavine Fruit, Radix Salviae Miltiorrhizae, Rhizoma Chiuanxiong and haws, with effects of replenishing Qi, restoring pulse, promoting blood circulation, removing blood stasis, nourishing yin and generating body fluid. With the development of medical technology, the inspection requirements for drugs are becoming more and more strict. Currently, there are few reports on the overall quality control of Yixinshu tablets. The control of only one or two indicator components could not reflect the overall quality of the Chinese medicine preparation. As a comprehensive quality control method, fingerprint technology has the characteristics of large amount of information, strong characteristics and integrity[1-2]. At present, the fingerprint established for Yixinshu tablets only reflects the quality of the indicator components with better absorption at a certain wavelength, and it does not reflect the quality of the components with no absorption or weak absorption at the wavelength. According to the absorption characteristics of different kinds of indicator components, in the corresponding position of the fingerprint, changing the absorption wavelength will allow for better detection of different chemical compositions[3-5]. Therefore, based on the characteristics of the indicator components of Yixinshu tablets, this paper established a fingerprint of Yixinshu tablets, which can better and more comprehensively reflect the quality of the preparation and improve the quality control of Yixinshu tablets.

2 Test materials

2.1 Apparatus and instrumentsThe used apparatus and instruments included high-performance liquid chromatograph (1200, Agilent, USA), digital ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd., China), electronic balance (MS204TS/02, METTLER TOLEDO, Switzerland), electronic analytical balance (accurate to 0.001%; XSE205DU, METTLER TOLEDO, Switzerland) and ultrapure water system (Milli-Q, Millipore, USA).

2.2 Reagents and drugsThe standards of caffeic acid (batch No.110885-201703, mass fraction 99.7%), chlorogenic acid (batch No.110753-201716, mass fraction 99.3%), salvianolic acid B (batch No.11562-201716, mass fraction 94.1%), rosmarinic acid (batch No.111871-201505, mass fraction 98.5%), schisandrol A (batch No.110857-201714, mass fraction 99.9%), cryptotanshinone (batch No.110852-200806, mass fraction 98.7%), tanshinone IIA (batch No.110766-200619), schizandrin A (batch No.110764-201714, mass fraction 99.3%), schisandrin B (batch No.110765-201311, mass fraction 98.8%) and tanshinone I (batch No.110867-201607) were purchased from National Institutes for Food and Drug Control. Yixinshu tablets were produced by Jingfukang Pharmaceutical Group Co., Ltd. Acetonitrile of chromatographically pure was purchased from Mreda Company (USA). Methanol of analytically pure and phosphoric acid of chromatographically pure were purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (China). Water used was self-made.

3 Methods and results

3.1 Chromatographic conditionsThe chromatographic conditions were as follows: column, Agilent 5 TC-C18(2) (250 mm×4.6 mm, 5 μm); mobile phase, acetonitrile (A)-0.02% phosphoric acid solution (B); gradient elution (0-5 min, 95%-95% B; 5-40 min, 95%-78% B; 40-60 min, 78%-73% B; 60-61 min, 73%-67% B; 61-70 min, 67%-62% B; 70-80 min, 62%-50% B; 80-110 min, 50%-30% B; 110-120 min, 30%-5% B; 120-125 min, 5%-95% B); detection wavelength 280 nm (0-62 min) and 260 nm (62-125 min); flow rate, 1.0 mL/min; column temperature, 25 ℃; and injection volume, 10 μL.

3.2 Solution preparation

3.2.1Test solution. Ten Yixinshu tablets were de-coated and ground finely. A certain amount (1 g) of the powder was transferred into a dry conical flask, added with 10 mL of 75% methanol, weighed, sonicated for 30 min, and then weighed again. The lost weight was supplemented with 75% methanol. The new solution was filtered, and the filtrate obtained was the prepared test solution.

3.2.2Negative sample solution. Referring to thePharmacopoeiaofthePeople’sRepublicofChina(2015 edition), Yixinshu tablets were prepared as negative sample. According to the description in Section3.2.1, the test solution of the negative sample was prepared.

3.2.3Reference solution. Accurate amounts of the standards were dissolved in methanol to a certain volume to prepare mixed reference solution, in which the concentrations of caffeic acid, chlorogenic acid, rosmarinic acid, salvianolic acid B, schisandrol, cryptotanshinone, tanshinone I, tanshinone IIA, schisandrin B and schisandrin A were 0.985, 0.863, 0.094 5, 0.113, 0.782, 0.125, 0.107, 0.164, 0.079 and 0.451 mg/mL, respectively.

3.3 Methodology

3.3.1Precision test. Under the chromatographic conditions described in Section3.1, the test solution was detected six times repeatedly. Taking salvianolic acid B as the reference, the relative area and relative retention time of each common peak were calculated. The results show that theRSDvalue of relative area of the common peaks was smaller than 2.6% (n=6), and theRSDvalue of relative retention time of the common peaks was smaller than 1.1% (n=6), indicating that the instrument has good precision.

3.3.2Stability test. After standing for 0, 2, 4, 6, 8, 10, 12 and 24 h, the test solution was detected under the chromatographic conditions in Section3.1, respectively. The relative area and relative retention time of each common peak were calculated. The results show that theRSDvalue of relative area of the common peaks was smaller than 2.8% (n=8), and theRSDvalue of relative retention time of the common peaks was smaller than 1.0% (n=8), indicating that the test solution was stable within 24 h.

3.3.3Reproducibility test. According to the method described in Section3.2.1, six test solutions were prepared. Under the chromatographic conditions described in Section3.1, the test solutions were detected. Taking salvianolic acid B as the reference, the relative area and relative retention time of each common peak were calculated. The results show that theRSDvalue of relative area of the common peaks was smaller than 2.9% (n=6), and theRSDvalue of relative retention time of the common peaks was smaller than 0.6% (n=6), indicating that the method established has good reproducibility.

3.4 Fingerprint establishment and results analysis

3.4.1Calibration of common peaks. Taking salvianolic acid B as the reference (S), based on the relative retention time, 25 peaks were shared by the nine batches of samples, and they were calibrated as common peaks, as shown in Fig.1.

Fig.1 HPLC fingerprints of the nine batches of Yixinshu tablets and identification of common peaks

3.4.2Similarity evaluation of fingerprints. The fingerprints of the nine batches of Yixinshu tablets were analyzed according to theSimilarityEvaluationSystemforChromatographicFingerprintofTCM(Version 2012), and the reference fingerprint was established by the median method, as shown in Fig.1. The similarities between the fingerprints of the nine batches of Yixinshu tablets and the reference fingerprint were 0.996, 0.991, 0.999, 0.969, 0.989, 0.999, 0.956, 0.998 and 0.986, respectively, all greater than 95%.

3.4.3Attribution of common peaks. The prepared negative sample was detected under the chromatographic conditions described in Section3.1to establish the fingerprint of the negative sample, as shown in Fig.2. By comparison, it was found that the main ingredients of the fingerprint come from Radix Salviae Miltiorrhizae and Chinese Magnoliavine Fruit, followed by Rhizoma Chiuanxiong. The remaining ingredients contribute less to the chromatogram.

Fig.2 Attribution of the common peaks in the HPLC fingerprints of Yixinshu tablets

3.4.4Identification of common peaks. The common peaks were identified according to the reference fingerprint, and ten common peaks were identified, as shown in Fig.3.

4 Discussion

In the fingerprint established in this paper, the cryptotanshinone, tanshinone I and tanshinone IIA from Radix Salviae Miltiorrhizae and the schisandrin A from Chinese Magnoliavine Fruit are negative and have no interference with others. The chromatographic peaks have good resolution and shape. The established method can be used for content determination of related chemical compositions. It should increase the amount of information on the fingerprint to provide a more comprehensive basis for the quality control of Yixinshu tablets.

Note: 6. chlorogenic acid; 7. caffeic acid; 12. rosmarinic acid; 14. salvianolic acid B; 15. schisandrol A; 19. cryptotanshinone; 20. tanshinone I; 22. schisandrin A; 23. tanshinone IIA; 25. schisandrin B.

Fig.3 HPLC fingerprints of Yixinshu tablet (A) and mixed reference (B)

Based on the absorption characteristics of related substances in Yixinshu tablets, in order to obtain more peaks and more information to better control the quality of the preparation, the detection wavelength was finally determined as 280 nm before the 62th min and 260 nm after the 62th min[6-7].

This study investigated four commonly used extraction solvents of ethanol, 100% methanol, 75% methanol, and 50% methanol. The extraction effect of 75% methanol was the best.

The four extraction times of 20, 30, 40 and 50 min were investigated. It was observed that there was no significant difference in the area of the peaks between ultrasonic extraction times of 30, 40 and 50 min. Therefore, the extraction solvent of 75% methanol and the ultrasonic extraction time of 30 min were finally used.

This study examined the three columns of Inertsil ODS-3 (250 mm×4.6 mm, 5 μm), Kromasil 100-5-C18(250 mm×4.6 mm, 5 μm) and Agilent 5 TC-C18(2) (250 mm×4.6 mm , 5 μm), the three chromatographic temperatures of 25, 30 and 35 ℃ and the four mobile phases of acetonitrile-0.02% phosphoric acid solution, acetonitrile-0.01% phosphoric acid solution, acetonitrile-water and methanol-water. Finally, the column of Agilent 5 TC-C18(2) (250 mm×4.6 mm, 5 μm), the column temperature of 25 ℃ and the mobile phase of acetonitrile-0.02% phosphoric acid solution were screened out.

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