Application of UPLC-QTOF-MS Technology in Quality Stability Evaluation of Moluodan Concentrated Pills

Huiyun LIU, Yingrui TIAN, Jianliang SUI, Menglei WANG, Miaomiao YIN, Zhen WANG

1. Handan Pharmaceutical Co., Ltd., Handan 056000, China; 2. School of Life Science, Fudan University, Shanghai 200030, China; 3. The Affiliated Hospital and Medical College, Hebei University of Engineering, Handan 056000, China

Abstract [Objectives] To use liquid chromatography-mass spectrometry technology to analyze the chemical composition of traditional Chinese medicine and explore its application in the evaluation of quality stability of traditional Chinese medicine. [Methods] Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to detect the samples of Moluodan concentrated pills. By comparing and analyzing the detection results of 10 different batches of Moluodan concentrated pills, combined with principal component analysis (PCA), the quality stability of Moluodan concentrated pills was evaluated. [Results] A total of 367 chemical components were identified in Moluodan concentrated pills. The average repetition rate of the chemical components contained in the 10 different batches of samples reached 92%. The overall quality stability of the Moluodan concentrated pills was good. [Conclusions] The UPLC-QTOF-MS technology combined with PCA provides a reference for the overall quality evaluation of Moluodan concentrated pills, and provides new detection methods and ideas for the analysis of the components of Chinese medicine.

Key words Moluodan concentrated pill, Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry, Quality stability, Principal component analysis

1 Introduction

The active ingredients of Chinese patent medicines are complex, and the material basis of efficacy is not clear. The quality stability is closely related to varieties, origins, processing methods, formulation and dosage of finished medicine, preparation processing technology, pharmaceutical equipment, quality management and control methods, packaging materials,etc. Dou Zhihua[1]pointed out that the most prominent problem of Chinese patent medicines is the instability of product quality, and the same product produced by different factories and even different batches of products of the same manufacturer have varying quality. Based on the national sample inspection work, Liu Jingetal.[2]proposed that the main factors affecting the quality and safety of Chinese patent medicines are the quality of raw materials and the production process.

Moluodan concentrated pills are a preparation composed of 18 traditional Chinese medicines, with good clinical efficacy[3-5]. The chemical composition of Moluodan concentrated pills is complex, and the preparation process is complicated. In addition, the material basis for the pharmacological activity is not yet clear. Testing any one of the components of Moluodan concentrated pills, as a compound preparation, cannot reflect its overall quality. The types and quantities of chemical components contained in traditional Chinese medicine can more comprehensively reflect the inherent quality of traditional Chinese medicine[6-7]. Therefore, by comparing the types and quantities of chemical components contained in different batches of samples, the quality stability of the samples can be directly reflected. Liquid chromatography-mass spectrometry technology is suitable for composition analysis of complex systems, and it is a powerful method for the rapid separation and identification of active ingredients in complex systems[8-10]. In addition, principal component analysis (PCA) is also widely used in the identification and quality analysis of Chinese herbal medicines and Chinese patent medicines[11-13]. In this study, ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) technology was used to analyze the chemical components contained in Moluodan concentrated pills, and combined with PCA, the quality stability of the samples was evaluated.

2 Materials and methods

2.1 Materials

2.1.1Instruments and equipment. Eksigent ekspert ultra performance liquid chromatograph (AB Sciex, USA); Triple TOF 5600 high-resolution mass spectrometer (AB Sciex, USA); Heraeus Fresco 17 centrifuge (Thermo Fisher Scientific, USA); BSA124S-CW balance (Sartorius, Germany); JXFSTPRP-24 grinder (Shanghai Jingxin Technology Co., Ltd., PRC); Mingche D24 UV pure water meter (Merk Millipore, USA); PS-60AL ultrasound system (Shenzhen Leaderbang Electronics Co., Ltd.).

2.1.2Reagents and drugs. Methanol, acetonitrile, formic acid (LC-MS grade, CNW Technologies, Germany);L-2-chlorophenylalanine (Shanghai Hengbai Biotechnology Co., Ltd., PRC; purity ≥ 98%).

A total of 10 batches of Moluodan concentrated pills (Lots: 00518072, 00518073, 00518074, 00518077, 00518084, 00518086, 00518088, 00518089, 00518090 and 00518097) were sampled. They were produced by Handan Pharmaceutical Co., Ltd.

2.2 Methods

2.2.1Sample solution preparation. A certain amount (100 mg) of Moluodan concentrated pill powder was added with 500 μL of extract (80% methanol, internal standard mass concentration 1 μg/mL) containing internal standard (L-2-chlorophenylalanine), vortexed for 30 s, ultrasonicated in ice water bath for 1 h, and centrifuged at 12 000 r/min (radius 859 mm), 4 ℃ for 10 min, and the supernatant was collected as test solution.

2.2.2Analysis methods. The chromatographic conditions were as follows: column, UPLC BEH C18(100 mm×2.1 mm, 1.7 μm); mobile phase, 0.1% formic acid aqueous solution (A) -0.1% formic acid-acetonitrile (B); gradient elution (0-6.5 min, 5%-30% B; 6.5-12.5 min, 30%-70% B; 12.5-22 min, 70%-100% B); column temperature, 40 ℃.

The mass spectrometry conditions were as follows: electrospray ion source; atomization pressure, 377.24 kPa; auxiliary pressure, 377.24 kPa; curtain pressure, 241.32 kPa; temperature, 550 ℃; spray voltage, 5 500 V (positive ion mode ) or -4 000 V (negative ion mode); bombardment energy, 40 eV; collision energy difference, 20 V; 15 secondary spectra, 50 ms for each. The mass spectrometer performed primary and secondary mass spectrometry data collection based on the information-dependent acquisition (IDA) function under the control of the control software. In each data collection cycle, the molecular ions with the strongest intensity greater than 100 were selected for the corresponding secondary mass spectrometry data collection.

2.2.3Data processing and statistics. (i) Data processing. The original mass spectrum data were imported into Progenesis QI software to perform retention time correction, peak identification, peak extraction, peak integration, peak alignment,etc. Through the accurate mass-to-charge ratio of the primary mass spectrum data and the matching between fragment information and corresponding fragmentation law of the secondary mass spectrum data, combined with the traditional Chinese medicine database and related literature, the MS/MS peaks were identified.

(ii) PCA. Logarithmic transformation and centralization (CTR) formatting processing were performed on the data using SIMCA V14.1 software. Then, automatic modeling and analysis were conducted.

2.2.4Methodological verification. After determining the preparation method of test solution and chromatographic separation conditions, by referring to relevant literature, the UPLC-QTOF-MS analysis of Moluodan concentrated pills was verified methodologically to investigate the stability of the instruments and the response of the internal standard[6-7].

(i) Stability. A total of 10 batches of Moluodan concentrated pills were sampled and prepared into test solutions in line with the method in Section2.2.1, respectively. An accurate volume (20 μL) of each of the test solutions was taken out and mixed as quality control (QC) sample, and three replicates were arranged to obtain 3 QC samples (QC-001, QC-002 and QC-003), which were detected under the conditions described in Section2.2.2. The detection of QC samples was interspersed with the detection of 10 test samples. After the detection was over, PCA was performed on the QC samples in accordance with Section2.2.3(ii).

(ii) Internal standard response. The internal standardL-2-chlorophenylalanine in the 3 QC samples was detected according to the analysis method in Section2.2.2.

3 Results and discussion

3.1 Internal standard response stabilityThe internal standardL-2-chlorophenylalanine in the 3 QC samples was detected, and 3 total ion current chromatograms were obtained. They were superimposed, and the results are shown in Fig.1.

As can be seen from Fig.1, in the positive and negative ion mode, the retention time and response intensity of the chromatographic peak of the internal standardL-2-chlorophenylalanine in the QC samples had a good overlap in the total ion chromatograms, indicating that the instrument is more stable in the data collection process.

Note: A. positive ion mode; B. negative ion mode. The same below.

The internal standard is a substance introduced from an external source. As the concentration of internal standard in the QC samples is the same, the smaller the response difference of the internal standard (RSD≤20%), the more stable the system, and the higher the quality of data. The response difference of the internal standard in the QC samples was analyzed. The results show that in the positive and negative ion modes, theRSDvalue of the response intensity of the internal standardL-2-chlorophenylalanine in the QC samples was 14.3% and 15.7%, respectively, both less than 20%, showing that the data quality of this experiment is high, and the system is relatively stable.

3.2 UPLC-QTOF-MS detection results of Moluodan concentrated pills and quality evaluation of different batches of samples

3.2.1Total ion current chromatogram of Moluodan concentrated pills. Because the QC samples were prepared with the test solutions of the 10 different batches of Moluodan concentrated pills, in order to eliminate the difference in chemical composition of different batches of samples, in the qualitative analysis of the chemical components contained in Moluodan concentrated pills, the QC samples were selected as the research object. Due to the good results of methodological verification, the sample QC-001 was detected and the total ion current chromatogram was obtained (Fig.2).

Fig.2 Total ion current chromatogram of Moluodan concentrated pill QC sample

UPLC-QTOF-MS was used to determine the samples of Moluodan concentrated pills, and the total ion current chromatogram of the sample QC-001 was obtained. Based on the total ion current chromatogram, each of the peaks was identified. In the positive ion mode, a total of 214 chemical components were identified in Moluodan concentrated pills; in the negative ion mode, a total of 164 chemical components were identified. As 11 chemical components were shared by the positive and negative ion modes, in total, 367 chemical components were identified.

As a large preparation, Moluodan concentrate pill contains more complex chemical components. According to the quality standards of the drug, there are only 3 identification items and 1 content determination item, far from enough to reflect the quality of the medicine. This is also the difficulty in quality control of traditional Chinese medicine. UPLC-QTOF-MS technology quickly identified the 367 chemical components contained in the drug at one time, more comprehensively reflecting the quality of the drug.

According to the structure type of the chemical components of Chinese medicine, the identified chemical components were further classified. In Moluodan concentrated pills, there are 64 kinds of triterpenoids, 38 kinds of flavonoids, 33 kinds of alkaloids, 33 kinds of terpenoids, 12 kinds of sugars and glycosides, 10 kinds of organic acids and fatty acids, 9 kinds of phenylpropanoids, 4 kinds of amino acids and 3 kinds of quinones. Among them, the amount of triterpenes, flavonoids, alkaloids and terpenes is large. The literature[14-15]shows that these types contain many anti-inflammatory, antibacterial, and antioxidant chemical components. Through further research on these chemical components, the mechanism of action of the drug will be further understood.

Based on the complex and diverse ingredients of Moluodan concentrated pills, in order to analyze the components more comprehensively and systematically, the general sources of the 367 chemical ingredients were identified using numerous database, including the Chemical Database of Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences (www.organchem.csdb.cn), Taiwan Traditional Chinese Medicine Information Database (China) (http://tcm. cmu.edu. tw/), Google Scholar, Medline, CNKI and PubMed. As shown in Table 1, the number of chemical components identified in the 18 medicinal materials is not the same. More chemical components come from Herba Artemisiae Capillariae, Radix Scrophulariae, Notoginseng Radix et Rhizoma and Rhizoma Cordalis, all more than 30 kinds.

Table 1 Attribution of chemical ingredients contained in Moluodan concentrated pills

3.2.2Repetition rate of chemical components contained in different batches of Moluodan concentrated pills. In order to study the quality stability of different batches of Moluodan concentrated pills, in this study, the 10 different batches of Moluodan concentrated pill test solutions were analyzed by UPLC-QTOF-MS, and the total ion current chromatogram of each batch of sample was obtained. The 10 chromatograms were superimposed, and the result is shown in Fig.3.

As can be seen from Fig.3, in the positive and negative ion modes, the total ion current chromatograms of 10 different batches of Moluodan concentrated pills could overlap well. From the perspective of overlap of retention time and signal intensity, the types and contents of chemical components contained in each batch of Moluodan concentrated pills were relatively similar, indicating that the quality of each batch of Moluodan concentrated pills had little difference, and the overall quality stability is good.

Fig.3 Superimposed total ion current chromatogram of 10 batches of Moluodan concentrated pills

UPLC-QTOF-MS technology can more comprehensively reflect the overall quality of the drug when used to obtain the total ion current chromatogram of the drug. This method is relatively stable, and the reproducibility is high.

In order to more clearly apply the UPLC-QTOF-MS technology to express the repetition rate of the chemical components contained in the samples of different batches, the types and quantities of chemical components identified in each batch of samples were further analyzed. The results are shown in Table 2.

As can be seen from Table 2, the number of compounds identified in the 10 different batches of Moluodan concentrated pills in the positive ion mode ranged from 193 to 209, and the total number of compounds shared by different batches of samples was 185. The average repetition rate of the chemical components contained was 90%. The number of compounds identified in the negative ion mode ranged from 154 to 159. The total number of compounds shared by different batches of samples was 151, and the average repetition rate of the chemical components contained reached 96%.

Table 2 Repetition rate of chemical ingredients contained in different batches of Moluodan concentrated pills in different ion modes

The difference in total number of chemical ingredients identified was small among the 10 batches of Moluodan concentrated pills, with a minimum of 341 and a maximum of 359. The number of common components among different batches of the samples was 325, and the average repetition rate of the common chemical components contained in each batch reached 92%. From the perspective of the repetition rate of the chemical components contained in different batches of samples, Moluodan concentrated pills had little difference in quality between batches, and the quality stability was better.

3.2.3PCA of different batches of Moluodan concentrated pills. In order to compare the quality stability of different batches of Moluodan concentrated pills as a whole, PCA was performed on the data of the 10 batches of Moluodan concentrated pills, and the scatter plot and one-dimensional distribution map of the quality scores are obtained (Fig.4-5).

In Fig.4, the ellipse represents the 95% confidence interval. As shown in Fig.4, in the positive ion mode, the batch 00518073 showed a tendency to separate from others on the first principal component; the batch 00518089 showed a tendency to separate from others on the second principal component; and the remaining batches were more concentrated. Combined with the one-dimensional distribution map in Fig.5, except for batch 00518073, the quality of other batches was less than 2 standard deviations. In the negative ion mode, it can be seen from Fig.4 that the distribution of the batches was relatively discrete, but in combination with Fig.5, it was observed that the quality of all the batches was less than 2 standard deviations. It shows that the difference between different batches of samples was small, and the overall quality was relatively stable. The results of PCA of the 10 batches of samples show that Moluodan concentrated pills had good overall quality stability, with slight differences between individual batches. UPLCQTOF-MS technology, combined with PCA method, can reflect the quality stability of medicines between batches from another angle.

Fig.4 Scatter plot of PCA quality scores of 10 batches of Moluodan concentrated pills (n=10)

Fig.5 One-dimensional distribution map of PCA quality score of 10 batches of Moluodan concentrated pills

4 Conclusions

In this study, UPLC-QTOF-MS technology was used to determine the chemical composition of Moluodan concentrate pills. A total of 367 chemical components were identified. Among them, triterpenoids, flavonoids, alkaloids and terpenes were large in amount. The effective material basis and quality of Moluodan concentrated pills was reflected more comprehensively. At the same time, it also provides reference for the future research on the pharmacological effect of the drug. The types and amounts of chemical components contained in the 10 batches of Moluodan concentrated pills were compared. The results show that the average repetition rate of the chemical components contained in different batches of samples reached 92%. Further combining the PCA results, UPLC-QTOF-MS technology was used to evaluate the overall quality stability of Moluodan concentrated pills more comprehensively. The deviation of quality among the 10 batches of samples, except one batch, was small, indicating that the overall quality of the drug was stable, and there were slight differences between individual batches. In this study, UPLC-QTOF-MS technology was applied to the quality evaluation of traditional Chinese medicine preparations, with strong applicability and high stability, providing ideas for the establishment of the quality stability evaluation system of traditional Chinese medicine preparations.