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Liquid-liquid chromatography in sample pretreatment for quantitative analysis of trace component in traditional Chinese medicines by conventional liquid chromatography
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Liquid-liquid chromatography in sample pretreatment for quantitative analysis of trace component in traditional Chinese medicines by conventional liquid chromatography Shanshan Zhao , Chaoyue Wang , Xiang Wang , Yang Jin , Wenyu Sun , Xingchu Gong , Shengqiang Tong PII: DOI: Reference:
S0021-9673(20)30094-7 https://doi.org/10.1016/j.chroma.2020.460917 CHROMA 460917
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
28 November 2019 19 January 2020 22 January 2020
Please cite this article as: Shanshan Zhao , Chaoyue Wang , Xiang Wang , Yang Jin , Wenyu Sun , Xingchu Gong , Shengqiang Tong , Liquid-liquid chromatography in sample pretreatment for quantitative analysis of trace component in traditional Chinese medicines by conventional liquid chromatography, Journal of Chromatography A (2020), doi: https://doi.org/10.1016/j.chroma.2020.460917
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Highlights
Liquid-liquid chromatography was studied in sample pretreatment for quantification.
Continuous-stirred tank reactors model was used to predict collection time of analytes.
Determination of wilforlide A in Tripterygium wilfordii and tablets was established.
A new method for sample pretreatment in analysis of trace component was developed.
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Liquid-liquid chromatography in sample pretreatment for quantitative analysis of trace component in traditional Chinese medicines by conventional liquid chromatography Shanshan Zhao1, Chaoyue Wang1, Xiang Wang1, Yang Jin1, Wenyu Sun1, Xingchu Gong2, Shengqiang Tong1,* 1
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310032,
China 2
Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, 310023, China
The telephone/fax number, email and postal address of the corresponding author: Prof. Shengqiang Tong Tel.: +86 571 88320984; fax: +86 571 88320984 E-mail: [email protected] Postal address: College of Pharmaceutical Science, Zhejiang University of Technology, Chaowang Road 18, Chaohui No. 6 District, Hangzhou, CHINA
Abstract A method for sample pretreatment using liquid-liquid chromatography combined with a conventional liquid chromatography was developed for quantitative determination of trace chemical components in traditional Chinese medicine. The main effective component, wilforlide A, in the traditional Chinese medicinal herb Tripterygium wilfordii as well as in its Chinese patent medicine glycosides tablets was successfully determined after sample pretreatment by liquid-liquid chromatography. A biphasic solvent system n-hexane-ethyl acetate-ethanol-water (6:4:6:4, v/v) was screened for crude sample treatment by liquid-liquid chromatography. The collection time of eluted fractions containing target components could be well predicted using a continuous-stirred tank reactors model after determination of its retention time. Then, quantitative 2
analysis of wilforlide A in Tripterygium wilfordii as well as in its tablets could be successfully determined by conventional reversed-phase high performance liquid chromatography with UV detector. Under the optimized conditions, the method showed good linearity (R 2 =0.9999) for wilforlide A in the range of 0.01 mg mL-1 -0.10 mg mL-1. The limit of detection and limit of quantity were 1.35 ng mL-1 and 4.50 ng mL-1, respectively. The average recovery rate, intra-day and inter-day precisions of wilforlide A were 96.43 %, 0.67 % and 1.14 %, respectively. Compared with previous studies, the present method showed advantages of complete recovery of target component in the sample pretreatment and good repeatability. Keywords: Liquid-liquid chromatography; Liquid chromatography; Sample pretreatment; Quantitative determination; Wilforlide A; Triperygium wilfordii Hook.f
1. Introduction In order to extract, separate and enrich target analytes from complex samples and to meet the requirements of chromatography and mass spectrometry, various sample pretreatment techniques have been developed. Traditional methods of sample preparation, such as liquid-liquid extraction and solid phase extraction, are time-consuming, tedious and high consumption of organic solvents [1]. In recent years, in order to overcome the deficiency of liquid-liquid extraction and solid phase extraction, the development of sample pretreatment technology is mainly focused on automation, simplification and miniaturization [2]. Some microextraction technologies such as single-drop microextraction, solid phase microextraction, dispersion liquid-liquid microextraction, magnetic solid phase extraction, headspace liquid phase microextraction, thin film microextraction and stir bar sorptive extraction have emerged successively [3]. They could be fully automated and connected to liquid chromatography, gas chromatography or capillary electrochromatography.
3
Compared with traditional methods, these new developed extraction strategies have made the whole sample pretreatment process simpler, faster, and more economical, efficient and environmental-friendly [4]. However, it is still a very challenging task for quantitative determination of chemical components with very low content in an extremely complex sample due to the difficulty of sensitivity, selectivity and limited recovery of solutes in the crude sample pretreatment. For instance, it is difficult to obtain complete recovery of analytes during the sample pretreatment by solid phase extraction, in which irreversible adsorption of solutes to solid matrix would be always involved [5]. For liquid-liquid extraction, it occurs in a single solution (one-step method) and it is difficult to set two or more extraction stages due to the increase in extraction volume and time [6]. So a complete recovery of analytes in the crude sample would be difficult to achieve. For dispersive liquid-liquid microextraction, although a high enrichment factor could be achieved in the sample pretreatment, it had the drawback of low recovery rate and poor selectivity, which greatly limited its application [7]. Liquid-liquid chromatography, generally known as centrifugal partition chromatography and countercurrent chromatography, is a support-free liquid-liquid partition chromatography, which relies on the partition of a solute between two immiscible solvents to achieve separation [8]. Liquid-liquid chromatography can be considered as an apparatus for multi-step liquid-liquid extractive process. Compared with the traditional solid phase chromatography, the support-free liquid stationary phase in liquid-liquid chromatography allows for complete recovery for the analytes. Since liquid-liquid chromatography are usually performed at ambient temperature and the sample would not be subjected to any physical sorption processes, the original physicochemical properties of the sample constituents are largely preserved [9]. To the best of our
4
knowledge, at present liquid-liquid chromatography has been mainly used for preparative separation and purification of chemical components from fermentation broth, synthetic mixtures and natural products [10]. Few report on sample pretreatment by liquid-liquid chromatography has been available in quantitative determination of trace chemical components in natural products [11]. Tripterygium wilfordii Hook. F, a Chinese herbal medicine (‘Lei gong teng’ in Chinese), has a long history of clinical use in China. Its whole root and the root xylem without bark are mainly used for medicinal purpose. Tripterygium wilfordii glycosides tablets (‘lei gong teng duo gan pian’ in chinese), a famous Chinese patent medicine, which has been used to treat rheumatoid arthriits, lupus erythematosus, and Henoch-Schonlein purpura nephriits and skin diseases for many years, constituting a large fraction of drugs sales for rheumatic disease in China [12]. Wilforlide A is one of the main bioactive chemical components in the tablets, and its content is regarded as the main chemical marker for quality control of this drug [13]. However, it was found that the content of wilforlide A in Tripterygium wilfordii glycosides tablets was extremely low and chemical composition of the tablets was very complicated. Therefore, it was a very challenging task to establish a simple and efficient method for quantitative determination of wilforlide A in the tablets. And it would be more difficult to determine wilforlide A in Tripterygium wilfordii. Two simple methods for simultaneous quantitative determination of five terpenoids in Tripterygium wilfordii were developed by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) [12] and by highperformance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD)[14], in which no specific sample pretreatment was employed. However, it was noticed that the content of
5
wilforlide A in root of Tripterygium wilfordii could not be accurately determined due to its limited content [12]. Meanwhile, analysis time was very long and peak resolution of wilforlide A was rather limited in the literature due to complex composition of the crude sample [14], resulting in low accuracy for quantitative determination of wilforlide A in Tripterygium wilfordii. Furthermore, quadrupole time-of-flight mass spectrometry and evaporative light scattering detection were not frequently-used detectors for conventional liquid chromatography. Therefore, sample pretreatment was necessary for determination of this trace effective component by conventional liquid chromatography. Several traditional methods for sample pretreatment were reported, including neutral alumina column chromatography [15-17] and silica gel column chromatography [18]. And solid phase extraction was necessary for sample pretreatment if HPLC-MS was used to determine content of wilforlide A in crude extracts of this medicinal plant[19], human urine[20] and human plasma[21]. However, no complete recovery of solutes could be obtained since irreversible adsorption of sample to the stationary phase was involved in the above methods. Meanwhile, it was difficult to accurately collect the eluted fractions containing the target component due to its limited content and its extremely complex composition when tradition column chromatography was used for sample pretreatment, which was also a great challenge for column chromatography in sample pretreatment due to the need for long analysis time and multiple-step analysis [22]. Various mathematical models have been used to simulate the adsorption process of substances, for predicting the breakthrough curve and collection time [23,24]. However, the elution curve of components with very limited content could not be effectively predicted due to many influencing factors [25]. For liquid-liquid chromatography, the elution behavior of solute was determined by the two-phase solvent system, mobile phase flow
6
rate, rotation speed of the column [26]. Several theoretical models have been reported for predicting the elution behavior of target solute, including elution countercurrent distribution model, compartment model, probability model and continuous stirred tank reactor model [27]. The continuous stirred tank reactor model could be used to predict the chromatogram of the target components by relating all model parameters directly to experimental settings and column dimensions. Most importantly, this model can forecast the chromatogram of target compound without using empirical values to calibrate [26]. In our present study, a new method for sample pretreatment by liquid-liquid chromatography using the continuous stirred tank reactor model was developed for quantitative determination of the trace component wilforlide A in Tripterygium wilfordii and in its Chinese patent medicine glycosides tablets. 2. Experimental section 2.1 Instruments Sample pretreatment using liquid-liquid chromatography was achieved by a model of TBE-200V high speed countercurrent chromatographic system (Tauto Biotech Co. Ltd., Shanghai, China). The separation column was 2.0 mm ID polytetrafluoroethylene tubing with a total capacity of 190 mL. The rotation speed of separation column could be adjusted in a range from 0 to 1000 rpm, where 800 rpm was used. A manual six-way injection valve with a 20 mL loop was used to introduce the crude sample into the column. The column temperature was controlled by SDC-6 constant-temperature controller (Nanjing Xinchen Biotechnology Co., Ltd., Nanjing, China). The solvent was pumped into the column by a model MPO 106 constant-flow pump (Shanghai Sanwei Science and Technology Co., Ltd., shanghai,China). The other instruments included a model HD-21C-B detector (Shanghai Kanghua biochemical instrument Manufacturing Factory, Shanghai,
7
China) operating at 254 nm and a SEPU3000 workstation (Hangzhou Puhui Technology, Hangzhou, China). Analysis and determination of the analytes were performed on a LabSolutions MS-20160509 system (Shimadzu, Japan) equipped with a Shimadzu SCL-10ASvp controller. The solvent was pumped by Shimadzu LC-20ATvp Multisolvent Delivery System. The detection signal was collected by a LabSolutions MS-20160509 workstation. 2.2 Reagents and materials Acetonitrile and methanol for HPLC analysis, formic acid and analytical grade chemicals were purchased from Huipu chemical instrument Co. Ltd., Hangzhou, China. The reference substance of wilforlide A was purchased from Chengdu Must Bio-Tech Co.Ltd., Chengdu, China. Chinese patent medicines of Tripterygium wilfordii glycosides tablets were purchased from six different pharmaceutical companies (Tablet 1, Fudan Fuhua Pharmaceutical Co. Ltd., Shanghai, China; Tablet 2, Xieli Pharmaceutical Co. Ltd., Hunan, China; Tablet 3, Deende Pharmaceutical Co. Ltd., Zhejiang, China; Tablet 4, YDYYHSFY Pharmaceutical Co. Ltd., Hubei, China; Tablet 5, Suzhou Yifan Pharmaceutical Co. Ltd., Anhui, China; Tablet 6, Hanfang Pharmaceutical Co. Ltd., Guizhou, China ). Specimens of Tripterygium wilfordii were collected from three different planting areas, including Xinchang (Zhejiang, China), Ji’an (Jiangxi, China) and Yuxi (Yunnan, China). 2.3 Preparation of crude extracts Each kind of tested tablet (1.5 g) and Tripterygium wilfordii (10.0 g) were ground into fine powder individually. Then each fine powder was extracted by ethyl acetate twice under ultrasonic with a volume ratio of 20 mL g-1 of dry weight of sample for 30 min. The ultrasonic power was set at 300 W and ultrasonic frequency was set at 40 KHz. After filtration, the combined ethyl acetate filtrates 8
were evaporated to dryness under vacuum to obtain the crude extracts. 2.4 Selection of biphasic solvent system for liquid-liquid chromatography The two-phase solvent system was selected according to the partition coefficient of wilforlide A. The biphasic solvent systems were prepared in advance and allowed to equilibrate in a separation funnel for 30 min. About 0.10 mg of reference substance was added in a 15 mL test tube with a stopper and it was dissolved with 3 mL of the lower phase and 3 mL of the upper phase of the biphasic solvent system. Then it was shaken vigorously for 5 min to ensure the sample was dissolved and partitioned between the two phases thoroughly. After settling, 1 mL of each phase was transferred to two test tubes and evaporated to dryness, respectively. The residues were dissolved with 1 mL of acetonitrile and it was analyzed by HPLC. The partition coefficient of wilforlide A was determined using the following equation: KD=A2/A1, A1 was the HPLC peak area of wilforlide A in the lower phase, and A2 represented the HPLC peak area of wilforlide A in the upper phase.
2.5 Preparation of biphasic solvent systems and sample solution for liquid-liquid chromatography
The selected two-phase solvent system consisting of n-hexane-ethyl acetate-ethanol-water (6:4:6:4, v/v) was placed into a separation funnel and it was thoroughly equilibrated by repeated shaking. After vigorous shaking, the solvent system was left to stand for 30 minutes and divided into upper phase and lower phase under room temperature 25 oC. The organic phase was used as the stationary phase while the aqueous phase was used as the mobile phase. The two phases were separated shortly before use.
Each crude extract obtained from section 2.3 was dissolved in 20 mL of mixture of the biphasic solvent system composed of equal volume of upper phase and lower phase. 9
2.6 Procedure for liquid-liquid chromatography The separation column was firstly filled with the upper phase. Then the revolution speed of the apparatus was adjusted to 800 rpm, and the lower phase was pumped into the column with a flow rate of 2.0 mL min-1. After hydrodynamic equilibrium was reached, as indicated by a distinct mobile phase eluting at the tail outlet, each sample solution was injected into the column using a manual sample injection loop. The effluent containing wilforide A was collected according the calculation of retention time and simulated peak width, and it was evaporated to dryness. The residue was dissolved by acetonitrile and transferred to 5 mL measuring flask, where it was diluted to the mark with acetonitrile. All the sample solutions were filtered through 0.22 μm membrane filters before HPLC analysis. 2.7 Validation of HPLC assay 2.7.1 HPLC conditions The analytes were separated on a phenomenex SYNERGI C18 column (250×4.6mm 4μm) at a temperature of 35 oC. The mobile phase consisted of water containing 0.1 % formic acid and acetonitrile (25:75, v/v), and was filtered through 0.22 μm membrane filters in a solvent filtration apparatus. The flow rate was kept at 1.0 mL min-1. The analytes volume injected was set at 20 μL. The detection wavelength was 210 nm. 2.7.2 Preparation of the calibration standards and calibration curves The standard substance of wilforlide A was accurately weighed and dissolved in ethanol to yield 0.10 mg mL-1 of stock solution. The stock solution was stored at 4 oC before use. A volume of 20μL of standard solution at seven different concentrations was respectively injected into HPLC system for analysis, and calibration curves were plotted based on linear regression analysis of the integrated peak areas (y) versus concentrations (x, mg mL -1) of the standard substance at seven 10
different concentrations. 2.7.3 Precision The analytical precision, expressed as relative standard deviation (RSD), was determined by intra-day and inter-day. For intra-day variability test, the standard solution of the reference substance wilforide A was analyzed for six times in one day, while for inter-day the standard solution of the wilforide A was examined twice a day on three consecutive days under the selected optimal conditions of HPLC system. 2.7.4 Stability The sample of Tablet 1 was stored at 4 oC for 24 h. According to the above chromatographic conditions, the stability of the sample solution was determined at 0, 2, 4, 6, 8, 12, 24 h, respectively. The RSD value of the peak area of different time injection was used as the parameter for stability evaluation. 2.7.5 Repeatability Five determinations were performed on Tablet 1 prepared in parallel according to Section 2.3-2.6. Repeatability was evaluated by the variation (RSD) within the five measurements. 2.7.6 Recovery The standard of wilforlide A was added into a certain amount of Tablet 1 sample. The mixture was extracted and enriched, and then injected directly into the HPLC system in accordance with the developed method. 3. Results and discussion 3.1 Methodology for sample collection To develop an efficient method for collecting the eluted fractions containing target components was very important for sample pretreatment using preparative liquid-liquid chromatography because it was difficult to detect the elution profile of the target component due to its extremely
11
limited content and the complex composition in the crude sample. As known, separation of components from mixture by liquid-liquid chromatography was based on distribution behavior of solutes in the two immiscible solvent systems. The solutes were eluted according to their partition coefficients in the two-phase solvent systems, which is usually defined as the solute concentration in the upper phase divided by the solute concentration in the lower phase. The adjusted retention time of solutes could be well defined by the following retention equation: t' R Vc[Sf ( KD 1) 1] F
(1)
Where t'R was the adjusted retention time of solutes, Vc was the column volume, F was the flow rate of mobile phase, Sf was the stationary phase retention and KD was the partition coefficient of the solutes. To get a complete collection of eluted fractions containing target components was still difficult although the retention time could be easily calculated because the elution profile of the target component was difficult to obtain in the preparative separation due to its very low content in the crude sample. In the same time, chemical components with similar structures would always be eluted together with the target component. It would be an efficient strategy to obtain a prediction of peak width of the eluted target component during the sample treatment by liquid-liquid chromatography. As known, it was difficult to predict peak width and peak resolution in chromatogram for conventional liquid chromatography in which calibration using empirical values based on experimental results was necessary. However, it was found that peak width of target for liquid-liquid chromatography solutes could be well predicted within high accuracy by a continuous stirred-tank reactors model when it was combined with calculation of theoretical plates of the separation column. The continuous stirred-tank reactors model was developed for modeling
12
countercurrent chromatography, in which the entire length of the column was taken as a series of fully equilibrated continuous stirred-tank reactors [26], which was a specific value for each column. It was found in this model that peak widths were primarily determined by the column length and the number of column loop, partition coefficient of the solute and the volume of continuous stirred-tank reactors. Therefore, peak width could be expected to be a constant value for separation of target component by countercurrent chromatography with a biphasic solvent system along with a specific apparatus. The number of continuous stirred-tank reactors for a specific separation column was determined by the following equation [26]: nCSTRs nLOOPS *180 COS1 1 4B
(2)
where nCSTRs indicated the number of continuous stirred-tank reactors in an entire separation column of countercurrent chromatography, nLoops was the number of column loops and β value was the ratio of planet radius (r) to the orbital radius (R). The number of loops for a specific separation column could be calculated by following equation [26]:
nLOOPs L 2r
（3）
where L was the length of the total column , and r was the average wound radius. The number of continuous stirred-tank reactors was determined by the number of column loops and the average β value. The β values and other column parameters for the present instrument was shown in Table 1. The number of continuous stirred-tank reactors was directly proportional to the number of instantaneous mixing zones in the separation column, which was quite familiar with the definition of the number of theoretical plates, and it was used for evaluating separation efficiency for a chromatographic column with the following equation:
N 16(t' R / Wb) 2
(4)
where N represented the number of theoretical plates and Wb was the peak with for a specific solute. It was found in our experiment that peak width Wb could be well predicted and calculated directly by the combination of equation (2) and (4) if N was replaced by nCSTRs. Furthermore, in a
13
typical Gauss peak, peak width was defined as Wb=4σ. Therefore, the collection time for eluted fractions containing the target component should be larger than 4σ, where 6σ could be selected. With the above calculated adjusted retention time of target analyte and peak width, collection time for eluted fractions containing target component could be well defined, even though the elution profile of target component could not be obtained during the separations. Furthermore, elution behavior and chromatogram of target analyte could be well simulated by the continuous stirred-tank reactors model using Matlab R2018a. The simulated chromatogram for separation of wilforlide A was shown in Fig. S2 (supporting information). Table 2 shows the calculated value of elution time using equation (1), simulated elution time using Matlab R2018a and the experimental value of elution time for wilforlide A The calculated collection time was determined by calculating peak width using equation (4), the simulated collection time was obtained using Matlab R2018a (supporting information) and the experimental value of elution time for wilforlide A was determined by HPLC analysis. All the retention times listed in Table 2 were the adjusted retention time, which does not include the extra retention time caused by the extra column volume composed of sample injection loop 20 mL and flying leads 4mL. The adjusted retention time for simulation 145.5min does not include extra retention time caused by the injection loop (5 min), and the adjusted retention time for experimental 146.5min does not include extra retention time caused by the injection loop (5 min) and flying leads (2 min). The results showed that the calculated and simulated values were very close to the experimental results. During the experiment, the actual collection time (tR) of the target component was calculated by following equation [28]:
tR t' R VCext F Vsa 2F
14
(5)
where t'R was the adjusted retention time of solutes, VCext was the extra column volume (flying leads), and Vsa was the volume of sample injection loop.
3.2 Sample treatment by liquid-liquid chromatography Preparative separation of various kinds of chemical components from partially purified sample of natural products by countercurrent chromatography have been well explored in the past decades, in which selection of biphasic solvent system is the key step for successful separation of target components. On one hand, an ideal partition coefficient for the target component is in the range of 0.5-2.0 [29]. Large partition coefficient generally leads to long retention time while limited peak resolution might be resulted from a very small partition coefficient for the target component. On the other hand, high solubility of crude sample would be favorable for preparative separation. Since the target component wilforlide A was a chemical component with moderate polarity, a typical biphasic solvent system composed of n-hexane-ethyl acetate-ethanol-water with moderate polarity was investigated. Table 3 shows the results of partition coefficient of wilforlide A in the tested biphasic solvent systems. The solvent system n-hexane-ethyl acetate-ethanol-water (6:4:6:4, v/v) was selected for the present work since a partition coefficient of 1.96 was obtained. Fig. 2 (a) and (b) were typical chromatograms for preparative enrichment of the target component from Tablet 1 and Tripterygium wilfordii (Jiangxi, China) by countercurrent chromatography using the above selected solvent system n-hexane-ethyl acetate-ethanol-water (6:4:6:4, v/v). Head-to-tail elution mode was employed, where the upper organic phase was used as the stationary phase while the lower aqueous phase was used as the mobile phase. Retention of stationary phase reached more than 58.3%. The shaded area in Fig.2 shows the collection time for the eluted fractions containing target component wilforlide A, in which the collection time was 15
equal to 6σ based on its adjusted retention time. HPLC analysis showed that complete collection of the eluted fractions containing the target component wilforlide A was achieved by the present collection methodology. As shown in Fig.2, the elution profile of the target component was difficult to obtain due to its extremely limited content in the crude sample. Fig. 3 showed two typical chromatograms for analysis of the combined fractions from countercurrent chromatography by high performance liquid chromatography. It was impossible to give a quantitative determination of wilforlide A in the crude sample extracted from Tablet and Tripterygium wilfordii because the target component could not be detected due to its limited content. However, a high peak resolution of wilforlide A could be obtained after its sample pretreatment by countercurrent chromatography.
3.3 Quantitative determination by HPLC In the present study, different solvents were investigated for extraction of wilforlide A, including water, ethanol, methanol, dichloromethane, chloroform, ethyl acetate and their combinations with changing volume ratio. It was found that ethyl acetate showed complete recovery of wilforlide A from glycoside tablets and Tripterygium wilfordii. Analysis of crude sample and the combined fractions containing wilforlide A by high performance liquid chromatography was optimized in the present work. In order to obtain a fast and specific detection of wilforlide A in the sample, the crude sample of Tablet 1 was used for optimization of analysis conditions. Results showed that acetonitrile-0.1 % formic acid (75:25, v/v) gave the optimal separation of wilforlide A in the sample. 3.3.1 Validation of HPLC assay Specificity and sensitivity: Wilforlide A was well separated with other components and it was 16
quantified under the optimized HPLC chromatographic condition and eluted at 19.8 min (Fig.S1) The calibration curve was established by plotting the concentration of reference substance against the corresponding peak area. The regression equation, linearity range (mg mL-1), correlation coefficient (r2), limit of detection (LOD) and limit of quantification (LOQ) of wilforlide A were shown in Table 4. The calibration curves showed good liner regression (r 2=0.9999, n=7) within the test ranges. The LOD and LOQ for wilforlide A were determined to be 1.35 ng mL -1 and 4.50 ng mL-1 based on signal-to-noise ratios (S/N) of 3 and 10, respectively. Precision, stability, repeatability and accuracy: Intra-day and inter-day variations were chosen to determine the precision of the developed method. The analytical precision from the data of the intra-day and inter-day determinations was indicated by the RSD values, and they were determined to be 0.67 % and 1.14 %, respectively (Table S1). As shown in Table S2, wilforlide A was found to be stable at 4oC with a deviation of less than 1.5 % for the stock solution. The repeatability of the method was shown in Table S3, where RSD for repeatability was found to be 1.19%. Table S4 indicated that a high recovery (94.52 % to 98.27 %) for wilforlide A could be obtained (RSD =1.58 %). 3.3.2 Content of wilforlide A in Tablets and Tripterygium wilfordii Table 5 shows the determined content of wilforlide A in six different Chinese patent medicines glycoside tablets and three different Tripterygium wilfordii. It was reported in the Chinese drug standard that the content of wilforlide A in the Chinese patent medicine glycoside tablets should not be less than 0.10 mg g-1 [13]. As shown in Table 4, the content of wilforlide A in each tested Tablet met the standard. However, it was found that the content of wilforlide A in Tripterygium wilfordii was much lower than that in the Chinese patent medicine, which might be the main
17
reason for that wilforlide A could not be accurately determined by UHPLC-QTOF-MS [12] and by HPLC-ELSD [14], in which no sample pretreatment was used. Compared with the tradition methods for sample pretreatment, the major advantage for the present method was the complete recovery of analytes from the crude sample and the trace component in Tripterygium wilfordii could be successfully determined. The target trace component in the crude sample could be selectively enriched by liquid-liquid chromatography using a selected biphasic solvent system. Although it was difficult to obtain the elution profile of interested analytes, it could be well predicted and simulated by a continuous stirred tank reactors model.
4 Conclusions In the present work, an approach for sample pretreatment by liquid-liquid chromatography was developed for quantitative determination of trace chemical components in traditional Chinese medicinal herb and its Chinese patent medicine. The major advantage of the present methodology was that a complete recovery could be obtained during the sample treatment since liquid-liquid chromatography was a partition chromatography with no solid support for the stationary phase. The collection time of eluted fractions containing target components could be well predicted by calculation of peak width and simulation of peak chromatogram for the target component using a continuous-stirred tank reactors model. With this method, successful quantitative determination of the trace component, wilforlide A, in its traditional Chinese medicinal herb and its Chinese patent medicine by conventional high performance liquid chromatography with UV detector could be achieved with high recovery, repeatability and accuracy. This method could be expected to be used for quantitative determination of components with extremely low content in a complex sample. 18
Acknowledgement This work was financially supported by national natural science foundation of China (21978266).
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Credit Author Statement Shanshan Zhao: Conceptualization, Methodology, Software, Data curation, Writing- Original draft preparation. Chaoyue Wang: Help during the experiment Xiang Wang: Writing- Reviewing Yang Jin: Writing- Reviewing Wenyu Sun: Writing- Reviewing Xingchu Gong: Software, Validation Shengqiang Tong: Guidance during the experiment, Writing- Reviewing and Editing.
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https://doi.org/10.1016/j.ultsonch.2018.08.010. [8] A. Frey, E. Hopmann, M. Minceva, Selection of biphasic liquid systems in liquid-liquid chromatography using predictive thermodynamic models, Chem. Eng. Technol. 37 (2014) 1663 1674. https://doi.org/10.1016/j.ultsonch.2018.08.010. [9] R. Morley, M. Minceva, Operating mode and parameter selection in liquid–liquid chromatography, J. Chromatogr. A. 2019. https://doi.org/10.1016/j.chroma.2019.460479. [10] K. Skalicka-Wozniak，I. Garrard, A comprehensive classification of solvent systems used for natural product purifications in countercurrent and centrifugal partition chromatography, Nat. Prod. Rep. 32 (2015) 1556–1561. https://DOI: 10.1039/c5np00061k. [11] C. Fan, M. Liu, W. Wang, X. Cao, Determination of alternaria mycotoxins in wine and juice using ionic liquid modified countercurrent chromatography as a pretreatment method followed by high-performance liquid chromatography, J. Chromatogr. A. 1436 (2016) 133-140. http://dx.doi.org/doi:10.1016/j.chroma.2016.01.069. [12] R. Zhuo, L. Zhang, L Wang, G. Shan, Q. Yang, X. Yuan, H. Xiao, Rapid simultaneous quantitative determination of terpenoids in Tripterygium wilfordii Hook F by ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, Anal. Methods. 5 (2013) 2046 - 2052. https://DOI: 10.1039/c3ay26501c. 20
[13] Pharmacopoeia Committee of the Ministry of Health of the people's Republic of China, Traditional Chinese medicine prescription preparation，Drug Standard of the Ministry of Health of the people's Republic of China, People's Health Publishing House., Beijing, 2015, pp.Z17 - 275. [14] X. Luo, Q. Shao, H. Qu, Y. Cheng, Simple method for determination of five terpenoids from different parts of Tripterygium wilfordii and its preparations by HPLC coupled with evaporative light scattering detection, J. Sep. Sci. 30 (2007) 1284 - 1291. https://DOI 10.1002/jssc.200600450. [15] H. Que, S. Deng, S. Lin, Improvement of the method for determination of triptolide a in February, Chin. Med. Mat. 28 (2005) 144 -145. [16] R. Zhang, Y. He, S. Shi, J. Qian, Y. Fan, Determination of six effective components, total diterpenoids, total alkaloids and total triterpenes in Tripterygium wilfordii Hook. f., China J. Tradit. Chin. Med. Pharm. 28 (2013) 224 - 229. [17] H. Zhang, Y. Jiang, M. Meng, L. Tang, F. Wan, J. Zhang, Content determination of wilforlide in tripterygium wilfordii from different producing areas by HPLC, China. Pharmacy. 25 (2014) 2916 - 2918. [18] L. Ni, J. Dai, F. Mu, L. Yu, J. Lu, Determination of wilforlide A in tripterygium wilfordii Hook. f. and Its Preparations by NP-HPLC, Drug Stand. China. 8 ( 2007) 32 - 34. [19] X. Ou Yang, M. Jin, C. He, Simultaneous Determination of triptolide and tripdiolide in extract of tripterygium wilfordii Hook. f. by LC–APCI-MS, Chromatographia. 65 (2007) 373 375. https://DOI: 10.1365/s10337-006-0156-80009-5893/07/03. [20] M. Jin, X. Chen, X. Ou Yang, Simultaneous determination of triptolide, tripdiolide and tripterine in human urine by high-performance liquid chromatography coupled with ion trap atmospheric-pressure chemical ionization mass spectrometry, Biomed. Chromatogr. 23 (2009) 289 - 294. https://DOI 10.1002/bmc.1112. [21] J. Yao, L. Zhang, X. Zhao, L. Hu, Z. Jiang, Simultaneous determination of triptolide, wilforlide A and triptonide in human plasma by high-performance liquid chromatography-electrospray ionization mass Spectrometr, Biol. Pharm. Bull. 29 (2006) 1483 1486.
21
[22] W. Liu , S. Zhang, Y. Zu, Y. Fu , W. Ma, D. Zhang, Y. Kong, X. Li, Preliminary enrichment and separation of genistein and apigenin from extracts of pigeon pea roots by macroporous resins, Bioresour. Technol. 101 (2010) 4667 - 4675. https://doi.org/10.1016/j.biortech.2010.01.058. [23] C. Jiang, X. Gong, H. Qu, Multivariate modeling and prediction of breakthrough curves for herbal medicine adsorption on column chromatography: A case study, Sep. Sci. Technol. 50 (2015) 1030 - 1037. https://doi.org/10.1080/01496395.2014.978458. [24] X. Du, Q. Yuan, Y. Li, Equilibrium, thermodynamics and breakthrough studies for adsorption of solanesol onto macroporous resins, Chem. Eng. Process. 47 (2008) 1420 - 1427. https://doi.org/10.1016/j.cep.2007.10.005. [25] K. Miyabe, Moment analysis of chromatographic behavior in reversed-phase liquid chromatography, J. Sep. Sci. 32 (2009) 757 - 770. https://doi.org/10.1002/jssc.200800607. [26] H. Guzlek, I. I.R. Baptista, P. L. Wood, Andrew Livingston. A novel approach to modelling counter-current chromatography, J. Chromatogr. A. 1217 (2010) 6230 - 6240. https://doi:10.1016/j.chroma.2010.08.011. [27] F. Wang, Y. Ito, Y. Wei, Recent progress on countercurrent chromatography modeling, J. Liq. Chromatogr. Relat. Technol. 38 (2015) 415 - 421. https://doi.org/10.1080/10826076.2014.941268 [28] J. Cazes, Encyclopedia of Chromatography, Third Edition, in: P. Wood, Stationary phase retention versus peak elution in CCC, E-Publishing Inc., New York, 2009, pp. 2231–2239. [29] A. Marston , K. Hostettmann, Developments in the application of counter-current chromatography to plant analysis, J. Chromatogr. A. 1112 (2006) 181–194. https://doi.org/10.1016/j.chroma.2005.10.018.
Figure captions:
22
Fig. 1 chemical structure of wilforlide A
.
Fig. 2 Chromatogram for preparative enrichment of wilforlide A from the crude sample by countercurrent chromatography. (a) 1.5 g of sample from Tablet 1; (b) 10.0 g of sample from Tripterygium
wilfordii.
Separation
conditions:
solvent
system:
n-hexane-ethyl
acetate-ethanol-water (6:4:6:4, v/v); stationary phase: the upper phase; mobile phase: the lower phase; flow rate: 2 mL min-1; column temperature: 25 oC; detection wavelength: 254nm; retention of stationary phase: 58.3%. The shaded area indicated the collection time for the eluted fractions containing wilforlide A.
23
Fig. 3 Chromatogram for analysis of the combined fractions from countercurrent chromatography by high performance liquid chromatography. (a ): The combined fractions containing wilforlide A from Fig. 2(a); (b): it’s the combined fractions containing wilforlide A from Fig. 2(b). Analytical conditions: column: phenomenex SYNERGI C18 column (250×4.6mm, 4µm); mobile phase: 0.1 % formic acid and acetonitrile (25:75, v/v), flow rate: 1.0 mL min -1; sample injection: 20 μL; detection wavelength: 210 nm; column temperature: 35oC.
24
Table 1 Various parameters of the instrument Column
Column
Column
Number
Extra
Sample
average
β value
Estimated
bore
length
volume
of
column
injection
wound
range
number
(mm)
(m)
(mL)
loops
volume
volume
radius
of
(mL)
(mL)
(cm)
CSTRs
4
20
0.33
2.0
60.5
190
288
25
0.36-0.69
836
Table 2 Retention time and collection time for wilforlide A Adjusted retention
Errors (%)
timea) (min)
Collection time
Errors (%)
(min)
Calculated
148.2
1.16
31.9
3.33
Simulation
145.5
0.68
35.5
7.5
Experimentalb)
146.5
-
33.0
-
a) Adjusted retention time does not include the extra retention time (7 min) caused by the extra column volume. b) Adjusted retention time and collection time denote average adjusted retention time and average collection time in experimental procedure
26
Table 3 Partition coefficient (KD) of wilforlide A in the tested biphasic solvent systems Solvent systems
Partition coefficient KD
n-hexane-ethanol-water= (6:5:1, v/v)
0.22
n-hexane-ethyl acetate-ethanol-water= (5:5:5:5, v/v)
7.63
n-hexane-ethyl acetate-ethanol-water =(9:1:9:1, v/v)
0.74
n-hexane-ethyl acetate-ethanol-water =(8:2:8:2, v/v)
0.50
n-hexane-ethyl acetate-ethanol-water =(7:3:7:3, v/v)
0.66
n-hexane-ethyl acetate-ethanol-water =(6:4:6:4, v/v)
1.96
n-hexane-ethyl acetate-ethanol-water =(13:7:13:7, v/v)
1.03
27
Table 4
Regression equations and correlation coefficient
Regression equation a)
Linearity range (mg ml-1)
R2 b)
LOD (ng mL-1)
LOQ (ng mL-1)
y =685.06x + 18.648
0.01- 0.10
0.9999
1.35
4.50
a) x denotes the concentration and y denotes the peak area. b) R2 is the correlation coefficient.
28
Table 5 Content of wilforlide A in the tested Tablets and Tripterygium wilfordii Samples
Contents (mg g-1)
Samples source
Tablet 1
0.5044
Fudan Fuhua Pharmaceutical Co. Ltd., Shanghai, China
Tablet 2
0.2105
Xieli Pharmaceutical Co. Ltd., Hunan, China
Tablet 3
0.2460
Deende Pharmaceutical Co. Ltd., Zhejiang, China
Tablet 4
0.2288
HSFY Pharmaceutical Co. Ltd., Hubei, China
Tablet 5
0.2138
Suzhou Yifan Pharmaceutical Co. Ltd., Anhui, China
Tablet 6
0.2881
Hanfang Pharmaceutical Co. Ltd., Guizhou, China
Root 1
Not detected
Xinchang, Zhejiang, China
Root 2
0.0027
Jian, Jiangxi, China
Root 3
0.0012
Yuxi, Yunnan, China
29
Liquid-liquid chromatography in sample pretreatment for quantitative analysis of trace component in traditional Chinese medicines by conventional liquid chromatography Shanshan Zhao , Chaoyue Wang , Xiang Wang , Yang Jin , Wenyu Sun , Xingchu Gong , Shengqiang Tong PII: DOI: Reference:
S0021-9673(20)30094-7 https://doi.org/10.1016/j.chroma.2020.460917 CHROMA 460917
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
28 November 2019 19 January 2020 22 January 2020
Please cite this article as: Shanshan Zhao , Chaoyue Wang , Xiang Wang , Yang Jin , Wenyu Sun , Xingchu Gong , Shengqiang Tong , Liquid-liquid chromatography in sample pretreatment for quantitative analysis of trace component in traditional Chinese medicines by conventional liquid chromatography, Journal of Chromatography A (2020), doi: https://doi.org/10.1016/j.chroma.2020.460917
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Highlights
Liquid-liquid chromatography was studied in sample pretreatment for quantification.
Continuous-stirred tank reactors model was used to predict collection time of analytes.
Determination of wilforlide A in Tripterygium wilfordii and tablets was established.
A new method for sample pretreatment in analysis of trace component was developed.
1
Liquid-liquid chromatography in sample pretreatment for quantitative analysis of trace component in traditional Chinese medicines by conventional liquid chromatography Shanshan Zhao1, Chaoyue Wang1, Xiang Wang1, Yang Jin1, Wenyu Sun1, Xingchu Gong2, Shengqiang Tong1,* 1
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310032,
China 2
Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, 310023, China
The telephone/fax number, email and postal address of the corresponding author: Prof. Shengqiang Tong Tel.: +86 571 88320984; fax: +86 571 88320984 E-mail: [email protected] Postal address: College of Pharmaceutical Science, Zhejiang University of Technology, Chaowang Road 18, Chaohui No. 6 District, Hangzhou, CHINA
Abstract A method for sample pretreatment using liquid-liquid chromatography combined with a conventional liquid chromatography was developed for quantitative determination of trace chemical components in traditional Chinese medicine. The main effective component, wilforlide A, in the traditional Chinese medicinal herb Tripterygium wilfordii as well as in its Chinese patent medicine glycosides tablets was successfully determined after sample pretreatment by liquid-liquid chromatography. A biphasic solvent system n-hexane-ethyl acetate-ethanol-water (6:4:6:4, v/v) was screened for crude sample treatment by liquid-liquid chromatography. The collection time of eluted fractions containing target components could be well predicted using a continuous-stirred tank reactors model after determination of its retention time. Then, quantitative 2
analysis of wilforlide A in Tripterygium wilfordii as well as in its tablets could be successfully determined by conventional reversed-phase high performance liquid chromatography with UV detector. Under the optimized conditions, the method showed good linearity (R 2 =0.9999) for wilforlide A in the range of 0.01 mg mL-1 -0.10 mg mL-1. The limit of detection and limit of quantity were 1.35 ng mL-1 and 4.50 ng mL-1, respectively. The average recovery rate, intra-day and inter-day precisions of wilforlide A were 96.43 %, 0.67 % and 1.14 %, respectively. Compared with previous studies, the present method showed advantages of complete recovery of target component in the sample pretreatment and good repeatability. Keywords: Liquid-liquid chromatography; Liquid chromatography; Sample pretreatment; Quantitative determination; Wilforlide A; Triperygium wilfordii Hook.f
1. Introduction In order to extract, separate and enrich target analytes from complex samples and to meet the requirements of chromatography and mass spectrometry, various sample pretreatment techniques have been developed. Traditional methods of sample preparation, such as liquid-liquid extraction and solid phase extraction, are time-consuming, tedious and high consumption of organic solvents [1]. In recent years, in order to overcome the deficiency of liquid-liquid extraction and solid phase extraction, the development of sample pretreatment technology is mainly focused on automation, simplification and miniaturization [2]. Some microextraction technologies such as single-drop microextraction, solid phase microextraction, dispersion liquid-liquid microextraction, magnetic solid phase extraction, headspace liquid phase microextraction, thin film microextraction and stir bar sorptive extraction have emerged successively [3]. They could be fully automated and connected to liquid chromatography, gas chromatography or capillary electrochromatography.
3
Compared with traditional methods, these new developed extraction strategies have made the whole sample pretreatment process simpler, faster, and more economical, efficient and environmental-friendly [4]. However, it is still a very challenging task for quantitative determination of chemical components with very low content in an extremely complex sample due to the difficulty of sensitivity, selectivity and limited recovery of solutes in the crude sample pretreatment. For instance, it is difficult to obtain complete recovery of analytes during the sample pretreatment by solid phase extraction, in which irreversible adsorption of solutes to solid matrix would be always involved [5]. For liquid-liquid extraction, it occurs in a single solution (one-step method) and it is difficult to set two or more extraction stages due to the increase in extraction volume and time [6]. So a complete recovery of analytes in the crude sample would be difficult to achieve. For dispersive liquid-liquid microextraction, although a high enrichment factor could be achieved in the sample pretreatment, it had the drawback of low recovery rate and poor selectivity, which greatly limited its application [7]. Liquid-liquid chromatography, generally known as centrifugal partition chromatography and countercurrent chromatography, is a support-free liquid-liquid partition chromatography, which relies on the partition of a solute between two immiscible solvents to achieve separation [8]. Liquid-liquid chromatography can be considered as an apparatus for multi-step liquid-liquid extractive process. Compared with the traditional solid phase chromatography, the support-free liquid stationary phase in liquid-liquid chromatography allows for complete recovery for the analytes. Since liquid-liquid chromatography are usually performed at ambient temperature and the sample would not be subjected to any physical sorption processes, the original physicochemical properties of the sample constituents are largely preserved [9]. To the best of our
4
knowledge, at present liquid-liquid chromatography has been mainly used for preparative separation and purification of chemical components from fermentation broth, synthetic mixtures and natural products [10]. Few report on sample pretreatment by liquid-liquid chromatography has been available in quantitative determination of trace chemical components in natural products [11]. Tripterygium wilfordii Hook. F, a Chinese herbal medicine (‘Lei gong teng’ in Chinese), has a long history of clinical use in China. Its whole root and the root xylem without bark are mainly used for medicinal purpose. Tripterygium wilfordii glycosides tablets (‘lei gong teng duo gan pian’ in chinese), a famous Chinese patent medicine, which has been used to treat rheumatoid arthriits, lupus erythematosus, and Henoch-Schonlein purpura nephriits and skin diseases for many years, constituting a large fraction of drugs sales for rheumatic disease in China [12]. Wilforlide A is one of the main bioactive chemical components in the tablets, and its content is regarded as the main chemical marker for quality control of this drug [13]. However, it was found that the content of wilforlide A in Tripterygium wilfordii glycosides tablets was extremely low and chemical composition of the tablets was very complicated. Therefore, it was a very challenging task to establish a simple and efficient method for quantitative determination of wilforlide A in the tablets. And it would be more difficult to determine wilforlide A in Tripterygium wilfordii. Two simple methods for simultaneous quantitative determination of five terpenoids in Tripterygium wilfordii were developed by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) [12] and by highperformance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD)[14], in which no specific sample pretreatment was employed. However, it was noticed that the content of
5
wilforlide A in root of Tripterygium wilfordii could not be accurately determined due to its limited content [12]. Meanwhile, analysis time was very long and peak resolution of wilforlide A was rather limited in the literature due to complex composition of the crude sample [14], resulting in low accuracy for quantitative determination of wilforlide A in Tripterygium wilfordii. Furthermore, quadrupole time-of-flight mass spectrometry and evaporative light scattering detection were not frequently-used detectors for conventional liquid chromatography. Therefore, sample pretreatment was necessary for determination of this trace effective component by conventional liquid chromatography. Several traditional methods for sample pretreatment were reported, including neutral alumina column chromatography [15-17] and silica gel column chromatography [18]. And solid phase extraction was necessary for sample pretreatment if HPLC-MS was used to determine content of wilforlide A in crude extracts of this medicinal plant[19], human urine[20] and human plasma[21]. However, no complete recovery of solutes could be obtained since irreversible adsorption of sample to the stationary phase was involved in the above methods. Meanwhile, it was difficult to accurately collect the eluted fractions containing the target component due to its limited content and its extremely complex composition when tradition column chromatography was used for sample pretreatment, which was also a great challenge for column chromatography in sample pretreatment due to the need for long analysis time and multiple-step analysis [22]. Various mathematical models have been used to simulate the adsorption process of substances, for predicting the breakthrough curve and collection time [23,24]. However, the elution curve of components with very limited content could not be effectively predicted due to many influencing factors [25]. For liquid-liquid chromatography, the elution behavior of solute was determined by the two-phase solvent system, mobile phase flow
6
rate, rotation speed of the column [26]. Several theoretical models have been reported for predicting the elution behavior of target solute, including elution countercurrent distribution model, compartment model, probability model and continuous stirred tank reactor model [27]. The continuous stirred tank reactor model could be used to predict the chromatogram of the target components by relating all model parameters directly to experimental settings and column dimensions. Most importantly, this model can forecast the chromatogram of target compound without using empirical values to calibrate [26]. In our present study, a new method for sample pretreatment by liquid-liquid chromatography using the continuous stirred tank reactor model was developed for quantitative determination of the trace component wilforlide A in Tripterygium wilfordii and in its Chinese patent medicine glycosides tablets. 2. Experimental section 2.1 Instruments Sample pretreatment using liquid-liquid chromatography was achieved by a model of TBE-200V high speed countercurrent chromatographic system (Tauto Biotech Co. Ltd., Shanghai, China). The separation column was 2.0 mm ID polytetrafluoroethylene tubing with a total capacity of 190 mL. The rotation speed of separation column could be adjusted in a range from 0 to 1000 rpm, where 800 rpm was used. A manual six-way injection valve with a 20 mL loop was used to introduce the crude sample into the column. The column temperature was controlled by SDC-6 constant-temperature controller (Nanjing Xinchen Biotechnology Co., Ltd., Nanjing, China). The solvent was pumped into the column by a model MPO 106 constant-flow pump (Shanghai Sanwei Science and Technology Co., Ltd., shanghai,China). The other instruments included a model HD-21C-B detector (Shanghai Kanghua biochemical instrument Manufacturing Factory, Shanghai,
7
China) operating at 254 nm and a SEPU3000 workstation (Hangzhou Puhui Technology, Hangzhou, China). Analysis and determination of the analytes were performed on a LabSolutions MS-20160509 system (Shimadzu, Japan) equipped with a Shimadzu SCL-10ASvp controller. The solvent was pumped by Shimadzu LC-20ATvp Multisolvent Delivery System. The detection signal was collected by a LabSolutions MS-20160509 workstation. 2.2 Reagents and materials Acetonitrile and methanol for HPLC analysis, formic acid and analytical grade chemicals were purchased from Huipu chemical instrument Co. Ltd., Hangzhou, China. The reference substance of wilforlide A was purchased from Chengdu Must Bio-Tech Co.Ltd., Chengdu, China. Chinese patent medicines of Tripterygium wilfordii glycosides tablets were purchased from six different pharmaceutical companies (Tablet 1, Fudan Fuhua Pharmaceutical Co. Ltd., Shanghai, China; Tablet 2, Xieli Pharmaceutical Co. Ltd., Hunan, China; Tablet 3, Deende Pharmaceutical Co. Ltd., Zhejiang, China; Tablet 4, YDYYHSFY Pharmaceutical Co. Ltd., Hubei, China; Tablet 5, Suzhou Yifan Pharmaceutical Co. Ltd., Anhui, China; Tablet 6, Hanfang Pharmaceutical Co. Ltd., Guizhou, China ). Specimens of Tripterygium wilfordii were collected from three different planting areas, including Xinchang (Zhejiang, China), Ji’an (Jiangxi, China) and Yuxi (Yunnan, China). 2.3 Preparation of crude extracts Each kind of tested tablet (1.5 g) and Tripterygium wilfordii (10.0 g) were ground into fine powder individually. Then each fine powder was extracted by ethyl acetate twice under ultrasonic with a volume ratio of 20 mL g-1 of dry weight of sample for 30 min. The ultrasonic power was set at 300 W and ultrasonic frequency was set at 40 KHz. After filtration, the combined ethyl acetate filtrates 8
were evaporated to dryness under vacuum to obtain the crude extracts. 2.4 Selection of biphasic solvent system for liquid-liquid chromatography The two-phase solvent system was selected according to the partition coefficient of wilforlide A. The biphasic solvent systems were prepared in advance and allowed to equilibrate in a separation funnel for 30 min. About 0.10 mg of reference substance was added in a 15 mL test tube with a stopper and it was dissolved with 3 mL of the lower phase and 3 mL of the upper phase of the biphasic solvent system. Then it was shaken vigorously for 5 min to ensure the sample was dissolved and partitioned between the two phases thoroughly. After settling, 1 mL of each phase was transferred to two test tubes and evaporated to dryness, respectively. The residues were dissolved with 1 mL of acetonitrile and it was analyzed by HPLC. The partition coefficient of wilforlide A was determined using the following equation: KD=A2/A1, A1 was the HPLC peak area of wilforlide A in the lower phase, and A2 represented the HPLC peak area of wilforlide A in the upper phase.
2.5 Preparation of biphasic solvent systems and sample solution for liquid-liquid chromatography
The selected two-phase solvent system consisting of n-hexane-ethyl acetate-ethanol-water (6:4:6:4, v/v) was placed into a separation funnel and it was thoroughly equilibrated by repeated shaking. After vigorous shaking, the solvent system was left to stand for 30 minutes and divided into upper phase and lower phase under room temperature 25 oC. The organic phase was used as the stationary phase while the aqueous phase was used as the mobile phase. The two phases were separated shortly before use.
Each crude extract obtained from section 2.3 was dissolved in 20 mL of mixture of the biphasic solvent system composed of equal volume of upper phase and lower phase. 9
2.6 Procedure for liquid-liquid chromatography The separation column was firstly filled with the upper phase. Then the revolution speed of the apparatus was adjusted to 800 rpm, and the lower phase was pumped into the column with a flow rate of 2.0 mL min-1. After hydrodynamic equilibrium was reached, as indicated by a distinct mobile phase eluting at the tail outlet, each sample solution was injected into the column using a manual sample injection loop. The effluent containing wilforide A was collected according the calculation of retention time and simulated peak width, and it was evaporated to dryness. The residue was dissolved by acetonitrile and transferred to 5 mL measuring flask, where it was diluted to the mark with acetonitrile. All the sample solutions were filtered through 0.22 μm membrane filters before HPLC analysis. 2.7 Validation of HPLC assay 2.7.1 HPLC conditions The analytes were separated on a phenomenex SYNERGI C18 column (250×4.6mm 4μm) at a temperature of 35 oC. The mobile phase consisted of water containing 0.1 % formic acid and acetonitrile (25:75, v/v), and was filtered through 0.22 μm membrane filters in a solvent filtration apparatus. The flow rate was kept at 1.0 mL min-1. The analytes volume injected was set at 20 μL. The detection wavelength was 210 nm. 2.7.2 Preparation of the calibration standards and calibration curves The standard substance of wilforlide A was accurately weighed and dissolved in ethanol to yield 0.10 mg mL-1 of stock solution. The stock solution was stored at 4 oC before use. A volume of 20μL of standard solution at seven different concentrations was respectively injected into HPLC system for analysis, and calibration curves were plotted based on linear regression analysis of the integrated peak areas (y) versus concentrations (x, mg mL -1) of the standard substance at seven 10
different concentrations. 2.7.3 Precision The analytical precision, expressed as relative standard deviation (RSD), was determined by intra-day and inter-day. For intra-day variability test, the standard solution of the reference substance wilforide A was analyzed for six times in one day, while for inter-day the standard solution of the wilforide A was examined twice a day on three consecutive days under the selected optimal conditions of HPLC system. 2.7.4 Stability The sample of Tablet 1 was stored at 4 oC for 24 h. According to the above chromatographic conditions, the stability of the sample solution was determined at 0, 2, 4, 6, 8, 12, 24 h, respectively. The RSD value of the peak area of different time injection was used as the parameter for stability evaluation. 2.7.5 Repeatability Five determinations were performed on Tablet 1 prepared in parallel according to Section 2.3-2.6. Repeatability was evaluated by the variation (RSD) within the five measurements. 2.7.6 Recovery The standard of wilforlide A was added into a certain amount of Tablet 1 sample. The mixture was extracted and enriched, and then injected directly into the HPLC system in accordance with the developed method. 3. Results and discussion 3.1 Methodology for sample collection To develop an efficient method for collecting the eluted fractions containing target components was very important for sample pretreatment using preparative liquid-liquid chromatography because it was difficult to detect the elution profile of the target component due to its extremely
11
limited content and the complex composition in the crude sample. As known, separation of components from mixture by liquid-liquid chromatography was based on distribution behavior of solutes in the two immiscible solvent systems. The solutes were eluted according to their partition coefficients in the two-phase solvent systems, which is usually defined as the solute concentration in the upper phase divided by the solute concentration in the lower phase. The adjusted retention time of solutes could be well defined by the following retention equation: t' R Vc[Sf ( KD 1) 1] F
(1)
Where t'R was the adjusted retention time of solutes, Vc was the column volume, F was the flow rate of mobile phase, Sf was the stationary phase retention and KD was the partition coefficient of the solutes. To get a complete collection of eluted fractions containing target components was still difficult although the retention time could be easily calculated because the elution profile of the target component was difficult to obtain in the preparative separation due to its very low content in the crude sample. In the same time, chemical components with similar structures would always be eluted together with the target component. It would be an efficient strategy to obtain a prediction of peak width of the eluted target component during the sample treatment by liquid-liquid chromatography. As known, it was difficult to predict peak width and peak resolution in chromatogram for conventional liquid chromatography in which calibration using empirical values based on experimental results was necessary. However, it was found that peak width of target for liquid-liquid chromatography solutes could be well predicted within high accuracy by a continuous stirred-tank reactors model when it was combined with calculation of theoretical plates of the separation column. The continuous stirred-tank reactors model was developed for modeling
12
countercurrent chromatography, in which the entire length of the column was taken as a series of fully equilibrated continuous stirred-tank reactors [26], which was a specific value for each column. It was found in this model that peak widths were primarily determined by the column length and the number of column loop, partition coefficient of the solute and the volume of continuous stirred-tank reactors. Therefore, peak width could be expected to be a constant value for separation of target component by countercurrent chromatography with a biphasic solvent system along with a specific apparatus. The number of continuous stirred-tank reactors for a specific separation column was determined by the following equation [26]: nCSTRs nLOOPS *180 COS1 1 4B
(2)
where nCSTRs indicated the number of continuous stirred-tank reactors in an entire separation column of countercurrent chromatography, nLoops was the number of column loops and β value was the ratio of planet radius (r) to the orbital radius (R). The number of loops for a specific separation column could be calculated by following equation [26]:
nLOOPs L 2r
（3）
where L was the length of the total column , and r was the average wound radius. The number of continuous stirred-tank reactors was determined by the number of column loops and the average β value. The β values and other column parameters for the present instrument was shown in Table 1. The number of continuous stirred-tank reactors was directly proportional to the number of instantaneous mixing zones in the separation column, which was quite familiar with the definition of the number of theoretical plates, and it was used for evaluating separation efficiency for a chromatographic column with the following equation:
N 16(t' R / Wb) 2
(4)
where N represented the number of theoretical plates and Wb was the peak with for a specific solute. It was found in our experiment that peak width Wb could be well predicted and calculated directly by the combination of equation (2) and (4) if N was replaced by nCSTRs. Furthermore, in a
13
typical Gauss peak, peak width was defined as Wb=4σ. Therefore, the collection time for eluted fractions containing the target component should be larger than 4σ, where 6σ could be selected. With the above calculated adjusted retention time of target analyte and peak width, collection time for eluted fractions containing target component could be well defined, even though the elution profile of target component could not be obtained during the separations. Furthermore, elution behavior and chromatogram of target analyte could be well simulated by the continuous stirred-tank reactors model using Matlab R2018a. The simulated chromatogram for separation of wilforlide A was shown in Fig. S2 (supporting information). Table 2 shows the calculated value of elution time using equation (1), simulated elution time using Matlab R2018a and the experimental value of elution time for wilforlide A The calculated collection time was determined by calculating peak width using equation (4), the simulated collection time was obtained using Matlab R2018a (supporting information) and the experimental value of elution time for wilforlide A was determined by HPLC analysis. All the retention times listed in Table 2 were the adjusted retention time, which does not include the extra retention time caused by the extra column volume composed of sample injection loop 20 mL and flying leads 4mL. The adjusted retention time for simulation 145.5min does not include extra retention time caused by the injection loop (5 min), and the adjusted retention time for experimental 146.5min does not include extra retention time caused by the injection loop (5 min) and flying leads (2 min). The results showed that the calculated and simulated values were very close to the experimental results. During the experiment, the actual collection time (tR) of the target component was calculated by following equation [28]:
tR t' R VCext F Vsa 2F
14
(5)
where t'R was the adjusted retention time of solutes, VCext was the extra column volume (flying leads), and Vsa was the volume of sample injection loop.
3.2 Sample treatment by liquid-liquid chromatography Preparative separation of various kinds of chemical components from partially purified sample of natural products by countercurrent chromatography have been well explored in the past decades, in which selection of biphasic solvent system is the key step for successful separation of target components. On one hand, an ideal partition coefficient for the target component is in the range of 0.5-2.0 [29]. Large partition coefficient generally leads to long retention time while limited peak resolution might be resulted from a very small partition coefficient for the target component. On the other hand, high solubility of crude sample would be favorable for preparative separation. Since the target component wilforlide A was a chemical component with moderate polarity, a typical biphasic solvent system composed of n-hexane-ethyl acetate-ethanol-water with moderate polarity was investigated. Table 3 shows the results of partition coefficient of wilforlide A in the tested biphasic solvent systems. The solvent system n-hexane-ethyl acetate-ethanol-water (6:4:6:4, v/v) was selected for the present work since a partition coefficient of 1.96 was obtained. Fig. 2 (a) and (b) were typical chromatograms for preparative enrichment of the target component from Tablet 1 and Tripterygium wilfordii (Jiangxi, China) by countercurrent chromatography using the above selected solvent system n-hexane-ethyl acetate-ethanol-water (6:4:6:4, v/v). Head-to-tail elution mode was employed, where the upper organic phase was used as the stationary phase while the lower aqueous phase was used as the mobile phase. Retention of stationary phase reached more than 58.3%. The shaded area in Fig.2 shows the collection time for the eluted fractions containing target component wilforlide A, in which the collection time was 15
equal to 6σ based on its adjusted retention time. HPLC analysis showed that complete collection of the eluted fractions containing the target component wilforlide A was achieved by the present collection methodology. As shown in Fig.2, the elution profile of the target component was difficult to obtain due to its extremely limited content in the crude sample. Fig. 3 showed two typical chromatograms for analysis of the combined fractions from countercurrent chromatography by high performance liquid chromatography. It was impossible to give a quantitative determination of wilforlide A in the crude sample extracted from Tablet and Tripterygium wilfordii because the target component could not be detected due to its limited content. However, a high peak resolution of wilforlide A could be obtained after its sample pretreatment by countercurrent chromatography.
3.3 Quantitative determination by HPLC In the present study, different solvents were investigated for extraction of wilforlide A, including water, ethanol, methanol, dichloromethane, chloroform, ethyl acetate and their combinations with changing volume ratio. It was found that ethyl acetate showed complete recovery of wilforlide A from glycoside tablets and Tripterygium wilfordii. Analysis of crude sample and the combined fractions containing wilforlide A by high performance liquid chromatography was optimized in the present work. In order to obtain a fast and specific detection of wilforlide A in the sample, the crude sample of Tablet 1 was used for optimization of analysis conditions. Results showed that acetonitrile-0.1 % formic acid (75:25, v/v) gave the optimal separation of wilforlide A in the sample. 3.3.1 Validation of HPLC assay Specificity and sensitivity: Wilforlide A was well separated with other components and it was 16
quantified under the optimized HPLC chromatographic condition and eluted at 19.8 min (Fig.S1) The calibration curve was established by plotting the concentration of reference substance against the corresponding peak area. The regression equation, linearity range (mg mL-1), correlation coefficient (r2), limit of detection (LOD) and limit of quantification (LOQ) of wilforlide A were shown in Table 4. The calibration curves showed good liner regression (r 2=0.9999, n=7) within the test ranges. The LOD and LOQ for wilforlide A were determined to be 1.35 ng mL -1 and 4.50 ng mL-1 based on signal-to-noise ratios (S/N) of 3 and 10, respectively. Precision, stability, repeatability and accuracy: Intra-day and inter-day variations were chosen to determine the precision of the developed method. The analytical precision from the data of the intra-day and inter-day determinations was indicated by the RSD values, and they were determined to be 0.67 % and 1.14 %, respectively (Table S1). As shown in Table S2, wilforlide A was found to be stable at 4oC with a deviation of less than 1.5 % for the stock solution. The repeatability of the method was shown in Table S3, where RSD for repeatability was found to be 1.19%. Table S4 indicated that a high recovery (94.52 % to 98.27 %) for wilforlide A could be obtained (RSD =1.58 %). 3.3.2 Content of wilforlide A in Tablets and Tripterygium wilfordii Table 5 shows the determined content of wilforlide A in six different Chinese patent medicines glycoside tablets and three different Tripterygium wilfordii. It was reported in the Chinese drug standard that the content of wilforlide A in the Chinese patent medicine glycoside tablets should not be less than 0.10 mg g-1 [13]. As shown in Table 4, the content of wilforlide A in each tested Tablet met the standard. However, it was found that the content of wilforlide A in Tripterygium wilfordii was much lower than that in the Chinese patent medicine, which might be the main
17
reason for that wilforlide A could not be accurately determined by UHPLC-QTOF-MS [12] and by HPLC-ELSD [14], in which no sample pretreatment was used. Compared with the tradition methods for sample pretreatment, the major advantage for the present method was the complete recovery of analytes from the crude sample and the trace component in Tripterygium wilfordii could be successfully determined. The target trace component in the crude sample could be selectively enriched by liquid-liquid chromatography using a selected biphasic solvent system. Although it was difficult to obtain the elution profile of interested analytes, it could be well predicted and simulated by a continuous stirred tank reactors model.
4 Conclusions In the present work, an approach for sample pretreatment by liquid-liquid chromatography was developed for quantitative determination of trace chemical components in traditional Chinese medicinal herb and its Chinese patent medicine. The major advantage of the present methodology was that a complete recovery could be obtained during the sample treatment since liquid-liquid chromatography was a partition chromatography with no solid support for the stationary phase. The collection time of eluted fractions containing target components could be well predicted by calculation of peak width and simulation of peak chromatogram for the target component using a continuous-stirred tank reactors model. With this method, successful quantitative determination of the trace component, wilforlide A, in its traditional Chinese medicinal herb and its Chinese patent medicine by conventional high performance liquid chromatography with UV detector could be achieved with high recovery, repeatability and accuracy. This method could be expected to be used for quantitative determination of components with extremely low content in a complex sample. 18
Acknowledgement This work was financially supported by national natural science foundation of China (21978266).
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Credit Author Statement Shanshan Zhao: Conceptualization, Methodology, Software, Data curation, Writing- Original draft preparation. Chaoyue Wang: Help during the experiment Xiang Wang: Writing- Reviewing Yang Jin: Writing- Reviewing Wenyu Sun: Writing- Reviewing Xingchu Gong: Software, Validation Shengqiang Tong: Guidance during the experiment, Writing- Reviewing and Editing.
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Figure captions:
22
Fig. 1 chemical structure of wilforlide A
.
Fig. 2 Chromatogram for preparative enrichment of wilforlide A from the crude sample by countercurrent chromatography. (a) 1.5 g of sample from Tablet 1; (b) 10.0 g of sample from Tripterygium
wilfordii.
Separation
conditions:
solvent
system:
n-hexane-ethyl
acetate-ethanol-water (6:4:6:4, v/v); stationary phase: the upper phase; mobile phase: the lower phase; flow rate: 2 mL min-1; column temperature: 25 oC; detection wavelength: 254nm; retention of stationary phase: 58.3%. The shaded area indicated the collection time for the eluted fractions containing wilforlide A.
23
Fig. 3 Chromatogram for analysis of the combined fractions from countercurrent chromatography by high performance liquid chromatography. (a ): The combined fractions containing wilforlide A from Fig. 2(a); (b): it’s the combined fractions containing wilforlide A from Fig. 2(b). Analytical conditions: column: phenomenex SYNERGI C18 column (250×4.6mm, 4µm); mobile phase: 0.1 % formic acid and acetonitrile (25:75, v/v), flow rate: 1.0 mL min -1; sample injection: 20 μL; detection wavelength: 210 nm; column temperature: 35oC.
24
Table 1 Various parameters of the instrument Column
Column
Column
Number
Extra
Sample
average
β value
Estimated
bore
length
volume
of
column
injection
wound
range
number
(mm)
(m)
(mL)
loops
volume
volume
radius
of
(mL)
(mL)
(cm)
CSTRs
4
20
0.33
2.0
60.5
190
288
25
0.36-0.69
836
Table 2 Retention time and collection time for wilforlide A Adjusted retention
Errors (%)
timea) (min)
Collection time
Errors (%)
(min)
Calculated
148.2
1.16
31.9
3.33
Simulation
145.5
0.68
35.5
7.5
Experimentalb)
146.5
-
33.0
-
a) Adjusted retention time does not include the extra retention time (7 min) caused by the extra column volume. b) Adjusted retention time and collection time denote average adjusted retention time and average collection time in experimental procedure
26
Table 3 Partition coefficient (KD) of wilforlide A in the tested biphasic solvent systems Solvent systems
Partition coefficient KD
n-hexane-ethanol-water= (6:5:1, v/v)
0.22
n-hexane-ethyl acetate-ethanol-water= (5:5:5:5, v/v)
7.63
n-hexane-ethyl acetate-ethanol-water =(9:1:9:1, v/v)
0.74
n-hexane-ethyl acetate-ethanol-water =(8:2:8:2, v/v)
0.50
n-hexane-ethyl acetate-ethanol-water =(7:3:7:3, v/v)
0.66
n-hexane-ethyl acetate-ethanol-water =(6:4:6:4, v/v)
1.96
n-hexane-ethyl acetate-ethanol-water =(13:7:13:7, v/v)
1.03
27
Table 4
Regression equations and correlation coefficient
Regression equation a)
Linearity range (mg ml-1)
R2 b)
LOD (ng mL-1)
LOQ (ng mL-1)
y =685.06x + 18.648
0.01- 0.10
0.9999
1.35
4.50
a) x denotes the concentration and y denotes the peak area. b) R2 is the correlation coefficient.
28
Table 5 Content of wilforlide A in the tested Tablets and Tripterygium wilfordii Samples
Contents (mg g-1)
Samples source
Tablet 1
0.5044
Fudan Fuhua Pharmaceutical Co. Ltd., Shanghai, China
Tablet 2
0.2105
Xieli Pharmaceutical Co. Ltd., Hunan, China
Tablet 3
0.2460
Deende Pharmaceutical Co. Ltd., Zhejiang, China
Tablet 4
0.2288
HSFY Pharmaceutical Co. Ltd., Hubei, China
Tablet 5
0.2138
Suzhou Yifan Pharmaceutical Co. Ltd., Anhui, China
Tablet 6
0.2881
Hanfang Pharmaceutical Co. Ltd., Guizhou, China
Root 1
Not detected
Xinchang, Zhejiang, China
Root 2
0.0027
Jian, Jiangxi, China
Root 3
0.0012
Yuxi, Yunnan, China
29