Analysis of phenolic acids by ionic liquid-in-water microemulsion liquid chromatography coupled with ultraviolet and electrochemical detector

Accepted Manuscript Title: Analysis of phenolic acids by ionic liquid-in-water microemulsion liquid chromatography coupled with ultraviolet and electrochemical detector Authors: Li-Qing Peng, Jun Cao, Li-Jing Du, Qi-Dong Zhang, Yu-Tin Shi, Jing-Jing Xu PII: DOI: Reference:

S0021-9673(17)30517-4 http://dx.doi.org/doi:10.1016/j.chroma.2017.03.086 CHROMA 358431

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

23-1-2017 23-3-2017 31-3-2017

Please cite this article as: Li-Qing Peng, Jun Cao, Li-Jing Du, Qi-Dong Zhang, Yu-Tin Shi, Jing-Jing Xu, Analysis of phenolic acids by ionic liquid-in-water microemulsion liquid chromatography coupled with ultraviolet and electrochemical detector, Journal of Chromatography Ahttp://dx.doi.org/10.1016/j.chroma.2017.03.086 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Analysis of phenolic acids by ionic liquid-in-water microemulsion liquid chromatography coupled with ultraviolet and electrochemical detector Li-Qing Peng, Jun Cao*, Li-Jing Du, Qi-Dong Zhang, Yu-Tin Shi, Jing-Jing Xu College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 310036, China * Corresponding author: Dr. Jun Cao E-mail: [email protected] Tel.: +86 571 2886 7909 Fax: +86 571 2886 7909 Highlights: 

An environmentally friendly IL/W ionic liquid-in-water microemulsion was established and applied as mobile phase in MELC microemulsion liquid chromatography.

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The proposed method involves the use of UV ultraviolet and ECD electrochemical detector.

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Highly sensitive and selective separation performance of phenolic acids in Danshen and its preparation was achieved.

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The proposed method is a simple, sensitive, effective and environmental friendly.

ABSTRACT An environmentally friendly ionic liquid-in-water (IL/W) microemulsion was established and applied as mobile phase in microemulsion liquid chromatography (MELC) with ultraviolet (UV) detection or electrochemical detector (ECD) for analysis of phenolic compounds in real samples. The optimal condition of the method

was using the best composition of microemulsion (0.2% w/v [HMIM]PF6, 1.0% w/v SDS, 3.0% w/v n-butanol, 95.8% v/v water, pH 2.5) with UV detection. The validation results indicated that the method provided high degree of sensitivity, precision and accuracy with the low limit of detections ranged from 17.89-238.10 17.9-238 ng/mL, satisfactory mean recovery values in the range of 80.11-105.21 80.1105% and good linearity (r2>0.9994). Additionally, this method exhibited high selectivity and resolution for the analytes and was more eco-friendly compared with traditional MELC method. Consequently, the established IL/W MELC method was successfully applied to simultaneously separate and determine target compounds in Danshen sample and its preparation. Keywords: Danshen injection; Ionic liquids; liquid-in-water microemulsion; Microemulsion liquid chromatography; Phenolic acids; Salvia miltiorrhiza 1. Introduction Ionic liquids (ILs), known as molten salt or fused salts, are composed of bulky organic cations such as alkyl imidazolium, alkyl ammonium, alkyl phosphonium, alkyl pyridinium, alkyl pyrrollidinium and various inorganic (such as chloride, bromide, hexafluorophosphate, tetrafluoroborate and triflate anion) or organic anions (such as [(CF3SO2)2N]- and [(CF3CO2)]-) [1-2]. ILs have an wide range of solubility for both polar and non-polar compounds, due to the wide variety of structures. In recent years, ILs have attracted increasing attention, due to their unique physicochemical properties, such as a variable viscosity range, low volatility, low

melting point, no effective vapor pressure, high chemical and thermal stabilities and recycling [2-4]. ILs have been receiving a “green” connotation (mainly due to their negligible volatility and non-flammability) and utilized as alternate eco-friendly solvents to conventional solvents, although some ILs are toxic and may cause soil and water pollution which may further exert toxic effects toward organisms [5-7]. Commonly, the ILs toxicity are strongly influenced by cation-anion structure, the design or selection of appropriate IL anion and cation is very significant [8]. The ILs are utilized as alternate eco-friendly nonvolatile solvents to conventional solvents mainly due to their negligible volatility and non-flammability. The physicochemical properties can be tuned and modulated by selecting the appropriate anion and cation. Moreover, ILs can interact with analytes by hydrogen bonding, π-π interactions, n-π interactions, electrostatic interactions and dipolar interactions [9]. Furthermore, due to their unique properties, ILs have widespread applications in various analytical fields, such as sample preparation [10-12], catalysis [13], and separation [14-15]. In chromatographic analysis, ILs can be employed as stationary phase in gas chromatography (GC), liquid chromatography (LC) and supercritical fluid chromatography (SFC), additive of mobile phase in LC and electrolyte solution in capillary electrophoresis (CE) [1,16-17]. Additionally, the application of ILs as additives in mobile phase in LC can improve the peak shapes and affect the column efficiency and theoretical plate number. Microemulsion is a thermodynamically stable, transparent, optically clear and isotropic liquid disperse system containing organic phase (oil) and aqueous phase

(water) stabilized by a surfactant and cosurfactant [18]. Both hydrophilic and hydrophobic substances can dissolve in microemulsion. The microemulsions have been used as pseudostationary phase in capillary electrophoresis (CE) [19] and mobile phase in liquid chromatography (LC) [20]. In addition, microemulsions can be classified as oil-in-water (O/W) and water-in-oil (W/O), where the O/W microemulsions which consist of oil droplets distributed in an aqueous phase are widely used as a mobile phase in reversed phase high performance liquid chromatography due to the high water content and low viscosity [21]. In recent years, the microemulsion liquid chromatography (MELC) has received growing attentions and be successfully applied in separation sciences [21-24, 25] due to its unique selectivity and high separation efficiency without the need of gradient elution and its capacity to overcome the environmental problem (consuming large amounts of organic solvents) caused by conventional LC methods. The commonly used oil phase in MELC are hydrophobic alkanes such as ethyl acetate, octane and cyclohexane which have a bad effect on the environment, using an environmentally friendly ionic liquid as the oil phase in MELC has not been reported to the best of our knowledge. Moreover, electrochemical detector has not been applied in MELC system while ultraviolet detector is commonly used. Salvia Miltiorrhiza Bunge (Chinese name Danshen) is a popular traditional Chinese herbal medicine due to its remarkable biological activity. The herb and its preparations such as Danshen tablets, Danshen injection, Danshen capsules and Compound Danshen dripping pill are widely used for the clinical treatment of various

diseases, including cerebrovascular diseases, coronary artery disease, atherosclerosis, hyperlipidemia, hypertension, hepatitis, chronic renal failure, hepatocirrhosis and bone loss[26-29]. The major bioactive constituents in Danshen can be classified into two main active groups: the hydrophilic phenolic acid compounds (such as danshensu, caffeic acid, protocatechualdehyde, rosmarinic acid and salvianolic acid B) and the lipophilic diterpenoids compounds (including dihydrotanshinone I, cryptotanshinone, tanshinone IIA and tanshinone I) [29-30]. Therefore, the extraction and determination of these active compounds from Danshen and its preparations is very significant. Currently, a various of analytical methods such as high performance liquid chromatography coupled with ultraviolet (HPLC-UV) [31], HPLC with diode array detection (DAD) [32], ultra high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) [33], have been reported for the determination of active compounds in Danshen and its preparations. However, hydro-organic mobile phase was used in these methods, large amounts of toxic organic solvents are consumed, resulting in an environmental impact. In consequence, establishing an MELC system which require less organic solvent in mobile phase and provide high separation capacity is quite meaningful for the analysis of chemical constituents in real samples. In this paper, a simple, effective and environmentally friendly MELC method using IL/W microemulsion as mobile phase coupled with UV or ECD was established and applied for determination of phenolic acids in Danshen and its preparation. Several important parameters that can influence the separation performance of the target

analytes including the type of oil phase, oil concentration, SDS concentration, nbutanol concentration and pH of microemulsion are evaluated and optimized. The validation of the method is performed in terms of linearity, precision, reproducibility, accuracy, limits of detection and limits of quantification. Ultimately, the established IL/W MELC method is successfully applied to the separation and determination of target compounds in Danshen sample and its preparation. 2. Experimental 2.1. Reagents and materials 1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF6), 1-hexyl-3methylimidazolium hexafluorophosphate ([HMIM]PF6), 1-octyl-3methylimidazolium hexafluorophosphate ([OMIM]PF6), 1-hexyl-3methylimidazolium tetrafluoroborate ([HMIM]BF4), 1-butyl-3-methylimidazolium bis [(trifluoromethyl) sulfonyl] imide ([BMIM]TF2N), and 1-octyl-3-methylimidazolium bis (trifluoromethyl sulfonyl) imide ([OMIM]TF2N) were provided by Shanghai Cheng Jie Chemical Co., Ltd. (Shanghai, China). HPLC grade n-butanol, ethyl acetate, sodium dihydrogen phosphate, phosphoric acid and sodium dodecyl sulfate (SDS) were all purchased from ANPEL Laboratory Technologies (Shanghai) Inc. (Shanghai, China). Chromatographic grade methanol was supplied by Tedia Company Inc. (Fairfield, US). The purified water used in this study was obtaimed from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China).

The 0.2 μm disposable nylon membrane was acquired from Jinteng Laboratory Equipment Co., Ltd. (Tianjin, China). Eight standard compounds inluding sodium danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, lithospermic acid, rosmarinic acid, salvianolic acid B and salvianolic acid A were purchased from Shanghai Winherb Medical Technology Co., Ltd. (Shanghai, China), with the purity of all the standards higher than 98%, the structures of these standard compounds were showed in Fig. S1. The Danshen sample and Danshen injection were obtained from local drugstore (Hangzhou, China). 2.2. Apparatus The analytes were separated and determined on an Agilent 1290 liquid chromatography system (Santa Clara, CA) equipped with a UV detector set at 286 nm. The separation was performed on an Agilent reverse-phase SB-C18 column (5 μm particle size, 150 × 4.6 mm i.d.). The temperature of the column was maintained at 30 oC. The flow rate was set at 0.6 mL/min and the injection volume was kept at 2 μL. The mobile phase was a microemulsion consisting 0.2% w/v [HMIM]PF6, 1.0% w/v SDS, 3.0% w/v n-butanol and 95.8% v/v water, the pH was adjusted to 2.5 by using phosphoric acid and 10% ammonium hydroxide. An Agilent 1290 system (Santa Clara, CA) with an Antec SDC ECD (Antec, Netherlands) was also used in this study. The analytes were separated on an Agilent SB-C18 column (5 μm, 4.6 mm i.d. × 150 mm) which was maintained at 30 °C. The mobile phase was a microemulsion composed of 25 mM sodium dihydrogen

phosphate, 0.2% w/v [HMIM]PF6, 1.0% w/v SDS, 3.0% w/v n-butanol and 95.8% v/v water, the pH was adjusted to 2.5 by using phosphoric acid and 10% ammonium hydroxide. The injection volume was set at 1 μL, and the flow rate was maintained at 0.4 mL/min. The electrochemical cell was a SenCell of a confined wall-jet design. ECD was performed at the cell potential of Ecell = +0.70 V, the detector was set at 500 nA range versus the Ag/AgCl reference electrode. 2.3. Preparation of microemulsion mobile phase, standard and sample solutions The microemulsion mobile phase was prepared by weighting 0.1-0.4% w/v of oil phase (ethyl acetate, [BMIM]PF6, [HMIM]PF6, [OMIM]PF6, [BMIM]TF2N, [HMIM]BF4 and [OMIM]TF2N), 0.5-2% w/v of SDS and 2-5% w/v of n-butanol. Then, the mixture was sonicated for 30 min (100 W, 40 kHz) to form a transparent and stable microemulsion. After that, the pH was adjusted by using phosphoric acid or 10% ammonium hydroxide. Finally, the microemulsion was filtered under vacuum through a 0.2 μm disposable nylon membrane. At room temperature, the IL/W microemulsion mobile phase stabilized within two weeks without stratification and emulsification. The standard stock solution was obtained by dissolving each appropriate amount of standards in methanol at a concentration of 1000 μg/mL, and stored at 4 oC. The working solutions were prepared by diluting the stock solutions with methanol before use.

The Danshen sample was powdered by a pulverizer and sieved through a 100 mesh sieve. Then 0.15 g of Danshen powder was precisely weighed and mixed with 50 mL methanol-water (80:20, v/v), then the mixture was sonicated for 30 min. After ultrasonic processing, 5 mL of the solution was diluted directly to 10 mL with methanol. The Danshen injection was diluted 5 times by methanol before analysis. After centrifugation (13000 rpm, 5 min), the supernatants of the two sample solutions were injected into the MELC system. 2.4. Calculation All measurements were performed in triplicate and the values for the retention factor (k) have been reported as the average of three replicates. The k can be expressed as k = (tR−t0)/t0, where t0 was the dead time, tR was the retention time of the test solute. The resolution (R) was defined as R=2 (tR2-tR1)/(W1+W2), Where tR2 and tR1 were the retention times of peak 1 and peak 2 which were adjacent to each other, respectively, W1 and W2 were the peak width of peak 1 and peak 2 at peak base, Respectively. 3. Results and discussion 3.1. Selection of oil The oil, as the core phase which was enclosed by the surfactant with the assistance of a co-surfactant, is dispersed into nanodroplets in water phase to form a nanoemulsion. The structure of the oil has a remarkable influence on the physicochemical properties of microemulsions. In MELC, the type of oil has an

obvious effect on the retention time and separation efficiency of target analytes. The commonly used oil phases were ethyl acetate, octane and cyclohexane. These oil phases were organic solvents which had negative effect on environmental protection. In this study, six hydrophobic ILs with the same cation ([BMIM]+, [HMIM]+, or [OMIM]+) or anion (PF6-, TF2N-, or BF4-) including [BMIM]PF6, [HMIM]PF6, [OMIM]PF6, [BMIM]TF2N, [HMIM]BF4 and [OMIM]TF2N were compared to select the optimal oil phase for the microemulsion droplets (the other conditions were: 0.2% w/v oil phase, 1% w/v SDS, 3% w/v n-butanol, pH 2.5). Six phenolic compounds including sodium danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, lithospermic acid, rosmarinic acid, salvianolic acid B and salvianolic acid A were selected as the tested analytes. The result in Fig. 1 show that the completely separation of all the tested analytes was achieved when the [HMIM]PF6 was used as the oil phase. It can also be observed that the retention times of the phenolic compounds were increased as a whole with the increase of the alkyl chain from butyl to octyl in the imidazolium cation of IL which had a same anion (PF6- or TF2N-) (Fig. 1a-c; d, f). The reason for this phenomenon may be that the longer alkyl chain exhibited the stronger hydrophobicity which can reduce the solubility of the polar phenolic compounds and resulted in the increased retention times due to the lower distribution of target analytes in the microemulsion droplets. Moreover, replacing the PF6- anion by TF2N- caused a shorter retention time and poor separation selectivity, especially for analytes 5, 6, 7 and 8. Compared with the [HMIM]PF6, the peaks of 5, 6 and 7, 8 obtained by [HMIM]BF4 were overlapped and the retention times were

increased, the possible reason may be that the molecular weight of [HMIM]BF4 was smaller than [HMIM]PF6 leading to the more hydrophobic [HMIM]PF6 molecular per mass which would lead to the lower distribution of polar analytes in the droplets. the number of hydrophobic [HMIM]+ molecular in [HMIM] BF4 was more than that in [HMIM] PF6, when use the [HMIM] PF6 and [HMIM] BF4 of equal mass. This may be lead to the lower distribution of analytes in [HMIM] BF4. Additionally, the elution order of analytes 6 and 7 was altered when using the different oil types (Fig. 1a, d). It was likely due to the difference of the solubility, association and distribution for the analytes in different oil phases. In conclusion, [HMIM]PF6 was selected as the optimal oil phase for its excellent separation selectivity of all the analytes in a short analysis time. 3.2. Concentration of oil The concentration of the oil phases can drastically influence the retention time, separation selectivity and resolution of the target analytes in the MELC system. For the purpose to assess the effect of the oil phase concentration on the separation performance in MELC, different concentrations of oil ([HMIM]PF6) in the microemulsion mobile phase were studied in the range of 0.1-0.4% w/v (the other conditions were: [HMIM]PF6 as the oil phase, 1% w/v SDS, 3% w/v n-butanol, pH 2.5). As can be seen from chromatograms showed in Fig. S2 and the line chart showed in Fig. 2A, when the concentration of oil was 0.1% w/v, the peaks of analytes 6 and 7 was overlapped into a single peak. The increase of the oil content increased the retention time factor of all the phenolic compounds, this may be due to the fact

that the increase of the oil concentration increased the hydrophobicity of the microemulsion mobile phase which led to the decreasing of the solubility and distribution of the phenolic compounds in mobile phase and increased the retention. What is more, the hydrophobic oil may be distributed on the surface of the column to some extent, which increased the amount of stationary phase and influenced the separation selectivity and retention of analytes. The optimal chromatographic performance was obtained using 0.2% w/v [HMIM]PF6 in the mobile phase. Continually increasing the oil content from 0.2% to 0.4%w/v gave longer retention times and the overlaps of analytes 5, 6 and 7, 8 were observed when the oil content was 0.3%. Moreover, the alteration of the elution order of analytes 5, 6, 7 and 8 was appeared when the oil content was 0.3% and 0.4%. It is possible due to the nature of the analytes and the change in the distribution and retention mechanisms which influenced the separation selectivity to some extent. Thus, 0.2% w/v [HMIM]PF6 was chosen as the oil phase with the shortest analysis time. 3.3. Concentration of SDS The surfactant molecules can adsorb onto the surface of the stationary phase in MELC which can fill up the silica pore volume and change the surface area and polarity of the stationary phase. For SDS, the long hydrophobic chain of SDS monomersis was associated to the C18 alkyl-chain bonded to the silica stationary phase, with the sulphate group oriented away from the surface which created a negatively charged stationary phase affecting the distribution of solutes on the column. The concentration of SDS in the microemulsions mobile phase had a significant effect on the retention of the

analysed compounds and their partition with the stationary phase in MELC. To evaluate the impact of the SDS concentration for the separation of phenolic compounds, different concentrations of SDS ranging from 0.5% to 2% w/v were investigated (Fig. S3 and Fig. 2B). It was observed that the retention time factor and separation selectivities of the eight phenolic compounds were decreased with the increase of concentration of SDS from 0.5% to 2% w/v. The possible reason for these observations may be that the increased SDS concentration distributed the target phenolic acids into an increased volume of microemulsion droplets or to the surface of the droplets which decreased solute retention. What is more, the anionic SDS monomers modified stationary phase could form electrostatic repulsive-force with the phenolic acids resulting in the reduction of the retention time. It was found that optimal separation performance was achieved by using 1% SDS in the mobile phase. Decreasing the concentration of SDS at 0.5% dramatically increased the retention time of the tested analytes which was too large to be measured. Further increasing the concentration of SDS to 1.5% and 2% decreased the resolution and separation selectivity of the analytes owing to the occurrence of undifferentiated partitioning of the analytes. Therefore, 1% (w/v) SDS was selected as the optimal surfactant concentration for further studies. 3.4. Concentration of cosurfactant The cosurfactant, which commonly was the short-chain alcohols such as n-butanol added to the microemulsion system to enhance and stabilize the microemulsion system by decreasing the surface tension between the oil phase and the aqueous phase, was a significant parameter in affecting the partition and retention of the analytes in MELC.

To further evaluate how the cosurfactant affected the chromatographic performance, different concentrations of cosurfactant n-butanol in the range of 2-5%w/v were investigated in the present study (the other conditions were: 0.2% w/v [HMIM]PF6 as the oil phase, 1% w/v SDS, pH 2.5). It was observed from the results displayed in Fig. 2C and Fig. S4 that the retention of the analytes enhanced as the concentration of the cosurfactant increased from 2 to 5% w/v. It was possible that an increase of the cosurfactant concentration in the microemulsion increased the proportion of the organic solvent in the mobile phase, which decreased the elution speed of hydrophilic solutes. Moreover, the n-butanol could be adsorbed onto the stationary phase by replacing the anion of SDS, which increased the polarity of the stationary phase and increased the retention of the analytes. The results also revealed that the optimum chromatographic performance was observed to occur by using 3% n-butanol in the solvent, lower and higher concentrations of n-butanol have both a negative effect on the resolution and the peak shape. When the concentration of n-butanol was lower than 3%, analytes 6 and 7 co-eluted into a single peak. Further increased the n-butanol concentration to 4% and 5% led to longer retention times and lower resolutions for analytes 7 and 8, which may be caused by an increase in the hydrophobicity of the microemulsion with increasing the n-butanol concentration, affecting the retention of tested analytes. Additionally, the elution order of analytes 5, 6 and 7, 8 changed when the n-butanol concentration was set at 4% and 5%, this phenomenon may be attributed to the increased viscosity of nbutanol and the change of distribution and solubility of analytes. Consequently, 3% w/v n-butanol was adopted as cosurfactant in subsequent experiments.

3.5. Effect of pH In RP-HPLC, the more hydrophobic analytes can be retained on stationary phase for longer. When the tested analyte was ionized, the compound became less hydrophobic and its retention decreased. In the same way, in MELC the retention of ionizable compound can be influenced by the pH of the microemulsion mobile phase. The effect of the pH of the mobile phase on the resolution and separation selectivity of eight phenolic compounds was investigated in the range of 1.5-5.5 using phosphoric acid and 10% ammonium hydroxide to adjust the pH value in the microemulsion eluent (the other conditions were: 0.2% w/v [HMIM]PF6 as the oil phase, 1% w/v SDS, 3% nbutanol). The experimental results presented in Fig. 2D and Fig. S5 demonstrate that the retention time factor of the target analytes decreased with increasing the pH value. It also can be observed that analytes 5, 6 and 7, 8 co-eluted into a single peak at pH 1.5 and 2.0. Additionally, when pH value increased to 5.0 and 5.5, the eight phenolic compounds did hard to separate, nearly overlapped into one or two peaks, hence, the pH, higher than 5.5, did not need to be investigated further. The possible reason for these phenomenons may be that the eight phenolic compounds were weak acid compounds due to their phenolic hydroxyl or carboxyl groups. The pH value determined the ionization extent of analytes. The ionization of acid analytes increased at higher pH values, resulting in less hydrophobicity and a decrease of analytes retention on the stationary phase. Therefore a pH value of 2.5 was the most appropriate pH, providing well resolutions and high separation selectivities with a reasonable run time. Based on the experiments discussed above, the mobile phase was adjusted to pH

2.5 in this study. 3.6. Method validation In order to evaluate the developed IL/W based MELC method, several parameters including linearity, precision (intra-day and inter-day), repeatability and sensitivity were investigated under the optimum conditions (0.2% w/v [HMIM]PF6 as the oil phase, 1% w/v SDS, 3% n-butanol, pH 2.5). Each parameter was assessed in triplicate. The results are showed in Table 1. 3.6.1. Linearity The calibration curves of eight analytes inluding sodium danshensu, protocatechuic acid, protocatechuic aldehyde, caffeic acid, lithospermic acid, rosmarinic acid, salvianolic acid B and salvianolic acid A, were obtained by plotting the corresponding peak areas versus the concentrations of eight phenolic compounds in the chromatogram. Excellent linearity was obtained by using a series concentrations of standard solutions over the linear range of 5-250 μg/mL for seven analytes and 5-500 μg/mL for salvianolic acid B with correlation coefficients (r2) ranging from 0.9994 to 0.9999 for all the target analytes. 3.6.2. Precision The precision of the method was determined by evaluating the intra- and inter-day precision using the same concentration of standard solution. The intra-day precision was obtained by injecting the solution six times during one day. The relative standard deviations (RSDs) of intra-day precision were 0.15-0.68% for retention times and 1.262.58% for peak areas, respectively. The RSDs of inter-day precision which was

determined by repeating the same study twice a day in 3 consecutive days were in the range of 0.53-0.87% and 1.38-3.95% for retention times and peak areas, respectively. The results indicated the good precision and reproducibility of the proposed method. 3.6.3. Repeatability and sensitivity The repeatability of microemulsion mobile phase was studied by performing three replicates of microemulsion. The RSDs ranged from 0.06 to 1.03% and 0.91 to 4.48% for retention times and peak areas, respectively. The results indicated that the method was very reliable and repeatable. The sensitivity of the present method was expressed by the limits of detection (LODs) and limits of quantification (LOQs). The LODs which were the concentrations at the ratio of signal to noise equal to three (S/N = 3) were in the range of 17.89-238.10 17.9-238 ng/mL for eight phenolic compounds. The LOQs (S/N = 10) of all the target analytes ranged from 59.04-785.73 59.0-785 ng/mL. The results showed the high sensitivity of the method. 3.7. Application of method In order to evaluate the application of the optimized MELC method, it was applied to separation and determination of the phenolic compounds in real samples. All the evaluation experiments were performed in triplicate. The HPLC chromatograms of the Danshen injection (A) and extracts of Danshen (B) are showed in Fig. 3. The experimental results are displayed in Table 2, as can be seen from the Table, only five analytes of sodium Danshensu, protocatechuic aldehyde, caffeic acid, lithospermic acid and rosmarinic acid were found in Danshen injection with the contents of 950.93, 411.53, 32.95, 93.60 and 218.92 951, 411, 33.0, 93.6 and 219 μg/mL, respectively.

While the Danshen sample contained three target analytes with the contents of 3.88, 3.85 and 62.38 mg/g for lithospermic acid, rosmarinic acid and salvianolic acid B, respectively. Recoveries were investigated by performing the spiked Danshen samples which was added by the standard stock solutions at two different concentration levels (1 and 50 μg/mL) to evaluate the accuracy of the established method. The Table 2 showed that the average recoveries of all the target compounds were in the range of 80.11-105.21 80.1-105% and 80.42-104.20 80.4-104% for Danshen injection and Danshen, respectively. The repeatability of the extraction method was investigated by performing three parallel extracts of Danshen sample, the RSDs of the retention time were in the range of 0.46 to 2.84%, and RSDs of peak areas were in the range of 0.56 to 4.57%. Thus, the established method was applicable and accurate for the simultaneous separation and determination of phenolic compounds in real samples. 3.8. Comparison of different microemulsion and detection methods The traditional microemulsion mobile phases using ethyl acetate, dichloromethane and octane as the oil phase replaced the [HMIM]PF6 in the optimal microemulsion system (0.2% w/v oil phase, 1% w/v SDS, 3% n-butanol, pH 2.5) were evaluated for the separation and determination of eight phenolic compounds. Compared with the [HMIM]PF6 based microemulsion (Fig. 1b), the ethyl acetate based microemulsion exhibited poor resolution of target analytes especially for analytes 4, 5, 6, 7 and 8 (Fig. 4A), the dichloromethane and octane based microemulsion showed worse separation performance for all the tested analytes (Fig. 4B-C). Additionally, Fig. 5A-E show the chromatograms of eight analytes obtained by five kinds of oil phase in microemulsion

(0.2% w/v oil phase, 1% w/v SDS, 3% n-butanol, pH 2.5, 25mM NaH2PO4) including ethyl acetate, [BMIM]PF6, [HMIM]PF6, [OMIM]PF6 and [BMIM]TF2N using electrochemical detector (ECD), which provided lower separation selectivity and resolution compared to that obtained by UV detector (Fig. 1a-d, Fig. 4A). For example, when [BMIM]PF6 was used as oil phase, the resolution of peaks 4, 5 and 5, 7 were 1.04 and 0.92 for ECD, 3.67 and 2.32 for UV detector, respectively. Similarly, the resolution of analytes 5 and 6 obtained by ECD (R=1.22) was lower than that obtained by UV detector (R=2.59). Moreover, the responses of all the analytes were decreased visibly when determined by ECD with different oil phases, even between two consecutive runs with same microemulsion mobile phase which had a decrease of 25%-49% for the peak height of all the analytes. The possible reason for the phenomenon may be that the IL in the mobile phase would adsorb on the electrode surface leading to a narrow electrochemical window, which caused the redox reaction of ions of ILs on the electrode surface affecting the detection of target analytes. Furthermore, the positively charged imidazolium groups of ILs might associate with the hydroxy groups on the phenolic compounds and influenced the ECD signal of target analytes [34-35]. The pulsed amperometric detection, which can clean and reactivate the electrode surface by using a triple-step potential waveform to combine amperometric detection with alternating anodic and cathodic polarization [36], may be can solve the reproducibility issues of IL/W microemulsion with ECD due to its unique properties. Thus, the ECD detector was not suitable for determining the analytes with the IL based microemulsion mobile phase. As a result, the UV detector was used in this work.

4. Conclusions In this study, a reliable, simple and environmentally friendly MELC method coupled with UV detector or ECD, using IL/W microemulsion as the mobile phase, was optimized and validated for simultaneous separation and determination of eight phenolic compounds from Danshen and its preparation. Several important parameters which could affect the separation performance of the target analytes, including the type of oil phase, concentration of oil, SDS concentration, n-butanol concentration and pH of microemulsion were evaluated systematically to obtain the optimal condition. The results reveal that the ECD was not suitable for this IL/W microemulsion system, while detecting the analytes by UV detector was very stable and sensitive. In addition, there may be some limiting characteristics of this method, such as the high viscosity of the IL/W microemulsion which may limit the flow rate to some extent, the ultraviolet absorption of ILs below 250 nm, and the difficulty to simultaneously separate hydrophobic and hydrophilic solutes at isocratic elution. Compared with traditional MELC (using ethyl acetate as the oil phase in microemulsion), the proposed green IL/W based MELC had unique advantages such as excellent separation selectivity, good resolution, less toxicity and low consumption of organic solvents. In addition, the validation data of the method demonstrated that the present method was sensitive, precise and accurate enough to separation and determination of phenolic compounds in Danshen sample and its preparation. Acknowledgements

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(2) protocatechuic acid, (3) protocatechuic aldehyde, (4) caffeic acid, (5) lithospermic acid, (6) rosmarinic acid, (7) salvianolic acid B, (8) salvianolic acid A. Fig. 2 Retention factors of eight analytes obtained by UV detector versus changes in different microemulsion parameters: oil concentration: 0.1% w/v; 0.2% w/v; 0.3% w/v; 0.4% w/v (A), SDS concentration: 0.5% w/v; 1.0% w/v; 1.5% w/v; 2.0% w/v (B), n-butanol concentration: 2.0% w/v; 3.0% w/v; 4.0% w/v; 5.0% w/v (C) and pH values: 1.5; 2.0; 2.5; 5.0; 5.5 (D). Analytes: (1) sodium Danshensu, (2) protocatechuic acid, (3) protocatechuic aldehyde, (4) caffeic acid, (5) lithospermic acid, (6) rosmarinic acid, (7) salvianolic acid B, (8) salvianolic acid A. Fig. 3 MELC-UV chromatograms of Danshen injection (A) and extract soultion of Danshen sample (B). Analytes: (1) sodium Danshensu, (3) protocatechuic aldehyde, (4) caffeic acid, (5) lithospermic acid, (6) rosmarinic acid, (7) salvianolic acid B. Fig. 4 MELC-UV chromatogram of standard solution using three different organic oil phase: (A) ethyl acetate; (B) dichloromethane; (C) octane. (other parameters: 0.2% w/v oil phase, 1% w/v SDS, 3% n-butanol, pH 2.5). Fig. 5 MELC-ECD chromatograms of standard solution using different type of oil phase (other parameters: 0.2% w/v oil phase, 1% w/v SDS, 3% n-butanol, pH 2.5, 25mM NaH2PO4). Type of oil phase: (A) ethyl acetate; (B) [BMIM]PF6; (C) [HMIM]PF6; (D) [OMIM]PF6; (E) [BMIM]TF2N. Analytes: (1) sodium Danshensu, (2) protocatechuic acid, (3) protocatechuic aldehyde, (4) caffeic acid, (5) lithospermic acid, (6) rosmarinic acid, (7) salvianolic acid B, (8) salvianolic acid A.

Table1 Linear Regression Data, Precision, Reproducibility, Limits of detection (LODs) and Limits of quantification (LOQs) of the Investigated Compounds

Calibration curve

Analyte

Calibration levels (n=5)

Precision（RSD%）

Intra-day n=6 Inter-day n=6

Reproducibilit y (Microemulsio n) LOD

LOQ

（RSD%） n=3

r2

Linear Reten Retenti Retenti Peak Peak Peak Interce ranges tion ng mL- ng mLon on Slopes 1 1 pts -1 μg mL time area time area time area

Sodium 0.999 2.69 Danshensu 5

4.07

5-250

0.39

1.76

0.71

2.21

0.64

4.48

48.0

158

0.999 5.06 5

18.2

5-250

0.15

2.16

0.48

2.24

0.06

2.33

25.0

82.4

Protocatech 0.999 14.5 uic aldehyde 4

-55.4

5-250

0.53

1.26

0.72

1.38

0.79

1.69

17.9

59.0

0.999 14.0 8

-40.4

5-250

0.60

1.67

0.83

1.68

0.22

3.34

22.0

72.4

0.999 3.59 9

-5.12

5-250

0.68

1.28

0.87

2.02

0.41

3.01

211

697

Rosmarinic 0.999 6.64 acid 7

-17.6

5-250

0.59

1.49

0.66

1.68

0.21

2.98

101

334

Salvianolic 0.999 3.55 acid B 5

4.52

5-500

0.47

2.52

0.53

2.65

1.03

0.91

238

786

Salvianolic 0.999 7.80 acid A 8

-7.70

5-250

0.51

2.58

0.72

3.95

0.44

3.55

173

571

Protocatech uic acid

Caffeic acid

Lithospermi c acid

Table 2 The Content, Average Recovery and Reproducibility of Samples Content

Recovery% Added (μg/mL)

Analyte

Sodium Danshensu

Protocatechui c acid

Danshen injection (μg/mL)

Danshen (mg/g)

951

－

－

Protocatechui c aldehyde

412

Caffeic acid

33.0

Lithospermic acid

93.6

Rosmarinic acid

219

Salvianolic acid B

－

Salvianolic acid A

－

Danshen injection

Danshen

1

88.6

102

50

101

104

1

96.2

101

50

98.6

86.1

1

101

91.5

50

99.0

91.4

1

93.8

85.8

50

85.2

89.0

1

105

102

50

98.7

99.8

1

85.5

80.4

50

96.3

88.1

1

80.1

95.9

50

96.1

83.8

1

85.4

82.8

50

97.2

90.8

－

－

－

3.88

3.85

62.4

－

Reproducibility (sample extraction) (RSD%) n=3 Retention Peak area time

－

－

－

－

－

－

－

－

0.46

4.57

1.98

2.40

2.84

0.56

－

－

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5