Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweeping-micellar electrokinetic chromatography

Analytica Chimica Acta xxx (xxxx) xxx

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Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweeping-micellar electrokinetic chromatography Li-Qing Peng a, Xin Dong b, Xiao-Ting Zhen b, Juan Yang b, Yan Chen b, Shu-Ling Wang a, Tian Xie a, **, Jun Cao a, b, * a b

Holistic Integrative Pharmacy Institutes, Medical College, Hangzhou Normal University, Hangzhou, 311121, People’s Republic of China College of Material Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou, 311121, People’s Republic of China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


An on-line CD assisted sweepingMEKC method is developed for separation and concentration of neutral analytes.
Satisfactory resolution and enrichment efficiency are achieved in a short analysis time.
Compared with conventional sweeping-MEKC and MEKC method, the proposed method shows higher enrichment efficiency.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 October 2019 Received in revised form 10 January 2020 Accepted 17 January 2020 Available online xxx

An on-line cyclodextrin assisted sweeping-micellar electrokinetic chromatography (CD assisted sweeping-MEKC) was developed for the simultaneous separation and concentration of four neutral analytes (erianin, dendrophenol, naringenin and scoparone) in Dendrobium officinale Kimura et Migo (D. officinale). The D. officinale was directly determined by this on-line stacking method after simple extraction and dilution. The optimized background solution (BGS) was 50 mM phosphoric acid (PA) containing 100 mM SDS and 30% (v/v) methanol. The best separation and concentration performance of analytes dissolved in 90 mM CD and 100 mM PA was achieved in a short analysis time when injected at 50 mbar for 100 s. Compared with conventional sweeping-MEKC and MEKC method, significant improvement in enrichment efficiency was achieved by using this proposed method. A series of validation studies of the present method was performed under the optimal conditions. Good linearities were obtained with the correlation coefficients in the range of 0.994e0.999, the detection limits were ranged from 13 to 40 ng/mL. Sensitivity enhancement factors (SEFs) were in the range of 28.5e46.8 compared with traditional injection (injection time 3 s). Therefore, the proposed method was successfully applied for the separation and concentration of neutral analytes in real samples. © 2020 Published by Elsevier B.V.

Keywords: Capillary electrophoresis Cyclodextrin Dendrobium officinale kimura et migo Micellar electrokinetic chromatography Sweeping

* Corresponding author. Holistic Integrative Pharmacy Institutes, Medical College, Hangzhou Normal University, Hangzhou, 311121, People’s Republic of China. ** Corresponding author. E-mail addresses: [email protected] (T. Xie), [email protected] (J. Cao).

1. Introduction Sweeping, an on-line sample concentration technique in

https://doi.org/10.1016/j.aca.2020.01.037 0003-2670/© 2020 Published by Elsevier B.V.

Please cite this article as: L.-Q. Peng et al., Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweepingmicellar electrokinetic chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.037

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micellar electrokinetic chromatography (MEKC), was first proposed by Quirino et al., in 1998 as an efficient method to enhance the method sensitivity [1]. In sweeping-MEKC, sample is prepared in a solution without adding pseudostationary phase (PSP) and with conductivity lower, similar or higher to the background solution (BGS) which contains a PSP. The analytes are picked up and accumulated by the PSP that penetrates the sample zone when the voltage is applied through the partitioning and interaction of analytes in the sample zone and PSP in the BGS [2e5]. The analyte zones are narrowed during the sweeping procedure, the enrichment efficiency is mostly determined by the retention factor (k) of the analytes [6]. Recently, because of its various advantages including high enrichment efficiency, simplicity and with no need for additional instrumentation, on-line sweeping-MEKC has become an effective technique to analyze trace compounds in different sample matrices. Liu et al. [7] applied sweeping-MEKC with high-conductivity sample solution for determination of trace-level paliperidone in human plasma. The sensitivity enhancement factor (SEF) of the method was 100 compared to the conventional MEKC method with the LOD of 10 ng/mL. Chen et al. [8] developed a cyclodextrin (CD) assisted dispersive liquid-liquid microextraction combined with sweeping-MEKC method for the concentration and detection of carbamazepine and clobazam in human urine samples. The SEFs were 3575 and 4675 with LODs of 0.6 and 0.5 ng/mL for carbamazepine and clobazam, respectively. It is necessary to develop new sweeping-MEKC methods for determination of complex active compounds in medical plants. CDs reported in 1891 by A. Villiers [9] are cyclic oligosaccharides obtained by enzymatic digestion of starch. They are made up of glucopyranose units linked by a-1,4 glycosidic bonds. There are three types of natural CDs including a-CD, b-CD and g-CD contain 6, 7 and 8 glucose units, respectively [10]. CDs have a truncated cone shape structure with a hydrophilic outer surface and hydrophobic inner cavity. The numerous hydroxyl groups on the CD structure can form hydrogen bonds with water in aqueous solutions increasing the solubility of CDs in water. The CDs can form inclusion complexes with a wide range of guest compounds by taking up the entire or part of the guest molecule into their hydrophobic cavity via noncovalent interaction, the formation of inclusion complexes improved the physicochemical properties of the guest compounds such as the chemistry stability and aqueous solubility [11e15]. Due to their special properties, the CDs have been widely used as sorbents in extraction fields and as additives in separation fields. In capillary electrophoresis (CE), CDs were frequently used as additive in BGS for separation, especially for enantiomer separation [16]. Kodama et al. [17] used hydroxypropyl-g-CD-modified MEKC to separate hydroxyeicosatetraenoic acid enantiomers. Complete resolution of enantiomers was achieved and the stereochemistry of analytes was evaluated successfully. Wang et al. [18] developed a CD-modified MEKC method for simultaneously separating and determining hirsutine and hirsuteine from Uncaria rhynchophylla and its formulations. Satisfactory resolution of hirsutine and hirsuteine was obtained within 13 min. For enrichment technique in CE, Ghiasvand et al. [19] developed a micelle to CD stacking-MEKC method for the separation and enrichment of cationic, neutral, and chiral compounds with the sensitivity enhancement factors of 171. However, research about using CD for enhancing the sensitivity in on-line concentration technique of CE was still rarely reported. Dendrobium officinale Kimu et Migo (D. officinale), one of the endangered and prized herbs, has been widely used as herbal medicine and functional food in China and other Asian countries for centuries because of its pharmacological and clinical effects [20,21]. The stem of D. officinale named “Tiepi Fengdou” has been used as a traditional Chinese tonic medicine since Qing dynasty [22]. It can nourish yin, benefit the stomach, moisten the lung and prolong life

Fig. 1. The structural formulas of four analytes: erianin, dendrophenol, naringenin, and scoparone.

[22]. Pharmacology studies indicate that D. officinale has diverse pharmacological properties, such as anti-inflammatory [23], antitumor [24], immunomodulatory [25] and hypoglycemic activities [26]. The major active constituents of D. officinale are polysaccharides and phenols. To this day, studies on D. officinale are mainly focused on the polysaccharides. Limited studies have focused on analyzing other compounds such as the phenols. Several detection methods including high performance liquid chromatography [27], near-infrared spectroscopy [28], and ultrahigh performance liquid chromatography-electrospray ionization mass spectrometry [29] were applied for determination of D. officinale in previous studies. In recent years, CE has been an efficient technique for determination of active constituents in natural medicines. It is significant and necessary to establish a sensitive CE method for separation and determination of active compounds in D. officinale. In this study, a CD assisted sweeping-MEKC method is established by dissolving target analytes in CD solution to separate and concentrate erianin, dendrophenol, naringenin, and scoparone. Several experimental factors including the type and concentration of CD, the percentage of methanol, the concentration of sodium dodecyl sulfate (SDS), and injection time of sample solution are evaluated in details. The validation of the method is investigated systematically. Comparison of the proposed method with other traditional methods is performed. Consequently, the CD assisted sweeping-MEKC method is successfully applied to separate and enrich of active compounds in D. officinale.

2. Experimental 2.1. Reagents and materials Phosphoric acid (PA) was obtained from ANPEL Laboratory Technologies (Shanghai) Inc. (Shanghai, China). a-CD, methyl-b-CD, 2-hydroxypropyl-b-CD, g-CD and sodium hydroxide (NaOH) (analytical reagent) were all purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). SDS was supplied by SigmaAldrich Shanghai Trading Co., Ltd. (Shanghai, China). Highperformance liquid chromatography-grade methanol was provided by Merck Darmstadt Ltd. (Darmstadt, Germany). The purified water was acquired from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China). All standards (98%) including erianin, dendrophenol, naringenin, and scoparone were purchased from Shanghai Winherb Medical Technology Co., Ltd. (Shanghai, China). The structural formulas of four analytes are shown in Fig. 1. The 0.22 mm nylon filter was provided by Jinteng Laboratory Equipment Co., Ltd. (Tianjin, China). The Dendrobium officinale Kimura et Migo

Please cite this article as: L.-Q. Peng et al., Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweepingmicellar electrokinetic chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.037

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Fig. 2. Schematic diagrams of the CD assisted sweeping-MEKC model. (A) Injection of a sample plug of a low-pH matrix with CDs into a capillary filled with low-pH BGE. (B) Application of voltage and stacking of analytes. (C) Separation of the stacked analytes by MEKC. And the chromatograms of target analytes obtained from the CD assisted sweepingMEKC (a), conventional sweeping-MEKC (b, c) and MEKC (d).

(D. officinale) produced from Zhejiang province was purchased from a local drugstore (Zhejiang, China). 2.2. Preparation of solutions and samples Stock solutions of PA and SDS were prepared in purified water at concentrations of 0.5 M and 0.2 M, respectively. The pH of 0.5 M PA stock solution was adjusted to 2.5 by using 10 M NaOH. Standard stock solutions of four analytes were prepared by dissolving appropriate amounts of each analyte in methanol at a final concentration of 1000 mg/mL. The working solutions used for sweepMEKC analysis were prepared by diluting the standard stock solutions in 30e130 mM CD in 100 mM PA. The BGSs used for sweeping and separation were prepared by mixing suitable volumes of 0.5 M PA, 0.2 M SDS, methanol and purified water. All solutions were sonicated and filtered through 0.22 mm disposable nylon membranes prior to injection to MEKC system. 1 g of D. officinale was weighted and mixed with 20 mL of methanol, then let it stand for 25 min and sonicated for 40 min. The extract was filtered through 0.22 mm nylon filters. The supernatant of the extract was diluted 10 times with 90 mM CD in 100 mM PA.

HP ChemStation (Agilent) software was used for control and analysis. The detection wavelengths used was set at 205 nm for determining wavelength and 360 nm for reference wavelength. The stacking and separation of analytes were carried out at a voltage of 20 kV and temperature maintained at 30  C. The uncoated fused-silica capillary (50 mm i.d.  360 mm o.d.) with 50 cm in total length and 41.5 cm in effective length used in this study was purchased from the Yongnian Optic Fiber Plant (Hebei, China). Prior to first use, each virgin capillary was conditioned by flushing with 1.0 M NaOH for 10 min, purified water for 10 min, methanol for 5 min and purified water for 5 min. Between two consecutive runs, the capillary was conditioned with 1.0 M NaOH for 1 min, purified water for 2 min, methanol for 1min, and BGS for 5 min prior to injection. Sample solutions were all injected into the capillary by hydrodynamic injection at constant pressure of 50 mbar. The present CD assisted sweeping-MEKC method was compared with conventional sweeping-MEKC (water as sample solvent) and MEKC (BGS as sample solvent). 3. Results and discussion 3.1. The CD assisted sweeping-MEKC mechanism for analytes

2.3. Sweeping-MEKC analysis The MEKC analysis was performed on an Agilent CE system (7100, Palo Alto, CA, USA) equipped with a diode array detector. A

In this work, an on-line sample enrichment technique termed CD assisted sweeping-MEKC was carried out under suppressed cathodic EOF conditions by employing a BGS containing SDS

Fig. 3. The effect of the type of CD (A) and the concentration of CD (B) on the resolution and enhancement efficiency of four target analytes.

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zone migrated to the anode during stacking. When all the CDs in the sample zone were exhausted by complexing with the SDS, the stacking ended. Under the applied voltage, the target analytes were carried to the detector by the SDS micelles and the separation was occurred when the BGS penetrated the stacked zone (Fig. 2C). In MEKC procedure, the effective electrophoretic mobility of analytes was depended on the retention factor (k) with SDS micelles. The k value was higher when the affinity of an analyte to the micelles was stronger. The effective electrophoretic mobility (mep(a)) of analytes is given by Eq. (1) [19]:

 

mep a ¼

   k mep mc 1þk



(1)

where the mep(mc) is the electrophoretic mobility of SDS micelles (mc). The mobility of target analytes is related to the mep(mc) and k. Besides, the length of the swept zone (Lsweep) is approximated by Eq. (2) [1]:

Lsweep ¼

1 Linj 1þk

(2)

where Linj is the injection length. The equation indicates that higher the k value, narrower the swept zone. Additionally, the present CD assisted sweeping-MEKC method (sample in CD solution) with optimized conditions was compared with the sample in water or in BGS without SDS (conventional sweeping-MEKC) and the sample in BGS (MEKC) with other conditions remained the same (Fig. 2). The results indicated that the enrichment efficiency of CD assisted sweeping-MEKC was much higher that other three methods. 3.2. Optimization of CD assisted sweeping-MEKC conditions

Fig. 4. Electropherograms of the optimization of the percentage of methanol (A), a: 10%, b: 20%, c: 30%, d: 40%; the concentration of SDS (B), a: 25 mM, b: 50 mM, c: 75 mM, d: 100 mM, e: 125 mM; and injection time of sample solution (C), a: 3 s, b: 50 s, c: 100 s, d: 150 s, e: 200 s. Analytes: (1) erianin, (2) dendrophenol, (3) naringenin, (4) scoparone.

micelles and methanol with a low pH value. Schematic diagrams of this procedure are shown in Fig. 2. The analytes were prepared in a CD solution with low pH which had similar conductivity with BGS and without adding PS (micelles). The electrophoretic mobility of the charged SDS micelles was faster than the EOF due to the low pH value. The voltage was applied at negative polarity and the detector was set at the anodic side of the capillary. The target analytes injected for a long time were combined with CDs through hostguest interactions in the sample zone (Fig. 2A). When the negative voltage was applied, the SDS micelles in the BGS penetrated the sample zone and interacted with CDs to form the stable CD-SDS complexes [19], then the analytes were released from CDs and interacted with the SDS micelles in the BGS (Fig. 2B). The continued transport and release of analytes as well as the partitioning or interaction of tested analytes with SDS micelles led to the narrowed analytes zones and enrichment of the target analytes. Besides, the SDS micelles collapsed at the boundary when the micelles penetrated the sample zone with CDs, the differential partitioning of the neutral analytes between the intact SDS micelles and CDs in the boundary might have an additional effect on the stacking performance. The analytes were first swept and then stacked by micelleto-cyclodextrin, then swept again. The SDS micelles from the BGS continuously penetrated the sample zone, the stacked analytes

In order to obtain the satisfactory separation and enhancement efficiency, several experimental factors including the type and concentration of CD, the percentage of methanol, the concentration of SDS, and injection time of sample solution which influenced the experimental performance of sweeping-MEKC were investigated in detail in this study. 3.2.1. Type of CD The type of CD is a crucial parameter which has a significant effect on the enrichment and separation efficiency of target analytes in this proposed CD assisted sweeping-MEKC procedure. Several kinds of CDs including a-CD, methyl-b-CD, 2hydroxypropyl-b-CD and g-CD were used in this study to evaluate the effect of the type of CD on the separation and stacking efficiency of target analytes while other experimental conditions were kept in constant (four target analytes were prepared in 50 mM CD in 100 mM PA at concentrations of 3 mg/mL for erianin and scoparone, and 10 mg/mL for dendrophenol and naringenin, respectively, the BGS consisted of 100 mM SDS and 30% methanol in 50 mM PA, and the sample solution was injected under 50 mbar for 50 s). As can be seen from the line graph in Fig. 3A, the highest peak areas of four analytes were obtained when 2-hydroxypropyl-b-CD was applied, and the satisfactory resolutions were also achieved as shown in Fig. S 2A (see supplementary material). It was suspected that the reason for this phenomenon might be that the interactions between the 2-hydroxypropyl-b-CD and SDS micelles were stronger than other CDs due to the breaking of intramolecular ring hydrogen bond and more possibility of reaction with other substance, which led to the narrowed stacking zones. Besides, the water solubility of 2-hydroxypropyl-b-CD was better than other three CDs, and 50 mM was almost the maximum soluble concentration of other three CDs. For further investigation of CD

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Table 1 Linear regression data, limits of detection (LODs) and limits of quantification (LOQs) of the investigated compounds. Analyte

Calibration curve

LOD

LOQ

ng/mL

ng/mL

21 40 36 13

71 132 118 44

Calibration levels (n ¼ 6) r2

Slopes

Intercepts

Linear ranges

mg/mL Erianin Dendrophenol Naringenin Scoparone

0.999 0.999 0.996 0.994

59.82 13.57 20.89 40.94

1.252 0.621 1.836 0.542

0.15e3 0.5e10 0.5e10 0.15e3

concentration, 2-hydroxypropyl-b-CD was more suitable. Therefore, 2-hydroxypropyl-b-CD was used in this study. 3.2.2. Concentration of CD The concentration of CD in sample solution is an important factor in the CD assisted sweeping-MEKC analysis for enhancement and separation of tested analytes. The influence of CD concentration on the resolution and enhancement of analytes was tested by varying the concentrations from 30 to 120 mM with other conditions remaining unchanged (target analytes were prepared in 100 mM PA with different CD concentrations at concentrations of 3 mg/mL for erianin and scoparone, and 10 mg/mL for dendrophenol and naringenin, respectively, the BGS consisted of 100 mM SDS and 30% methanol in 50 mM PA, and the sample solution was injected under 50 mbar for 50 s). The results are displayed in Fig. 3B and Fig. S 2B. When the concentration of CD increased from 30 to 90 mM, the migration times and peak areas of target analytes were increased gradually. The possible reason might be that the weak interaction between analytes and CDs with less amount of CD would lead to a lack of release procedure which had a negative effect on the stacking efficiency. However, further increasing the CD concentration from 90 to 130 mM, the peak intensities of target analytes were decreased slightly. This might be due to the fact that the lower conductivity of sample solution with higher CD concentration would produce an enhanced electric field in the sample zone which was stronger than that in BGS region. The concentration of SDS penetrated into the sample zone was lower than its concentration in BGS which led to a decreased retention factor (k) between the analytes and SDS micelles resulting in the decreased focus effect. As a result, CD concentration of 90 mM which provided baseline separation and highest peak areas of analytes was selected as the optimal concentration of CD. 3.2.3. Percentage of methanol The interactions between the SDS micelles in BGS and analytes can generate high stacking efficiency in sweeping-MEKC but may result in poor separation efficiency. Adding organic additives to the BGS can improve separation selectivity in MEKC due to their ability to change the solubility of target analytes in the solution and cause different affinity between PSP and analytes. To obtain the best resolution and concentration efficiency for target analytes, the influence of the percentage of methanol on the separation efficiency was investigated by changing its concentration to 10%, 20%, 30%, and 40% (v/v), respectively, with other conditions remained constant (target analytes were prepared in 90 mM CD in 100 mM PA at concentrations of 3 mg/mL for erianin and scoparone, and 10 mg/mL for dendrophenol and naringenin, respectively, the BGS consisted of 100 mM SDS and different percentage of methanol in 50 mM PA, and the sample solution was injected under 50 mbar for 50 s). The experimental results are showed in Fig. S1A and Fig. 4A. As can be seen from the charts, the peak areas, resolutions and migration

Fig. 5. Electropherograms of four analytes obtained from the CD assisted sweepingMEKC (A-a), conventional sweeping-MEKC (A-b, c) and MEKC (A-d). Target analytes were prepared in 90 mM CD in 100 mM PA (a), in BGS without SDS (b), in water (c) and in BGS (d) at concentrations of 3 mg/mL for erianin and scoparone, and 10 mg/mL for dendrophenol and naringenin, respectively. BGS consisted of 100 mM SDS and 30% methanol in 50 mM PA, and the sample solution was injected under 50 mbar for 100 s. Electropherograms of the D. officinale sample extracts obtained by the injection time of 100 s (B-a) and 3s (B-b). Other conditions were the optimal conditions. Analytes: (1) erianin, (2) dendrophenol, (3) naringenin, (4) scoparone.

times of target analytes were increased with the increasing of the concentration of methanol. Satisfactory baseline separation and enrichment efficiency were obtained when 30% (v/v) of methanol was used. The reason for the increase of migration time with the increase of methanol concentration was because of the increased partition of analytes in the solution phase. However, further increasing the methanol content to 40% (v/v), the migration time of analytes became too long and the peaks became broader. Consideration of all the conditions, 30% (v/v) of methanol was selected as the optimal concentration in the BGS for the subsequent experiments. 3.2.4. Concentration of SDS The concentration of SDS in BGS has a significant influence on the resolution and enhancement performance, because it can affect the electroosmotic flow, conductivity of BGS and the k of analytes by altering the content of analytes partitioned into micelles. To assess the effect of SDS concentration on the stacking and separation efficiency of target analytes, several experiments were carried out by adding different SDS concentrations ranging from 25 to 125 mM to the BGS. Other conditions were kept unchanged (target analytes were prepared in 90 mM CD in 100 mM PA at concentrations of 3 mg/mL for erianin and scoparone, and 10 mg/mL for dendrophenol and naringenin, respectively, the BGS consisted of different concentration of SDS and 30% methanol in 50 mM PA, and the sample solution was injected under 50 mbar for 50 s). The results in Fig. S1B and Fig. 4B demonstrated that the migration time and peak area of analytes were decreased with the increased SDS concentration. The decreased migration time of target analytes caused by the increased SDS content might be due to the fact that more tested analytes were partitioned into the micellar phase and

Please cite this article as: L.-Q. Peng et al., Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweepingmicellar electrokinetic chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.037

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Table 2 The content, average recovery and reproducibility of samples. Analyte

Erianin Dendrophenol Naringenin Scoparone

Content

Recovery%

Reproducibility (RSD%) n ¼ 3

Dendrobium officinale Kimura et Migo from Zhejiang (mg/g)

Dendrobium officinale Kimura et Migo from Zhejiang

Retention time

Peak area

e e 0.227 0.120

80.5 93.8 85.10 94.47

0.68 0.46 0.58 0.26

2.32 2.21 2.08 1.47

migrated toward the positive electrode with a faster electrophoretic mobility by SDS micelles. The peak areas of anaytes were increased when the SDS concentration was decreased, the reason for this phenomenon might be that the lower SDS content resulted in the lower conductivity and enhanced electric field in BGS region which led to the stacked SDS in the boundary of sample zone and BGS. Thus, the target analytes were accumulated by the stacked SDS micelles and resulting in a narrowed sample zone. The highest peak areas of analytes were obtained at the SDS concentration of 25 mM, however the analysis time was prolonged obviously and the peak shapes were being broadened. When the SDS content was set at 50, 75, and 125 mM, the baseline separation between the peak of target analyte and impurity peak could not be achieved. High separation efficiency and good peak shape were obtained when the concentration of SDS was at 100 mM with a relatively short analysis time. Given consideration to the separation efficiency, short analysis time and stacking performance, 100 mM SDS was selected for further experiments. 3.2.5. Sample injection time The injection time of sample solution is an important factor for achieving a good peak shape, satisfactory separation efficiency and high stacking performance of target analytes in sweeping-MEKC, as the injection time can influence the amounts of sampling directly. The effect of injection time on the enhancement and separation of four target analytes was evaluated in the range of 50e200 s, with other conditions remained constant (target analytes were prepared in 90 mM CD in 100 mM PA at concentrations of 3 mg/mL for erianin and scoparone, and 10 mg/mL for dendrophenol and naringenin, respectively, the BGS consisted of 100 mM SDS and 30% methanol in 50 mM PA, and the sample solution was injected under 50 mbar for different times). The results in Fig. S1C and Fig. 4C demonstrated that the migration time and peak intensity of target analytes were increased with the increase of injection time from 3 to 200s. Poor separation efficiency between the peaks of target analytes and the peaks of impurities as well as relatively long analysis time were obtained at the injection time of 150 and 200 s, while low enrichment efficiency and some overlaps between analyte peak and impurity peak was observed at the injection time of 50 s. The peaks of four analytes were nearly undetectable when the injection time was 3 s. In terms of peak areas, the SEFs were in the range of 28.5e46.8 compared the injection time of 100 and 3 s. Additionally, the peak shape and current became unstable when the injection time was above 150 s. Complete baseline separation of target analytes and satisfactory stacking efficiency were achieved with relatively short time when the injection time of 100 s was applied. Consequently, 100 s was chosen as the optimized sample injection time based on a consideration of the short analysis time, satisfactory stacking efficiency and good resolution. 3.3. Method validation To evaluate the analytical performance of the proposed CD assisted sweeping-MEKC method for simultaneous separation and concentration of neutral analytes in D. officinale, the validation of

this method was performed in terms of calibration curves, linear ranges, limits of detection (LODs) and quantification (LOQs). All the experiments were implemented in triplicate under the above optimized experimental conditions (target analytes were prepared in 90 mM CD in 100 mM PA at concentrations of 3 mg/mL for erianin and scoparone, and 10 mg/mL for dendrophenol and naringenin, respectively, the BGS consisted of 100 mM SDS and 30% methanol in 50 mM PA, and the sample solution was injected under 50 mbar for 100 s). The parameters including the calibration levels, LOD and LOQ are listed in Table 1. The calibration curves were constructed by plotting the peak areas of target analytes against the corresponding concentration of analytes. A series of standard solutions used for calibration curves were prepared at the linear concentration range of 0.15e3 mg/mL for erianin and scoparone, and 0.5e10 mg/mL for dendrophenol and naringenin, respectively. As can be observed from Table 1, determination coefficient (r2) ranged from 0.994 to 0.999 for all the four analytes, and good linearity of the method was obtained. The LODs and LOQs were in the range of 13e40 ng/mL and 44e132 ng/mL, respectively, which were calculated as the analyte concentration yielding a signal-to-noise ratio of 3 and 10, respectively. These results reflected that the proposed method had good linearity and satisfactory sensitivity for separation and enrichment of target analytes. Additionally, the present CD assisted sweeping-MEKC method (90 mM CD in 100 mM PA as sample solvent) (Fig. 5 A-a) was compared with the use of BGS without SDS or water as sample solvent (conventional sweeping-MEKC) ((Fig. 5 A-b,c) and using BGS as sample solvent (MEKC) (Fig. 5 A-d) with other conditions remained the same. Four electropherograms in Fig. 5A demonstrated that the enhancement efficiency of the proposed method was much higher that other three methods.

3.4. Analysis of samples The proposed CD assisted sweeping-MEKC method was applied to analyze the active compounds in D. officinale under the optimum conditions established above. Fig. 5B shows the chromatograms of the sample of D. officinale injected for 100 s (Fig. 5Bea) and injected for 3 s (Fig. 5Beb) under the same conditions. The result indicated that the present method had obvious stacking efficiency for target analytes. Naringenin and scoparone were detected in the real sample. The contents of the two analytes displayed in Table 2 were determined to be 0.227 and 0.120 mg/g in the sample of D. officinale. The accuracy of this method was validated by performing the recovery study. The recoveries of four analytes in the sample were determined by adding 1.5 mg/mL of erianin and scoparone, and 5 mg/mL of dendrophenol and naringenin into the sample solution. The average recoveries of all the analytes were in the range of 80.5e94.47%. The reproducibility of the present method was obtained by determining three parallel replicate analyses. The RSDs of the retention time and peak areas were ranged from 0.26 to 0.68% and 1.47e2.32%, respectively. These results demonstrated that the established method possessed good accuracy and repeatability for separation and enhancement of the active compounds in real samples.

Please cite this article as: L.-Q. Peng et al., Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweepingmicellar electrokinetic chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.037

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4. Conclusions A simple, effective and sensitive online CD assisted sweepingMEKC method was developed and validated for separation and concentration of erianin, dendrophenol, naringenin and scoparone in D. officinale with DAD detection. Several crucial factors which influenced the separation and stacking performance of target analytes including the type and concentration of CD, the percentage of methanol, the concentration of SDS, and injection time of sample solution were evaluated systematically. Under the optimized conditions, effective concentration and separation of target analytes in real sample were achieved with SEFs in the range of 28.5e46.8. Satisfactory recovery and repeatability were also obtained. Compared with the conventional sweeping-MEKC (BGS without SDS or water as sample solvent) and MEKC method (BGS as sample solvent), the established online CD assisted sweeping-MEKC method (90 mM CD in 100 mM PA as sample solvent) offered higher concentration sensitivity. Thus, the present sensitive and accurate method was applicable to separate and concentrate neutral analytes in real samples.

[5]

[6]

[7]

[8]

[9] [10]

[11]

[12] [13]

Declaration of competing interest [14]

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.

[16]

CRediT authorship contribution statement

[17]

Li-Qing Peng: Writing - original draft, Writing - review & editing, Data curation. Xin Dong: Investigation, Validation. XiaoTing Zhen: Data curation, Validation. Juan Yang: Project administration, Validation, Formal analysis. Yan Chen: Software, Validation. Shu-Ling Wang: Supervision. Tian Xie: Funding acquisition. Jun Cao: Conceptualization, Methodology.

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Acknowledgements

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This study was supported by the Public Welfare Research Project of Zhejiang Province (LGF18H280006), National Natural Science Foundation of China (81573552), Hangzhou Social Development of Scientific Research projects (20191203B13), Hangzhou 131 Middleaged and Young Talent Training Plan (China, 2017).

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2020.01.037.

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Please cite this article as: L.-Q. Peng et al., Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweepingmicellar electrokinetic chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.01.037