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Sweeping with Electrokinetic Injection and Analyte Focusing by Micelle Collapse in Two-Dimensional Separation via Integration of Micellar Electrokinetic Chromatography with Capillary Zone Electrophoresis Zhaoxiang Zhang, Xiuzhen Du, and Xuemei Li* State Key Laboratory Base of Eco-chemical Engineering, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China ABSTRACT: A novel integrated concentration/separation approach involving online combination of sweeping with electrokinetic injection and analyte focusing by micelle collapse (AFMC) with heart-cutting twodimensional (2D) capillary electrophoresis (CE) in a single capillary was developed for analysis of Herba Leonuri and mouse blood samples. First, a new sweeping with an electrokinetic injection preconcentration method was developed to inject a large volume sample solution and significantly enhance detection sensitivity. Then, the preconcentration scheme was integrated to the 2D-CE to provide significant analyte concentration and extremely high resolving power. The sample was preconcentrated by sweeping with electrokinetic injection and separated in first dimension micellar electrokinetic chromatography (MEKC). Then, only a desirable fraction of the first dimension separation was transferred into the second dimension of the capillary by pressure and further analyzed by capillary zone electrophoresis (CZE) acting as the second dimension. As the key to successful integration of MEKC and CZE, an AFMC step was integrated between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The injected sample plug lengths for flavonoids under 15 kV for 60 min were experimentally estimated as 546 cm. The dual concentration methods resulted in the increased detection factors of 6000-fold relative to the traditional pressure injection method. The relative standard deviation (RSD) values of peak height, peak area, and migration time were 2.7-4.5%, 1.9-4.3%, and 4.7-6.8% (n = 10), respectively. The limits of detection (S/N = 3) were in the range of 7.3-36.4 ng/L, and the theoretical plate numbers (N) were in the range of 1.7-4.3 104 plates/m. This method has been successfully applied to determine flavonoids in Herba Leonuri and postdosing mouse blood samples. The pharmacokinetic study also demonstrated that the proposed concentration/separation method was convenient and sensitive and would become an attractively alternative method for online sample concentration and separation in complex samples.
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apillary electrophoresis (CE) is an effective separation tool and has been used in different areas of chemistry, biology, medicine, pharmaceutics, etc. However, the sensitivity and peak capacity is limited for the analysis of low abundance and complex samples. Online sample preconcentration has become a vital part in CE in order to obtain a decent signal for analyte quantitation. A number of online concentration strategies have been developed, for example, field amplified sample stacking,1 isotachophoresis (ITP),2 dynamic pH junction,3 sweeping,4 etc. Sweeping is an effective and convenient way for doing online sample preconcentration in micellar electrokinetic chromatography (MEKC). The usual procedure for sweeping includes a hydrodynamic injection step followed by the subsequent sweeping and separation processes. The limitation is that the maximum volume of sample that can be injected can only be as high as the total capillary volume and improvements in sensitivity are, therefore, very limited. To overcome this sweeping barrier, electrokinetic injection has been developed. Terabe’s group developed cation- and anion-selective exhaustive injection r 2011 American Chemical Society
(C/ASEI) and sweeping to improve the sensitivity by nearly 1 million-fold.5,6 Palmer et al. developed an electrokinetic stacking injection process for sweeping with sodium dodecyl sulfate (SDS) for neutral analytes, which has the capability of injecting 7-fold the effective capillary length of sample solution.7 Gong et al. used electroosmotic flow (EOF) to balance electrophoretic migration of cationic micelles with an improvement in the sensitivity of 4500-fold.8 A similar system, described as “electrokinetic accumulation”, was presented by Horakova et al.9 Weak acids were continuously electrokinetically injected into a low-pH electrolyte and accumulated on the sample/electrolyte interface. Mobilization of the neutral analytes after injection was achieved via sweeping, and an improvement in sensitivity of 4600 was obtained with a 120 min injection. Another interesting online sample preconcentration, which was named as “analyte focusing Received: September 3, 2010 Accepted: December 28, 2010 Published: January 19, 2011 1291
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Analytical Chemistry by micelle collapse”, was reported by Quirino et al.10,11 The focusing mechanism was based on the transport, release, and accumulation of molecules bound to micelle carriers that are made to collapse into a liquid phase zone. The accumulated analytes was subsequently separated by MEKC10 or capillary zone electrophoresis (CZE).11 More than 2 orders of magnitude improvement in detection sensitivity for model steroidal compounds using SDS micelles was demonstrated. In summary, the improvement in sensitivity is mainly influenced by the amount of sample that can be injected and focused. Moreover, if the stacking boundary continues to move during injection, there is a practical limitation imposed on the extent of injection since a part of the capillary must remain for subsequent separation. Counterbalancing the electrophoretic movement of a stacking boundary is a potential way in which longer electrokinetic injections can be made while ensuring that sufficient capillary length remains for separation. For complex samples, two-dimensional (2D)-CE has aroused great interest in recent years due to the high separation power and peak capacity. Dovichi’s group developed a cross interface with aligned capillaries for proteomic analysis by 2D-CE.12-17 Their setup combined submicellar CE at pH 7.5 with electrophoresis at pH 11.1,12 capillary sieving electrophoresis (CSE) with MEKC,13-16 and laser-induced fluorescence detection. Then, a CE-microreactor-CE-MS/MS instrument was presented for a fully automated bottom-up approach to protein characterization.17 Sheng and Pawliszyn developed a comprehensive 2D separation based on coupling MEKC with capillary isoelectric focusing (cIEF) by a 10-port valve with a two conditioning loop interface for protein digest analysis.18 A microdialysis interface was designed by Zhang’s group to couple cIEF with capillary gel electrophoresis (CGE),19 CZE,20 and CSE.21 A similar interface was employed by Mohan and Lee for proteome analysis by cIEF-CZE coupling with a transient ITP step between the two dimensions.22,23 Further, an etched fusedsilica porous junction was constructed by Zhang’s group for coupling of cIEF with CZE.24 Zhang and El Rassi used a nanoinjector valve for coupling of cIEF to capillary electrochromatography (CEC) for separation of proteomics.25 Sahlin reported a CZE-MEKC separation of peptides using tangentially connected capillaries.26 Weak basic drug mixtures were subjected to CZE-MEKC by Zhang et al. using a microhole interface for connecting the two separation modes.27 In summary, the success of the above-mentioned 2D-CE separations lies in the construction of a reliable interface to switch the effluents from the firstdimension column to the second one. However, the dead volume and sample loss could not be avoided at the interface, which decreases the resolution of 2D separation. Cottet’s group demonstrated the interest of an alternative approach based on the use of a single capillary for performing heart-cutting 2D-CE.28-30 The methodology has been applied for the charge- and size-based characterization of synthetic polymer mixtures by CZECGE28,29 and then used for online purification and separation of 12 derivatized amino acids using CZE-MEKC separations.30 Recently, the heart-cutting 2D-CE in a single capillary has been applied for blood samples by Zhang et al. with electrochemical detection.31 However, the use of hydrodynamic flow between the two dimensions reduced peak efficiency due to peak broadening by Taylor dispersion.30,31 To limit the loss in peak efficiency related to the hydrodynamic mobilization, a 10 μm id capillary and high sensitive capacitively coupled contactless conductivity detector were used.32
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In a previous work, we incorporated sample preconcentration and 2D CZE-MEKC separation for simultaneous enhancement of resolution and sensitivity.33 CSEI and tITP preconcentration methods were used to introduce a large amount of analytes into the first dimensional capillary and simultaneously narrow the long length of analyte band. At the same time, stacking was utilized between the two dimensions to avoid the primarily preconcentrated and separated analyte band diffusion at the interface. This methodology demonstrated the availability of the online concentration method to overcome the problems in 2DCE such as dead volume, peak broadening, low sensitivity, etc. In this paper, we developed a new sweeping with an electrokinetic injection preconcentration scheme with anionic surfactants. Keeping the surfactants at a stationary state, analyte solution was continually injected into capillary and sweeping by SDS micelle for essentially a large volume. Then, the preconcentration scheme was incorporated into the heart-cutting 2D MEKCCZE separation for simultaneous enhancement of sensitivity and separation power of CE. To release analytes from the interior of SDS micelle and to counteract the primarily preconcentrated and separated analyte band diffusion during the hydrodynamic mobilization process, an analyte focusing by micelle collapse (AFMC) method was utilized between the two dimensions. Herba Leonuri was chosen as model sample due to its significance and complexity. To the best of our knowledge, this work represented the first demonstration of heart-cutting 2D-CE couple with sweeping with electrokinetic injection and AFMC for a Herba Leonuri sample as well as pharmacokinetic study.
’ EXPERIMENTAL SECTION Chemicals. Apigenin, kaempferol, quercetin, phlorizin, hyperoside, quercitrin, rutin, hesperetin, isorhamnetin, and Herba Leonuri were obtained from Zelang medicine Ltd. (Nanjing, China). The isorhamnetin was used as an internal standard. Sodium hydroxide, sodium tetraborate, sodium acetate, sodium carbonate, ethyl acetate, phosphate, etc. were obtained from Shanghai Chemical Plant (Shanghai, China). SDS was from Sigma. Standard stock solutions of flavonoids at concentration of 1.0 mg/mL were prepared in ethanol and diluted to the desired concentration with the running buffer just prior to use. All standard solutions were kept in a refrigerator and could be stable for 2 months. The phosphate buffer solution was prepared by adjusting the acidity of sodium dihydrogen phosphate (concentrations ranging from 5 to 150 mM) solution to a desired pH with concentrated sodium hydroxide. All aqueous solutions were prepared with double-distilled water and filtered through 0.22 μm cellulose acetate membrane filters (Shanghai Yadong Resin Co., Shanghai, China) before use. Apparatus. 2D-CE separation and concentration was performed in a model MPI-A CE setup (Xi’an Remax Electronics Inc., Xi’an, China), equipped with a high-voltage power supplier (0-30 kV) for electronic sampling and separation, and an electrochemical (EC) potentiostat (0-2.5 V) for EC detection. The anodic high-voltage end was isolated in a Plexiglas box fitted with an interlock for operator safety. A 60 cm fused-silica capillary with dimensions of 50 μm I.D. and 375 μm O.D. (Yongnian Optical Conductive Fiber Plant, Yongnian, China) was used as concentration and separation capillary. The oncolumn EC detection system, including a field decoupler joint and a three-electrode system, was similar to that described elsewhere.31 Briefly, 5 mm of capillary wall started 1.5 cm from 1292
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Analytical Chemistry the capillary outlet was etched by laser to construct the field decoupler joint. The field decoupler joint and a Pt electrode serving as the ground electrode of electrophoresis were inserted into the ground cell filled with buffer solution. The threeelectrode system consisted of a 20 μm diameter Pt wire as working electrode, Ag/AgCl as reference electrode, and a Pt wire as auxiliary electrode. The Pt working electrode was inserted into the capillary outlet at a depth of 0.5 cm and adjusted to align with the decoupler and separation capillary. Preparation of Herba Leonuri Sample. After being air-dried and crushed into powder, six aliquots of 0.200 g Herba Leonuri was extracted with 20 mL of ethanol by sonication for 30 min, respectively. The extracts were filtered with a 0.22 μm acetic cellulose membrane and, then, concentrated to about 2.0 mL at 50-60 C. These extracts were combined and then stored in the refrigerator. The extract was diluted to a desired concentration with sodium phosphate buffer just prior to concentration and 2D-CE separation analysis. Preparation of Mouse Blood Sample. Six adult mice were housed in a cage with a 12-h light/dark cycle. Food and water were available at discretion. The mice were treated with Herba Leonuri extracts at an oral dose of 5 mL/kg of body weight. Four microliters of blood was withdrawn at certain time intervals from the tail vein using a serial bleeding design34 and, then, was mixed in an Eppendorf tube with 20 μL of ethanol, followed by the addition of 20 μL of internal standard at a concentration of 10.0 μg/L. The blood samples were purified by liquid-liquid extraction35 as follows: Blood samples were mixed with 1 mL of 0.1 M Na2CO3, and, then, with 3 mL of ethyl acetate. The tubes were capped, and the mixtures were vortexed for 5 min. The tubes were centrifuged for 10 min at 3000 rpm. The top organic layers were transferred to clean tubes and, then, concentrated to about 10 μL at 50-60 C. The purified blood samples were diluted to 50 μL with 90 mM sodium phosphate buffer solution (pH 5.0). The samples were stored at 4 C and were equilibrated to room temperature prior to injection. Aliquots of supernatants were injected for concentration and 2D MEKC-CZE separation analysis. Hyphenation of Sweeping with Electrokinetic InjectionMEKC and AFMC-CZE Procedure. The capillary was flushed daily in the order of H2O (1 min), 1.0 M NaOH (15 min), and H2O (1 min) and conditioned with buffer 1 (5 mM SDS in 90 mM sodium phosphate buffer, pH 5.0) for 10 min by pressure. Between two runs, the capillary was conditioned with buffer 1 for 6 min. The main steps of sweeping with electrokinetic injectionMEKC and AFMC-CZE were illustrated in Figure 1. In step 1, the capillary was initially filled with buffer 1. Flavonoids are phenolic substances, their pKas are larger than 7.0.36 Therefore, the flavonoids are neutral compounds when the pH is less than 7.0. The Herba Leonuri sample was electrokinetically pumped into the capillary with EOF at 15 kV and captured by the SDS micelle from buffer 1 (Figure 1a). The electrophoretic velocity (Vep) of SDS micelle was the reverse of electroosmotic velocity (Veo), and the migration velocity of the micelle is zero at pH 5.0.37 Consequently, the SDS micelle at the capillary inlet stayed in a stationary state with an apparent velocity of zero. The slow EOF at pH 5.0 and the stationary state of SDS micelle was maintained for up to 60 min of the sample injection. After 60 min for sweeping with electrokinetic injection preconcentration, the sample and buffer 1 were all exchanged with the MEKC separation buffer (5 mM SDS in 90 mM sodium phosphate buffer, pH 7.0). The same voltage was
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Figure 1. Evolution of sweeping with electrokinetic injection and AFMC with 2D MEKC-CZE separation. Step 1: sweeping with electrokinetic injection preconcentration and MEKC separation of sample in the first dimension (a-c); Step 2: elimination of interfering fractions (fraction A and C) and return of the fraction of interest (fraction B) to the inlet end of the capillary (d); Step 3: AFMC and CZE separation of the purified sample in the second dimension (e,f). Veo is velocity of electroosmotic flow. Vep is electrophoretic velocity of SDS micelle. Vhf is hydrodynamic flow. Xb is the boundary which separates the sample zone and the left CZE buffer zone. MDZ is the zone closest to Xb in the left CZE buffer solution. Buffer 1 is 5 mM SDS in 90 mM sodium phosphate buffer, pH 5.0 (conductivity κ = 2.54 S 3 cm-1); MEKC buffer is 5 mM SDS in 90 mM sodium phosphate buffer, pH 7.0 (conductivity κ = 3.86 S 3 cm-1); CZE buffer is 40 mM sodium phosphate buffer, pH 8.5 (conductivity κ = 1.71 S 3 cm-1). The pink circle, yellow circle, and red circle represent the components in the Herba Leonuri sample; the star represents the SDS micelle.
applied and hydroxyl ions in MKEC buffer solution entered the capillary from the cathode side and migrated toward the anode (Figure 1b), resulting in an increase of the EOF. The stationary state of SDS micelle was destroyed, and the micelle was pumped toward the detection end since the Veo was larger than the Vep of SDS micelle. Eventually, the pH in capillary was equal to that of MEKC buffer, and the Herba Leonuri sample was separated by MEKC in the first dimension (Figure 1c). During this separation stage, the sample was separated into three main fractions. The first one (fraction A) corresponded to the undesirable hydrophobic species such as secoiridoid glycosides, diterpenoids, the cationic alkaloids, etc. The fraction (B) was related to the flavonoid compounds, and the last one (fraction C) was some hydrophilic neutral species that comigrated with EOF. At the end of step 1, the unwanted fraction C was evacuated out of capillary by the outlet end. Once the fraction B reached detection end, the EC response signal appeared and the separation voltage was stopped immediately. In step 2, the detection cell was changed with CZE buffer (40 mM sodium phosphate buffer, pH 8.5), and a 2.0 psi nitrogen pressure was applied to the detection cell. The unwanted fraction A was evacuated out of the capillary by the inlet end. At the same time, the CZE buffer solution entered the capillary by hydrodynamic flow directed from the outlet to 1293
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Figure 2. Electropherograms showing the monodimensional separation of Herba Leonuri sample by CZE (a) or by MEKC (b) with oncolumn EC detection. Experimental conditions: fused-silica capillary, 50 μm I. D. 375 μm O. D., 60 cm length; separation voltage, 15 kV; detection potential, 0.85 V, the Herba Leonuri extract solution (without dilution) was electrokinetically injected at 15 kV for 8 s. (a) CZE separation of Herba Leonuri extract, running buffer, 40 mM sodium phosphate buffer (pH 8.5). (b) MEKC separation of Herba Leonuri extract, running buffer, 5 mM SDS in 90 mM sodium phosphate buffer (pH 7.0). Peak identification: 1, phlorizin; 2, kaempferol; 3, hesperetin; 4, apigenin; 5, rutin; 6, hyperoside; 7, quercitrin; 8, quercetin.
the inlet end of the capillry (Figure 1d). For a 50 μm I. D. capillary, the linear velocity of hydrodynamic flow was 0.2 cm 3 s-1. The fraction B was pushed to the inlet end by the bulk hydrodynamic flow after 297.5 s. To avoid any loss in sample from the inlet end of the capillary at the end of step 2, the nitrogen pressure at the detection end was stopped after 294 s. The very small amount of MEKC buffer in capillary was not influenced the second dimension separation. The partial separated analytes in fraction B (see Figure 2b) recombined again during the hydrodynamic mobilization step due to dispersion.30 In step 3, the capillary inlet reservoir was changed with CZE buffer and 15 kV separation voltage was applied. The high electrophoretic mobility anionic phosphate and SDS micelle moved in the direction of the anode relative to the Xb boundary (Figure 1e). The zone closest to Xb in the left CZE buffer solution was called the micellar dilution zone (MDZ).10,11 At the MDZ, the phosphate anions and SDS micelles from sample zone progressively replaced the original phosphate anions of the left CZE buffer solution. Initially, the MDZ was completely filled by high electrophoretic mobility phosphate anions from the sample zone. Then, SDS micelles moved into this zone, and its concentration increased from an initial value of zero. The approximated critical micelle concentration (cmc) of SDS in the systems we used was 3 mM measured by the conductivity method. Therefore, the SDS micelle in MDZ collapsed due to the being concentration less than its cmc. Subsequently, analyte molecules bound hydrophobically to the interior of the micelle were released to the MDZ. With more and more micelles from the sample zone reaching the MDZ and collapsing, neutral analytes of fraction B were all transported and accumulated to the boundary. After the AFMC, the neutral analytes were negatively charged in the CZE buffer and separated by the CZE mode (Figure 1f).
’ RESULTS AND DISCUSSION CE Separation of Herba Leonuri Sample. Herba Leonuri, a commonly used medicinal herb in traditional Chinese medicine,
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has the effects of invigorating blood circulation and regulating the menstrual function, dissolving blood stasis and promoting tissue regeneration, enhancing urine excretion and reducing swelling, and therefore, is used to treat such diseases as menoxenia, dysmenorrheal, dystocia, and postpartum blood stasis, to name only a few. Flavonoids including Apigenin, kaempferol, quercetin, phlorizin, hyperoside, quercitrin, rutin, hesperetin, and so on are one of the largest groups of naturally occurring phenols in Herba Leonuri and have antiinflammatory, antitumor, antivirus, antibacteria, and antioxidation functions.38 Xu et al. determined that flavonoids were in Herba Leonuri by CZE.39 Since the Herba Leonuri extract was diluted with running buffer prior to analysis, there were only four flavonoid peaks observed from the electropherogram. In this experiment, the Herba Leonuri extract was directly introduced into the capillary without dilution; the typical electropherogram was shown in Figure 2a. The flavonoid compounds in the Herba Leonuri sample were accurately identified by comparing their migration times with those of standards and by adding a pure standard to the sample. From the electropherogram, we could see that a number of interfering ingredients, noted with asterisks in Figure 2a, comigrated with the solutes of interest. These undesirable compounds were related to some alkaloids, diterpenoids, secoiridoid glycosides, and organic acids that existed in the Herba Leonuri sample in a large quantity. Although these compounds were not all identified, some examples of Leonurine hydrochloride, ferulic acid, etc. were identified by adding a pure standard to the sample. Except for some alkaloids such as Leonurine hydrochloride, most of the interfering compounds were neutral or anionic in the running buffer and comigrated with the flavonoid compounds. Furthermore, some flavonoid compounds have been separated by MEKC with SDS micelle40 or CEC41 due to hydrophobic interactions with micellar pseudostationary phase or silica-C18 material. The separation of target analytes in a Herba Leonuri sample was attempted by MEKC in a sodium phosphate-SDS background electrolyte. A typical electropherogram was shown in Figure 2b. The SDS micelles interacted strongly with the hydrophobic secoiridoid glycosides, diterpenoids, and cationic alkaloids (peaks 6*-10*) and weakly with the hydrophilic neutral species (peaks 1*-5*). The separation of the target analytes was better by CZE compared to MEKC, and the quercitrin (peak 7 in Figure 2a) was not detected by MEKC. However, the flavonoids were nicely isolated from the interfering ingredients by MEKC (Figure 2b). Therefore, the design of heart-cut 2D-CE with online preconcentration was motivated by the desire to analyze Herba Leonuri samples by evacuating the interfering species in a first dimension with MEKC mode and, next, to separate the flavonoid in a second dimension with CZE mode. The main idea of online integration of sweeping with electrokinetic injection and AFMC with 2D MEKC-CZE separation was illustrated in Figure 1. Optimization of the Sweeping with Electrokinetic Injection Preconcentration and MEKC Separation. MEKC was employed as the first dimension to separate the Herba Leonuri sample. In order to optimize the MEKC conditions, buffer solutions at different concentrations and pH values, including sodium phosphate, sodium borate, sodium acetate, sodium citrate, sodium tartrate, etc., were examined for separation of the Herba Leonuri sample. The results showed that sodium phosphate buffer solution was more suitable because the peak-topeak resolution (R) of flavonoids and interfering components excel more than those of other buffer solutions. The relationships 1294
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of resolution to the concentration of sodium phosphate (varied from 5 to 150 mM) and the buffer pH (ranging from 3.0 to 10.0) were tested. The best resolution was obtained using 90 mM phosphate at the pH range between 6.0 and 7.0. SDS micelle (3-40 mM) was added to 90 mM phosphate (pH 7.0) buffer solution to carry out MEKC. As higher concentrations of SDS were added to buffer solution, improvement in the resolution was observed. When the concentration of SDS was 4 mM, the flavonoids and interfering components could be separated on baseline. When the concentration of SDS was increased to 5 mM, larger resolution between the flavonoids and interfering components was observed. Further increase of the SDS concentration, the migration time, and current were observed to obviously increase in spite of improvement in the resolution. To eliminate interfering components out of capillary and avoid loss in sample from the inlet and outlet end of the capillary, as well as consider the subsequent AFMC between the two dimensions, 5 mM SDS in 90 mM phosphate was used in subsequent studies. To obtain higher detection sensitivity while maintaining resolution during the separation, a sweeping with electrokinetic injection scheme was developed for preconcentration in MEKC. The SDS micelles are negatively charged, so they have anodic Vep under the applied voltage. When the sign of the migrating direction was taken into account, the relationship between the Veo, Vep, and the migration velocity of the SDS micelle (Vmc) is given as V mc ¼ V eo þ V ep
ð1Þ
The sign of each velocity is defined as positive when the migration is toward the negative electrode. When Vmc = 0, the micelle is at a stationary state although the bulk flow is still on going. The EOF is low at pH 5.0, and thus, a longer injection time is necessary to introduce a larger volume of sample inside the capillary. However, the limited retention factor (k) of analytes in the micellar phase limits the large-volume sweeping since the swept zone is controlled by eq 2,42 1 lsweep ¼ ð2Þ linj 1þk where lsweep and linj are the lengths of the swept zone and the injected sample solution, respectively. When the interaction between the analytes and micelles is too weak, the analytes may “leak” from the concentrated zone and flow to the detection end during the sweeping with the electrokinetic injection process.43 Therefore, the sweeping with electrokinetic injection duration needs to be optimized through experiments to determine the optimal values according to the analyte retention factor. Figure 3 shows the variation of peak current and separation efficiency with different sample injection time. In the figure, the increased peak current could be observed up to the injection time of 80 min, but the separation efficiency had decreased obviously when the injection time was longer than 60 min. The effective sample volume injected into the capillary by the sweeping with electrokinetic injection is directly related to the apparent velocity of the analyte and the injection duration. Since the diameter of the capillary is fixed, the injected amount of an analyte can be quantified by the capillary length (linj) that would be occupied by the analyte at its original concentration. For neutral analytes, the apparent velocity is equal to the Veo, so the injected length is
Figure 3. Effect of sample injection time on peak current (a, b) and separation efficiency (c, d) of hyperoside (a, c) and hesperetin (b, d) (other flavonoids display similar results). The concentrations of hyperoside and hesperetin are 5.0 μg/L in 90 mM sodium phosphate buffer, pH 5.0; the preconcentration buffer solution (Buffer 1) is 5 mM SDS in 90 mM sodium phosphate buffer, pH 5.0; MEKC separation buffer is 5 mM SDS in 90 mM sodium phosphate buffer (pH 7.0); other conditions are the same as in Figure 2.
determined by the following equation, linj ¼ V eo t ¼ μeo Et
ð3Þ
where μeo is the EOF mobility. Under the experimental conditions, the μeo was 5.92 10-4 cm2/V 3 s using a neutral unretained probe mesityl oxide as EOF marker. For 60 min sample injection, the sample solution entering the capillary is about 546 cm. Optimization of the AFMC and CZE Separation. CZE was used as the second dimension to separate flavonoids in the purified Herba Leonuri sample. The effect of different types and concentrations of running buffer including sodium acetate, sodium phosphate, sodium borate, sodium citrate, NH3/NH4Cl, etc. were studied. Among them, 40 mM sodium phosphate buffer solution was proved to be best in terms of resolution and separation efficiency and was used in subsequent studies. The pH of 40 mM sodium phosphate buffer solution was optimized. The experimental results showed that, at pH 8.5, these compounds could obtain the best resolution. The operation of heart-cutting 2D-CE in a single capillary overcomes the difficulties in interfacing to capillaries. However, the use of pressure to hydrodynamically mobilize fraction B and introduce the second dimension buffer solution in step 2 led to peak broadening. To overcome the peak broadening and to achieve better separations, AFMC was carried out between the two dimensions of the separation. AFMC is based on the micelles to transport, release, and accumulate analytes into the MDZ according to the Kohlrausch inequality (viz., the following conservation eq 4) rather than the Kohlrausch regulating equation44 becuase a nonsteady state reaction boundary exists in the miceller collapse focusing system.45 X X X Fi, in ¼ Fi, out þ ΔQi, zone ð4Þ where F is the ionic flux and ΔQ is the net increase of quantity of an ion in the nonsteady state zone per unit time. The sample molecules are transported inside micelles under an applied electric field in a micellar electrolyte solution containing an anionic surfactant (SDS) and an additional anion having high electrophoretic mobility (phosphate). With an increase in the 1295
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Figure 4. Effect of AFMC on the second dimension separation by heart-cutting 2D-CE of the Herba Leonuri sample. The Herba Leonuri extract was 40-fold diluted with 90 mM sodium phosphate buffer (pH 5.0) prior to analysis. Figure A is the electropherogram of of flavonoids in the Herba Leonuri sample without AFMC in heart-cutting 2D-CE. Buffers: sweeping with electrokinetic injection preconcentration, 30 mM SDS in 90 mM sodium phosphate, pH 5.0; first dimension, 30 mM SDS in 90 mM sodium phosphate, pH 7.0 (conductivity κ = 2.78 S 3 cm-1); second dimension, 40 mM sodium phosphate, pH 8.5. High voltage program: t = 0-73.26 min, 15 kV; t = 73.26-78.16 min, 0 kV; t > 78.16 min, 15 kV. Nitrogen pressure program: 2.0 psi at detection end of the capillary, t = 73.26-78.16 min. Line B is the electropherogram of of flavonoids in the Herba Leonuri sample with AFMC between the two dimensions in heart-cutting 2D-CE. Buffers: sweeping with electrokinetic injection preconcentration, 5 mM SDS in 90 mM sodium phosphate, pH 5.0; first dimension, 5 mM SDS in 90 mM sodium phosphate, pH 7.0; second dimension, 40 mM sodium phosphate, pH 8.5. High voltage program: t = 0-69.42 min, 15 kV; t = 69.42-74.32 min, 0 kV; t > 74.32 min, 15 kV; Nitrogen pressure program, 2.0 psi at detection end of the capillary, t = 69.42-74.32 min. Peak identification and other conditions were as in Figure 2.
concentration of anions in the sample zone, the surfactant micelles are continuously diluted to below its cmc and collapsed into a liquid phase zone, thereby releasing and accumulating the transported molecules. The effect of AFMC on the second dimension separation can be contemplated in Figure 4. The first dimension separation time increased with increasing SDS concentration. For the electrochromatogram A in Figure 4, with 30 mM SDS micelles in the sample zone, the conductivity ratio (background solution/sample solution) = 0.62 which means that the concentration of SDS from the sample zone is above the cmc in the MDZ. Therefore, the SDS micelles persist at the MDZ, and no analyte accumulation and further separation by CZE were observed because the analytes were not released but were retained and transported by the micelle. Correspondingly, with 5 mM SDS micelles in the sample zone, the conductivity ratio (background solution/sample solution) is 0.44 and the concentration of SDS in the MDZ is below the cmc, so that the micelles collapsed and the analyte was focused and further separated by CZE as shown in the electrochromatogram B of Figure 4. This AFMC between the two dimensions has the functions of coupling MEKC with CZE for 2D separation by release of analytes from micelle to a liquid phase zone, effective conteraction of the peak broadening related to the hydrodynamic mobilization in step 2, and further compression of the analyte zone to improve the separation efficiency and sensitivity.
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Figure 5. Entire process of online integration of sweeping with electrokinetic injection and AFMC with hearting cutting 2D MEKC-CZE separation of flavonoids in the Herba Leonuri sample. The Herba Leonuri extract was 40-fold diluted with 90 mM sodium phosphate buffer (pH 5.0). The process includes sweeping with electrokinetic injection preconcentration, the first dimension separation until the first peak of fraction B is detected, the mobilization step, AFMC, and the second dimension separation. Experimental conditions: High voltage program (the plain line on the upper part of the graph), t = 0-69.42 min, 15 kV; t = 69.42-74.32 min, 0 kV; t > 74.32 min, 15 kV; Nitrogen pressure program (the dotted line on the upper graph), 2.0 psi at detection end of the capillary, t = 69.42-74.32 min, no pressure when not specified; Buffers: sweeping with electrokinetic injection preconcentration, 5 mM SDS in 90 mM sodium phosphate, pH 5.0; first dimension, 5 mM SDS in 90 mM sodium phosphate, pH 7.0; second dimension, 40 mM sodium phosphate, pH 8.5. Peak identification and other conditions were the same as in Figure 2.
Hyphenation of Sweeping with Electrokinetic InjectionMEKC and AFMC-CZE. After optimization of sweeping with
electrokinetic injection-MEKC and AFMC-CZE, respectively, the two concentration and separation methods were integrated for the analysis of the Herba Leonuri sample. First, the Herba Leonuri sample extract (40-fold dilution with 90 mM sodium phosphate buffer, pH 5.0) was preconcentrated by sweeping with electrokinetic injection and separated in first dimension MEKC. Then, only the flavonoid fraction of the first dimension separation was transferred into the second dimension of the capillary by pressure and further concentrated by AFMC and separated by CZE acting as the second dimension. The corresponding electropherogram of sweeping with electrokinetic injection-MEKC and AFMC-CZE separation of flavonoids in Herba Leonuri was shown in Figure 5. As expected, the sensitivity by sweeping with electrokinetic injection and AFMC concentration was higher than the sensitivity without concentration. The Herba Leonuri sample was well purified by the first dimension MEKC, and the interfering components were not detected. To demonstrate the concentrating power of the proposed sweeping with the electrokinetic injection-MEKC and AFMCCZE methods, the detection enhancement was tested using 60 min of electrokinetic injection for 5.0 μg/L hyperoside and 5.0 μg/L hesperetin relative to the normal pressure injection for 5.0 mg/L hyperoside and 5.0 mg/L hesperetin at 0.2 psi for 5 s. The enhancement factor was calculated by simply using the peak 1296
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Table 1. Analytical Characteristic Parameters of Analytes analytes
regression equation (nA ∼ μg/L)
correlation coefficient (r)
linear range (μg/L)
LOD (ng/L)
separation efficiency (N 104)
apigenin
I = 25.8C þ0.38
0.9919
0.05-40
25.1
1.7
phlorizin
I = 20.7C þ0.95
0.9966
0.08-50
31.6
2.7
quercetin
I = 31.6C þ0.44
0. 9953
0.05-30
18. 2
3.6
kaempferol
I = 26.1C þ 0.46
0.9945
0.05-40
23.7
2.4
hyperoside
I = 44.5C þ0.95
0.9971
0.02-20
9.8
3.1
quercitrin
I = 15.3C þ0.42
0.9984
0.08-60
36.4
4.3
rutin
I = 47.5x þ 0.51
0.9969
0.02-30
7.3
2.2
hesperetin
I = 37.3C þ0.91
0.9955
0.02-30
14.5
1.9
Figure 6. Electropherograms of blank (a) and postdosing (b) mouse blood samples. Experimental conditions and peak identification are the same as in Figure 5.
height ratio multiplied by the dilution factor. Signal enhancements of 6500- and 6300-fold were achieved for hyperoside and hesperetin, respectively. This more than 6000-fold improvement is the result of the combination of sweeping with electrokinetic injection and AFMC concentration methods. The width of analyte zone is less than that of normal CE separation. It is mainly due to the dual concentration effect that sweeping allowed large volume injection of samples and accumulated into a narrow band while AFMC not only counteracted the broadening effect caused by the mobilization process but also further compressed the analyte zone. The equation of linear regression, concentration linear range, and limit of detection (LOD) (S/N = 3) were investigated and listed in Table 1. The calibration curves were acquired by plotting the peak currents against individual analyte concentrations.
In regression equations, I was peak current and C was analyte concentration (μg/L). The calibration curves for all analytes using the proposed system at each concentration level were linear with a correlation coefficient greater than 0.99. To determine the precision of the proposed method, ten Herba Leonuri samples were determined and the results indicated that the migration times and peaks shapes of analytes were reproducible. The relative standard deviation (RSD) values of peak height, peak area, and migration time were 2.7-4.5%, 1.9-4.3%, and 4.7-6.8%, respectively. Application to Mouse Blood Sample. As an example, the proposed online integration of sweeping with electrokinetic injection and AFMC with 2D MEKC-CZE separation method was applied for the simultaneous determination of flavonoids in mouse blood to demonstrate the realistic suitability of analysis. The mouse was treated with Herba Leonuri extracts at an oral dose of 5 mL/kg of body weight. Figure 6 shows the electropherograms of drug-free mouse blood samples (Figure 6a) and postdosing mouse blood samples (Figure 6b) for the analytes and the internal standard. From Figure 6a,b, the endogenous ingredients did not interfere with the flavonoid peaks under experimental conditions due to the first dimension separation and purification. The liquid-liquid extraction of drugs in blood samples gave clean chromatograms with no other interfering peaks present. The model compounds in mouse blood sample were accurately identified by comparing their migration times with those of standards and by adding a pure standard to the sample so that the peak height and area of the corresponding compound was increased significantly. Also, six lots of blank mouse whole blood were screened to determine the selectivity of the assay. No detectable signal was observed at the migration times of the flavonoids which suggested a good specificity of the methods. The flavonoid concentrations in mouse blood following an oral dose at 5 mL/kg Herba Leonuri extracts were plotted against postdosing times (Figure 7). Table 2 showed the pharmacokinetic parameters based on the blood concentrations determined using the proposed method. Pharmacokinetic parameters were calculated as follows:46,47 “One-phase exponential decay” was chosen for nonlinear curve fitting of the last three quantified plasma concentrations of flavonoids and for calculation of the elimination constant Kel and plasma half-life time. The area under the curve from 0 to 8 h (AUC0-8 h) was calculated from time zero to 8 h postdosing Herba Leonuri extracts. The area under the curve 0 h-¥ (AUC0h-¥) was calculated from AUC0-8h þ (C8 h/Kel), both by the linear trapezoidal rule. The maximum mouse blood concentration of some rapidly absorbed and metabolized flavonoids including rutin, kaempferol, hesperetin, and quercitrin were 9.65, 2.76, 1.87, and 0.38 μg/L, respectively. 1297
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Table 2. Pharmacokinetic Parameters of Flavonoids after an Oral Dose of 5 mL/kg Herba Leonuri Extracts in Six Mice x ( SD pharmacokinetic parameters in mouse blood
apigenin
phlorizin
quercetin
kaempferol
hyperoside
quercitrin
rutin
hesperetin
Cmax (μg/L)
0.92 ( 0.35 0.56 ( 0.23
3.25 ( 0.66
2.76 ( 0.54
6.50 ( 0.92 0.38 ( 0.16
9.65 ( 1.32
1.87 ( 0.49
tmax (h)
1.86 ( 0.32 3.64 ( 0.43
2.75 ( 0.51
1.07 ( 0.29
1.68 ( 0.37 1.13 ( 0.26
0.83 ( 0.46
0.98 ( 0.21
Kel (R2 of curve fitting)a
0.40 (0.994) 0.16 (0.993)
0.32 (0.997)
0.40 (0.996)
0.19 (0.998) 0.24 (0.994)
0.29 (0.998)
0.35 (0.995)
t1/2 (h)a
3.24 ( 0.42 4.66 ( 0.34
5.58 ( 0.26
2.40 ( 0.41
3.72 ( 0.19 2.05 ( 0.30
1.57 ( 0.47
2.37 ( 0.23
AUC0-8 h (μg 3 h/L)b
4.25 ( 0.64 3.47 ( 0.39 18.78 ( 0.77 10.16 ( 0.86 27.82 ( 0.89 1.06 ( 0.72 34.93 ( 0.63
8.01 ( 0.58
AUC0-¥ (μg 3 h/L)c
6.42 ( 0.58 4.95 ( 0.48 24.13 ( 0.64 14.78 ( 0.75 35.34 ( 0.80 2.15 ( 0.63 43.58 ( 0.71 11.63 ( 0.56
a Calculated from the last three data points on the basis of one-phase exponential decay curve fitting. b Sum of the areas 0-8 h post dose. c Sum of the areas 0-8 h post dose þ Cplasma(8h)/Kel.
dimension and AFMC in the CZE dimension. Sweeping allowed large volume injection of samples while AFMC counteracted the broadening effect caused by the mobilization by pressure in the switch from MEKC to CZE. The feasibility and performance of the proposed system was demonstrated by the pharmacokinetic study in the mouse blood sample.
’ AUTHOR INFORMATION Corresponding Author
*Fax: þ86-532-84022750. E-mail:
[email protected].
Figure 7. Blood concentration (ng/L) vs postdosing time (h) of flavonoids. Conditions as in Figure 5.
The time at which the maximum analyte concentration in mouse blood was achieved was at 1 h, whereas some other flavonoids including hyperoside, quercetin, apigenin, and phlorizin were gradually absorbed and metabolized, and the time at which the maximum analyte concentration in rat blood was from 2 to 4 h. The apigenin, kaempferol, and hesperetin were more rapidly eliminated from the mouse body (Kel > 0.35 h-1) in comparison to other flavonoids such as phlorizin and hyperoside (Kel < 0.20 h-1). Biological elimination half time (t1/2) of all flavonoids varied from 1.57 ( 0.47 to 5.58 ( 0.26 h, and the area under the curve (AUC0-8h) varied from 1.06 ( 0.72 to 34.93 ( 0.63 μg 3 h/L. The results confirmed an applicability of the method for pharmacokinetic studies.
’ CONCLUSIONS A novel concentration and 2D separation system was demonstrated to Herba Leonuri and mouse blood samples. The sweeping with electrokinetic injection preconcentration combined with MEKC separation, which permitted injection of a large sample volume while maintaining separation efficiency, was straightforward and easy to perform. AFMC was employed to interface MEKC and CZE, which not only released analytes bound to micelle into a liquid phase zone but also accumulated analytes and was effective at counteracting analyte diffusion during the mobilization pressure step. A 2D CE with MEKC and CZE in the first and second dimensions was described for the analysis of flavonoids found in Herba Leonuri. More than 6000-fold improvement was obtained using sweeping with electrokinetic injection in the MEKC
’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 20875052), the Foundation for Outstanding Young Scientist in Shandong Province (BS2009HZ009), and the Program for New Century Excellent Talents in Universities (No. NCET-08-0878). ’ REFERENCES (1) Fang, H.; Zeng, Z.; Liu, L.; Pang, D. Anal. Chem. 2006, 78, 1257– 1263. (2) Baidoo, E. E. K.; Benke, P. I.; Neus€uss, C.; Pelzing, M.; Kruppa, G.; Leary, J. A.; Keasling, J. D. Anal. Chem. 2008, 80, 3112–3122. (3) Britz-McKibbin, P.; Otsuka, K.; Terabe, S. Anal. Chem. 2002, 74, 3736–3743. (4) Quirino, J. P.; Terabe, S.; Bocek, P. Anal. Chem. 2000, 72, 1934– 1940. (5) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023–1030. (6) Kim, J. B.; Otsuka, K.; Terabe, S. J. Chromatogr., A 2001, 932, 129–137. (7) Palmer, J.; Burgi, D. S.; Landers, J. P. Anal. Chem. 2002, 74, 632– 638. (8) Gong, M.; Wehmeyer, K. R.; Limbach, P. A.; Heineman, W. R. Anal. Chem. 2006, 78, 6035–6042. (9) Horakova, J.; Petr, J.; Maier, V.; Tesarova, E.; Veis, L.; Armstrong, D. W.; Gas, B.; Sevcik, J. Electrophoresis 2007, 28, 1540–1547. (10) Quirino, J. P.; Haddad, P. R. Anal. Chem. 2008, 80, 6824–6829. (11) Quirino, J. P. Electrophoresis 2009, 30, 875–882. (12) Michels, D. A.; Hu, S.; Schoenherr, R. M.; Eggertson, M. J.; Dovichi, N. J. Mol. Cell. Proteomics 2002, 1, 69–74. (13) Hu, S.; Michels, D. A.; Fazal, M. A.; Ratisoontorn, C.; Cunningham, M. L.; Dovichi, N. J. Anal. Chem. 2004, 76, 4044–4049. (14) Michels, D. A.; Hu, S.; Dambrowitz, K. A.; Eggertson, M. J.; Lauterbach, K.; Dovichi, N. J. Electrophoresis 2004, 25, 3098–3105. (15) Kraly, J. R.; Jones, M. R.; Gomez, D. G.; Dickerson, J. A.; Harwood, M. M.; Eggertson, M. J.; Paulson, T. G.; Sanchez, C. A.; Odze, R.; Feng, Z. D.; Reid, B. J.; Dovichi, N. J. Anal. Chem. 2006, 78, 5977– 5986. 1298
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