Enrichment and separation of cationic, neutral, and chiral analytes by

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Enrichment and separation of cationic, neutral, and chiral analytes by micelle to cyclodextrin stacking - micellar electrokinetic chromatography Alireza Ghiasvand, Zikai Feng, and Joselito P Quirino Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03542 • Publication Date (Web): 24 Dec 2018 Downloaded from http://pubs.acs.org on December 25, 2018

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Analytical Chemistry

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Enrichment and separation of cationic, neutral, and chiral analytes by micelle to cyclodextrin stacking - micellar electrokinetic chromatography Alireza Ghiasvand1,2, Zikai Feng1,3, Joselito P. Quirino1,* 1Australian

Centre for Research on Separation Science (ACROSS), School of Natural

Sciences-Chemistry, University of Tasmania, Hobart, Tasmania 7001, Australia 2Department 3School

of Chemistry, Lorestan University, Khoramabad, Iran

of Medicine-Pharmacy, University of Tasmania, Hobart, Tasmania 7001, Australia

corresponding author: *[email protected] additional corresponding author: Prof. Alireza Ghiasvand ([email protected])

1 ACS Paragon Plus Environment

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Abstract Analyte focusing by micelle to cyclodextrin stacking (MCDS) in micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) and fused silica capillaries is demonstrated for neutral, cationic, and chiral analytes. The stacking was at a dynamic boundary formed between the injected charged SDS micelles and neutral -cyclodextrin (CD) zones, where the analytes bound inside micelles were released due to the formation of stable SDS-CD inclusion complexes. The complex formation reduced or eliminated the affinity of the analytes to the micellar phase. There was reversal (for charged) or nulling (for neutrals) of the analyte’s effective electrophoretic mobility that caused the analytes to accumulate at the boundary. Under the conditions where the SDS micelles velocity is faster than the electroosmotic flow (using acidic buffer), MCDS was conducted by injection of a long plug of sample in a micellar diluent after injection of a CD solution plug into a capillary that was filled with MEKC background solution. By simply extending the length of the CD plug, chiral separations of chlorpheniramine and phenoxyacid herbicides were achieved without optimizing the MEKC conditions. The analytical figures of merit including linearity and repeatability for the tested compounds were found acceptable and the sensitivity enhancement factors were up to 171. The stacking strategy in MEKC was applied to metabolic stability studies of small molecules with HepG2 cell line, where the samples were only treated with acetonitrile and then diluted with the micellar diluent (demonstrating the reduction of tedious sample preparation requirements for biological samples prior to chemical analysis).

keywords: micelle to cyclodextrin stacking; micellar electrokinetic chromatography; neutral analytes; chiral separation; small molecules



Capillary electrophoresis (CE) is a family of green analytical separation techniques for small and large molecules.

Micellar electrokinetic chromatography (MEKC) has merged the

characteristics of electrophoresis and chromatography and is one of the most important modes of CE for small molecule analysis1-2. MEKC improved the separation selectivity of charged and allowed the separation of neutral analytes by using the beneficial features of micelles, which are formed by the addition of a surfactants above the critical micelle concentration (cmc) into the background solution (BGS)3. MEKC with UV detection generally needs sensitivity improvement for its application for analysis of real samples, due to its inherent sensitivity limitations4-5. Thus, different stacking techniques have been rigorously developed to improve MEKC sensitivity including field‐enhancement/amplification6-10, sweeping11-14, dynamic pH junction15-17, analyte focusing by micelle collapse18-21, and micelle-to-solvent stacking (MSS)22-24. In particular, MSS was initially developed for the CE mode of capillary zone electrophoresis (CZE)25-28. The above stacking techniques have also been implemented in CZE29-32. Micelle-to-cyclodextrin stacking (MCDS) was recently reported in CZE33. Cationic and anionic analytes were enriched by using the appropriate long-chain ionic surfactant, anionic sodium dodecyl sulfate (SDS) and cationic hexadecyltrimethyl ammonium bromide (CTAB), respectively. Peptides from a protein digest that contained SDS were successfully analysed without off-line sample preparation. Note that SDS is commonly used in proteomics for protein solubilization34-35, which improves protein identifications. In MCDS, the analyte focusing from the reversal in the analyte’s effective electrophoretic mobility was similar to MSS but was facilitated by cyclodextrins (CDs) instead of organic solvents. Native CDs form stable inclusion complexes with long chain ionic surfactants36-38, increasing the cmc and causing the release and enrichment of the analytes that are transported by micelles at the socalled dynamic stacking boundary. 3 ACS Paragon Plus Environment

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In this work, MCDS is demonstrated in the MEKC of cationic, neutral, and chiral analytes. Neutral and chiral analytes are not amenable by CZE, and additives or pseudophases such as micelles and/or CDs are required for separation. The sample was prepared in a micellar diluent and was injected as a long plug after injecting a CD solution plug. The native CDs (αCD, β-CD, and γ-CD) have been implemented for MCDS-CZE33. However, γ-CD was used because of its water solubility at the acidic pH used in this study with MCDS-MEKC. The mechanisms of stacking especially for the neutral and chiral analytes (including separation) which were enriched for the first time by MCDS are elaborated. The sensitivity enhancement factor (SEF) and analytical figures of merit were determined. The SEF values were calculated by dividing the peak height obtained from MCDS by the peak height obtained from typical injection, the ratio is then multiplied by the concentration factor (concentration in typical injection/concentration in MCDS). Quaternary ammonium herbicides (difenzoquat, paraquat, and diquat) and steroids (prednisolone, cortisone, prednisone, and triamcinolone) were employed as the model cationic and neutral analytes, respectively. The model chiral species were fenoprop, dichlorprop, mecoprop, and chlorpheniramine. Finally, a MCDS-MEKC method was applied to the metabolic stability studies of small molecules with HepG2 cell line, with emphasis on circumventing the tedious sample preparation steps required for biological samples before chemical measurement.



Materials and methods Chemicals and solutions Purified water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). HPLC-grade γ-CD hydrate (99%) was from AK Scientific, Inc. (Union City, CA, USA). HPLC-grade methanol (MeOH) and acetonitrile (ACN), analytical grade SDS, sodium hydroxide (NaOH), phosphoric acid (PA, 85%), and analytes (≥98%) prednisolone (pl), cortisone (ct), prednisone (pn), triamcinolone (tc), and (±)-chlorpheniramine maleate salt (cp) were obtained from Sigma-Aldrich (St. Louis, MA, USA). The herbicides (> 98%) difenzoquat (dfq), paraquat (pq), diquat (dq), fenoprop (fp), dichlorprop (dp), and mecoprop (mp) were obtained from Fluka Analytical (St. Louis, MO, USA) or Chem Service (West Chester, PA, USA). All analytes were used as received. Standard stock solutions (1000 µg/mL) of analytes were prepared by dissolving appropriate amounts in pure water, water/MeOH (50/50) or MeOH. 0.5 M PA and 0.2 M SDS stock solutions were prepared in purified water. The 0.5 M PA was adjusted to pH of 2.5 using 0.1 M NaOH. The ionizable analytes were all ionized in the pH used in this study. The BGSs were prepared by mixing appropriate volumes of 0.5 M PA, 0.2 M SDS, and purified water. The CD solution was prepared by weighing an appropriate amount of γ-CD which was dissolved in 100 mM PA. Based on a previous study33, the highest concentration of γ-CD (50 mM) that can be dissolved in the buffer was used to make the CD solution. All solutions were filtered using a 0.45 μm nylon filter and then sonicated prior to use.

MEKC analysis All MEKC analysis (with or without stacking) were performed on an Agilent HP G1600AX 3D CE system (Agilent Technologies, Waldbronn, Germany), equipped with a UV


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detector. A 3D-CE Chemstation (Rev.B.03.01.317) software was used for control and analysis. The detection wavelengths used are in the figure captions. 50 cm long (41.5 cm from inlet to the detection window) uncoated fused silica capillaries (50 µm i.d. × 360 µm o.d.) from Polymicro Technologies (Phoenix, AZ, USA) were used. Each new capillary was conditioned by flushing with NaOH (1.0 M, 10 min), purified water (10 min), MeOH (5 min), and water (5 min). Inter-run conditioning was performed by flushing with NaOH (1.0 M, 1 min), purified water (2 min), MeOH (1 min) and BGS (5 min). All injections of the CD and sample solutions were carried out hydrodynamically at 50 mbar. Typical injection was for 5s. In the analysis of each group of analytes in MCDS, the conductivities of CD solution, sample solution, and BGS were similar, thus excluding the effect of stacking by field enhancement6 in all experiments. Voltage for stacking and separation of 20 kV (which was within the linear range of the Ohm’s law plot of voltage vs. current) was applied at negative polarity (anode at detector end). The sample was prepared in 10 or 20 mM SDS in 100 mM PA. These SDS concentrations were above the cmc, allowing the sufficient transport of the analytes by the micelles to the stacking boundary. Higher concentrations did not lead to efficient stacking.

HepG2 cell line for small molecule metabolism study HepG2 (a human liver cancer cell line) was seeded in the edge wells of 6-well plates one day prior to the assay (2 ml of the cell culture was seeded at 1×105 cells/mL). After one day, the exhausted media was removed. Quaternary ammonium herbicides mixture (6.25 µg/mL of each herbicide difenzoquat, diquat, and paraquat) was prepared in Dulbecco Modified Eagle Medium. A 2 mL aliquot of the mixture was delivered into each well. Incubation was for up to 6 hours. From each well, 1.5 mL solution was taken and precipitated with 1.5 mL ACN, vortexed for 10 sec, and centrifuged for 10 min at 1500 rpm. Each



supernatant was filtered (0.45 μm filter) and 1 part was diluted with 5 parts of 20 mM SDS in 100 mM PA. %Recovered was calculated by dividing the concentration of each herbicide found at incubation time = 0, 1, 2, and 6 hr by the initial concentration (incubation time = 0 hr) of each herbicide, then multiplied by 100%. %Recovered is related to the amount of analyte that was left in the media after the metabolic stability test and at incubation time = 0 hr is equal to 100%.


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Result and discussion MCDS-MEKC mechanism for cationic and neutral analytes In this work, MEKC experiments were conducted under suppressed cathodic electroosmotic flow (EOF) conditions by using a BGS with low pH. This is reversed migration MEKC, where the electrophoretic mobility of the SDS micelles was faster than the EOF. The analytes were carried to the detector by the micelles. The analytes were prepared in a low pH diluent with SDS micelles. SDS is used because it is also the pseudophase in MEKC, and SDS and CDs have been demonstrated for the stacking of cationic analytes by MCDS-CZE33. The MCDS process in MEKC is similar to CZE33. Briefly, a CD zone was injected before the sample solution (see Figure 1(a)). The detector is at the anodic side of the capillary to allow MEKC analysis.

When the voltage was applied (see Figure 1(b)), the MCDS boundary

formed between the S and CD zones inside the capillary. The migration of the analytes due to the micelles in the S was dictated by the retention factor (k). The higher the k value, the greater the affinity of an analyte to the micelles. The analytes were transported to the boundary, where the SDS micelles were disrupted due to the formation of stable CD-SDS complexes. Less micelles were present, causing a decrease in the k. The formation of the inclusion complexes also caused an increase in the cmc, and thus more SDS monomers were required to form micelles. The micelles collapsed when the concentration of SDS became lower than the cmc and caused the k of all analytes to be equal to zero. The analytes were released at the boundary. The continued transport and release of the transported analytes caused the analyte enrichment at the MCDS boundary. The SDS from the sample continuously penetrated the CD zone and the MCDS boundary migrated to the anode (right) during stacking. The length of the injected CD zone also decreased during stacking. Once all the injected CDs were complexed with the SDS from the sample zone, the stacking or enrichment ended. With continued application of



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voltage, the separation was initiated when the micelles from the BGS penetrated the stacked zone (see Figure 1(c)). The MCDS mechanism for cationic analytes in MEKC relies on the reversal in the analyte’s effective electrophoretic mobility (µ*ep(a’)), given in equation (1). µ*ep(a’) = [1/(k+1)]µep(a’) + [k/(k+1)]µep(mc)

(1)

Where µep(a’) and µep(mc) is the electrophoretic mobility of cationic analyte (a’) and SDS micelles (mc), respectively. The µep(a’) and µep(mc) is directed to the cathode and anode, respectively. The µ*ep(a’) of the analytes in the micellar zone is directed to the anode, or the migration is dictated by the micelles or µep(mc) (see Figure 1(b)). At the MCDS boundary, the k of the analyte decreases to a point where the migration of the analyte will be dictated by the µep(a’), as per eqn. 1. This causes the µ*ep(a’) to be directed to the cathode. The collapse of the micelles will lead to the complete reversal in the µ*ep(a’), since k becomes equal to zero. For neutral analytes (a) with zero electrophoretic mobility, the effective electrophoretic mobility (µ*ep(a)) is given in equation (2). µ*ep(a) = [k/(k+1)]µep(mc)

(2)

While the effective electrophoretic mobility of cations is the sum of the µep(a’) and µep(mc) with consideration of the k (see equation 1), for neutrals the mobility is only related to the µep(mc) and k. For analyte focusing, the µ*ep(a) must become zero at the boundary or a complete collapse of the micelles (k = 0) is required (see Figure 1(b)). The obtainable enrichment factor or SEF in MCDS depends on the MCDS boundary. Stacking will occur as long as the MCDS boundary is maintained inside the capillary. The enrichment factor is theoretically infinity, if the sample is loaded continuously. The transported (by micelles) analytes will be trapped and enriched at the boundary. However, this is not the case in this work, where the amount of sample loaded is limited by the effective capillary length and the boundary was maintained by adjusting the amount of CD and micellar sample solution


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injection. In practice, the SEF with stacking performed with hydrodynamic injection are in the range from 10 to a few hundreds. Thus, for a 10x longer injection than typical, the SEF is 10.



Figure 1. Mechanism of MCDS of cationic and neutral analytes in reversed migration MEKC using SDS. (a) To a fused silica capillary that was initially conditioned with an acidic MEKC BGS, the CD solution was injected. This was followed by the injection of sample solution (S) that was prepared in a micellar diluent. (b) The application of an electric field created the MCDS boundary, found between the S and CD zones. The anode is at the detector side of the capillary. The retention factor (k) decreased or became = 0 at the MCDS boundary and caused the enrichment of the analytes at the boundary. (c) The stacking ended and the separation occurred due to the micelles from the BGS. More explanation in the text.


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Proof-of-concept for MCDS in MEKC- cationic and neutral analytes A typical injection-MEKC of the model cationic herbicides and neutral steroids is shown in Figure 2(a)(i) and Figure 2(b)(i), respectively. A long 100 s injection of the herbicides and steroids prepared using the BGS or 20 mM SDS in 100 mM PA resulted in broad peaks, with a representative result using the 20 mM SDS in 100 mM PA sample diluent is shown in Figure 2(a)(ii) and Figure 2(b)(ii), respectively. The 30% MeOH which was required in the BGS for MEKC separation was not sufficient to allow the analyte focusing by MSS of the injected sample. By simply injecting a CD solution plug (50 s for herbicides and 75 s for steroids) before the 100 s sample injection in Figure 2(a)(ii) and Figure 2(b)(ii) (see procedure in Figure 1), the herbicides (1 µg/mL each) and steroids (5 µg/mL each) were stacked as shown in Figure 2(a)(iii) and Figure 2(b)(iii), respectively. The stoichiometric ratio for -CD and SDS complexes is likely 2:139. Therefore, the electrophoretically migrating micelles from 20 mM SDS in the sample collapsed at the MCDS boundary that initially contained 50 mM -CD. Note that the concentration ratio for -CD and SDS in Figure 2(a)(iii) and Figure 2(b)(iii) is 5:2, thus there was excess -CDs for SDS sequestration. These results prove the concept of MCDS for cationic and neutral analytes in MEKC. The SEFs for the herbicides and steroids were 47-120 and 8-49, respectively. Note that the concentrations of herbicides and steroids in MCDS (Figure 2(a)(iii) and Figure 2(b)(iii)) were 10 and 2 times lower than in typical injection (Figure 2(a)(i) and Figure 2(b)(i)), correspondingly. The retention times in MCDS (see Figure 2(a)(iii) and 2(b)(iii)) were longer when compared to those in typical injection (see Figure 2(a)(i) and 2(b)(ii)). This was because of the additional time required to finish MCDS, which occurred before the MEKC separation.



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Figure 2. (a) Proof-of-concept for the MCDS of cationic analytes. Injection was 5 s of sample (i), 100 s of sample (ii), and 100 s of sample after 50 s of CD solution (iii).

Sample diluent

was BGS (i) and 20 mM SDS in 100 mM PA (ii and iii). Sample concentration was 10 µg/mL (i and ii) and 1 µg/mL (iii) of each herbicide. Detection was at 214 nm. (b) Proof-of-concept for the MCDS of neutral analytes. Injection was 5 s of sample (i), 100 s of sample (ii), and 100 s of sample after 75 s of CD solution (iii).

Sample diluent was BGS (i) and 20 mM SDS in

100 mM PA (ii and iii). Sample concentration was 10 µg/mL (i and ii) and 5 µg/mL (iii) of each steroid. Detection was at 247 nm. (a and b) The BGS used was 100 mM SDS and 30% MeOH in 50 mM PA, that allowed sufficient separations. The CD solution was 50 mM γ-CD in 100 mM PA. Other information in Materials and methods.


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MCDS-MEKC mechanism for chiral analytes The initial condition for MCDS-MEKC for chiral analytes is the same as in Figure 1(a) except a longer CD solution plug was required. After MCDS of chiral analytes that were transported to the boundary by the micelles from the sample solution, a CD zone remained inside the capillary (see Figure 3(a)(i)). The MCDS boundary disappeared. The micelles from the BGS eventually penetrated the stacked analytes zone. The additional CD zone injected did not create a MCDS boundary with the SDS micelles from the BGS. This allowed the analytes to be transported by the BGS micelles through the CD zone (see Figure 3(a)(ii)). The stacked analytes that migrated through the CD zone were then separated by known mechanisms in CD modified MEKC40.

Proof-of-concept for MCDS-chiral-MEKC A typical injection-MEKC of the model chiral fenoprop is shown in Figure 3(b)(i). A long 50 s injection of sample prepared using BGS or 10 mM SDS in 100 mM PA resulted in broad peaks, with a representative result using the latter sample diluent is shown in Figure 3(b)(ii). The stacking and chiral separation was achieved by increasing the CD solution injection that was performed before the 50 s injection of fenoprop (similar to Figure 3(b)(ii)). The results for the 20 to 100 s of CD solution are shown in Figures 3(b)(iii) to 3(b)(viii). With the 20 s injection, complete stacking by MCDS and a single sharp peak was observed (see Figure 3(b)(iii)). With the 30 s injection, splitting of the stacked racemic peak was obvious (see Figure 3(b)(iv)). The chiral separation improved as the injection of CD solution was increased to 75 s (see Figure 3(b)(v to vii)) and complete separation was obtained with the 100 s injection (see Figure 3(b)(viii)), proving the concept of MCDS chiral-MEKC described in Figure 3(a). The retention time increased as the CD solution injection was increased, again due to the time required to finish MCDS before MEKC separation occurs. The SEFs in terms



of peak height for the enantiomers were ~50, noting that the concentration of the racemate in typical injection (Figure 3b(i)) was 10 higher than in MCDS-chiral-MEKC (Figure 3b(iv)).


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Figure 3. a. MCDS-chiral MEKC mechanism. (i) A CD solution plug remained inside the capillary after MCDS. (ii) The micelles from the BGS transported the analytes into the CD zone where chiral separation occurred. b. Proof-of-concept for MCDS-chiral-MEKC using fenoprop. The BGS was 50 mM SDS in 50 mM PA. The CD solution was 50 mM γ-CD in 100 mM PA. Sample diluent was BGS (i) and 10 mM SDS in 100 mM PA (ii to viii). Sample concentration was 50 µg/mL (i and ii) and 5 µg/mL (iii to viii) of fenoprop. Injection was 5 (i) and 50 (ii to viii) s of sample. A CD solution plug was injected for 20 (iii), 30 (iv), 40 (v), 50 (vi), 75 (vii), and 100 (viii) s before the sample. Detection was at 200 nm. Other information in Materials and methods.



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Optimization of CD and sample solution injection time ratio and sample injection time The optimum injection time ratio of CD and sample solution that provided complete stacking by MCDS was evaluated. The MCDS-MEKC conditions for the cationic, neutral, and chiral analytes are described in Figure 2(a), Figure 2(b), and Figure 3(b), respectively. For the cationic and neutral analytes and using a constant sample injection time of 100 s, the injection time of the CD solution was varied (0 to 150 s). The injection that resulted to the highest and sharpest peaks for the analytes was 50 s and 75 s for the cationic (see Figure 2(a)(iii)) and neutral (see Figure 2(b)(iii)) analytes, respectively. The optimum injection time ratio (CD solution:sample solution) was therefore 1:2 and 3:4, correspondingly. With the set optimum injection time ratio, the injection time of the samples were then increased. No further improvements in peak heights were obtained and the maximum injection time for both cations and neutrals was 100 s under the experimental conditions used.

MCDS is like any other

stacking technique in CE with hydrodynamic injection. The maximum sample injection time is limited by the length of the capillary that can accommodate the sample (and here including the CD solution injection) for complete stacking and separation of analytes. The determination of the optimum injection time ratio for the racemates also considers the additional length of the CD solution plug required for chiral separation. For fenoprop, the injection time ratio for complete MCDS was 2:5 or 20 s injection of CD solution was needed to stack 50 s injection of sample (see Figure 3(b)(iii)). An additional 80 s injection of CD solution was required to provide efficient chiral separations (see Figure 3(b)(viii), thus the optimum injection time ratio was 2:1 (100 s of CD solution: 50 s of sample solution) for MCDS-chiral-MEKC of fenoprop. Three other racemates were tested using the same BGS and sample conditions used for fenoprop. For the other herbicides dichlorprop and mecoprop, the injection time ratio for complete MCDS, which was evaluated using a 50 s injection of sample was 2:1 (see Supporting Information (SI) Fig. S1(a)(i)) and 1:1 (see SI Fig. S1(b)(i)),


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respectively. The optimum injection time ratio for MCDS-chiral-MEKC of dichlorprop and mecoprop was found to be 5:1 (see SI Fig. S1(a)(ii)) and 4:1 (see SI Fig. S1(a)(ii)), respectively. For chlorpheniramine, injection time ratio for complete MCDS using 100 s injection of sample was 1:4 (see SI Fig. S1(c)(i)) while the optimum injection time ratio for MCDS-chiral-MEKC was 3:2 (see SI Fig. S1(c)(ii)). The use of other CDs that are easily soluble in low pH solutions (a future work) will be required to allow better chiral separations. The maximum sample injection time of 50 and 100 s was found for the MCDS-chiral-MEKC of the herbicides and chlorpheniramine, respectively, again due to the limited effective capillary length to perform both stacking and separation.

The SEFs for dichlorprop, mecoprop, fenoprop, and

chlorpheniramine were found to be 146, 171, 54, and 94, respectively.

MCDS-(chiral)-MEKC analytical figures of merit The analytical figures of merit (linear dynamic ranges (LDRs), limits of detection (LODs), relative standard deviations (RSDs) are summarized in SI Table S1. The values are all typical to those obtained in CE methods that utilize a stacking strategy. The SEF values (one to two orders of concentration magnitude) in SI Table S1 are also typical to stacking methods that use hydrodynamic injection.

Recovery and determination of small molecules in HepG2 cell line metabolism media Samples from the in-vitro metabolism of small molecules using HepG2 cell line was used to evaluate the applicability of MCDS-MEKC for the analysis of complex real samples without the need for rigorous sample extraction or clean-up. For this purpose, difenzoquat, diquat, and paraquat were added to the HepG2 cell line metabolism media. To prevent the death of the cells during incubation, the concentration of each herbicide was low at 6.3 g/mL. The analytes were then determined at different incubation times (0 to 6 hours) (see Materials 18 ACS Paragon Plus Environment


and methods for sample processing). After final dilution with 20 mM SDS in 100 mM PA, the resulting concentration of SDS in the sample for injection was 17 mM, which was completely amenable by MCDS-MEKC as described in Figure 2(a)(iii). It is noted that samples from cell incubates are typically processed using one or two extraction techniques (e.g., liquid-liquid and solid-phase extraction) prior to chemical analysis by e.g., liquid chromatography or CE41-42. Typical injection of the sample did not show any peak for the herbicides (see SI Figure S2(a)) while MCDS-MEKC allowed the reliable detection of the small molecules (see SI Figure S2(b)). Finally, the metabolism or decrease in the concentration of the small molecules with the increase in the incubation time from 0 to 6 hours was also successfully observed (see SI Figure S3, where %Recovered was indirectly proportional to the incubation time), demonstrating the applicability of MCDS-MEKC for complex real sample analytes with very minimal sample preparation. The metabolic stability of the model small molecules was very similar, with the herbicides degrading to around 50% after 6 hr of incubation.


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Conclusion The new sample concentration technique in CE called MCDS was demonstrated in MEKC. The mechanism of MCDS-MEKC with SDS is chromatographic (interaction between pseudophase/SDS micelles and analytes) and was facilitated by the presence of an electric field. The analytes in the sample solution with micelles were transported to the ‘dynamic' stacking boundary by micelles. At the stacking boundary, the CDs form stable inclusion complexes with SDS. The cmc of SDS increased and the concentration of SDS micelles decreased. The concentration of free SDS could fall below the cmc and this caused the collapse of micelles. The decrease in the concentration of SDS and collapse of micelles released the analytes from the micelles at the boundary. At the boundary, the effective electrophoretic velocity of analytes reversed in direction (for cations) or became zero (for neutrals). The effective electrophoretic mobility reversed in sign (negative to positive) for the cations and became zero for neutrals (negative to zero). MCDS was applied for the first time for the analysis of neutral analytes, which was due to the loss in the effective electrophoretic mobility of the analyte from the complete collapse of the micelles. Chiral analytes were stacked and separated by simply optimizing the CD (i.e., -CD) solution injection and without changing the MEKC conditions. The application of MCDS-MEKC to real samples without off-line sample extraction was shown in the metabolic stability testing of model small molecules in-vitro using living cells. The SEFs in MCDS-MEKC reached >100, which is normal for stacking procedures with hydrodynamic injection. The use of MCDS in MEKC with cationic surfactants (e.g., CTAB), and combination of MCDS to other stacking techniques in CE for greater improvements in sensitivity is a future investigation for samples containing salts and/or micelles that are difficult to stack in CE.



Acknowledgments The authors thanks Assoc/Prof. Nuri Güven (Pharmacy) at the University of Tasmania for providing the HepG2 cell line metabolic assay facilities. JPQ thanks the Australian Research Council (ARC) for an ARC Discovery Grant (DP180102810).

Conflict of interest The authors have no conflict of interest to declare.


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Enrichment and separation of cationic, neutral, and chiral analytes by

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