Use of cyclodextrin‐modified gold nanoparticles for enantioseparations of drugs and amino acids based on pseudostationary phase‐capillary electrochromatography

The application of chemical‐modified gold nanoparticles (GNPs) as chiral selector for the enantioseparation based on pseudostationary phase‐CEC (PSP‐CEC) is presented. GNPs modified by thiolated β‐CD were characterized by NMR and FT‐IR. The nanoparticle size was determined to be of 9.5 nm (+2.5 nm) by Transmission Electron Microscopy (TEM) and UV spectra. Four pairs of dinitrophenyl‐labeled amino acid enantiomers (DL‐Val, Leu, Glu and Asp) and three pairs of drug enantiomers (RS‐chlorpheniramine, zopiclone and carvedilol) were analyzed by using modified GNPs as the chiral selector in PSP‐CEC. Good theoretical plate number (up to 2.4×10⁵ per meter) and separation resolution (up to 4.7) were obtained even with low concentration of modified GNPs (0.8–1.4 mg/mL). The corresponding concentration of β‐CD in the buffer was only 0.30?0.53 mM, which was much lower than the optimum concentration of 15 mM if pure β‐CD was used as chiral selector. Our results showed that thiolated β‐CD modified GNPs have more sufficient interaction with the analytes, resulting in significant enhancement of enantioseparation. The study shed light on potential usage of chemical modified GNPs as chiral selector for enantioseparation based on PSP‐CEC.     

Sugar‐Decorated Sugar Vesicles: Lectin–Carbohydrate Recognition at the Surface of Cyclodextrin Vesicles

An artificial glycocalix self‐assembles when unilamellar bilayer vesicles of amphiphilic β‐cyclodextrins are decorated with maltose and lactose by host–guest interactions. To this end, maltose and lactose were conjugated with adamantane through a tetra(ethyleneglycol) spacer. Both carbohydrate–adamantane conjugates strongly bind to β‐cyclodextrin (K_a≈4×10⁴ M ^?1). The maltose‐decorated vesicles readily agglutinate (aggregate) in the presence of the lectin concanavalin A, whereas the lactose‐decorated vesicles agglutinate in the presence of peanut agglutinin. The orthogonal multivalent interaction in the ternary system of host vesicles, guest carbohydrates, and lectins was investigated by using isothermal titration calorimetry, dynamic light scattering, UV/Vis spectroscopy, and cryogenic transmission electron microscopy. It was shown that agglutination is reversible, and the noncovalent interaction can be suppressed and eliminated by the addition of competitive inhibitors, such as D ‐glucose or β‐cyclodextrin. Also, it was shown that agglutination depends on the surface coverage of carbohydrates on the vesicles.     

Analysis of 3‐quinuclidinol derivatives by capillary electrophoresis

Capillary electrophoresis with indirect UV detection was applied to the analysis of a mixture of 3‐quinuclidinol and its four quaternary derivatives (N‐methyl, N‐ethyl, N‐propyl, and N‐isopropyl derivative). 10 mM imidazole acetate buffer, pH = 4.0 offers suitable detection sensitivity (LOD = 1 μmol L^–1) and permits separation of the mixture except for the pair 3‐quinuclidinol–N‐methyl derivative. The separation of all analytes was achieved on addition of 15 mmol L^–1 β‐cyclodextrin or 25% (w/w) polyethylene glycol 2000 to the background electrolyte. The optimized method was employed for the analysis of pond water spiked with these analytes. Actual ionic mobilities of the studied compounds were measured using mobility standards (potassium, sodium, tetramethyl‐ and tetrabutylammonium). The migration index was derived as another identification parameter based on migration data and the precision of the obtained values is discussed in brief.     

Determination of brompheniramine enantiomers in rat plasma by cation‐selective exhaustive injection and sweeping cyclodextrin modified electrokinetic chromatography method

A method consisting of cation‐selective exhaustive injection and sweeping (CSEI‐sweeping) as online preconcentration followed by a cyclodextrin modified electrokinetic chromatography (CDEKC) enantioseparation has been developed for the simultaneous determination of two brompheniramine enantiomers in rat plasma. In this method, analytes were electrokinetically injected at a voltage of 8 kV for 80 s in a fused‐silica capillary. Prior to the injection, the capillary was rinsed with 50 mM phosphate buffer of pH 3.5, followed by a plug of a higher conductivity buffer (150 mM phosphate pH 3.5, 20 psi, 6 min) and a plug of water (0.5 psi, 5 s). Separation was carried out applying –20 kV in 50 mM phosphate buffer, pH 3.5, containing 10% v/v ACN and 30 mg/mL sulfated‐β‐cyclodextrin (S‐β‐CD). Analytical signals were monitored at 210 nm. The detection sensitivity of brompheniramine enantiomers was enhanced by about 2400‐fold compared to the normal injection mode (hydrodynamic injection for 3 s at 0.5 psi, with a BGE of 50 mM phosphate buffer containing 20 mg/mL S‐β‐CD at pH 3.5), and LLOQ of two enantiomers were both 0.0100 μg/mL. In addition, this method had fairly good repeatability and showed promising capabilities in the application of stereoselective pharmacokinetic investigations for brompheniramine enantiomers in rat.     

Surfactant‐assisted electromembrane extraction combined with cyclodextrin‐modified capillary electrophoresis for the separation and quantification of Tranylcypromine enantiomers in biological samples

Surfactant‐assisted electromembrane extraction coupled with cyclodextrin‐modified capillary electrophoresis was developed for the separation and determination of Tranylcypromine enantiomers in biological samples. This combination would provide a new strategy for selective and sensitive determination of target analytes. The addition of surfactant in the donor solution improved the analyte transport into the lumen of hollow fiber that resulted in an enhancement in the analytes migration into acceptor solution. Optimization of the variables, affecting proposed method, was carried out and best results were achieved with a 175 V potential as driving force of the electromembrane extraction, 2‐nitrophenyloctylether as the supported liquid membrane, donor solution containing 0.2 mM Triton X‐100 with pH 3 and 0.1 M HCl for acceptor solution. Then, the extract was analyzed using cyclodextrin‐modified capillary electrophoresis method for separation of Tranylcypromine enantiomers. The best results were obtained with a phosphate running buffer (100 mM, pH 2.0) containing 7% w/v hydroxypropyl‐α‐cyclodextrin. Under the optimum conditions, a low limit of detection (3.03 ng/mL), good linearity (R² > 0.9953), and relative standard deviations below 4.0% (n = 5) were obtained. Finally, this procedure was applied to determine the concentration of Tranylcypromine enantiomers in urine samples with satisfactory results.     

Chiral capillary electrophoresis with cationic pyrrolidinium‐β‐cyclodextrin derivatives as chiral selectors

New single‐isomer, cationic β‐cyclodextrins, including mono‐6‐deoxy‐6‐pyrrolidine‐β‐cyclodextrin chloride (pyCDCl), mono‐6‐deoxy‐6‐(N‐methyl‐pyrrolidine)‐β‐cyclodextrin chloride (N‐CH₃‐pyCDCl), mono‐6‐deoxy‐6‐(N‐(2‐hydroxyethyl)‐pyrrolidine)‐β‐cyclodextrin chloride (N‐EtOH‐pyCDCl), mono‐6‐deoxy‐6‐(2‐hydroxymethyl‐pyrrolidine)‐β‐cyclodextrin chloride (2‐MeOH‐pyCDCl) were synthesized and used as chiral selectors in capillary electrophoresis for the enantioseparation of carboxylic and hydroxycarboxylic acids and dansyl amino acids. The unsubstituted pyCDCl exhibited the greatest resolving ability. Most analytes were resolved over a wide range of pH from 6.0 to 9.0 with this chiral selector. In general, increasing pH led to a decrease in resolution. The effective mobilities of all the analytes were found to decrease with increasing CD concentration. The optimal concentration for most carboxylic acids and dansyl amino acid was in the range 5–7.5 mM and >15 mM for hydroxycarboxylic acids. ¹H NMR experiments provided direct evidence of inclusion in the CD cavity.     

An aminoquinoline based fluorescent probe for sequential detection of Znic (II) and inorganic phosphate and application in living cell imaging

A novel fluorescent probe 5‐(diethylamino)‐2‐(((2‐(hydroxymethyl)quinolin‐8‐yl)imino)methyl)phenol ( QS) was synthesized by condensation reaction of 8‐aminoquinoline derivative and 4‐(diethylamino)salicylaldehyde. It was found that the probe QS was capable of high selectivity and sensitivity about specific color and fluorescence changes towards Zn²⁺ ion in EtOH‐H₂O (v/v = 4/1, 0.01 M, Tris–HCl buffer, pH = 7.30) solution. The interaction of QS with Zn²⁺ ion illustrated a “turn‐on” fluorescence response at 550 nm (λ_ex: 458 nm), moreover, after the subsequent addition of inorganic phosphate (Pi) into the solution above, a “turn‐off” fluorescence response was observed. The sensing ability of the probe QS towards Zn²⁺ was confirmed by fluorescence titration, UV–Vis titration and HRMS analysis. Besides, the intracellular sensing behavior of QS with Zn²⁺ and Pi were captured in living PC12 cells. The limit of detection (LOD) for Zn²⁺ and Pi sensing was found to be 0.03 μM and 0.08 μM, respectively.     

Capillary electrophoresis/visible‐LED induced fluorescence of tryptophan: What's new?

Tryptophane (Trp) labelled by 3‐(4‐carboxybenzoyl)‐2‐quinolinecarboxaldehyde (CBQCA) is very difficult to identify using CE and fluorescence detection (480 nm). Why in this article some mass spectrometry experiments show that Trp is really labelled by CBQCA as Leucine (Leu)? If the maximum of UV absorption (λ_max) is the same between Leu‐CBQCA and Trp‐CBQCA, the molar extinction coefficient is around 2 fold higher for Trp‐CBQCA. The fluorescence of the Leu‐CBQCA derivative is 50 times more important than for Trp‐CBQCA. The addition of 7.5 mM of β‐cyclodextrin (β‐CD) was found to be a good mean to improve 2.1 fold the sensitivity of the Trp‐CBQCA fluorescence. Using a buffer containing SDS and β‐CD in CE, a LOD of 0.7 µM of L‐Trp can be reached and the ratio of the intensities between Leu, Isoleucine, Valine, Trp is 100, 21, 15, 1. Negative ESI^/MS and MS/MS of the labeled amino acids show that a loss of the carboxylate function takes place. In the presence of two enantiomers of Trp‐CBQCA, we have shown that this decarboxylation is not due to the derivatization process in the solution but rather occurs in the source of the mass spectrometer.     

Dual‐Modal Magnetic Resonance/Fluorescent Zinc Probes for Pancreatic β‐Cell Mass Imaging

Despite the contribution of changes in pancreatic β‐cell mass to the development of all forms of diabetes mellitus, few robust approaches currently exist to monitor these changes prospectively in vivo. Although magnetic‐resonance imaging (MRI) provides a potentially useful technique, targeting MRI‐active probes to the β cell has proved challenging. Zinc ions are highly concentrated in the secretory granule, but they are relatively less abundant in the exocrine pancreas and in other tissues. We have therefore developed functional dual‐modal probes based on transition‐metal chelates capable of binding zinc. The first of these, Gd ?1 , binds Zn^II directly by means of an amidoquinoline moiety (AQA), thus causing a large ratiometric Stokes shift in the fluorescence from λ_em=410 to 500 nm with an increase in relaxivity from r₁=4.2 up to 4.9 mM ^?1 s^?1. The probe is efficiently accumulated into secretory granules in β‐cell‐derived lines and isolated islets, but more poorly by non‐endocrine cells, and leads to a reduction in T₁ in human islets. In vivo murine studies of Gd ?1 have shown accumulation of the probe in the pancreas with increased signal intensity over 140 minutes.     

Enantioselective determination of cathinones in urine by high pressure in‐line SPE–CE

This work presents a strategy based on the in‐line coupling of SPE and CE for the chiral determination of cathinones (R,S‐mephedrone, R,S‐4‐methylephedrine, and R,S‐ methylenedioxypyrovalerone) in urine samples, using a sample pretreatment based on liquid‐liquid extraction. The chiral separation of the compounds is achieved by adding a mixture of 8 mM 2‐hydroxypropil β‐CD and 5 mM β‐CD to the BGE, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Oasis HLB was the selected sorbent for the in‐line SPE device, and to reduce analysis time and LODs, several parameters affecting the in‐line SPE system were evaluated, such as pressure and time of sample injection and dimensions of the SPE device. The highest preconcentration factors were achieved by using 3 bar of injection pressure for 20 min with an in‐line SPE device of 2 mm length and 150 µm of i.d. The developed method was applied to determine the presence of the compounds in spiked urine samples. The LODs obtained were between 3 and 8 ng/mL, and these levels were below the usual concentrations at which these drugs are present in urine from cathinone abusers. Thus, the optimized method has the potential to be applied for toxicological and forensic purposes.     

Use of MEKC for the analysis of reactant and product of Baylis–Hillman reaction

A facile, fast and high efficiency micellar EKC has been explored for the analysis and UV detection of p‐nitrobenzaldehyde and 2‐[hydroxy(4‐nitrophenyl)methyl]‐2‐cyclopenten‐1‐one with a buffer electrolyte of 30.0 mM tetraborate and 50.0 mM sodium taurodeoxycholate at pH 9.3. Under the optimal conditions, a linear range from 7.8×10^–2 to 5.0×10² mM for those analytes (r² > 0.99) was achieved. The LOD was 3.9 μM for 2‐[hydroxy(4‐nitrophenyl)methyl]‐2‐cyclopenten‐1‐one and 7.8 μM for p‐nitrobenzaldehyde, respectively (S/N = 3). The applicability of this new method for the analysis of reactants (p‐nitrobenzaldehyde and cyclopent‐2‐enone), catalysts (imidazole or N‐methyl imidazole or 1‐benzyl‐imidazole) and product (2‐[hydroxy(4‐nitrophenyl)methyl]‐2‐cyclopenten‐1‐one) on offline Baylis–Hillman reaction was examined. The relationship between the reaction time and the amount of product has been studied. Meanwhile, three different kinds of catalysts were investigated for getting the desired moderate to good amount products. It was found that comparing with N‐methyl imidazole or 1‐benzyl‐imidazole catalyst, imidazole‐catalyzed reaction could produce more products within the same reaction time. Furthermore, the results indicated that the rate law for the investigated Baylis–Hillman reaction was second‐order reaction. The rate constant for the reaction is 1.34 (±0.01)×10^–3 mol^–1 m³/s.     

Dynamic double coating,electrophoretic method with indirect detection for the simultaneous quantification of mono‐ and divalent cations in various water samples

An indirect UV detection method based on CE was developed and validated to determinate 12 metal cations, including alkali, alkaline earth, transition metal, and ammonium. In this paper, a new electrolyte system (pH 4.22) contained 20 mM benzimidazole (as co‐ion), 75 mM acetic acid (as a counter‐ion) as well as 0.6 mM 18‐crown‐6 ether was applied. The metal ions were completely separated within 8 min under hydrodynamic mode injection with a running voltage of 20 kV at 25 ± 0.1°C. Additional use of the dynamic double coating method enabled to get an excellent repeatability of migration times and quantitative parameters for all analytes. The repeatability of migration times for analytes were less than 0.9% and peak areas and peak heights ranged from 3.7 to 7.2 and 3.9 to 7.7%, respectively (n = 6). The proposed technique proved to be definitely faster and less expensive in comparison to currently employed methods. In this work, we discuss also the linear range, method detection limits as well as precision and accuracy. The applicability of the elaborated method was authenticated by the quantification of metal ions in commercially available mineral water, tap water, and selected medical injection samples.     

Use of Ionic liquids as additives in ion exchange chromatography for the analysis of cations

The behavior of acids (citric acid, nitric acid, oxalic acid, tartaric acid) as a mobile phase and imidazolium ionic liquids (the bromides, tetrafluoroborates and hexafluorophosphates of 1‐ethyl, 1‐butyl, and 1‐hexyl‐3‐methylimidazolium) as additives in ion exchange chromatography for cations (Na⁺, K⁺, Mg²⁺, Ca²⁺) separation were studied. The results showed that nitric acid and 1‐hexyl‐3‐methyl‐imidazolium hexafluorophosphate offered the most interesting features in the separation of cations, such as lower retention time and better resolution. The selected optimal conditions were achieved by adding 0.10 mM 1‐hexyl‐3‐methyl‐imidazolium hexafluorophosphate in 4.0 mM HNO₃ mobile phase for the separation of four cations with the flow rate of 0.9 mL/min at room temperature (25°C). The linear regression equations of Na⁺, K⁺, Mg²⁺, Ca²⁺ were S  = 4.4763c  + 0.0209, S  = 3.8903c  – 0.0087, S  = 6.3974c  – 0.0173, and S  = 7.601c  – 0.0339 and the limits of detection of Na⁺, K⁺, Mg²⁺, Ca²⁺ were 0.296, 4.98, 0.0970, and 1.22 μg/L, respectively. In this work, four cations in samples were successfully detected.     

Synthesis of a New Modification of Lithium Chloride Confirming Theoretical Predictions

In accordance with prior calculations, the new polymorph β‐LiCl (wurtzite structure type) has been synthesised, by the low‐temperature atomic‐beam‐deposition (LT–ABD) technique, in a mixture with α‐LiCl (rock salt structure type) by depositing LiCl vapour (2 to 5.3 × 10^–4 mbar) onto a cooled substrate (–30 to –60 °C). The maximum β‐LiCl fraction of 53 % was obtained using a sapphire (0001) substrate at –50 °C and 3.7 × 10^–4 mbar LiCl vapour pressure. The proportion of the new polymorph contained in the bulk sample decreases as temperature or vapour pressure deviate from these values, until finally the rock salt type LiCl is found exclusively. When the samples are warmed up to room temperature, β‐LiCl irreversibly transforms to α‐LiCl. The X‐ray diffraction pattern of the two phase LiCl sample measured at –50 °C has been indexed and refined based on a hexagonal unit cell for β‐LiCl with the lattice constants a = 3.852(1) Å and c = 6.118(1) Å and a cubic unit cell for α‐LiCl with the lattice constant a = 5.0630(8) Å. By Rietveld refinement the wurtzite type ofstructure (P6₃mc, No. 186) was suggested for the new hexagonal modification of LiCl with the Li–Cl distances (2.32 and 2.34 Å) being 8 % smaller than those of α‐LiCl. Moreover, the cell volume decreases as much as 16 % during the transition from β‐LiCl to α‐LiCl. Both the shifts in bond lengths and volume correspond well with the situation encountered for LiBr and LiI. Besides the variation of LiCl vapour pressure and substrate temperature, also different substrate materials were employed for testing their influence on formation of the β‐LiCl polymorph.     

Enantiomeric separation by MEKC using dodecyl thioglycoside surfactants: Importance of an equatorially oriented hydroxy group at C‐2 position in separation of dansylated amino acids

To investigate the influence of stereogenic centers of sugar‐based surfactants for enantiomeric separation, four n‐dodecyl thioglycoside sulfates (CMC 1.5–3.6 mM) were chosen as micelle‐forming surfactants and five dansylated hydrophobic amino acids were used as test analytes. The analytes were mutually separated by these micelles exhibiting almost similar migration times independent of the used surfactant. Baseline separations of all enantiomers were achieved using both β‐D ‐glucose and β‐D ‐galactose derivates that have an equatorially oriented hydroxy group at C‐2 position. In contrast, the ability of enantioseparation was markedly decreased in the case of β‐D ‐mannose and 2‐deoxy‐β‐D ‐glucose derivatives. These results suggested that the structure of C‐2 position of the sugar unit, namely presence of an equatorially oriented hydroxy group, is highly important for the enantiomeric separation of the chosen hydrophobic dansylated amino acids.     

Amperometric Biosensor for Hydrogen Peroxide,Using Supramolecularly Immobilized Horseradish Peroxidase on the β‐Cyclodextrin‐Coated Gold Electrode

Horseradish peroxidase, previously modified with 1‐adamantane moieties, was supramolecularly immobilized on gold electrodes coated with perthiolated β‐cyclodextrin. The functionalized electrode was employed for the construction of an amperometric biosensor device for hydrogen peroxide using 1 mM hydroquinone as electrochemical mediator. The biosensor exhibited a fast amperometric response (6 s) and a good linear response toward H₂O₂ concentration between 12 μM and 450 μM. The biosensor showed a sensitivity of 1.02 mA/M cm², and a very low detection limit of 5 μM. The electrode retained 97% of its initial electrocatalytic activity after 30 days of storage at 4 ⁰C in 50 mM sodium phosphate buffer, pH 7.0.     

Pressure‐assisted capillary electrophoresis frontal analysis for faster binding constant determination

Adding external pressure during the process of capillary electrophoresis usually add to the band broadening, especially if the pressure induced flow is significant. The resolution is normally negatively affected in pressure‐assisted capillary electrophoresis (PACE). Frontal analysis (FA), however, can potentially benefit from using an external pressure while avoiding the drawbacks in other modes of CE. In this work, possible impact from the external pressure was simulated by COMSOL Multiphysics®. Under a typical CE‐FA set‐up, it was found that the detected concentrations of analyte will not be significantly affected by an external pressure less than 5 psi. Besides, the measured ligand concentration in PACE‐FA was also not affected by common variables (molecular diffusion coefficient (10⁻⁸ to 10⁻¹¹ m²/s), capillary length etc). To provide an experimental proof, PACE‐FA is used to study the binding interactions between hydroxypropyl β‐cyclodextrin (HP‐β‐CD) and small ligand molecules. Taking the HP‐β‐CD /benzoate pair as an example, the binding constants determined by CE‐FA (18.3 ± 0.8 M⁻¹) and PACE‐FA (16.5 ± 0.5 M⁻¹) are found to be similar. Based on the experimental results, it is concluded that PACE‐FA can reduce the time of binding analysis while maintaining the accuracy of the measurements.     

Chiral separation of brompheniramine enantiomers by recycling high‐speed countercurrent chromatography using carboxymethyl‐β‐cyclodextrin as a chiral selector

A recycling high‐speed countercurrent chromatography protocol was proposed for the enantioseparation of brompheniramine by employing β‐cyclodextrin derivatives as a chiral selector. The two‐phase solvent system of n‐hexane/isobutyl acetate/0.10 mol/L phosphate buffer solution with a volume ratio of 2:4:6 was selected by a series of extraction experiments. Factors that affected the distribution of the enantiomers over the two‐phase system (e.g., the type and concentration of β‐cyclodextrin derivatives = pH value of the aqueous solution, and the separation temperature) were also investigated. In addition, the theory of thermodynamics is applied to verify the feasibility of the enantioseparation process and the corresponding results demonstrate that this separation process is feasible. The optimized conditions include carboxymethyl‐β‐cyclodextrin concentration of 0.010 mol/L, pH of 7.5, and temperature of 5°C. Under the optimal conditions, the purities of both monomer molecules were over 99%, and the recovery yields were 88% for (+)‐brompheniramine and 85% for (–)‐brompheniramine, respectively.     

Rapid analysis of alkylphosphonate drugs by capillary zone electrophoresis using indirect ultraviolet detection

A rapid capillary electrophoretic method for the analysis of three alkylphosphonate drugs (i.e. fosfomycin disodium (FOS), clodronate disodium (CLO) and alendronate sodium (ALN)) was developed by using multiple probe BGE and indirect UV detection. BGE containing 30 mM benzoic acid, 5 mM salicylic acid and 0.5 mM CTAB (pH 3.8), temperature of 30°C, applied voltage of ?30 kV and detection at 220 nm provided baseline separation of all analytes (resolution (R)>2.2) in 3.2 min. EOF reversal by addition of CTAB and negative voltage polarity leading to the co‐EOF flow and short analysis time. Two probe BGE greatly improved peak symmetry. The method showed good linearity (r²>0.999 in ranges of 20–1000 μg/mL for FOS, 100–1000 μg/mL for CLO and 100–750 μg/mL for ALN) repeatablitiy (RSD<2.15%), recovery (99.3–101.1%) and sensitivity (LOD<50 μg/mL). Freshly prepared BGE and sample solutions are essential for the method precision and accuracy. This new method can be utilized for routine analysis of FOS, CLO and ALN in dosage forms because of its efficiency, reliability, speed and simplicity.     

Determination of peimine and peiminine in Bulbus Fritillariae Thunbergii by capillary electrophoresis by indirect UV detection using N-(1-naphthyl)ethylenediamine dihydrochloride as probe

A simple and inexpensive CE method was developed for the determination of peimine and peiminine. Because of the lack of an UV chromophore of peimine and peiminine, the detection method chosen was indirect UV detection, with N‐(1‐naphthyl)ethylenediamine dihydrochloride (NED) as the UV absorbing probe. It was thought that NED, a chromophoric ion, may form hydrogen bonding pairs with the analytes to cause significant changes in separation selectivity. Additionally, the hydrophobic interactions between analytes and the probe also play a crucial role in achieving a resolution between the two analytes. The analyses were carried out with a background electrolyte composed of 66% MeOH–ACN (1:1, v/v), 34% aqueous buffer containing 15 mM NaH₂PO₄, 2.5 mM NED, 4 mM H₃PO₄. MeOH–ACN mixtures used as organic modifiers can not only reduce the adsorption of NED to the capillary wall, but also decrease the baseline noise and drift. The method provided a linear response ranging from 5 to 200 μg/mL. The limits of detection (LODs) for peimine and peiminine were 3.9 and 4.1 μg/mL, respectively. The repeatabilities (n = 3) reached relative standard deviation values (RSDs) of 3.4 and 4.1% for the peak areas, 4.0 and 4.4% for the peak heights, and 0.29 and 0.30% for the migration time of peimine and peiminine, respectively. Regression equations revealed linear relationships (r = 0.9995–0.9996) between the peak area of each analyte and the concentration. The method developed was successfully applied to quantify peimine and peiminine in chloroform extracts of the ground Bulbus Fritillariae Thunbergii.