Cationic double-chained surfactant as pseudostationary phase in micellar electrokinetic capillary chromatography for drug separations

The cationic double-chained surfactant didodecyldimethylammonium bromide (DDAB) was used as pseudostationary phase (PSP) in micellar electrokinetic capillary chromatography (MEKC). Its performance on the three kinds of drugs, i.e., basic, acidic, and neutral drugs, was systematically investigated. Nicotine, cotinine, caffeine, lidocaine, and procaine were selected as the model basic drugs. Good baseline separation and high efficiency were obtained under the optimal separation condition that consisted of 50mM phosphate (pH 4.0) and 0.08 mM DDAB. Three basic phenylenediamine isomers can also be well separated with DDAB in buffer. In addition, DDAB can form cationic bilayer on the capillary wall, thus the wall adsorption of basic analytes was greatly suppressed. Compared with commonly used CTAB, the separation of basic drugs was significantly improved with a much lower amount of DDAB present in the buffer. The DDAB-involved MEKC also showed superiority to CTAB upon the separation of acidic drugs, amoxicillin and ampicillin. In the case of neutral compounds, a good separation of resorcinol, 1-naphthol and 2-naphthol was achieved with 0.1mM DDAB and 30% (v/v) acetonitrile (ACN) present in buffer. Hence, it was concluded that the double-chained cationic surfactant DDAB can be a good substitute for traditional single-chained surfactant CTAB in MEKC.     

The two-phase amphiphilic preconcentration based on surfactants to enrich phenolic compounds from diluted plant extracts and rat urine by micellar electrokinetic chromatography

A novel technology by two-phase amphiphilic preconcentration based on surfactants was established for enriching phenolic compounds by micellar electrokinetic chromatography (MEKC). The cationic surfactant cetyltrimethylammonium chloride (CTAC) was combined with the anionic analytes that existed in the sample solution before injection. The boundary was formed between CTAC and sodium dodecyl sulfate (SDS) in the background solution when the sample solution was injected into the capillary, where the analytes bound inside micelles were released due to the stronger electrostatic force between SDS and CTAC. This procedure accelerated the separation of analytes from CTAC and greatly improved the enrichment efficiency. The optimal conditions were obtained after a series of optimizations, and the sensitivity enrichment factors of the four analytes were in the range of 39–93 compared to typical injections in capillary zone electrophoresis. Good linearity for matrix-matched calibrations was established for all analytes with R² values of 0.9993–0.9997. The limits of detection (S/N = 3) for kaempferol, quercetin, salvianolic acid C, and salvianolic acid B were 0.0166, 0.0292, 0.0215, and 0.0195 µg/ml, respectively. The intracapillary RSDs of the analytes ranged from 0.8% to 1.3% for migration time and from 0.4% to 1.8% for peak areas. The developed method was successfully applied to the determination of phenolic compounds, the main compounds of Salvia miltiorrhiza Bge., and had been validated for the determination of spiked recoveries in rat urine.     

Separation of alkamides from Echinacea purpurea extracts by cyclodextrin-modified micellar electrokinetic chromatography

Separation of nine important alkyl methylbutyl- and isobutylamides (known as alkamides) obtained from Echinacea purpurea extracts was investigated by using cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC). Hydrophobic alkamides interact strongly with the micelles from the most common surfactants used in MEKC and this lead to predominant partition of the analytes into the micellar phase, resulting in poor resolution. The addition of neutral CDs to the alkaline (10 mM phosphate buffer pH 8.0) micellar system of sodium dodecyl sulfate (SDS), sodium cholate (SC) and sodium deoxycholate (SDC) was found to improve the separation of the studied alkamides. Among the several combinations surfactant/CD, three different systems showed to be particularly effective: SDS/hydroxypropyl-beta-CD (110 mM/100 mM) and SC/heptakis (2, 3, 6-tri-O-methyl)-beta-CD (200 mM/40 mM) which provided a complete separation of the studied compounds, and SDC/heptakis (2, 6-di-O-methyl)-beta-CD. The importance of appropriate surfactant vs. CD concentration ratio as well as that of total concentration of both surfactant and CD was considered. The optimization of the separation was performed by focussing the need for a rapid separation of nine alkamides diagnostically useful to define the fingerprint of Echinacea species.     

Separation Study of Eight Isoflavones by MEKC with Different Surfactants

Isoflavones are a very important group of natural products. This study investigated the separation of eight isoflavones, namely ononin, daidzin, genistin, biochanin A, formononetin, puerarin, genistein, and daidzein, from pueraria by micellar electrokinetic chromatography (MEKC) with different surfactants. The following micellar systems of MEKC were systematically compared for the analysis of these isoflavones: (1) a single surfactant comprising the anionic surfactant sodium dodecyl sulfate (SDS), the cationic surfactant hexadecyltrimethylammonium bromide, the neutral surfactant polyoxyethylene sorbitan monolaurate (Tween 20), and the ionic liquid-type surfactant (also a cationic surfactant) 1-dodecyl-3-methylimidazolium tetrafluoroborate (C₁₂MIMBF₄); (2) different single surfactants with 1-butyl-3-methylimidazolium tetrafluoroborate (BMImBF₄) as an additive (modifier); and (3) mixed micelles of SDS + Tween 20 and C₁₂MIMBF₄ + Tween 20. Both SDS with BMImBF₄ as additive and mixed micelles of SDS + Tween 20 had the highest separation efficiency for the eight investigated compounds. Furthermore, the SDS with BMImBF₄ as additive was more stable (good repeatability of retention time and peak shape of analytes) than mixed micelles of SDS + Tween 20, which may be the result of a stabilizing effect of BMImBF₄. Therefore, the final analytical conditions were 15 mM SDS added with 50 mM BMImBF₄ in 30 mM sodium tetraborate (STB, pH 9.5) as running buffer; applied voltage, 20 kV; injection, 50 mbar for 5 s; cartridge temperature, 25 °C; compounds were detected at 260 nm. The developed method was fully validated (limit of detection, limit of quantification, intraday precision, inter-day precision, and recovery) and successfully applied to determine the eight analytes in three Radix Puerariae samples. The present study indicated that SDS with ionic liquids as additive in MEKC was suitable for the analysis of isoflavones.

     

Determination of nonylphenol and nonylphenol ethoxylates in wastewater using MEKC

Nonylphenol ethoxylates (NPEO_x) are surfactants which are used worldwide and can be transformed in the environment by microorganisms to form nonylphenol (NP). Analysis of these compounds was carried out with micellar electrokinetic capillary chromatography (MEKC). Different parameters such as background electrolyte (BGE) solution, pH, type of surfactant, and sample stacking were optimized. The use of CHES (20 mM, pH 9.1) in combination with 50 mM sodium cholate as a surfactant as BGE solution, together with sample stacking using 50 mM NaCl in the sample and an injection time of 20 s, provided the best separation of the compounds studied. The method was applied to the determination of target analytes in two types of sludge water coming from two steps of a wastewater treatment plant. Liquid–liquid extraction was carried out using toluene as solvent, resulting in recoveries around 100% for all studied analytes. The presence of NPEO_x was observed in the first step of the sludge water treatment, based on migration time and UV spectra. Identification was confirmed using tandem MS. LOQs of the studied compounds were in the range of 12.7 to 30.8 ng/mL, which is satisfactory for the analysis of real wastewater samples.     

Cationic surfactants for micellar electrokinetic chromatography: 2. Representative applications to acidic, basic, and hydrophobic analytes

Changes in MEKC chemical selectivity that are induced by changes in the headgroup structure of cationic surfactants are examined. Separations of acidic, basic, and hydrophobic solutes are examined. The acidic analytes are comprised of methoxyphenols, which are of interest due to their prevalence in wood smoke. The basic solutes consist of compounds often found in forensic urine analysis, and represent typical basic pharmaceuticals. The hydrophobic solutes are six pharmaceutical corticosteroids used in replacement therapy of adrenocortical insufficiency and nonspecific treatment of inflammatory and allergic conditions. The role of the headgroup was found to be quite significant when analyzing acidic compounds with not all the surfactants being able to resolve all of the analytes. The headgroup also induced migration order switches among the acidic analytes. All of the surfactants examined here in were found to be suitable for the analysis of basic analytes with each surfactant providing unique selectivity. The hydrophobic solutes were separated best with the larger more hydrophobic surfactant headgroups. The steroid separation with these two surfactants was achieved without the use of organic modifiers or a mixed micellar phase.     

Use of capillary electrophoresis as a versatile tool to measure interaction constants between a KDR‐binding PEGylated lipopeptide and pegylated phospholipid micelles

In the frame of our molecular imaging activities, a PEGylated lipopeptide has been developed as a specific ligand for the human vascular endothelial growth factor receptor 2, which is considered as one of the important molecular marker of angiogenesis. In this study, the potential of affinity capillary electrophoresis (ACE) is evaluated to measure the interactions of an active PEGylated lipopeptide, its hydrolysis product and its precursor consisting of a peptide structure with different micelles including Brij‐35, Tween‐20, and pegylated phospholipids. Given the amphiphilic structure of the PEGylated lipopeptide, a MEKC method allowing the simultaneous separation of the compounds of interest was set up, using low percentages of acetonitrile. Analytes were resolved using a BGE consisting of 100 mM borate buffer pH 9.0, 1 mM Brij, and 25% acetonitrile. Optimized conditions were then used to perform ACE experiments. The affinity constants of the analytes with the micelles were calculated on the basis of their mobility decrease when surfactant concentration increased in the electrolyte. The use of different linearization models to estimate affinity constants was discussed and comparison of different surfactants was reported. PEGylated lipopeptide interacted more strongly with pegylated phospholipid micelles than with Brij‐35 or Tween‐20. Moreover, it is likely that the chemical structure of the compounds, and particularly the lipidic part of the molecules, significantly affects the interaction with micelles. In conclusion, the ACE method can be readily applied to investigate interactions of our targeting lipopeptides with various micelles currently used for the preparation of pharmaceutical vehicles.     

Novel anionic copolymerized surfactants of mixed achiral and chiral surfactants as pseudostationary phases for micellar electrokinetic chromatography

One disadvantage of amino acid-based chiral selectors for micellar electrokinetic chromatography (MEKC) is that either they have very low solubility or are insoluble at acidic pHs. In order to increase solubilities at lower pHs, we have synthesized a highly water-soluble achiral surfactant and copolymerized it with an amino acid-based chiral surfactant. These two surfactants were polymerized either separately or at various molar rations of binary solutions, yielding pure molecular or copolymerized surfactant (CoPS), respectively. All surfactants were characterized by use of several analytical techniques prior to using them as novel pseudostationary phases in MEKC. The chromatographic performance of the CoPS in MEKC was tested with chiral and achiral analytes. The highly soluble sulfate head group significantly increased the solubility of amino acid-based CoPS over a wide range of pH. Three chiral binaphthyl derivatives were tested and each surfactant system was found to have different selectivity.     

Towards a general approach for the impurity profiling of drugs by micellar electrokinetic chromatography

A general micellar electrokinetic chromatographic (MEKC) strategy for the impurity profiling of drugs was developed involving a sodium dodecyl sulfate (SDS) and a cetyltrimethylammonium bromide (CTAB) MEKC system. With this combination, in principle, each sample component passes the detector in at least one of the two MEKC systems provided that separation buffers of the same pH are used in both systems. In order to select the proper MEKC systems, the electroosmotic flow (EOF) and micelle migration time (t(mc)) were determined for separation buffers of several pH values, containing various amounts of surfactant and organic modifier. The selectivity of the MEKC systems was studied using a mixture of compounds with a wide range of physico-chemical properties. The final selection of two adequate MEKC systems for this approach was based on the requirements that the t(mc) (i.e., analysis time) of both systems was below 20 min and that the t(mc)/t(eof) ratio was above 3 or 2 for the SDS and CTAB system, respectively. Furthermore, the systems should provide high efficiency, exhibit differences in selectivity and use moderate concentrations of modifier and surfactant, so that, if needed, further optimization is possible. The selected MEKC systems contained 60 mM SDS or 10 mM CTAB, respectively, in a phosphate buffer (pH 7.5) with 10% acetonitrile. Some test compounds with extreme mobilities were used to demonstrate the suitability of the MEKC approach to detect each component of a sample. The potential of the proposed MEKC combination for impurity profiling was demonstrated by the analysis of fluvoxamine with several impurities at the 0.1% level.     

Using the cationic surfactants N-cetyl-N-methylpyrrolidinium bromide and 1-cetyl-3-methylimidazolium bromide for sweeping–micellar electrokinetic chromatography

This paper describes a sweeping–micellar electrokinetic chromatography (sweeping–MEKC) technique for the determination of seven benzodiazepines, using, as sweeping carriers, the ionic liquid-type cationic surfactants 1-cetyl-3-methylimidazolium bromide (C₁₆MIMBr) and N-cetyl-N-methylpyrrolidinium bromide (C₁₆MPYB). These surfactants resemble the commonly employed cationic surfactant cetyltrimethylammonium bromide (CTAB), but they provide different separation efficiencies. We optimized the separation and sweeping conditions, including the pH, the concentrations of organic modifier and surfactant, and the sample injection volume. Adding C₁₆MIMBr or C₁₆MPYB to the background electrolyte enhanced the separation efficiency and detection sensitivity during the sweeping–MEKC analyses of the benzodiazepines. C₁₆MIMBr enhanced the sensitivity for each benzodiazepine 31–59-fold; C₁₆MPYB, 86–165-fold. In the presence of C₁₆MPYB, the limits of detection for the seven analytes ranged from 4.68 to 9.75 ng/mL. We adopted the sweeping–MEKC conditions optimized for C₁₆MPYB to satisfactorily analyze a human urine sample spiked with the seven benzodiazepines. To minimize the matrix effects, we subjected this urine sample to off-line solid phase extraction (SPE) prior to analysis. The recoveries of the analytes after SPE were satisfactory (ca. 77.0–88.3%). Our experimental results reveal that the cationic surfactant C₁₆MPYB exhibits superior sweeping power relative to those of C₁₆MIMBr and CTAB and that it can be applied in sweeping–MEKC analyses for the on-line concentrating and analyzing of benzodiazepines present in real samples at nanogram-per-milliliter concentrations.     

Micelle to trapping solution stacking in micellar electrokinetic chromatography

An analytical strategy micelle to trapping solution stacking (MSS) was developed in acidic buffer in micellar electrokinetic chromatography (MEKC). The stacking mechanism is based on the transport, release, capturing of molecules bound to micelle carriers that are made to collapse into trapping solution (TS) to serve as the medium to contain and stacking the analytes. Tetrandrine and fangchinoline were selected as model mixture using sodium dodecyl sulfate (SDS) micelles as carrier to demonstrate this stacking method. The experiments by MSS-MEKC were carried out and further compared with those by normal MEKC. The results reveal that 113–123-fold improvements in the detection sensitivity was obtained for the analytes, and separation and determination of tetrandrine and fangchinoline in Stephaniae tetrandrae S. Moore and Fengtongan capsules were finished under optimum conditions using the sample matrix containing 8.0 mM SDS and TS containing 50 mM H₃PO₄–55% (v/v) ethanol.     

Dodecyl thioglycopyranoside sulfates: novel sugar-based surfactants for enantiomeric separations by micellar electrokinetic capillary chromatography

Four novel chiral anionic surfactants having carbohydrate hydrophilic heads, sodium n-dodecyl 1-thio-beta-D-glucopyranoside 6-hydrogen sulfate (6-betaGlcD), sodium n-dodecyl 1-thio-beta-L-glucopyranoside 6-hydrogen sulfate (6-betaGlcL), sodium n-dodecyl 1-thio-beta-L-fucopyranoside 3-hydrogen sulfate (3-betaFucL), and sodium n-dodecyl 1-thio-alpha-L-rhamnopyranoside 3-hydrogen sulfate (3-alphaRhaL), were synthesized by selective sulfation of the corresponding thioglycosides. Their CMC determined by fluorescence using pyrene as a probe in water was 1.3-2.7 mM. These surfactants found to be useful as chiral selectors for enantiomeric separation by MEKC. The enantiomeric separation was optimized with respect to pH, buffer concentration, and surfactant concentration. Under the optimized conditions (50 mM phosphate buffer at pH 6.5, 30 mM surfactant, 20 kV), the enantiomeric separations of five dansylated amino acids (Dns-AAs) were achieved within approximately 20 min with the migration order of Val相似文献   

Widening of the elution window in micellar electrokinetic chromatography with cationic surfactants. II. Cationic additives and modifiers of the electroosmotic flow.

In micellar electrokinetic chromatography (MEKC) with cationic surfactants the migration window is significantly narrower than with anionic surfactants. In order to overcome this disadvantage of cationic surfactants, it is investigated whether it is possible to widen the migration window by reducing the velocity of the aqueous phase while the electrophoretic mobility of the micelles is maintained. Short chain alkylammonium compounds, hexamethonium bromide and hydroxypropylmethylcellulose are tested as additives to the separation electrolyte with the potential to improve the migration window via reducing the velocity of the electroosmotic flow. It will be shown that these modifiers can be successfully used in order to widen the migration window in MEKC with cationic surfactant employing an alkyltrimethylammonium bromide as micelle forming agents. Influence of the modifiers selected on retention of neutral and acidic solutes and on efficiency of the separation system is investigated.     

Simultaneous determination of phosphorus-containing amino acid-herbicides by nonionic surfactant micellar electrokinetic chromatography with laser-induced fluorescence detection

The potential of micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection for the separation and determination of phosphorus-containing amino acid-herbicides (glufosinate and glyphosate), and aminomethylphosphonic acid (the major metabolite of glyphosate), involving derivatization with fluorescein isothiocyanate (FITC) isomer I, was investigated. Different variables that affect derivatization (pH, FITC concentration, time and temperature) and separation (pH and concentration of the buffer, kind and concentration of surfactants and applied voltage) were studied. The analysis was conducted within about 8 min and the use of the nonionic surfactant Triton X-100 improved the selectivity, thus indirectly enhancing sensitivity by shifting of the interfering peaks of the FITC excess. Dynamic ranges of 2.0-3,000 microg/L, limits of detection at microgram or submicrogram-per-liter level, and relative standard deviations from 4.7 to 6.4% were obtained. The ensuing method--nonionic surfactant MEKC-- is a useful choice for the determination of these herbicides as it provides limits of detection similar or lower than those reported by existing chromatographic alternatives without the use of an additional preconcentration technique such as solid-phase extraction. The separation of a mixture of nine FITC-derivatized amino acids, selected as target compounds, was also carried out to assess the discrimination power of the nonionic surfactant MEKC method for the analysis of closely related anionic analytes.     

Analysis of organic components of smokeless gunpowders: high-performance liquid chromatogaphy vs. micellar electrokinetic capillary chromatography

The aim of the present study was to verify the analytical performances of high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MEKC) for the separation and qualitative determination of a selected group of organic components of smokeless gunpowders. The HPLC method was based on a gradient reversed-phase elution with a mobile phase composed of 0.17 M H(3)PO(4)/methanol; detection was performed by UV absorption at the wavelengths of 220, 254, and 270 nm. The MEKC experiments were carried out by using uncoated fused-silica capillaries (50 microm inside diameter, 50 cm effective length) and a running buffer composed of 10 mM sodium tetraborate at pH 9.24 added with 25 mM sodium dodecyl sulfate (SDS); the applied voltage was 25 kV; detection was either at a fixed wavelength UV of 214 nm or with a diode-array detector operating in the wavelength range from 190 to 350 nm. Both reversed-phase HPLC and MEKC techniques succeeded in resolving the tested standard mixtures of organic components of smokeless powders. Although the sequence of elution of the different analytes was slightly different between HPLC and MEKC, a statistical analysis based on the Spearman's rank correlation test showed that the two separation patterns were highly correlated. HPLC and MEKC were comparable in terms of elution/migration time precision, whereas MEKC showed higher reproducibility of peak areas. The interfacing of capillary electrophoresis with diode array UV detection provided distinct UV spectra of the individual analytes, thus improving, on the detection side, the analytical selectivity and identification power of capillary electrophoresis.     

Analysis of bacitracin by micellar electrokinetic capillary chromatography with mixed micelle in acidic solution

A method used for quantitative analysis of bacitracin with micellar electrokinetic capillary chromatography (MEKC) is described. As capillary zone electrophoresis gave poor separation selectivity, MEKC was preferable. It was found that a zwitterionic surfactant, 3-(N,N-dimethylhexadecylammonium)-propanesulfonate (PAPS) gave the best selectivity among the several surfactants studied. As the analytes tend to adsorb onto the capillary wall due to their positive charge, an acidic solution composed of Tris-phosphate buffer at pH 2.5 was necessary to diminish such adsorption. The peak tailing caused by relatively strong ion pair interaction between the analyte and PAPS micelle could be reduced by adding nonionic surfactant Brij 35 to the PAPS solution. This phenomenon is possibly explained by a mixed micelle mechanism. In order to obtain the optimal conditions and to test the method robustness, a central composite experimental design was performed. The optimal conditions are as follows: 44 cm length of fused-silica capillary with 50 microm inner diameter, 90 mM Tris-phosphate buffer (pH 2.5) containing 17 mM PAPS and 0.3% w/v-Brij 35, 18 kV applied voltage, UV detection at 192 nm and 25 degrees C column temperature. Under the optimal conditions, more than 50 peaks could be obtained in 30 min. The method had a linearity range from 1 to 0.05 mg/mL (concentration of bacitracin A). The limit of quantitation (LOQ) and limit of detection (LOD) were 0.005 and 0.0012 mg/mL, respectively.     

Separation and determination of haloperidol, parabens and some of their degradation products by micellar electrokinetic chromatography

A micellar solution containing phosphate buffer, anionic surfactant, and water-miscible organic solvent was employed as a migration solution for the separation and the quantification of eleven analytes by micellar electrokinetic chromatography (MEKC): the analytes examined were haloperidol, methylparaben, ethylparaben, n-propylparaben, iso-propylparaben, n-butylparaben, iso-butylparaben, sec-butylparaben, 4-(4-chlorophenyl)-4-hydroxypiperidine, 4-fluorobenzoic acid and 4-hydroxybenzoic acid. In order to provide good separation between micelle and haloperidol, which showed strongest interaction with the micelle among the analytes, surfactant concentrations and organic modifier percentages were studied with phosphate buffer at pH 7.0. All the analytes were successfully resolved when 10 mM sodium dodecylsulfate and 15% ethanol were contained in the migration solution; the time window was very wide in the range from 14.8 to 65.5 min. Optimized applied voltage at 30 kV and capillary temperature at 45 degrees C enable analyze all compounds in less than 17 min with the best resolution, the shorter migration time window, the highest precision and lowest detection limit.     

Direct coupling of micellar electrokinetic chromatography to mass spectrometry using a volatile buffer system based on perfluorooctanoic acid and ammonia

Direct coupling of micellar electrokinetic chromatography (MEKC) to mass spectrometry (MS) without employing partial filling is considered to be a challenge. One way of solving the problem would be the use of an MS-compatible surfactant. In the present study, the applicability of a series of surfactants (sodium dodecyl sulfate (SDS), lauric acid, cholic acid and perfluorated carboxylic acids) have been investigated both in terms of separation performance and MS compatibility. It was found that a MEKC system based on perfluorooctanoic acid (PFOA) and ammonia gave excellent results. The separation performance of the suggested system is comparable to the one obtained with standard systems based on SDS and sodium borate buffer although the selectivity is different. The electrospray ionization MS signal of the analytes is not seriously suppressed even at a PFOA concentration of 100 mM. Clusters are formed but their intensities are relatively low and comparable to those obtained with acetic acid. PFOA is volatile enough to allow long-term use, 30 h of continuous use has been recorded without any signs of decreasing performance. After use residual PFOA is easily removed from the ion-source (no memory effects). Furthermore, quantitation of trace impurities is possible at 25 ppb level when employing selected ion monitoring.     

Detection of caffeine and its main metabolites for early diagnosis of Parkinson's disease using micellar electrokinetic capillary chromatography

Caffeine (CA) is a common xanthine alkaloid found in tea leaves, coffee beans, and other natural plants, and is the most widely used psychotropic substance in the world. Accumulating evidence suggests that low plasma levels of CA and its metabolites may serve as reliable diagnostic markers for early Parkinson's disease (PD) patients. In this study, we demonstrated a new MEKC method for determining CA and its three main downstream metabolites, paraxanthine (PX), theobromine (TB), and theophylline (TP), in human plasma. Plasma samples were collected, and analyzed using MEKC, after SPE. The running buffer was composed of 35 mM phosphate, pH of 10.5, and 25 mM SDS. The separation voltage was 15 kV and the detection wavelength was at 210 nm. Under the optimum conditions, four distinct analytes were completely separated and detected in less than 12 min. Method limits of detection were as low as 7.5 ng/mL for CA, 5.0 ng/mL for TB, and 4.0 ng/mL for both PX and TP. The recoveries were between 88.0% and 105.9%. This method was successfully applied to 27 human plasma samples. The results indicate that the plasma concentrations of the four analytes are significantly lower in patients with early PD than in control subjects (p < 0.05). The area under curve was improved to 0.839 when CA and its three main metabolites were included, suggesting that MEKC testing of CA, TP, TB, and PX may serve as a potential method for early diagnosis of PD.