In-capillary derivatization and analysis of ephedrine and pseudoephedrine by micellar electrokinetic chromatography with laser-induced fluorescence detection

This paper describes an automatic rapid approach for in-capillary derivatization of ephedrine (E) and pseudoephedrine (PE) and subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as fluorescent reagent. The unique feature of this method is the capillary being used as a small reaction chamber, in which the sample, derivatization buffer and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step (5 kV, 15s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 1 min for reaction, the derivatives were then immediately separated and determined. Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated. Under these optimized conditions, a baseline separation of the two analytes was achieved within 10 min and the derivatization concentration limits of detection were found to be 4.8 ng mL(-1) for E and 1.6 ng mL(-1) for PE, respectively. The method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of the two alkaloids in ephedra herb and its preparations.     

Microemulsion electrokinetic chromatography with laser-induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive microemulsion electrokinetic chromatography with laser-induced fluorescence detection method was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol. By a series of optimization, a running buffer composed of 20 mM borate + microemulsion (23.3 mM Sodium dodecyl sulfate/180.85 mM 1-butanol/16.4 mM n-heptane) +8% acetonitrile was applied for the separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.058-11.58 microg.mL(-1) (correlation coefficient: 0.9993 for E, 0.9995 for PE), and the detection limits for E and PE were 5.3 and 3.9 ng.mL(-1). The method was applied to the analysis of the two alkaloids in Chinese traditional herbal preparations with recoveries in the range of 96.9-105.4%.     

Micellar electrokinetic chromatography with laser-induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive micellar electrokinetic chromatography method with laser-induced fluorescence detection was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. After conducting a series of optimizations, a running buffer of 10 mM sodium borate + 16 mM SDS was used for separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.044-6.6 microg mL(-1) (correlation coefficient: 0.9943 for E, 0.9946 for PE), and the detection limits for E and PE were 0.70 and 0.30 ng mL(-1), respectively. The sensitivity of E and PE was improved by several multiples of ten over those of CZE-LIF method. The method was applied to the analysis of the two alkaloids in ephedra herbal medicine and preparations with recoveries in the range of 98.3-107.1%.     

Rapid and sensitive determination of ephedrine and pseudoephedrine by micellar electrokinetic chromatography with on-line regenerating covalent coating

A rapid, sensitive and reproducible micellar electrokinetic chromatographic method using hexamethyldisilazane as on-line regenerating covalent coating was developed for the quantification of ephedrine (E) and pseudoephedrine (PE). E and PE were derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol for laser-induced fluorescence detection. The on-line regenerating covalent coating formed a combinative double coating with the subsequently produced dynamic SDS coating. The total coating can be easily removed and conveniently regenerated on-line. The simple coating procedure was described. By a series of optimization, a running buffer of 20 mm Na(2)B(4)O(7) + 16 mm SDS was applied for the separation of the derivatives. Linear relationships for E and PE were obtained in the range of 0.044-6.60 microg mL(-1) (correlation coefficients: 0.9975 for E, 0.9981 for PE), and the detection limits for E and PE were 1.71 and 0.67 ng mL(-1), respectively. The separation speed, the reproducibility and the sensitivity were much improved over those of other capillary electrophoresis methods more recently reported. The method was applied to the analysis of the two alkaloids in traditional herbal preparations with recoveries in the range 92.8-104.8%.     

Analytical potential of fluorescein analogues for ultrasensitive determinations of phosphorus-containing amino acid herbicides by micellar electrokinetic chromatography with laser-induced fluorescence detection

The analytical potential of three fluorescein analogues, fluorescein isothiocyanate isomer I (FITC), 5-(4,6-dichlorotriazinylamino) fluorescein (DTAF) and 5(6)-carboxyfluorescein N-succinimidyl ester (CFSE), as labelling reagents for the ultrasensitive determination of phosphorus-containing amino acid herbicides (glufosinate and glyphosate) and aminomethylphosphonic acid (the major metabolite of glyphosate) by nonionic surfactant micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection was investigated. Practical aspects related to label chemistry and MEKC separation showed that DTAF is the best choice for the determination of these herbicides; in addition, the most important features of these reagents for the derivatization of amino compounds are discussed. The optimum procedure includes a derivatization step of the herbicides at 40 degrees C with DTAF for 1 h and a 2-fold dilution prior to MEKC analysis, which is conducted within about 10 min using Brij-35 in the running buffer. This nonionic surfactant improves the selectivity and therefore the sensitivity of the method at low analyte concentrations by shifting the interfering peaks of the DTAF excess. The lowest detectable analyte concentration ranged from 0.06 to 0.16 microg/L with a precision of 2.1-3.2%. These results indicate that nonionic surfactant MEKC-LIF is useful as a selective, rapid and sensitive tool for the determination of these herbicides showing a great potential for their analysis in environmental samples without previous enrichment steps. The proposed method surpasses other chromatographic alternatives in terms of limit of detection and sample requirements for the analysis.     

Capillary zone electrophoresis hyphenated with laser-induced fluorescence detection for sensitive determination of noradrenaline and dopamine with 5-(4, 6-dichloro-s-triazin-2-ylamino) fluorescein as fluorescent label

A new capillary zone electrophoresis method with laser-induced fluorescence detection is presented for the determination of noradrenaline (NA) and dopamine (DA). The fluorescent reagent, 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein, which readily reacts with amine group under mild conditions, was initially used to label the two compounds. Under optimized separation and labeling conditions, linear relationship between peak area and analyte concentration was obtained with high sensitivity (nM level). The applicability of the method for complex sample analysis was validated by determining the two compounds in a human serum and an herb, Portulaca oleracea L with recoveries ranging from 89% to 102%.     

Sensitive determination of ephedrine and pseudoephedrine by capillary electrophoresis with laser-induced fluorescence detection

A CE-LIF method was developed for the separation and sensitive detection of ephedrine and pseudoephedrine after derivatization by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD-C1). The derivatization and separation conditions were investigated in detail and the optimum conditions were obtained. Under the optimum experiment conditions, good linearity relationships (correlation coefficients: 0.9942 for ephedrine and 0.9970 for pseudoephedrine) between the peak heights and concentrations of the analytes were obtained (0.7-140 microM). The detection limits were 0.16 microM for ephedrine and 0.17 microM for pseudoephedrine, which indicated that the sensitivities were at least ten times improved over those reported in the literature obtained by UV detection. The method was applied to the analysis of ephedrine and pseudoephedrine in ephedra herb plants and preparations with good results.     

Simultaneous determination of catecholamines and amino acids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A selective and sensitive method was developed for separation and simultaneous determination of catecholamines and amino acids by MEKC with LIF. Interestingly enough, such work has been firstly performed on catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole and the detailed derivatization mechanism was discussed. After derivatization at 60 degrees C for 20 min, NBD-labeled catecholamines and amino acids were separated in a buffer system containing 10 mM sodium tetraborate-Na2HPO4, 20 mM SDS, and 10% v/v ACN at pH 9.75. SDS micelles were employed to improve the fluorescence intensity of catecholamine derivatives efficiently. Under optimum conditions, two catecholamines and 11 amino acids were separated in a short 13 min analysis time and the RSDs for migration time and peak area were less than 0.60 and 6.50%, respectively. The method was successfully applied for the quantification of catecholamines and amino acids in Portulaca oleracea L., human urine sample, and mixed injection sample.     

Rapid and simple determination of adenine and guanine in DNA extract by micellar electrokinetic chromatography with indirect laser-induced fluorescence detection

This paper describes a micellar electrokinetic chromatography with indirect laser-induced fluorescence detection method for the simultaneous determination of adenine and guanine in DNA extracts from fungus, maize and soybean. The baseline separation was achieved within 2.5min with running buffer (pH 9.3) composed of 10mM borate, 20mM SDS, 3.0x10(-7)M fluorescein sodium as background reagent, applied voltage of 27.5kV, cartridge temperature of 25.0 degrees C. Good linearity relationships (correlation coefficients>0.9917) between the second-order derivative peak heights (RFU) and concentrations of the analytes (mgml(-1)) were obtained. The detection limits in second-order derivative electrophoregrams were 1.16x10(-3)mgml(-1) for adenine and 0.29x10(-3)mgml(-1) for guanine, respectively. The RSD data of intra-day for migration times and second-order derivative peak heights were less than 0.59 and 4.09%, respectively. This developed method was applied to the analysis of the two purines in DNA extracts with recoveries in the range of 85.3-110.2%. In this work, although the detection sensitivity was lower than that of direct LIF, yet the method has the advantages of rapidness and simplicity. And it would also extend the application range of LIF detector.     

Determination of catecholamines and serotonin by micellar electrokinetic chromatography with laser-induced fluorescence detection

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein, a new synthesized fluorescent reagent, was established for the first time as a label for the sensitive analysis of catecholamines (CAs) and serotonin (5-HT) by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. After careful study on the derivatization conditions such as pH value, reagent concentration, temperature and reaction time, the labeling reaction was accomplished as quickly as 7 min with stable yield. The separation parameters for the CAs and 5-HT were also optimized in detail. The derivatives were baseline separated in a running buffer containing 30 mM boric acid and 15 mM sodium dodeculsulfate at pH 9.0. The detection limits ranged from 5 x 10(-10) to 2 x 10(-9) M (signal-to-noise ratio = 3). The rapid and sensitive method was also applied to the determination of the CAs and 5-HT of urine samples.     

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.     

Determination of phosphoamino acids by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

A micellar electrokinetic capillary chromatography (MEKC) method with laser-induced fluorescence detection (LIF) was developed for analyzing three phosphoamino acids including phosphotyrosine (P-Tyr), phosphoserine (P-Ser), and phosphothreonine (P-Thr). 3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for derivatization of these phosphoamino acids. Results indicated that the complete baseline resolution of each phosphoamino acid was obtained within 10 min, using 20 mmol l⁻¹ sodium borate buffer (pH 9.35) containing 20 mmol l⁻¹sodium deoxycholate (SDC) and 10 mmol l⁻¹ Brij35. Other common amino acids, especially Glu and Asp, did not disturb the assay of these phosphoamino acids. There was a linear relationship between the peak area for analyte and its concentration, with correlation coefficients in the range of 0.9966-0.9996. The concentration detection limits (signal-to-noise = 3) for P-Tyr, P-Ser, and P-Thr were 10, 40, and 75 nmol l⁻¹, respectively. The developed method was successfully applied for determining phosphoamino acids in the hydrolysis sample of a phosphorylated protein kinase.     

Determination of biogenic amines in HeLa cell lysate by 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein and micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

An MEKC-LIF method using 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxy-carbonyl) fluorescein (SAMF) newly synthesized in our lab as a labeling reagent for the separation and determination of eight typical biogenic amines was proposed. After careful study of the derivatization condition such as pH value, reagent concentration, temperature, and reaction time, derivatization reaction was accomplished as quickly as 10 min with stable yield. Optimal separation of SAMF-labeled amines was achieved with a running buffer (pH 9.3) containing 30 mM boric acid, 25 mM SDS, and 20% v/v ACN. The proposed method allowed biogenic amines to be determined with LODs as low as 0.25-2.5 nmol/L and RSD values from 0.4 to 4.5%. The present method has been successfully used to monitor biogenic amines in HeLa cells and fish samples. This study exploits the potential of MEKC-LIF with SAMF labeling as a tool for monitoring biogenic amines involved in complex physiological and behavioral processes in various matrices.     

Selective fluorometric detection of polyamines using micellar electrokinetic chromatography with laser-induced fluorescence detection

The polyamines putrescine, cadaverine, spermine and spermidine were separated and quantified by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. The derivatization reagent, 1-pyrenebutanoic acid succinimidyl ester (PSE), allowed for the selective detection of the polyamines at 490 nm. Multiple labeling of the polyamines with PSE allows the formation of intramolecular excimers that emit at longer wavelengths (450-520 nm) than mono-labeled analytes (360-420 nm). Optimal separation of the labeled polyamines was achieved using a separation buffer consisting of 10 mM phosphate pH 7.2, 30 mM cholate, and 30% acetonitrile. Using these conditions, the four polyamines were separated in under 10 min. Limits of detection for putrescine, cadaverine, spermine and spermidine were 6, 5, 15 and 13 nM, respectively. These are superior or comparable to those previously reported in the literature using fluorescence detection.     

Rapid determination of aniline metabolites of chlorpropham in potatoes by micellar electrokinetic chromatography using negative-charged mixed micelles and laser-induced fluorescence detection

A rapid, reliable method has been developed for the multi-residue analysis of aniline metabolites of chlorpropham in potato samples. The method involves the precolumn derivatization of aniline metabolites with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein (DTAF) and their subsequent separation and determination by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF). The optimum procedure includes a derivatization step of the aniline metabolites (3-chloroaniline, 3-chloro-4-hydroxyaniline and 3-chloro-4-methoxyaniline) at 40 degrees C for 40 min and a 5-fold dilution prior to MEKC analysis, which is conducted within about 7 min using negative-charged mixed micelles (SDS/Triton X-100) in the running buffer. Under these conditions, the DTAF-anilines were readily detected at 0.3-3.1 microg/L level with a precision of 4.8-6.4%. These results indicate that negative-charged mixed surfactant MEKC-LIF is useful as a selective, rapid, and sensitive tool for the determination of these anilines and surpasses other electrophoretic alternatives based on the use of fluorescein-isothiocyanate (FITC) as label reagent. Finally, the potato matrix showed no significant effects on the derivatization and determination of these analytes, since the analytical figures of merit for the real samples were similar to those obtained in aqueous solutions, and the average recovery at fortification levels of 10-250 microg/kg was over 97%.     

Trace determination of short-chain aliphatic amines in biological samples by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

In this study, a new capillary electrophoresis (CE) method is described originally for the sensitive and selective determination of short-chain aliphatic amines in biological samples. These amines were converted into their N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA) derivatives and measured by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection. The derivatization conditions and separation parameters for the aliphatic amines were optimized in detail. The SIFA-labeled amines were fully separated within 8.5 min using 25 mM pH 9.6 boric acid electrolyte containing 60 mM sodium dodecyl sulfate (SDS). The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.02 to 0.1 nM, which was the lowest value reported by CE methods. The developed method was successfully employed to monitor aliphatic amines in serum and cells samples. After comparison of other CE methods using different fluorescent probes, the present method represents a powerful tool for the trace determination of aliphatic amines in complex biological samples.     

Profiling of amine metabolites in human biofluids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A rapid and sensitive capillary electrophoresis (CE) method has been developed for profiling organic metabolites containing amine functional groups in mammalian biofluids. Metabolites containing an amine group were derivatized with 4-fluoro-7-nitrobenzo-2,1,3-oxadiazol (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence detection. The optimized MEKC background electrolyte conditions were: 50 mmol L⁻¹ sodium cholate, 5 mmol L⁻¹ β-cyclodextrin, and 20 mmol L⁻¹ Brij 35 in 20 mmol L⁻¹ aqueous borate buffer, pH 9.3, containing 7% methanol. Under these conditions all the amine compounds in mammalian biofluids, for example plasma, saliva, and urine, were derivatized directly, without extraction, in a minimum volume of 100 nL and the derivatives could be separated within 16 min. Up to 90% of the amine-containing metabolites in plasma and saliva could be identified by reference to standard compounds. For twenty amine standards linearity of calibration was better than R ² = 0.99. Migration-time and peak-area reproducibility were better than RSD 1.5% and 15% respectively. In replicate analysis of human plasma bioanalytical precision ranged between 0.7 and 3.8 RSD% for a 5.0-μL volume and between 1.7 and 5.5 RSD% for 100-nL volume. The concentrations measured were found to be in agreement with literature values.     

Analysis of aminophospholipid molecular species by methyl-beta-cyclodextrin modified micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

Micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence detection is used for the analysis of three classes of aminophospholipids: phosphatidylethanolamine (PE), phosphatidylserine (PS), and lysophosphatidylethanolamine (LPE) molecular species. 3-(2-Furoyl) quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for labeling of these phospholipids. The FQ-labeled lipid species were then separated by sodium deoxycholate MEKC modified with methyl-beta-cyclodextrin. Baseline resolution of each class of phospholipids was achieved within 7 min. The migration time in each class increased with the carbon number of their side aliphatic chain. Separation efficiencies of approximately 3x10(5) plates were observed for most of these species. Concentration detection limits (3 sigma) were from 10(-9) to 10(-10) M for PE and LPE species and from 10(-8) to 10(-9) M for PS species. The relative standard deviations for migration time and peak area were less than 0.9% and 4.5%, respectively, for seven PE species. This method was applied to the separation of PE isolated from HT29 human colon cancer cells and roughly 30 PE species were resolved.     

Determination of alkylphosphonic acids using micellar electrokinetic chromatography with laser-induced fluorescence detection and high-salt stacking

Methyl-, ethyl- and propylphosphonic acids (MPA, EPA, and PPA, respectively) were derivatized with panacyl bromide in dry N,N-dimethylformamide (DMF). After mixing with a high-salt dilution buffer, the derivatives were separated by micellar electrokinetic chromatography in 35 min and detected by laser-induced fluorescence (He-Cd laser excitation at 325 nm and detection at 500 nm). Baseline resolution was achieved using a separation buffer containing 50 mM sodium cholate, 40% (v/v) of acetonitrile and 50 mM borate. Addition of 400 mM NaCl to the dilution buffer allowed the injection time to be increased to 30 s while still maintaining baseline resolution. Limits of detection for MPA, EPA, and PPA were 0.13 microM (12 ppb), 0.13 microM (14 ppb) and 0.14 microM (17 ppb) injected, respectively. The reproducibility of corrected peak area at 15 microM was 3.7 approximately 4.3%.     

Methyl-beta-cyclodextrin modified micellar electrokinetic capillary chromatography with laser-induced fluorescence for separation and detection of phospholipids

Micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection was applied to the separation of amino group-containing phospholipids including phosphatidylethanolamine (PE), phosphatidylserine (PS), lysophosphatidylethanolamine (LysoPE), and lysophosphatidylserine (LysoPS). A fluorogenic dye, 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), was successfully used to fluorescently label these phospholipids. 4-Fluoro-7-nitrobenzofurazan only produced fluorescent product from LysoPE and PE; signals were not observed from LysoPS and PS. A borax buffer containing sodium deoxycholate modified with methyl-beta-cyclodextrin (methyl-beta-CD) was an excellent MECC system for these phospholipids. Under the optimum conditions, four FQ-labeled phospholipid classes were separated within 8 min. Moreover, each of the PE, PS, LysoPE and LysoPS peaks split into two components corresponding to subclasses with different lengths of the fatty acid chains, but these subclasses were completely resolved only for LysoPE. Detection limits ranged from 0.18 to 1.1 fg (10(-9) - 10(-10) M), which was four- to five-orders of magnitude superior to previously reported CE methods.