Competitive immunoassay of phenobarbital by microchip electrophoresis with laser induced fluorescence detection

A microchip electrophoresis method with laser induced fluorescence detection was developed for the immunoassay of phenobarbital. The detection was based on the competitive immunoreaction between analyte phenobarbital and fluorescein isothiocyanate (FITC) labeled phenobarbital with a limited amount of antibody. The assay was developed by varying the borate concentration, buffer pH, separation voltage, and incubation time. A running buffer system containing 35 mM borate and 15 mM sodium dodecyl sulfate (pH 9.5), and 2800 V separation voltage provided analysis conditions for a high-resolution, sensitive, and repeatable assay of phenobarbital. Free FITC-labeled phenobarbital and immunocomplex were separated within 30s. The calibration curve for phenobarbital had a detection limit of 3.4 nM and a range of 8.6-860.0 nM. The assay could be used to determine the phenobarbital plasma concentration in clinical plasma sample.     

Determination of 1-aminocyclopropane-1-carboxylic acid in apple extracts by capillary electrophoresis with laser-induced fluorescence detection

A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues has been described. This method is based on the derivatization of ACC with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), and separation and quantification of the resulting FQ-ACC derivative by capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF). Our results indicated that ACC derivatized with FQ could be well separated from other interfering amino acids using 20 mM borate buffer (pH 9.35) containing 40 mM sodium dodecyl sulfate and 10 mM Brij 35. The linearity of ACC was determined in the range from 0.05 to 5 microM with a correlation of 0.9967. The concentration detection limit for ACC was 10 nM (signal-to-noise = 3). The sensitivity and selectivity of this described method allows the analysis of ACC in crude apple extracts without extra purification and enrichment procedure.     

Hyphenated techniques in anticancer drug monitoring. II. Liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry

A micellar electrokinetic chromatographic (MEKC) method was developed for the separation of ten phenolic acids including cichoric acid and caftaric acids, specific marker phytochemicals of Echinacea purpurea. The MEKC method involved the use of 70 mM sodium deoxycholate (SDC) in 40 mM borate (pH 9.2) buffer and UV detection at 300 nm. The bile acid was used as biosurfactant able to provided a micellar system with different and more selective properties than sodium dodecyl sulfate. The effects of SDC and borate concentration and buffer pH on the analyte resolution were evaluated. The validated method was applied to the determination of cichoric acid and related compounds in E. purpurea root extracts, and in commercial E. purpurea based dried extracts and tablets.     

Rapid analysis of amino acids in Japanese green tea by microchip electrophoresis using plastic microchip and fluorescence detection

Microchip electrophoresis for the short-time analysis of amino acids in Japanese green tea was developed. The amino acids in Japanese green tea were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The derivatives were filtered and directly analyzed by electrophoresis on a plastic microchip with a 31-mm long separation channel with fluorescence detection. Amino acid analysis of Japanese green tea was improved by removing polyphenols using a polyvinylpolypyrrolidone pretreatment. Elution profiles of NBD-amino acids were examined under different running buffer conditions, and the sodium dodecyl sulphate in the running buffer exhibited a dramatically high-separation efficiency of amino acids by inhibiting their adsorption on the channel walls. Under the optimized conditions (5 mM phosphate buffer (pH 5.5) containing 0.05 mM sodium dodecylsulfate as running buffer), the main amino acids contained in Japanese green tea were well separated within 2 min, and theanine (1475 mg/100 g tea leaf), Arg (408 mg/100 g tea leaf) and Gln (217 mg/100 g tea leaf) were detected in Japanese green tea.     

Simultaneous analysis of sulfur-containing excitatory amino acids using micellar electrokinetic chromatography with diode array and laser-induced fluorescence detection

The suitability of micellar electrokinetic chromatography (MEKC) coupled with diode array or laser-induced fluorescence (LIF) detection to analyze the four sulfur-containing excitatory amino acids (SEAA), homocysteine sulfinic acid (HCSA), homocysteic acid (HCA), cysteine sulfinic acid (CSA), and cysteic acid (CA) was investigated. 5-Carboxy-fluorescein succinimidyl ester was chosen as fluorescent reagent to derivatize HCSA, HCA, CSA, and CA. During method development, the yield of reaction dependent on pH and incubation time as well as the stability of the products were analyzed. The maximum yield was obtained after 30 min using a 0.1 M borate buffer (pH 8.9) as derivatization buffer. Each labeled amino acid exhibited high stability at room temperature over a period of 5 days. Baseline separation of labeled HCSA, HCA, CSA, and CA was obtained using a buffer consisting of 0.1 M borate, 50 mM sodium dodecyl sulfate (SDS), and 5% v/v methanol (pH 9.0). By applying LIF detection, limits of detection ranged from 0.9 x 10(-10) M for HCSA to 6.0 x 10(-10) M for CA, respectively. Slightly modified separation conditions enabled the analysis of SEAA in cerebrospinal fluid in the presence of the neurotransmitters glutamate and aspartate. In conclusion, MEKC coupled with LIF detection is a suitable technique for the simultaneous and sensitive analysis of SEAA. Further work will focus on the validation of the method with cerebrospinal fluid as sample matrix.     

Amino acid analysis by micellar electrokinetic chromatography with laser-induced fluorescence detection: application to nanolitre-volume biological samples from Arabidopsis thaliana and Myzus persicae

Amino acids were derivatised 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. The optimised MEKC background electrolyte conditions were: 40 mM sodium cholate, 5 mM beta-cyclodextrin in 20 mM aqueous borate buffer, pH 9.1, with 7% v/v acetonitrile. Using these conditions, 19 amino acids were separated within 17 min. The limits of detection were in the range of 7.6-42.2 pmol/mL and limits of quantitation from 0.05-0.14 nmol/mL. The method was systematically validated for injection volume error, migration time variation, calibration linearity, accuracy, precision, and recovery. Nanolitre volume samples of phloem sap of individual sieve element cells from the plant Arabidopsis thaliana and honeydew from the aphid Myzus persicae were directly analysed with this method. Quantitative amino acid concentrations in these two biological matrices were profiled for the first time. This method is particularly important because it allows the complete profile of the amino acids obtained from individual phloem elements, allowing cell to cell and plant to plant variation to be quantified, which to date has not been possible with Arabidopsis thaliana.     

Characterization of amino acids in silk sericin protein from Gonometa rufobrunnae by MEKC with phenyl isothiocyanate derivatization

An MEKC method was developed for the separation and characterization of phenyl-isothiocyanate (PITC)-labeled amino acids derived from Gonometa rufobrunnae silkworm after microdialysis sample cleanup. The influence of the buffer and SDS concentration on the resolution of the amino acids was investigated. A buffer system consisting of 25 mM phosphate, 10 mM borate buffer at pH 9.00, and 70 mM SDS showed the best results, with 13 PITC-amino acid derivatives being resolved out of 15 possible amino acids that were under study. Microdialysis sampling demonstrated its efficiency as a sample cleanup technique. Sericin protein from G. rufobrunnae was found to be characterized by at least 11 positively identified amino acids. These included His, Tyr, Ser, Ala, Phe, Lys, Gly, Arg, Cys, Glu, and Asp. Leu/Met and Val/Thr were coeluting pairs and hence could not be positively confirmed.     

High-performance liquid chromatographic analysis of free amino acids in fruit juices using derivatization with 9-fluorenylmethyl-chloroformate

A simple, rapid, and reliable reversed-phase high-performance liquid chromatographic method for the analysis of 16 amino acids of main interest in commercial fruit juices (pear, orange, grapefruit, pineapple, peach, and apricot) is described. No sample cleanup is required. The pH of the fruit juices is adjusted to alkaline value (8.5) using 200 mM borate buffer, then amino acid is converted to stable derivatives using 9-fluorenylmethyl-chloroformate. The excess of derivatization reagent is removed by a hydrophobic amine, 1-amino-adamantane hydrochloride. The derivatization procedure is simple, fast, and described in detail. Amino acids are detected at 263 nm and eluted within 35 min. The calibration, precision (< or = 6.1%), and recovery (102% +/- 4%) of the method are reported. The conditions of separation are optimized; however, serine partially overlapped with aspartic acid. The amino acid profile of fruit juices is consistent with data from the literature.     

Analysis of amino acids in human vascular endothelial (ECV-304) cells by microchip electrophoresis with fluorescence detection

A rapid and sensitive method was developed for the analysis of amino acids by microchip electrophoresis with Hg-lamp excitation fluorescence detection. Fluorescein-isothiocyanate (FITC) was chosen to estimate the sensitivity of this system, and the detection limit (S/N = 3) with FITC was 1.7 nM, which showed that the system was sensitive as well as simple. Two derivatizing agents, FITC and ortho-phthalaldehyde (OPA) were employed to label amino acids and were compared in the same fluorescence detection system with an Hg lamp as the excitation source. The separation parameters were optimized in detail. Optimum separation of OPA-labeled amino acids was obtained in less than 200 s with 20 mM borate buffer (pH 9.0) containing 20% acetonitrile and 10 mM beta-cyclodextrin. Detection limits for amino acids (alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp)) of 0.38-1.0 muM were achieved. The method was successfully applied to analysis of amino acids in human vascular endothelial cells (ECV-304). The average amount of amino acids in single ECV-304 cells is estimated to be 5.84 fmol for Ala, 2.78 fmol for Tau, 1.15 fmol for Gly, 3.10 fmol for Glu, and 1.30 fmol for Asp.     

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.     

Simultaneous determination of multiple constituents in real beer samples of different origins by capillary zone electrophoresis

Simultaneous determination of alcohols, amines, amino acids, flavonoids, and purine and pyrimidine bases in bottled beer samples directly without any pre-treatment was carried out by capillary zone electrophoresis with diode-array detection. Electrolyte conditions such as pH, composition and concentration of the buffer, working voltage and type and time of injection were checked. The best separation of the cited analytes was achieved in 70 mM sodium borate solution and pH 10.25. The detection limits were from 2.1 to 5.6 mg L^–1 for the 18 compounds studied. The developed method is rapid, sensitive and quantitative and has been applied to seven types of international bottled beers of different origins bought locally.     

Pre-capillary derivatisation and capillary zone electrophoresis for amino acids analysis in beverages

A simple and rapid method for the simultaneous analysis of amino acids has been developed. Amino acids were derivatised based on pre-capillary derivatisation with 1,2-naphthoquinone-4-sulfonate (NQS) in basic medium (pH 10.0) and developed reaction at 70 degrees C. Their derivatives were analysed by capillary zone electrophoresis (CZE). The parameters affecting CZE separation were investigated including buffer (pH, type and concentration), organic modifier and separation voltage. The optimum condition was 70 mmol L(-1) borate (pH 10.0) containing 10% acetonitrile, separation voltage of 12 kV, and sample injection (0.5 psi, 5s) and on-capillary detection at 240 nm. The separation of seven amino acids was achieved within 17 min. The detection limit was 1.0 mg L(-1) for all studied amino acids. The calibration curves were linear in the concentration range of 1.0-100.0 mg L(-1). The repeatability, intra-day and inter-day analysis were < or = 1.0% and < or = 2.0% for migration time and < or = 5.0% and 6.0% for peak area. The proposed method has been applied to several beverage samples with only a simple dilution and filtration treatment of sample before derivatisation and analysed by CZE.     

Assay of bradykinin-related peptides in human body fluids using capillary electrophoresis with laser-induced fluorescence detection

A CE with LIF detection was developed for separation and determination of bradykinin (BK)-related peptides, such as BK, kallidin (Kal), and neurokinin A (NKA). BK-related peptides were derivatized with FITC prior to CE-LIF analysis. Sodium borate 10 mmol/L at pH 9.5 was selected as derivatization media in order to get the high efficiency. Three peptides were baseline-separated within 10 min by using 110 mmol/L sodium borate-sodium hydroxide solution at pH 10.0 as the running buffer. Concentration detection limits (S/N = 3) for BK, Kal, and NKA were 0.08, 0.5, and 0.2 nmol/L, respectively. Meanwhile we have also developed a simple, quick, and sensitive large-volume sample stacking (LVSS) technique for CE-LIF detection of BK, Kal, and NKA. By using this stacking technique, the detection limits (S/N = 3) for BK, Kal, and NKA were 0.02, 0.05, and 0.04 nmol/L, respectively. This method has been applied to the assay of human saliva and cerebrospinal fluid with satisfactory results.     

Selective determination of gamma-aminobutyric acid, glutamate and alanine by mixed micellar electrokinetic chromatography and fluorescence detection

A mixed micellar electrokinetic chromatography method with fluorescence detection was developed to simultaneously monitor gamma-aminobutyric acid (GABA), glutamate (Glu) and alanine (Ala) in biological samples. Amino acids were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA). The separation of three NDA-labeled isomers (GABA, alpha-ABA, beta-ABA) was studied in detail with different micelles solutions such as sodium dodecyl sulfate (SDS), beta-cyclodextrin (beta-CD) and sodium cholate (SC). Simultaneous resolution of GABA, Glu and Ala from 21 amino acids was achieved within 5 min using 20 mM phosphate buffer at pH 8.7 containing 24 mM SC and 26 mM SDS. The detection limits were 4.0 x 10(-8), 1.1 x 10(-8) and 1.3 x 10(-8) M, for GABA, Glu and Ala, respectively, with S/N = 2. The method was applied to monitor the changes of amount of GABA, Glu and Ala in tobacco leaf in response to cold and dark stress.     

Analysis of amino acid neurotransmitters by capillary electrophoresis and laser-induced fluorescence using a new fluorescein-derived label

A capillary electrophoresis laser-induced fluorescence detection method (CE-LIF) was developed for the separation of eight neurotransmitters tagged on their amino function with 6-oxy-(N-succinimidyl acetate)-9-(2′-methoxycarbonyl) fluorescein (SAMF), a new fluorescent reagent synthesized in our lab. Derivatization was performed in boric acid buffer (pH = 7.75) at 37 °C over 15 min. The pH-independent fluorescence of SAMF (pH 4–9) permits background buffers over a wide range of pH. It was demonstrated that an acidic running buffer offers a better resolution compared to basic medium in terms of resolution and peak shapes. Employing Cu²⁺ as the additive, the molecules were baseline-separated using a running buffer consisting of 40 mM sodium acetate and 2 mM Cu²⁺ (pH 6.0). The detection limits ranged from 1 to 2 × 10⁻¹⁰ M. The method has been validated for the characterization of lymphocyte samples. The results obtained illustrate the advantages of combining SAMF derivatization with CE-LIF for determining neurotransmitters.     

Enantioseparation of baclofen with highly sulfated beta-cyclodextrin by capillary electrophoresis with laser-induced fluorescence detection

The enantioseparation of baclofen (4-amino-3-p-chlorophenylbutyric acid) was achieved by CE-LIF with highly sulfated beta-CD (HS-beta-CD) as chiral selector. Naphthalene-2,3-dicarboxaldehyde was used for the derivatization of nonfluorescent baclofen. HS-beta-CD (2%) containing 50 mM borate buffer at pH 9.5 was chosen as the optimal running electrolyte and applied to the analysis of baclofen enantiomers in human plasma. The linearity of calibration curves (R2 > or = 0.998) for R-(-) and S-(+)-baclofen was in the 0.1-2.0 microM concentration range. After a simple ACN-protein precipitation, the LOD of baclofen in plasma sample was found as low as 50 nM.     

Capillary electrophoretic determination of the constituents of Artemisiae Capillaris Herba

Two capillary electrophoretic methods, a micellar electrokinetic electrophoretic (MEKC) one and a capillary zone electrophoretic (CZE) one, were developed for the separation of 12 constituents in Artemisiae Capillaris Herba. Detection at 254 nm with 20 mM sodium dodecyl sulfate and 20 mM sodium borate buffer (pH 9.82) in MEKC or with 25 mM sodium borate and 6.75 mg/ml 2,3,6-tri-O-methyl-beta-cyclodextrin buffer in CZE was found to be the most suitable approach for this analysis. Within 42 min, the MEKC method could successfully separate 12 authentic constituents, whereof chlorogenic acid, however, appeared as a broad and split peak, and capillarisin and chlorogenic acid overlapped partially with other coexisting substances in crude extract of the herb. The CZE method could completely overcome these problems and was used to determine the amounts of capillarisin, chlorogenic acid, scopoletin and caffeic acid in the extract. The effect of buffers on the constituent separation and the validation of the two methods were discussed.     

Separation and determination of B vitamins and essential amino acids in health drinks by CE-LIF with simultaneous derivatization

An efficient and sensitive method for the separation and determination of three essential amino acids and three B vitamins by CE-LIF with a simultaneous derivatization procedure was developed. The conditions for derivatization and separation of these micronutrients were investigated. FITC was used as the reagent for fluorescence tagging of arginine (Arg), valine (Val), tryptophan (Trp), folic acid (FA), and niacinamide (NA). Riboflavin (RF) was detected without derivatization. Derivatization of analytes dissolved in borate solution was performed by successive introduction of fluorescence reagent and analytes followed by water bathing at 43°C. The molar ratio of sample/reagent (S/R), derivatization temperature, and incubation time significantly influenced the efficiency of derivatization. To maximize the fluorescence yield, a high S/R (≥20) was required. The nonderivatized RF and five derivatized analytes were separated in the optimized CE-LIF system with the application of 22 kV voltage and 25 mM borate buffer at pH 9.85. Validation of the method showed good linearity for the corrected peak areas versus standard concentrations for the six analytes. The RSDs (n = 3) of the migration time and the peak area obtained for the analytes ranged from 0.4 to 1.1% and from 1.9 to 4.4%, respectively. The developed method, with the lowest LOD of 0.5 nM, was successfully applied for the efficient derivatization and determination of B vitamins in four health drink samples.     

On-column labeling technique and chiral CE of amino acids with mixed chiral selectors and UV detection

A novel method has been developed for the on-column labeling of amino acid enantiomers with 9-fluoroenylmethyl chloroformate (FMOC), followed by chiral CE with a binary chiral selector system and UV detection. Efficient labeling was achieved by sequential injection of amino acids, borate buffer, and FMOC labeling solution at 0.2 psi for 6 s. After injection, the sandwich sections were electrically mixed at 250 V/cm for 6 s and allowed to react (electric field-free) at room temperature for 2 min. With this procedure, successful online-labeling and chiral CE separation of 19 pairs of amino acids (AA) have been conducted, giving 17 pairs fully enantioresolved (R(s) = 1.73-5.79) and two pairs partially resolved (Ala, R(s) = 0.39 and Arg, R(s) = 1.15) using a running buffer of 150 mM borate containing 30 mM beta-CD, 30 mM sodium taurodeoxycholate (STDC), and 15% isopropanol (IPA) at pH 9.0. Chiral CE of some mixed pairs was also demonstrated, much the same as using precolumn labeling. Surprisingly, Met, Asp, Asn, Gln, and His gained even higher enantioresolution (up to 2.5%) compared with the case of precolumn labeling. As validated by both artificially prepared solutions and serum samples, the method was applicable to the quantitative determination of AA, with LODs down to 4.0 microM. The method allowed the determination of D-AA at the ratio of 1:100 (D:L).     

Dynamic pH junction-sweeping for on-line focusing of dipeptides in capillary electrophoresis with laser-induced fluorescence detection

In this paper, we developed a simple and effective on-line focusing technique combining dynamic pH junction and sweeping by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Dynamic pH junction-sweeping is defined when the sample has a different buffer pH (dynamic pH junction condition) and is devoid of micelles (sweeping condition) relative to the background electrolyte (BGE). This hyphenated focusing mode was applied to the sensitive and selective focusing of four dipeptides: Tyr-Phe, Tyr-Leu, Trp-Gly, and Ala-Gln. Picomolar detectability of these dipeptides by CE-LIF detection was demonstrated through effective focusing of large sample volumes (up to 39% capillary length) using the dual pH junction-sweeping focusing mode. 25 mmol L(-1) sodium dihydrogen phosphate, pH 2.5 was used as the sample matrix, and 100 mmol L(-1) borate, 21 mmol L(-1) sodium dodecylsulfate (SDS), 16 mmol L(-1) Brij35, pH 9.0 as the background solution (BGS). The concentration detection limits (S/N = 3) of the four dipeptides were in the range of 1.0-5.0 pmol L(-1). The developed method has been successfully used for the determination of dipeptides in human serum samples.