Short-end Injection Research Articles

Determination of deferasirox in human plasma by short-end injection and sweeping with a field-amplified sample stacking and micellar electrokinetic chromatography

A field-amplified sample stacking-sweeping micellar electrokinetic chromatography with short-end injection was established for determination of deferasirox (DFX) in plasma. DFX was extracted from plasma and reconstituted with deionized water (lower conductivity solution). Capillary (effective length, 10cm) was filled with background electrolyte (40mM phosphate buffer, pH 4.5, containing 20% methanol). After sample loading from outlet end at 5psi for 15s, separation was carried out by applying high voltage at 15kV for 10min. Sodium dodecyl sulfate (SDS) was used to sweep DFX for enhancing sensitivity. The optimal CE separation conditions were 40mM phosphate buffer at pH 4.5 containing 100mM SDS and 20% methanol. The analysis time was about 3.5min for DFX. The calibration curve of DFX was ranged from 1 to 20μg/ml. The linearity (r) was more than 0.998. RSD and RE in intra– and inter–day assays were all below 12.14%. The limit of detection (LOD, S/N=3) for DFX was 0.3μg/ml. The sensitivity enhancement factor between sweeping-FASS MEKC and capillary zone electrophoresis is 3.3. Finally, the method was applied for determination of DFX in β-thalassemia patients.

Myrosinase Compatible Simultaneous Determination of Glucosinolates and Allyl Isothiocyanate by Capillary Electrophoresis Micellar Electrokinetic Chromatography (CE-MEKC).

The functional food Cruciferous vegetables contain glucosinolates which are decomposed by the myrosinase enzyme upon tissue damage. The isothiocyanates are the most frequent decomposition products. Because of their various bioactivities, these compounds and the myrosinase is of high interest to many scientific fields. Development of a capillary electrophoresis method capable of myrosinase-compatible, simultaneous quantification of glucosinolates and isothiocyanates. Capillary electrochromatography parameters were optimised, followed by optimisation of a myrosinase-compatible derivatisation procedure for isothiocyanates. Vegetable extracts (Brussels sprouts, horseradish, radish and watercress) were tested for myrosinase activity, glucosinolate content and isothiocyanate conversion rate. Allyl isothiocyanate was quantified in some food products. The method allows quantification of sinigrin, gluonasturtiin and allyl isothiocyanate after myrosinase compatible derivatisation in-vial by mercaptoacetic acid. The chromatograhpic separation takes 2.5 min (short-end injection) or 15 min (long-end injection). For the tested vegetables, measured myrosinase activity was between 0.960-27.694 and 0.461-26.322 µmol/min/mg protein, glucosinolate content was between 0-2291.8 and 0-248.5 µg/g fresh weight for sinigrin and gluconastrutiin, respectively. The possible specificity of plants to different glucosinolates was also shown. Allyl isothiocyanate release rate was different in different vegetables (73.13 - 102.13%). The method could also be used for quantification of allyl isothiocyanate from food products. The presented capillary electrophoresis method requires a minimal amount of sample and contains only a few sample preparation steps, and can be used in several applications (glucosinolate determination, myrosinase activity measurement, isothiocyanate release estimation). Copyright © 2016 John Wiley & Sons, Ltd.

Assaying human neutrophil elastase activity by capillary zone electrophoresis combined with laser-induced fluorescence

Skin aging is a progressive process determining the ultimate skin appearance. Human neutrophil elastase (HNE) has been shown to play an important role in the degradation of the extracellular matrix. In order to assay HNE kinetics, a novel online capillary zone electrophoresis (CZE) assay has been developed in this study for the determination of the maximum velocity (Vmax) and of the Michaelis–Menten constant (Km) of HNE regarding several potential substrates. These assays are based on short-end injection to shorten analysis time, on transverse diffusion of laminar flow profiles (TDLFP) for in-capillary reactant mixing, and on UV or laser-induced fluorescence (LIF) detection. Kinetic constants for a referenced peptidic substrate were determined using not only online assays but also offline (pre-capillary) mode. The results obtained were cross compared and compared to the literature in order to validate the developed assays. The hydrolysis of three new potential fluorogenic substrates by HNE was also monitored. Two new peptidic substrates for HNE were identified through this study. Km values of these novel substrates were successfully determined using online CZE assay (Km ∼0.07mM). This value was in the same order of magnitude of that of the referenced substrate despite the presence of the labeling group 5-carboxyfluorescein (5-FAM). HNE activity has never been assessed using online CZE-based assay, neither with UV nor with LIF detection. The developed assay conducted with the new labeled substrates is particularly sensitive (LOQ of few nM), does not require the presence of micelles in the BGE (which is the case for the reference substrate) and only necessitates few nanoliters of reactants making it particularly adapted for screening studies.

Sub-minute method for simultaneous determination of aspartame, cyclamate, acesulfame-K and saccharin in food and pharmaceutical samples by capillary zone electrophoresis

This paper reports the development of a sub-minute separation method by capillary zone electrophoresis for the determination of aspartame, cyclamate, acesulfame-K and saccharin in food products and pharmaceutical samples. Separations were performed in a fused uncoated silica capillary with UV detection at 220nm. Samples and standards were injected hydrodynamically using the short-end injection procedure. The electrophoretic system was operated under constant voltage of −30kV. The background electrolyte was composed of 45mmolL−1 2-amino-2-(hydroxymethyl)-1,3-propanediol and 15mmolL−1 benzoic acid at pH 8.4. The separation time for all analytes was less than 1min. Evaluation of analytical parameters of the method showed good linearity (r2>0.9972), limit of detection of 3.3–6.4mgL−1, intermediate precision better than 9.75% (peak area of sample) and recovery in the range of 91–117%.

Genotyping of exons 1 to 20 in Duchenne muscular dystrophy by universal multiplex PCR and short-end capillary electrophoresis.

One rapid CE method was established to diagnose Duchenne muscular dystrophy (DMD). DMD is a severe recessive inherited disorder frequently caused by gene deletions. Among them, exons 1-20 account for nearly 30% of occurrences. In this study, the universal multiplex PCR was used to enhance the fluorescently labeling efficiency, which was performed only by one universal fluorescent primer. After PCR, a short-end injection CE (short-end CE) speeded up the genotyping of the DMD gene. This method involved no extra purification, and was completed within 9min. The CE conditions contained a polymer solution of 1.5% hydroxylethylcellulose in 1× TBE buffer at 6kV for separation. This method was applied to test six DMD patients and one healthy male person. The results showed good agreement with those of multiplex ligation-dependent probe amplification. This method can be applied for clinical diagnosis of DMD disease. Accurate diagnosis of the DMD gene is the best way to prevent the disease.

Genotyping of intron 22 inversion of factor VIII gene for diagnosis of hemophilia A by inverse-shifting polymerase chain reaction and capillary electrophoresis.

This is the first capillary electrophoresis (CE) analysis for diagnosis of hemophilia A (HA). The intron 22 inversion of factor VIII gene (F8) causes 40-50% of severe bleeding disorder of HA in all human populations. Consequently, identification of the disease-causing mutations is becoming increasingly important for accurate genetic counseling and prenatal diagnosis. In this study, the key steps of inverse-shifting polymerase chain reaction (IS-PCR) and of short-end injection capillary electrophoresis were used for more specific and rapid genotyping of intron 22 inversion of F8. In IS-PCR, three specific primers were used to amplify 512-bp amplicon for wild type and 584-bp amplicon for patients with intron 22 inversion. The capillary gel electrophoresis (CGE) system was performed using 1× Tris-borate-EDTA (TBE) buffer containing 0.3% (w/v) polyethylene oxide (PEO). The PCR amplicons were electrokinetically injected at 10kV for 10s at a temperature of 25°C. The optimal short-end injection CGE was applied to detect the F8 gene of HA patients and carriers within 5min. Intron 22 inversion was indeed found on some HA patients (13/35, 37.1%). All genotyping results showed good agreement with DNA sequencing method and long-distance polymerase chain reaction (LD-PCR). The IS-PCR combined with short-end injection CGE method was feasible and efficient for intron 22 inversion screening of F8 in the HA populations.

Screening of neuraminidase inhibitors from traditional Chinese medicines by integrating capillary electrophoresis with immobilized enzyme microreactor

A simple and effective neuraminidase-immobilized capillary microreactor was fabricated by glutaraldehyde cross-linking technology for screening the neuraminidase inhibitors from traditional Chinese medicines. The substrate and product were separated by CE in short-end injection mode within 2min. Dual-wavelength ultraviolet detection was employed to eliminate the interference from the screened compounds. The parameters relating to the separation efficiency and the activity of immobilized neuraminidase were systematically evaluated. The activity of the immobilized neuraminidase remained 90% after 30 days storage at 4°C. The immobilized NA microreactor could be continuously used for more than 200 runs. The Michaelis–Menten constant of neuraminidase was determined by the microreactor as 136.6±10.8μM. In addition, six in eighteen natural products were found as potent inhibitors and the inhibition potentials were ranked in the following order: bavachinin>bavachin>baicalein>baicalin>chrysin and vitexin. The half-maximal inhibitory concentrations were 59.52±4.12, 65.28±1.07, 44.79±1.21 and 31.62±2.04 for baicalein, baicalin, bavachin and bavachinin, respectively. The results demonstrated that the neuraminidase-immobilized capillary microreactor was a very effective tool for screening neuraminidase inhibitors from traditional Chinese medicines.

Selective separation of ferric and non-ferric forms of human transferrin by capillary micellar electrokinetic chromatography

The previously published method allowing the separation of non-ferric (iron-free) and ferric (iron-saturated) forms of human serum transferrin via capillary electrophoresis has been further developed. Using a surface response methodology and a three-factorial Doehlert design we have established a new optimized running buffer composition: 50mM Tris–HCl, pH 8.5, 22.5% (v/v) methanol, 17.5mM SDS. As a result, two previously unobserved monoferric forms of protein have been separated and identified, moreover, the loss of ferric ions from transferrin during electrophoretic separation has been considerably reduced by methanol, and the method selectivity has been yet increased resulting in a total separation of proteins exerting only subtle or none difference in mass-to-charge ratio. The new method has allowed us to monitor the gradual iron saturation of transferrin by mixing the iron-free form of protein with the buffers with different concentrations of ferric ions. It revealed continuously changing contribution of monoferric forms, characterized by different affinities of two existing iron binding sites on N- and C-lobes of protein, respectively. Afterwards, the similar experiment has been conducted on-line, i.e. inside the capillary, comparing the effectiveness of two possible modes of the reactant zones mixing: diffusion mediated and electrophoretically mediated ones. Finally, the total time of separation has been decreased down to 4min, taking the advantage from a short-end injection strategy and maintaining excellent selectivity.

New multilayer coating using quaternary ammonium chitosan and κ-carrageenan in capillary electrophoresis: application in fast analysis of betaine and methionine.

The aim of this study was to develop a new multilayer coating with crosslinked quaternary ammonium chitosan (hydroxypropyltrimethyl ammonium chloride chitosan; HACC) and κ-carrageenan for use in capillary electrophoresis. A new semi-permanent multilayer coating was formed using the procedure developed and the method does not require the presence of polymers in the background electrolyte (BGE). The new capillary multilayer coating showed a cathodic electroosmotic flow (EOF) of around 30×10(-9) m(2) V(-1) s(-1) which is pH-independent in the range of pH 2 to 10. The enhanced EOF at low pH obtained contributed significantly to the development of a fast method of separation. The multilayer coating was then applied in the development of a fast separation method to determine betaine and methionine in pharmaceutical formulations by capillary zone electrophoresis (CZE). The BGE used to determine the betaine and methionine concentrations was composed of 10 mmol L(-1) tris(hydroxymethyl) aminomethane, 40 mmol L(-1) phosphoric acid and 10% (v/v) ethanol, at pH 2.1. A fused-silica capillary of 32 cm (50 µm ID×375 µm OD) was used in the experiments and samples and standards were analyzed employing the short-end injection procedure (8.5 cm effective length). The instrumental analysis time of the optimized method was 1.53 min (approx. 39 runs per hour). The validation of the proposed method for the determination of betaine and methionine showed good linearity (R(2)>0.999), adequate limit of detection (LOD <8 mg L(-1)) for the concentration in the samples and inter-day precision values lower than 3.5% (peak area and time migration). The results for the quantification of the amino acids in the samples determined by the CZE-UV method developed were statistically equal to those obtained with the comparative LC-MS/MS method according to the paired t-test with a confidence level of 95%.

Very fast electrophoretic determination of creatinine and uric acid in human urine using a combination of two capillaries with different internal diameters

A capillary system formed by combining 25 and 100 μm id capillaries was used in the short-end injection mode to determine creatinine and uric acid in human urine. The separation was performed at an electric field intensity of 2.3 kV/cm. Creatinine was determined in a BGE with a composition of 20 mM citric acid/NaOH (pH 3.0), and uric acid was determined in 20 mM MES/NaOH (pH 6.0). Under these conditions, migration times of 12.2 s for creatinine and 8.6 s for uric acid were achieved. The LOD value is 2.4 mg/L for creatinine and 0.9 mg/L for uric acid; the RSD for the migration time varies in the range 0.7-1.1% (intra day) to 1.0-7.5% (inter day); RSDs for the peak areas equalled 3.4-4.0% (intra day) and 4.3-4.7% (inter day). The determined creatinine values in seven urine samples vary in the range 221-1394 mg/L for creatinine and 87-615 mg/L for uric acid. t-Test did not reveal any statistically significant difference between the developed CE methodologies and reference methods - Jaffé reaction for creatinine and enzymatic uricase test for uric acid.

Contactless conductivity detection for screening myrosinase substrates by capillary electrophoresis

Myrosinase is a unique enzyme that catalyzes the hydrolysis of glucosinolates (GLS) to isothiocyanate (ITC), glucose and sulfate. Isothiocyanates display a diversified very interesting biological activity. In this study, capillary electrophoresis (CE) was used for the first time for evaluating myrosinase kinetics (maximum velocity Vmax and Michaelis–Menten constant Km) and to assess the affinity of a variety of substrates toward this enzyme.The pre-capillary approach was chosen since it is very simple to conduct. For this, the enzymatic reaction was performed in a micro-vial. The reaction mixture volume was of only 100μL and the incubation lasted only 5min at 37±1°C. Short-end injection of few tens of nanoliters (∼25nL) of the reaction mixture was performed which decreased analysis time without using any electroosmotic modifier. The sulfate produced was detected and quantified with a contactless capacitively coupled conductivity detector (C4D) allowing the evaluation of myrosinase kinetics.This study shows, that capillary electrophoresis with contactless conductivity detection can be very useful for monitoring myrosinase activity. Comparing to the conventional spectrophotometric method (1982), the CE method developed here is simple, automated, economic, rapid (incubation for few minutes) and robust. Results compared very well with those reported in literature using the conventional method. Moreover, the affinity of a variety of natural and synthetic glucosinolates toward this enzyme has been assessed for the first time.

Determination of artemether and lumefantrine in anti-malarial fixed-dose combination tablets by microemulsion electrokinetic chromatography with short-end injection procedure

BackgroundArtemether-lumefantrine (AL) combination therapy is now the most used anti-malarial treatment in the world. Quality control of AL formulations is still a major challenge in developing countries. Until now, only liquid chromatographic methods have been reported in the literature for their analysis. Capillary electrophoretic methods, which present various advantages (low price of capillary, low volumes of electrolyte consumption), may be an alternative to liquid chromatography methods. In this paper, a reliable method was developed and validated for the determination of AL in commercial fixed-dose combination tablets commercialized in Côte d’Ivoire.MethodsArtemether and lumefantrine were determined by microemulsion electrokinetic chromatography using short-end injection procedure. The two analytes were extracted from tablets by acidified methanol. Pyrimethamine was used as internal standard. Separation was carried out in an uncoated fused silica capillary, 30 cm long × 50 μm internal diameter, using an effective length of 10 cm and a microemulsion composed of octane, butanol, sodium dodecyl sulfate and borate buffer as background electrolyte, a - 500 V.cm-1 electric field and a detection wavelength of 214 nm.ResultsArtemether, lumefantrine and pyrimethamine were separated in 6 min. The method was reliable with respect to selectivity towards formulation excipients, linearity of the response function (r2 > 0.998), recovery studies from synthetic tablets (in the range 99–101%), repeatability (relative standard deviation 1–3%, n = 7 analytical procedures). Application to four commercial formulations containing 20/120 mg of AL per tablet gave a content in good agreement with the declared content. However, the electropherogram of one tablet formulation showed the presence of an ingredient which was not declared.ConclusionThe developed MEEKC method can be proposed as an alternative method to liquid chromatography for the determination of artemether and lumefantrine in fixed-dose combination tablet formulations.

Simultaneous determination of seven hydrophilic bioactive compounds in water extract of Polygonum multiflorumusing pressurized liquid extraction and short-end injection micellar electrokinetic chromatography

BackgroundPolygoni Multiflori Radix, He-Shou-Wu in Chinese, is a widely used traditional Chinese medicine. Clinically, water decoction is the major application form of He-Shou-Wu. Therefore, simultaneous determination of bioactive compounds in water extract is very important for its quality control.ResultsA pressurized liquid extraction and short-end injection micellar electrokinetic chromatography (MEKC) were first developed for simultaneous determination of seven hydrophilic bioactive compounds in water extract of He-Shou-Wu. The influence of parameters, such as pH, concentration of phosphate, SDS and HP-β-CD, capillary temperature and applied voltage, on the analysis were carefully investigated. Optimum separation was obtained within 14 min by using 50 mM phosphate buffer containing 90 mM SDS and 2% (m/v) HP-β-CD (pH 2.5) at 15 kV and 20°C. All calibration curves showed good linearity (R2>0.9978) within test ranges. The overall LOD and LOQ were lower than 2.0 μg/mL and 5.5 μg/mL, respectively. The RSDs for intra- and inter-day of seven analytes were less than 3.2% and 4.6%, and the recoveries were 97.0%-104.2%.ConclusionThe validated method was successfully applied to the analysis of He-Shou-Wu samples, which is helpful for its quality control.

Application of short‐end injection procedure in CE

CE is well known for its many advantages, including the speed of the analyses; most of which can be accomplished in less than 10 min. However many potential fields of application such as pharmaceutical research and industry and clinical diagnostics require analyses of large numbers of samples and so need an even shorter analysis time to be practical. There are generally three main options for enhancing the throughput of electromigration methods - using microchip or multi-capillary systems or modifying the method preformed on single capillary instruments. The latter option will probably be the method of choice for most laboratories, since they are only equipped with classical commercial CE instrumentation. Although several approaches can be used - higher electric field, EOF modification, external pressure application during analysis, this article reviews one of the simplest and at the same time most used, the so-called short-end injection procedure.

Very fast electrophoretic separation on commercial instruments using a combination of two capillaries with different internal diameters

A capillary formed by connecting a 9.7 cm-long separation capillary with id 25 μm with an auxiliary 22.9 cm-long capillary with id 100 μm (coupled capillary) was tested for electrophoretic separation at high electric field intensities. The coupled capillary was placed in the cassette of a standard electrophoresis apparatus. It was used in the short-end injection mode for separation of a mixture of dopamine, noradrenaline, and adrenaline in a BGE of 20 mM citric acid/NaOH, pH 3.2. An intensity of 2.7 kV/cm was attained in the separation part of the capillary at a separation voltage of 30 kV, which is 2.9 times more than maximum intensity value attainable in a capillary with the same length with uniform id. At these high electric field intensities, the migration times of the tested neurotransmitters had values of 12.3-13.3 s and the attained separation efficiency was between 2350 and 2760 plates/s. It is thus demonstrated that an effective separation instrument - a coupled capillary - can be used for very rapid separation in combination with standard, commercially available instrumentation.

Rapid separation of synthetic oligonucleotides on polymer modified capillary surfaces using short-end injection capillary electrophoresis in free solution

Here we use short-end electrokinetic injection capillary electrophoresis (CE) to investigate the free solution mobility of short strands of double-stranded oligonucleotides (dsODNs) on polymer modified capillaries. Single base pair (bp) resolution (Rs) of dsODNs ranging from 16-20 bp was achieved in free solution on an 8 cm capillary dynamically coated with poly(ethylpyrrolidine methacrylate-co-methyl methacrylate) (PEPyM-co-PMMA) random copolymer. Interestingly, separation of a dsODN mixture containing two 16 bp strands of different sequences resulted in partial resolution (0.52) implying that the free solution mobility of dsODNs was sequence dependent. The single bp resolution achieved for the complementary sequence strands (the sequence of all strands in the mixtures contained the same 16 bp sequence) was improved by up to 37% for separation of dsODNs containing non-complementary sequences. The 16 bp peak was not additive within each mixture, indicating the presence of ODN-ODN interactions. Investigation of these interactions (and ODN-buffer interactions) showed that they can be influenced by the ionic strength and conductivity of the background electrolyte (BGE). Increasing the ionic strength reduced the ODN-ODN interactions and improved the resolution, whereas, increasing the conductivity reduced ODN-buffer interactions, increasing the mobility, at the consequence of promoting ODN-ODN interactions, and hence decreasing the resolution.

A fast method for simultaneous analysis of methyl, ethyl, propyl and butylparaben in cosmetics and pharmaceutical formulations using capillary zone electrophoresis with UV detection

The development and optimization of a novel methodology for simultaneous analysis of methyl, ethyl, propyl, and butylparaben using capillary zone electrophoresis under direct UV detection at 297 nm was proposed. The background electrolyte optimization was performed with the aid of Peakmaster® software simulation. The optimized background electrolyte was composed of 20 mmol L−1 of 2-hydroxyisobutyric acid, 30 mmol L−1 of triethylamine, and 0.3 mmol L−1 of hexane-1,6-bis(triethylammonium) bromide at pH 10.6. A capillary with a total length of 32 cm, an effective length of 8.5 cm and 50 μm internal diameter was used. Analysis was performed at 25 °C and 30 kV with positive polarity in the injection side. The optimized method presented acceptable linearity within the range of 0.5 to 30 mg L−1 (R2 > 0.99), limit of detection of 0.1 mg L−1 and limit of quantification from 0.3 to 0.4 mg L−1. The optimized method was successfully applied to analysis of these parabens in several cosmetics and pharmaceutical formulation samples in the absence of pre-treatment and a run time of about 1.0 min, being the fastest in the literature. For this, short-end injection mode was used. At the end, the results found in real samples were compared to LC-MS/MS, which proved the accuracy of the method.

Development and validation of a sensitive enantiomeric separation method for new single enantiomer drug levornidazole by CD-capillary electrophoresis

A fast and sensitive chiral capillary electrophoresis method has been developed to determine levornidazole and its enantiomeric impurity at a 0.05% level in levornidazole injection solution. Several chemical and instrumental parameters which have an effect on chiral separation were investigated, including chiral selectors, buffer composition and pH, applied voltage, capillary length, temperature and rinsing procedure. After optimizing all the effective parameters, the ideal separation conditions were 20mM Tris phosphate buffer at pH 2.1, containing 2.0% (w/v) sulfated-α-cyclodextrin with short end injection at 0.5psi for 5.0s. Online UV detection was performed at 277nm. A voltage of 30kV was applied and the capillary temperature was kept at 25°C. 2,4,6-triaminopyrimidine was chosen as internal standard to improve the injection precision. The total analysis time is less than 7min, which is faster than the existing chiral HPLC method (65min). The validation of the method was performed in terms of factorial analysis, stability of the solution, different cyclodextrin batches study, selectivity, linearity (from 2.5μg/mL to 6000μg/mL, y=0.0015 x+0.0304; R2=0.9999 and the residuals were randomly scattered around 0), LOD and LOQ (0.3 and 1.0μg/mL, respectively), precision and accuracy. The proposed method was then applied to the enantiomeric purity control of the starting material and injection solution of levornidazole (0.5mg/100mL).

Development and validation of a capillary electrophoresis method for the determination of escitalopram and sensitive quantification of its enantiomeric impurity in formulations

A rapid capillary zone electrophoresis method has been developed capable of quantifying 0.05% of R-enantiomer and assaying the main component in escitalopram formulations. Many parameters influencing enantioseparation were investigated, which include chiral selectors, buffer composition and pH, applied voltage, capillary length, temperature, and rinsing procedure. Optimal separation conditions were obtained by using a 25 mM phosphate buffer at pH 7.0, containing 1.6% (w/v) sulfated-β-cyclodextrin with short-end injection at 0.5 psi for 5 s. Online UV detection was performed at 205 nm. A voltage of -20 kV was applied and the capillary temperature was kept at 25°C. Separation was achieved in less than 2 min. The method was further validated, including robustness, stability of the solution, selectivity, linearity (escitalopram from 0.25 μg/mL to 600 μg/mL, y = 1528.3 × +1812.9; R² = 0.9999), LOD and LOQ (0.08 and 0.25 μg/mL, respectively), precision and accuracy. The proposed method was then applied to the quality control of the bulk sample and tablets of escitalopram (10 mg).

A fast and sensitive method for the determination of nitrite in human plasma by capillary electrophoresis with fluorescence detection

Analysis of nitrite, the indicator of nitric oxide (NO) generation in vivo, provides a useful tool to study NO synthesis in vivo. A fast and sensitive fluorometric CE method was developed for determination of nitrite in human plasma through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite in human plasma was easily reacted with DAN under acid conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). Fluorescence detection was optimized to achieve subnanomolar detection which allows a direct analysis of plasma samples unlike most CE–UV methods using sample stacking. Acetonitrile was used to remove the protein. Short-end injection and a high voltage (−30kV) were used to shorten the analysis time. The good separation was achieved with 20mM borate buffer at pH 9.23. The separation of NAT was obtained within 1.4min. The deproteinized plasma sample was injected hydrodynamically for 5s at −50mbar into a 60cm×75μm internal diameter uncoated fused-silica capillary. Excitation wavelength was selected with a broad-band filter (240–400nm), and the emitted light was measured at 418nm by the use of a cutoff filter. A good linearity (R2=0.9975) was obtained in the range from 2 to 500nM. The detection limit of nitrite was 0.6nM in original plasma samples, which is 750 times lower than our previous CE–UV method. The developed fluorometric CE method offers the advantages of more simple system and lower cost compared with the current fluorometric HPLC methods without losing sensitivity. The detected mean nitrite concentration in human plasma by this method was consistent with the most frequently reported values.