Determination of sertraline and N-desmethylsertraline in human plasma by CE with LIF detection

A method has been developed for the analysis of the antidepressant drug sertraline together with its main metabolite N-desmethylsertraline (DMS) in human plasma. It is based on CE with LIF detection (lambda = 488 nm). A SPE procedure is employed for biological sample pretreatment, followed by a derivatization step with FITC; reboxetine was the internal standard. The effect of CD, acetone and N-methyl-D-glucamine (GLC) as constituents of the BGE for analyte separation was investigated. The final BGE consisted of 20 mM carbonate buffer, pH 9.0, with 2.5 mM heptakis(2,6-di-O-methyl)-beta-CD, 50 mM GLC and 20% v/v acetone. With 30 kV applied voltage, the electrophoretic run is completed in 7.5 min. Linearity was observed in the plasma concentration range from 3.0 to 500 ng/mL for sertraline and 4.0 to 500 ng/mL for DMS. Extraction yield was >97.1%, precision - expressed as RSD% - was <3.7, accuracy (recovery) was >95.6%. Due to its sensitivity and selectivity, the method was suited for the analysis of plasma samples from patients undergoing therapy with sertraline.     

Determination of vigabatrin in human plasma and urine by high-performance liquid chromatography with UV-Vis detection

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV-Vis detection has been developed and validated for the determination of vigabatrin (VG) in human plasma and urine. The samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A good chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10 mM orthophosphoric acid (pH 2.5) gradient elution. Tranexamic acid was used as an internal standard (I.S.). The method was linear over the concentration range of 0.8-30.0 microg/ml for both samples. The method is precise (relative standard deviation (R.S.D.) <9.13%) and accurate (relative mean error (RME) <-8.75%); analytical recoveries were 81.07% for plasma and 83.05% for urine. The assay was applied to pharmacokinetic study in a healthy volunteer after a single oral administration of 1 g of vigabatrin.     

Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro‐RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE‐LIF) using fluorescence‐labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA‐499 was found when hybridization was conducted at 40°C in 50 mM Tris‐acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA‐499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA‐499 was completed within 1 h using CE‐LIF. These results showed the potential of CE for fast, specific, and sensitive high‐throughput analysis of low‐abundance miRNAs in cell extracts, biofluids, and tissues.     

Analysis of DNA complexes with small solutes by CE with LIF detection

In this article, we describe the analysis of aptamers for Hg²⁺ ions through CE with LIF (CE‐LIF) detection using 2% poly(ethylene oxide) solutions containing OliGreen (fluorophore). In the presence of an EOF, DNA strands migrating against the EOF were detected at the cathode end. Four DNA strands – T₃₃, T₅C₂₈, T₅C₅T₂₃, and T₁₅C₅T₁₃ – could not be separated through CE‐LIF in the absence of Hg²⁺. At 0.3 mM Hg²⁺, however, all four were partially separated within 20 min, with SDs of the migration times all being less than 2.5%. From the CE, fluorescence, and ellipticity data, we concluded that the conformations of these four DNA strands all changed from random‐coil to folded structures as a result of T–Hg²⁺–T bonding. In addition, we found that this CE approach provided different electropherograms patterns for T₇, T₁₅, and T₃₃ in the absence and presence of Hg²⁺, indicating various interactions of the DNA strands with Hg²⁺. Using this simple, high‐resolution CE approach, we also demonstrated that adenosine triphosphate has a stronger interaction with the adenosine triphosphate aptamer than with either the platelet‐derived growth factor aptamer or T₃₃. This CE approach holds great potential for screening aptamers for small solutes, studying the catalytic activity of DNAzymes, and evaluating the biological functions of microRNA.     

LIF detection of peptides and proteins in CE

CE- and microchip-based separations coupled with LIF are powerful tools for the separation, detection and determination of biomolecules. CE with certain configurations has the potential to detect a small number of molecules or even a single molecule, thanks to the high spatial coherence of the laser source which permits the excitation of very small sample volumes with high efficiency. This review article discusses the use of LIF detection for the analysis of peptides and proteins in CE. The most common laser sources, basic instrumentation, derivatization modes and set-ups are briefly presented and special attention is paid to the different fluorogenic agents used for pre-, on- and postcapillary derivatization of the functional groups of these compounds. A table summarizing major applications of these derivatization reactions to the analysis of peptides and proteins in CE-LIF and a bibliography with 184 references are provided which covers papers published to the end of 2005.     

Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments

Inverse-flow derivatization is a novel approach to obtain fluorescent DNA derivatives in DNA analysis based on CE with LIF detection. In the present work, we want to explore the feasibility of the application of this method into the mutation detection based on constant denaturant capillary electrophoresis (CDCE) and SSCP analysis. The DNA fragments were first amplified by PCR using a pair of common primers without fluorescent label, and then the mutations were determined by CDCE or SSCP analysis based on CE-LIF with inverse-flow derivatization of DNA fragments. The experimental conditions were investigated systematically, and different labeling modes including inverse-flow derivatization, on-column derivatization and fluorescent labeled primer technique were compared. The inverse-flow derivatization was successfully used in the detection of C677T mutation in the methylenetetrahydrofolate reductase gene by CDCE or SSCP analysis. Our preliminary results demonstrate that inverse-flow derivatization is very simple, inexpensive and sensitive and well suitable for the genetic analysis in clinical diagnosis.     

Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detection

This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2‐aminoacridone and subjected to reversed polarity CE‐LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE‐LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE‐LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.     

Simultaneous LIF and retro-reflected beam interference detection for CE

This Short Communication describes a novel optical detection method for CE, based on the combination of LIF detection and retro-reflected beam interference detection. By the use of a side-illuminated laser beam, an on-column multifunctional detection for CE has been developed, and some key elements in the scheme have been optimized. In addition, two miscellaneous samples including fluorescent dyes, carbohydrates and amino acids have been determined to evaluate its performance. Without any additional pretreatment, sufficient LOD and linear ranges have been achieved for most analytes. Its performance in retro-reflected beam interference detection is better than those mentioned in a former report, and its fluorescent sensitivity is comparable with the achievable sensitivity by conventional LIF systems, all of which together combine high sensitivity and universal analysis to a certain extent.     

Determination of catecholamines by CE with direct chemiluminescence detection

A rapid and sensitive method to detect three catecholamines, isoprenaline, epinephrine, and dopamine, by CE coupled with direct luminol-potassium periodate chemiluminescence (CL) detection is described. The conditions for CE separation and CL reaction were systematically optimized. Under the optimum conditions, the baseline separation of three catecholamines was achieved within 6.5 min. The LODs obtained in standard solution were 5.3 x 10(-8 )mol/L for isoprenaline, 4.7 x 10(-8 )mol/L for epinephrine, and 1.5 x 10(-7 )mol/L for dopamine. The RSD of the migration time and peak area were less than 1.8 and 3.6% (n = 5), respectively. The present method was applied to the determination of the dopamine in urine samples of cigarette smokers and nonsmokers. The results obtained indicate that there is a close relationship between the content of dopamine in human urine and the amount of cigarettes smoked daily; the level of dopamine in smokers is higher than in nonsmokers.     

Determination of bencyclane in human plasma by means of capillary gas chromatography and nitrogen-phosphorus selective detection

A sensitive and specific method for the determination of bencyclane in human plasma is presented. Bencyclane was extracted from human plasma with two 3-ml volumes of isooctane and was shaken for 10 min. The organic phase was separated and evaporated to dryness at 40 degrees C under a nitrogen stream. The residue was dissolved and an aliquot was injected into the gas chromatograph. The separation was performed with a DB-17 column with helium as the carrier gas. Nitrogen-selective detection was performed. The quantification was performed with the signal output. The limit of detection was 1 ng/ml.     

Determination of galactose in human plasma by HPLC with electrochemical detection

Galactose in plasma from patients with hepatic diseases who had undergone low level galactose infusion was determined by using HPLC with electrochemical detection (LCEC). Agreement between galactose concentration determined by the LCEC and a fluorometric method was remarkably good at moderate levels of galactose in plasma. However, the fluorometric method is not suitable for samples containing very small amounts of galactose (blood from hepatic veins) and even for a few samples at moderate galactose content (blood from peripheral veins), suggesting the presence of an endogenous interference. There was no interference for the quantitation of galactose by the LCEC method, by virtue both of the specificity involved in the electrochemical detection and the separation by liquid chromatography. The detection limit of the LCEC method was 0.4 mg galactose/L blood.     

Determination of formaldehyde in single cell by capillary electrophoresis with LIF detection

As a small molecule gas, formaldehyde (FA) is the simplest carbonyl active material and plays an important role in the carbon cycle of metabolism. However, due to the volatile nature of the gas, it is difficult to accurately quantify its content, which limits the study of the mechanism of action in life activities. Thus, an efficient approach to quantitative detection of FA in cells especially in single cell is urgent needed. Nevertheless, no method for quantifying FA in single cell has been reported to date. In this work, a fluorescent probe N‐propyl‐4‐hydrazino‐naphthalimide (NPHNA), which has highly desirable attributes and has been applied to living biological samples, was chosen as labeling reagent to detect endogenous FA at single cell level. After optimization of separation conditions, fast baseline separation of the FA derivative N‐propyl‐4‐hydrazone‐naphthalimide product (NPHNA‐FA) and NPHNA was achieved in about 5 min by CE with LIF detection. The detection limit for FA was 5 amol (S/N ratio of 3). The developed method was validated by the measurements of intracellular levels of FA in single cell.     

Determination of fendiline in human plasma by means of capillary gas chromatography and nitrogen-phosphorus selective detection.

A sensitive and specific method for the determination of fendiline in human plasma is presented. Fendiline was extracted from human plasma after the addition of phosphate buffer two times with 4 ml of n-hexane. The organic phase was separated and evaporated to dryness at 40 degrees C under a stream of nitrogen. The residue was dissolved and an aliquot was injected into the gas chromatograph. Chromatographic separation was performed with a DB-1 column with helium as carrier gas. Nitrogen-selective detection was performed. Quantification was performed with the signal output. The limit of detection was 1 ng/ml of plasma.     

Determination of PCR products by CE with contactless conductivity detection

The use of CE with contactless conductivity detection for the determination of PCR products is demonstrated for the first time. The separation of specific length PCR products according to their size could be achieved using 5% PVP as a sieving medium in a separation buffer consisting of 20 mM Tris and 20 mM 2‐(cyclohexylamino)ethansulphonic acid (pH 8.5). A fused silica capillary of 60 cm length and 50 μm id and an applied separation voltage of –15 kV were employed and separations could be completed within 20–50 min. PCR amplified DNA fragments of different sizes obtained from different bacterial plasmid templates as well as a fragment from genomic DNA of genetically modified soybeans could be successfully identified.     

Determination of busulfan in human plasma by gas chromatography with electron-capture detection

A simple and highly sensitive gas chromatographic method has been developed for the determination of busulfan in human plasma. After extraction of plasma specimens (clinical or spiked) with ethyl acetate, busulfan and the internal standard [1,8-bis(methanesulfonyloxy)octane] were derivatized with 2,3,5,6-tetrafluorothiophenol to yield compounds monitored by a 63Ni electron-capture detector. Sample recoveries from extraction and derivatization were greater than 78 and 91%, respectively. The limit of quantitation was 0.01 microgram/ml (0.04 microM) in 1 ml of plasma with a linear relationship over the 0.01-1.0 micrograms/ml (0.04-4 microM) concentration range. The method has been applied to analyze the plasma versus time profile of busulfan in human subjects following administration of an oral dose of 4 mg/kg per day as a marrow ablative chemotherapy for bone marrow transplantation.     

Determination of mercury ion by MEKC with on-column derivatisation and LIF detection

A simple and automatic method for the determination of mercury ion by MEKC with on-column derivatisation and LIF detection is described in the present paper. In this method, solutions of a nonfluorescent rhodamine derivative and mercury ion were injected individually and mixed by applying a short voltage. Subsequently, the mercury ions reacted with the nonfluorescent rhodamine derivative to produce strongly fluorescent product. The resulting product was then removed by EOF and micelles towards the detection window and detected by LIF detector. The experimental conditions in terms of the concentration and injection volume ratios between mercury ion and derivatisation reagent, the mixing time and waiting time for the on-column reaction were optimised. The optimal conditions were determined as follows: the concentration and injection volume ratios between mercury ion and derivatisation reagent were 1:20 and 10:1, respectively; the mixing time was 40 s under the applied voltage of 5 kV; the waiting time was proved unnecessary. The detection limit for mercury ion was 5 x 10(-8) M, and the total analysis time was less than 10 min.     

Determination of pyrimethamine in human plasma after administration of fansidar of fansidar-mefloquine by means of high-performance liquid chromatography with fluorescence detection

A sensitive, rapid and selective high-performance liquid chromatographic (HPLC) method has been developed to measure plasma levels of pyrimethamine in human subjects dosed with the antimalarials Fansidar or Fansidar and mefloquine. The drug was extracted from plasma at basic pH with n-butyl chloride-dichloromethane (96:4, v/v) and quantified on a normal-phase HPLC column with fluorescence detection (excitation 290 nm, emission 345 nm). Pyrimethamine was almost quantitatively extracted from plasma in the concentration range 20-200 ng/ml. The sensitivity limit was about 10 ng/ml of plasma, using a 0.5-ml specimen. The method was shown to be specific with respect to the other two components in the antimalarial combinations, namely sulfadoxine and mefloquine, and their metabolites. The assay was applied to pharmacokinetic studies of pyrimethamine in man following the oral administration of Fansidar of Fansidar and mefloquine.     

Determination of medroxyprogesterone acetate residues by CE immunoassay with chemiluminescence detection

A rapid and simple method is developed for the determination of medroxyprogesterone acetate (MPA) by CE immunoassay with chemiluminescence (CL). This method is based on the competitive reactions between horseradish peroxidase (HRP)-labeled MPA (MPA-HRP) and free MPA with anti-MPA antiserum. The influencing factors on the electrophoresis and CL detection were studied completely and the optimal conditions of separation and determination were obtained. The linear range was 2.0-50 nmol/L and the LOD for MPA was 0.9 nmol/L. The present method was applied to the analysis of pork tissues.