Determination of monophosphate nucleotides by capillary electrophoresis inductively coupled plasma mass spectrometry

A preliminary study of a modified microconcentric nebulizer (CEI-100, CETAC) as the sample introduction device of capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS) for the determination of monophosphate nucleotides is described. The monophosphate nucleotides studied include adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate (UMP) and inosine 5'-monophosphate (IMP). The species studied were well separated using a 70 cm length x 75 microm id fused silica capillary while the applied voltage was set at -22 kV and a 20 mmol l(-1) ammonium citrate/citric acid buffer (pH 4.0) containing 0.1% m/v cationic polymer (hexadimethrine bromide, Polybrene) was used as the electrophoretic buffer. The electroosmotic flow was reversed by flushing the fused silica capillary with 0.2% m/v Polybrene to accelerate separation. The detection limit of various species studied was in the range of 0.036-0.054 microg P ml(-1), which corresponded to the absolute detection limit of 1.1-1.6 pg P based on the injection volume of 30 nl. We determined the concentrations of nucleotides in two IG-enriched monosodium glutamates purchased from the local market. The recovery was in the range of 100-112% for various species, and the concentrations of IMP and GMP in these samples were in the range of 0.15-0.18% m/m.     

Intracellular adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate detection by short-end injection capillary electrophoresis using methylcellulose as the effective electroosmostic flow suppressor

We present a new rapid CE method to measure adenine nucleotides adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in cells. The short-end injection mode allows a decrease in the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, reducing the migration distance. Moreover, the use of methylcellulose (MC) as run buffer additive to suppress EOF permits to further reduce the migration times of analytes. Thus, when a capillary with an effective length of 10.2 cm was used with a 60 mmol/L sodium acetate buffer pH 3.80 in the presence of 0.01% of MC, the migration time of analytes were 1.35 min for ATP, 1.85 min for ADP, and 4.64 min for AMP. These conditions gave a good reproducibility for intra- and interassay (CV <4 and 8%, respectively) and all the procedure demonstrated an excellent analytical recovery (from 98.3 to 99 %). The method suitability was proved both on red blood cells and in spermatozoa. We compared our proposed method to a spectrophotometric assay, by measuring ATP levels in 40 spermatozoa samples. The obtained data were analyzed by the Passing and Bablok regression and Bland-Altman test.     

Nucleoside monophosphates recognition using macrocyclic polyamine bonded phase in capillary electrochromatography

An open-tubular wall-coated macrocyclic polyamine capillary column (70 cm x 75 microm ID) with 50 cm effective length for the separation of nucleoside monophosphates is described. Some parameters with respect to concentration, pH, composition of the buffer, and voltage in order to optimize the separation were studied. The coated capillary showed reversed electroosmotic flow (EOF), allowing anions to be separated in the co-EOF mode. Baseline separations were achieved for the eight nucleotides in less than 26 min using a background electrolyte consisting of H(3)PO(4)-NaH(2)PO(4) (30 mM, pH 3.10), an applied voltage of -15 kV, and detection at 254 nm. The macrocyclic polyamine on the capillary wall introduced anion coordination for the interaction with the analytes, the strength of which could be moderated by the type and concentration of the competing ion used in the background electrolyte (BGE). With a low concentration of the competing ion (phosphate ion), the migration behavior followed that obtained in the electrophoretic system. Increasing the concentration of the competing ion resulted in a faster migration and more complete elution of the analyte. The method established was also employed for the analysis of nucleotides in mushrooms. Aqueous extracts of mushrooms from different species and various extraction methods were injected directly for the analysis. Uridine 5'-monophosphate, guanosine 5'-monophosphate, adenosine 5'-monophosphate, and cytidine 5'-monophosphate, were found in the sample tested.     

Large‐volume sample stacking with polarity switching for monitoring of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) reactions by capillary electrophoresis

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is a membrane glycoprotein involved in the hydrolysis of extracellular nucleotides. Its main substrate is ATP yielding AMP and pyrophosphate. NPP1 has been proposed as a novel drug target, for diabetes type 2 and the treatment of calcium pyrophosphate dihydrate deposition disease leading to inflammatory arthritis. The monitoring of NPP1 reactions is difficult because its velocity is very slow requiring highly sensitive analytical procedures. In this study, a method of large‐volume sample stacking with polarity switching was developed, and separations were optimized. Large sample volumes were loaded by hydrodynamic injection (5 psi, 13 s) followed by removal of a large plug of sample matrix from the capillary using polarity switching (?10 kV). The stacked analytes were subsequently separated in phosphate buffer (100 mM, pH 9.2) at 20 kV. The validated method was found to be linear (R² = 0.9927) in the concentration range of 0.05–50 μM of AMP, with high accuracy and precision. The determined LOD and LOQ of AMP were 18 nM and 60 nM, respectively. Compared to a previously reported CE procedure using sweeping technique, a fivefold improvement of sensitivity was achieved. Moreover, the new technique was faster, and reproducibility of migration times was improved (RSD value = 1.2%). Importantly, adenine nucleotide analogs and derivatives tested as NPP1 inhibitors could be completely separated from the substrate ATP and the enzymatic product AMP. The method was applied to NPP1 inhibition assays investigating nucleotide‐derived inhibitors in the presence of ATP.     

Separation of nucleoside mono-, di- and triphosphates by capillary electrophoresis via cadmium complexation

Summary The CE separation of twelve nucleotides (5′-mono-, di-, triphosphates of adenosine, guanosine, cytidine and uridine) was improved by adding cadmium ion to the ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM). Cadmium ion acts as a complexing agent for some nucleotides (ATP, CTP, GTP, UTP, GDP). In order to accelerate the separation, the electroosmotic flow was reversed by flushing the fused-silica capillary with 0.2 % aqueous solution of the polycationic surfactant hexadimethrine bromide. A good separation of the twelve nucleotides studied was then achieved on a dynamically coated capillary in less than 5 min by using an ammonium citrate/citric acid buffer (pH 5, ionic strength 100 mM) to which 2 mM cadmium ion has been added. High peak efficiencies were obtained (210 000 theoretical plates) and the resolution between two adjacent peaks was always greater than 1.5.     

Capillary electrophoresis-electrospray-mass spectrometry of nucleosides and nucleotides: application to phosphorylation studies of anti-human immunodeficiency virus nucleosides in a human hepatoma cell line

We report the use of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) for the determination of antiretroviral dideoxynucleosides (ddNs), their nucleotides, and a set of ribonucleosides and ribonucleotides. A CE system for separation of most commonly used antiretroviral ddNs has been developed based on a basic buffer with a volatile electrolyte suitable for ESI-MS detection in an untreated capillary column. Positive and negative ionization modes are investigated and compared for sensitive and stable electrospray performance. A 14-compound mixture of nucleosides and nucleotides is profiled in a single capillary zone electrophoresis separation with a distinct elution order: electroosmotic flow, ddNs, mononucleotides, dinucleotides, and trinucleotides in less than 18 min. The fragmentation pathways of the nucleosides and nucleotides in ESI-MS have been interpreted. Concentration limits of detection are 100 to 200 nM with an injection volume of approximately 10 nL. This technique has been used to detect naturally occurring nucleotides and to study the metabolism of lamivudine (3TC) in the human hepatoma cell line Hep G2. 3TC and its metabolites 3TC-monophosphate, 3TC-diphosphate, and 3TC-triphosphate were detected after 10 h of incubation of 3TC with the cells.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Capillary electrophoresis-atmospheric pressure ionization mass spectrometry for the characterization of peptides. Instrumental considerations for mass spectrometric detection.

On-line capillary electrophoresis (CE) separations are shown for a synthetic peptide mixture and a tryptic digest of human hemoglobin in an uncoated fused-silica capillary with detection using atmospheric pressure ionization mass spectrometry (API-MS). The CE system utilized a 1-m capillary column of either 75- or 100-microns I.D. These somewhat larger inside diameters allow higher sample capacities for MS detection and the 1-m length facilitates connecting the CE column to the liquid junction-ion spray interface and MS system. Low volatile buffer concentrations (15-20 mM) of ammonium acetate or ammonium formate, and high organic modifier content (5-50%) of methanol or acetonitrile facilitates ionization under electrospray conditions. This study shows that peptides separated by CE may be transferred to the API-MS system through a liquid junction coupling to the pneumatically assisted electrospray (ion spray) interface at low buffer pH when the electroosmotic flow is low (0-0.04 microliter/min). CE-MS as described herein is facilitated by features in modern CE instrumentation including robotic cleaning and pressurization of the capillary inlet. The latter is particularly useful for repetitive rinsing and conditioning of the capillary column between analyses in addition to continuous 'infusion' of sample to the mass spectrometer for tuning purposes. In addition to facile molecular weight determination, amino acid sequence information for peptides may be obtained by utilizing on-line tandem MS. After the tryptic digest sample components enter the API-MS system, the molecular ion species of individual peptides may be focussed and transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of protonated peptide molecules yielded structural information for their characterization following injection of 10 pmol of a tryptic digest from human hemoglobin.     

Separation and determination of 10-hydroxy-2-decenoic acid in royal jelly by capillary electrophoresis

Summary The application of capillary electrophoresis (CE) to the separation and determination of the active ingredient, 10-hydroxy-2-decenoic acid, in royal jelly with direct on-column UV detection at 214 nm is described. Using a cathodic injection and anodic detection scheme, 10-hydroxy-2-decenoic acid (10-HDA) was separated and detected in less than 10 min in a fused silica capillary column with a phosphate buffer at pH 7.3 with an applied voltage of 20 KV followed by direct UV detection. The use of cetyltrimethylammonium bromide (CTAB) as electroosmotic flow modifier allows the rapid separation of 10-HDA from other constituents in royal jelly by reversing the direction of electroosmotic flow. The influence of organic solvents in the electrolyte on separation selectivity is also discussed.     

Cationic polymer coatings for design of electroosmotic flow and control of DNA adsorption

A difficulty with the design and operation of an electrokinetically operated DNA hybridization microfluidic chip is the opposite direction of the electroosmotic flow and electrophoretic mobility of the oligonucleotides. This makes it difficult to simultaneously deliver targets and an appropriate hybridization buffer simultaneously to the probe sites. In this work we investigate the possibility of coating the inner walls of the microfluidic system with hexadimentrine bromide (polybrene, PB) and other cationic polymers in order to reverse the direction of electroosmotic flow so that it acts in the same direction as the electrophoretic transport of the oligonucleotides. The results indicated that the electroosmotic flow (EOF) in channels that were coated with the polymer could be reversed in 1× TBE buffer or 1× SSC buffer. Under these conditions, the DNA and EOF move in the same direction, and the flow can be used to deliver DNA to an area for selective hybridization within the channel. The effects of coating the surface of a nucleic acid microarray with polybrene were also studied to assess non-selective adsorption and stability. The polybrene coating significantly reduced the extent of non-selective adsorption of oligonucleotides in comparison to adsorption onto a glass surface, and the coating did not alter the extent of hybridization. The results suggest that use of the coating makes it possible to achieve semi-quantitative manipulation of nucleic acid oligomers for delivery to an integrated microarray or biosensor.     

Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix

The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.     

Flow manipulation for sweeping with a cationic surfactant in microchip capillary electrophoresis

Flow manipulation in sweeping microchip capillary electrophoresis (CE) is complicated by the free liquid communication between channels at the intersection, especially when the electroosmotic flows are mismatched in the main channel. Sweeping in traditional CE with cationic micelles is an effective way to concentrate anionic analytes. However, it is a challenge to transfer this method onto microchip CE because the dynamic coating process on capillary walls by cationic surfactants is interrupted when the sample solution free of surfactants is introduced into the microchip channels. This situation presents a difficulty in the sample loading, injection and dispensing processes. By adding surfactant at a concentration around the critical micelle concentration and by properly designing the voltage configuration, the flows in a microchip were effectively manipulated and this sweeping method was successfully moved to microchip CE using tetradecyltrimethylammonium bromide (TTAB). The sweeping effect of cationic surfactant in the sample solution was discussed theoretically and studied experimentally in traditional CE. The flows in a microchip were monitored with fluorescence imaging, and the injection and sweeping processes were studied by locating the detection point along the separation channel. A detection enhancement of up to 500-fold was achieved for 5-carboxyfluorescein.     

High-sensitivity capillary electrophoresis method for monitoring purine nucleoside phosphorylase and adenosine deaminase reactions by a reversed electrode polarity switching mode

A simple, efficient, and highly sensitive in-line CE method was developed for the characterization and for inhibition studies of the nucleoside-metabolizing enzymes purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) present in membrane preparations of human 1539 melanoma cells. After filling the running buffer (50 mM borate buffer, 100 mM SDS, pH 9.10) into a fused-silica capillary (50 cm effective length × 75 μm), a large sample volume was loaded by hydrodynamic injection (5 psi, 36 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-20 kV). The current was monitored and the polarity was reversed when 95% of the current had been recovered. The separation of the neutral analytes (nucleosides and nucleobases) was performed by applying a voltage of 15 kV. An about 10-fold improvement of sensitivity for the five investigated analytes (adenosine, inosine, adenine, hypoxanthine, xanthine) was achieved by large-volume stacking with polarity switching when compared with CE without stacking. For inosine and adenine detection limits as low as 60 nM were achieved. To the best of our knowledge, this represents the highest sensitivity for nucleoside and nucleobase analysis using CE with UV detection reported so far. The Michaelis-Menten constants (K(m)) for PNP and ADA and the inhibition constants (K(i)) for standard inhibitors determined with the new method were consistent with literature data.     

Use of dynamically coated capillaries with added cyclodextrins for the analysis of opium using capillary electrophoresis

A rapid, precise, accurate, and robust method using capillary electrophoresis (CE) with dynamically coated capillaries for the analysis of the major opium alkaloids in opium is presented. Dynamic coating of the capillary surface is accomplished using a commercially available reagent kit (polycation coating followed by polyanion coating). The addition of dual cyclodextrins (hydroxypropyl-beta-cyclodextrin and dimethyl-beta-cyclodextrin) to the run buffer imparts excellent selectivity for the opium alkaloids. For the determination of morphine, papaverine, codeine, noscapine and thebaine in opium gum and opium latex samples (using tetracaine as an internal standard) good agreement with values obtained by gradient high-performance liquid chromatography is obtained. Compared to the latter technique, CE affords better resolution with significantly faster analysis time (12 min versus 29 min). Dynamically coated capillaries, which give rise to a relatively high and robust electroosmotic flow (EOF) at the background electrolyte pH of 2.5, allow for rapid analysis and excellent migration time and peak area precision (RSDs < or = 0.12% and < or = 1.2%, respectively). Reproducible separations (relative migration times) for over 500 samples have been obtained on a single capillary. The nature of the injection solvent, the injection time and the contents of the waste vials have a profound effect on the pressure injection precision of the relatively hydrophobic solutes. The CE conditions reported in this study are also applicable to the analysis of lysergic acid diethylamide (LSD) exhibits.     

Analysis of drying oils used as binding media for objects of art by capillary electrophoresis with indirect UV and conductivity detection

Capillary electrophoresis (CE) was applied to analyse the long-chain fatty acid composition of vegetable oils, and their degradation products formed upon ageing when drying oils are used as binding media. The analytes were detected with contactless conductivity detection (CCD) and indirect UV absorption, both detectors positioned on-line at the separation capillary. The long-chain fatty acids were resolved in a background electrolyte (BGE) consisting of phosphate buffer (pH = 6.86, 15 mM) containing 4 mM sodium dodecylbenzensulfonate, 10 mM Brij 35, 2% (v/v) 1-octanol and 45% (v/v) acetonitrile. As in this system dicarboxylic analytes, the products of oxidative degradation of unsaturated fatty acids, cannot be determined, a suitable background electrolyte was developed by the aid of computer simulation program PeakMaster. It makes use of a 10 mM salicylic acid, 20 mM histidine buffer, pH 5.85, which combines buffering ability with the optical properties obligatory for indirect UV detection. This buffer avoids system eigenpeaks, which are often impairing the separation efficiency of the system. Separation of the dicarboxylic analytes was further improved by a counter-directed electroosmotic flow (EOF), obtained by dynamically coating the capillary wall with 0.2 mM cetyltrimethylammonium bromide. Long-chain fatty acids and their decomposition products could be determined in recent and aged samples of drying oils, respectively, and in samples taken from two paintings of the 19th century.     

Analysis of selected mycotoxins by capillary electrophoresis

Summary The separation of the mycotoxins ochratoxin A, ochratoxin B, zearalenone and moniliformin by standard capillary zone electrophoresis (CZE) and cyclodextrins modified CE is described. In addition, reversed electroosmotic flow (EOF) conditions via quarternary ammonium running buffer additives have been briefly examined. Parameters influencing selectivity and mobility as well as spectroscopic properties of the analytes have also been investigated. Separations performed at pH values from 5 to 11 show a marked pH dependency of the mobilities accompanied by pronounced shifts of the UV/VIS and/or fluorescence spectra of the compounds. In general, the on-line recording of spectra by diodearray detection (DAD), proved to be highly versatile for peak tracking simultaneously with the structure elucidation and thus for the optimization of sample introduction, peak resolution and detection conditions.     

Analysis of cytokinin nucleotides in coconut (Cocos nucifera L.) water using capillary zone electrophoresis-tandem mass spectrometry after solid-phase extraction

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is described for the separation and determination of six cytokinin nucleotides in coconut water. The best CZE separation for the six cytokinin nucleotide standards was achieved using a 25 mM ammonium formate/formic acid buffer (pH 3.8) and 2% (v/v) methanol with an applied gradient separation voltage (25 kV for 32 min, and then a linear gradient to 30 kV in 5 min, finally 30 kV to the end of separation) in less than 60 min. MS/MS with multiple reaction monitoring (MRM) detection was carried out to obtain sufficient selectivity and sensitivity for the cytokinin nucleotides. The combined use of on-line sample stacking and CZE-MS/MS achieved limits of detection (LODs) in the range of 0.06-0.19 microM for the six cytokinin nucleotides at a signal-to-noise ratio of 3. Furthermore, a novel dual-step SPE procedure was developed for the pre-concentration and purification of cytokinin nucleotides using Oasis HLB and Oasis MAX cartridges. The recoveries of the cytokinin nucleotides after the dual-step SPE were in the range of 44-71%. The combination of off-line SPE, on-line sample stacking and CZE-MS/MS approach was successfully applied to screen for endogenous cytokinin nucleotides present in coconut water sample. trans-Zeatin riboside-5'-monophosphate (ZMP) was detected and quantified in coconut water by CZE-MS/MS after SPE and on-line sample stacking.     

Fast enantiomeric separation with vancomycin as chiral additive by co-electroosmotic flow capillary electrophoresis: increase of the detection sensitivity by the partial filling technique

A fast and sensitive method is described by using vancomycin as a chiral additive for enantiomeric separation by capillary electrophoresis (CE). In order to overcome disadvantages associated with use of vancomycin as chiral additive in CE, several strategies including the dynamic coating technique, the co-electroosmotic flow technique, and the partial filling technique were employed sequentially in this method. Using the polycationic polymer hexadimethrine bromide (HDB) as a buffer additive, the capillary wall was dynamically coated with a thin film formed by the adsorbed HDB. Consequently, the adsorption of vancomycin onto the capillary wall was minimized via electrostatic repulsion between the coating of the capillary wall and the vancomycin molecule. In addition, the reversed electroosmotic flow (from cathode to anode) produced by the positively charged capillary wall migrates in the same direction of negatively charged analytes (co-electroosmotic flow electrophoresis). Thereby the electrophoretic mobility of negatively charged analytes were drastically accelerated leading to a short separation time of less than 3.4 min. The separation time was further reduced by the use of a short-end-injection technique. For example, the analysis time was achieved by as short as 55 s for a baseline separation of dansyl-alpha-amino-n-butyric acid. Concurrently, the partial filling technique was used to avoid the loss of detection sensitivity caused by the presence of vancomycin in the running buffer. The effect of several parameters, such as HDB concentration, buffer pH, plug length of the chiral selector, concentration of the chiral selector and applied voltage, on enantioselectivity were investigated toward optimization. Besides the advantage of a very short separation time, the method is characterized by high detection sensitivity, high selectivity, and high efficiency.     

Analysis of nucleotides by pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n=6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme.     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。