Analysis of double-stranded DNA by capillary electrophoresis using poly(ethylene oxide) in the presence of hexadecyltrimethylammonium bromide

The impact of hexadecyltrimethylammonium bromide (CTAB) on the separation of ds-DNA by capillary electrophoresis in conjunction with laser-induced fluorescence (CE-LIF) detection using poly(ethylene oxide) (PEO) solution is described. The use of CTAB for improved separation reproducibility and efficiency of DNA has not been demonstrated although it is widely used for controlling the magnitude and direction of electroosmotic flow in CE. With increasing CTAB concentration, the interactions of DNA with ethidium bromide (EtBr) and with the capillary wall decrease. For the separation of DNA fragments with the sizes ranging from several base pairs (bp) to 2,176 bp, a polymer solution consisting of 0.75% poly(ethylene oxide), 100 mM TB buffer (pH 8.0), 25 microg/mL EtBr, and 0.36 microg/mL CTAB is proper. Using the PEO solution, we separated a mixture of DNA markers V (pBR 322/HaeIII digest) and VI (pBR 328/BglI digest and pBR 328/HinfI digest) within 8 min at -375 V/cm, with the limit of detection of 2.0 ng/mL based on the peak height for the 18-bp DNA fragment. The method is highly efficient (>10(6)plate/m), repeatable (RSD of the migration times <1.5%), and sensitive. In addition, it is convenient to fill a capillary (75 microm in diameter) with such a low-viscosity PEO solution by syringe pushing.     

Electrophoretic behavior of individual nuclear species as determined by capillary electrophoresis with laser-induced fluorescence detection

We determined the feasibility of using capillary electrophoresis with postcolumn laser-induced fluorescence (CE-LIF) detection to characterize electrophoretic properties of isolated cell nuclei and impurities present in nuclear fractions. These fractions were isolated from NS-1 mouse hybridoma cells, stained with hexidium iodide, a DNA intercalating dye, and analyzed by CE-LIF detection. The corresponding electropherograms display two features: (i) broad peaks (6-90 s wide) caused by the cell culturing media and by free-DNA intercalated with hexidium iodide, and (ii) a large number of narrow peaks (180 ms wide), resulting from DNA associated with individual intact or disrupted nuclei. We confirmed that the narrow peaks were not caused by contaminating mitochondria. The overall electrophoretic mobility range of disrupted nuclei is 0 to -5 x 10(-4)cm(2)/Vs, while intact nuclei seem to have mobilities in the -1.5 to -3.5 x 10(-4)cm(2)/Vs range. Furthermore, the highly sensitive CE-LIF method reveals a high abundance of disrupted nuclei that cannot be directly observed by confocal microscopy.     

Speciation of arsenic compounds in fish and oyster tissues by capillary electrophoresis-inductively coupled plasma-mass spectrometry

A capillary electrophoresis-inductively coupled plasma-mass spectrometric (CE-ICP-MS) method for the speciation of six arsenic compounds, namely arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid, dimethylarsinic acid, arsenobetaine and arsenocholine is described. The separation has been achieved on a 70 cm length x 75 microm ID fused-silica capillary. The electrophoretic buffer used was 15 mM Tris (pH 9.0) containing 15 mM sodium dodecyl sulfate (SDS), while the applied voltage was set at +22 kV. The arsenic species in biological tissues were extracted into 80% v/v methanol-water mixture, put in a closed centrifuge tube and kept in a water bath, using microwaves at 80 degrees C for 3 min. The extraction efficiencies of individual arsenic species added to the sample at 0.5 microg As/g level were between 96% and 107%, except for As(III), for which it was 89% and 77% for oyster and fish samples, respectively. The detection limits of the species studied were in the range 0.3-0.5 ng As/mL. The procedure has been applied for the speciation analysis of two reference materials, namely dogfish muscle tissue (NRCC DORM-2) and oyster tissue (NIST SRM 1566a), and two real-world samples.     

Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry

Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with > or =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.     

Quantitative nuclear and cytoplasmic localization of antisense oligonucleotides by capillary electrophoresis with laser-induced fluorescence detection.

We demonstrate the use of simple extraction procedures to separate nuclear and cytoplasmic material from cell extracts, which have been scrape-loaded with a 2-O-methyl phosphorothioate antisense oligonucleotide. Separation and quantitation of the fluorescein-labeled antisense and the flourescein isothiocyanate (FITC)-dextran (molecular weight 40000) as an internal standard is done using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). The bulky FITC-dextran is unable to penetrate the nuclear membrane thereby making it a quantitative indicator of any overlap between the nuclear and cytoplasmic materials during separation of the two phases. Using this procedure, the fluorescein-labeled phosphorothioate oligomer was quantitated at 4.1 x 10(-13) and 3.4x 10(-14) mol antisense/microg-total cellular protein in the nuclear and cytoplasmic extracts respectively following scrape-load delivery of the phosphorothioate to a batch of confluent HeLa cells at a concentration of 0.5 microM (5 x 10(-10) total moles of oligomer). Additionally, gene expression was monitored by measurement of the luciferase reporter protein activity. Scrape-load, spontaneous and liposomal delivery were investigated and compared for subcellular distribution of the oligomer and subsequent gene expression.     

Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection

A rapid method using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed to determine free and protein-bound glutathione (GSH) in human HepG2 hepatocarcinoma cells. The samples were derivatized with 5-iodoacetamidofluorescein (5-IAF), and analyzed at 22 kV using sodium phosphate buffer (10mM, pH 11.4) and an uncoated 58 cm x 75 microm I.D. fused silica capillary. The analysis time was less than 10 min and N-acetylcysteine was used as internal standard. The derivatization conditions, such as reaction time, 5-IAF concentration, running buffer and cartridge temperature were optimized. Argon gas was used in the study to prevent the oxidization of GSH during sample preparation. The optimized method required only 30-40 nl sample per analysis and was fast and sensitive. The method was applied to the analyses of HepG2 cells treated with the small metal chelating agent, pyrrolidine dithiocarbamate (PDTC). The results demonstrate that the amount of protein-bound GSH, which reflects the amount of protein S-glutathionylation, increased in a time-dependent manner upon cell treatment with PDTC, reaching a maximum of over 50% increase 2h post-PDTC.     

聚环氧乙烷无胶筛分毛细管电泳分离宽分子量范围DNA片段

在无胶筛分毛细管电泳中,以聚环氧乙烷为筛分介质,用硅烷化处理的毛细管柱（31.2 cm×75 μm有效长度21.0 cm）分离DL5000 DNA Marker（DNA长度为100~5000 bp）,研究筛分介质浓度、缓冲液pH、分离电压和溴化乙锭浓度对分离双链DNA片段的影响,优化出分离100~5000 bp DNA片段的最佳条件。毛细管电泳的最佳条件为PEO浓度0.5%、缓冲液pH值8.0、电压12 kV、溴化乙锭浓度3.0 μg/mL。此条件下,对山梨醇脱氢酶基因（SDH）和乙烯受体基因（ETR1）的聚合酶链式反应（PCR）扩增产物同时检测,分离、鉴定效果良好。     

Iron protoporphyrin-modified monolithic support for on-line preconcentration of angiotensin prior to CE analysis

An on-line preconcentration method using a polymeric monolithic support is proposed for the retention of the decapeptide angiotensin I and its subsequent analysis by CZE. Monolithic capillary columns were prepared in fused-silica (FS) capillaries of 150 microm id by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iron protoporphyrin IX (Fe-ProP). Monolithic microcolumns (8 mm long) were coupled on-line to the inlet of the separation capillary (FS capillary, 75 microm id x10 cm from the inlet to the microcolumn and 27 cm from the microcolumn to the detector). Angiotensin I was released from the sorbent by a 50 mM sodium phosphate, pH 2.5/ACN, 75:25 v/v solution and then analyzed by CZE with UV absorption detection at 214 nm. The concentration LOQ (CLOQ) was 0.5 ng/mL. The Fe-ProP-derivatized monolithic microcolumn coupled to the separation capillary exhibited a high retention capacity for peptide angiotensin I, and showed as much as 10,000-fold improvement in concentration sensitivity.     

Determination of aristolochic acid in Chinese herbal medicine by capillary electrophoresis with laser-induced fluorescence detection

We have demonstrated the analysis of aristolochic acids (AAs) that are naturally occurring nephrotoxin and carcinogen by capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs (OAAs), reduction of the analytes by iron powder in 10.0 mM HCl is required prior to CE analysis. The reduced AAs (RAAs) exhibit fluorescence at 477 nm when excited at 405 nm using a solid-state blue laser. By using 50.0 mM sodium tetraborate (pH 9.0) containing 10.0 mM SDS, the determination of AA-I and AA-II by CE-LIF has been achieved within 12 min. The CE-LIF provides the LODs of 8.2 and 5.4 nM for AA-I and AA-II, respectively. The simple CE-LIF method has been validated by the analysis of 61 Chinese herbal samples. Prior to CE analysis, OAAs were extracted by using 5.0 mL MeOH, and then the extracts were subjected to centrifugation at 3,000 rpm for 5 min. After reduction, extraction, and centrifugation, the supernatants were collected and subjected to CE analysis. Of the 61 samples, 14 samples contain AA-I and AA-II, as well as 10 samples contain either AAI or AAII. The relative standard deviation (RSD) values of the migration times for AA-I and AA-II are less than 2.5% and 2.1% for three consecutive measurements of each sample. The RSD values for the peak heights corresponding to AA-I and AA-II in most samples are about 8.0% and 10.0%, respectively. The result shows that the present CE-LIF approach is sensitive, simple, efficient, and accurate for the determination of AAs in real samples.     

Determination of Riboflavin in Cereal Grains by Capillary Electrophoresis with Laser-Induced Fluorescence Detection with On-Line Concentration

A very simple, sensitive, and reliable method for the routine determination of riboflavin in cereal grains by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was established. Two on-line concentration techniques, namely, stacking in reverse migrating micelles (SRMM) and sweeping, were examined to enhance the detection signal. The optimum separation buffer consisted of 20 mM phosphoric acid and 140 mM sodium dodecyl sulfate (SDS), and a hydrodynamic injection of 30 s at 0.5 psi was confirmed for the introduction of a sample. In addition, it was found that salt in sample matrix influenced the performance of SRMM, showing the standard addition method was required for the quantitative analysis. The linearity of the method was good with a range of 0.5–500 ng/mL (r = 0.9990). The limit of detection (LOD) was 0.29 ng/mL at a signal-to-noise ratio of 3. This procedure presented was successfully applied to determine riboflavin in 18 samples of 9 types of cereal grains. The recovery rates varied from 94% to 98%, and the relative standard deviation (RSDs) was less than 4.1%.     

Preparation of an IMI dye (imidazole functional group) containing a 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole fluorophore for labeling of phosphomonoesters

We are studying dye-imidazole conjugates ("IMI dyes") as reagents for labeling phosphomonoesters such as nucleotides. Previously we have employed a BODIPY dye in our IMI reagents, and analyzed the labeled products by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) involving an argon ion laser. (The BODIPY fluorophore is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). Here we broaden the technology by preparing a DBD-IMI dye [DBD = 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole], and using a helium-cadmium laser. While DBD-IMI (IMI3) is about 50x more stable photolytically than a BODIPY-IMI dye (IMI2, a conjugate of a BODIPY dye with histamine, was tested), the detection limit for IMI2 (5.10(-11) M; S/N = 5, CE-LIF with an argon ion laser) is tenfold better than that for IMI3 (5.10(-10) M, S/N = 5, helium-cadmium laser). IMI3 conjugates of the four major DNA nucleotides were prepared and detected by CE-LIF.     

Identification and quantification of natural isoxazolinone compounds by capillary zone electrophoresis.

A capillary zone electrophoresis (CZE) method that is specific, simple, rapid and also cheap was developed to analyse some natural UV-absorbing isoxazolinone compounds with toxic potential present in legume seedlings. The six most common natural isoxazolinone compounds were separated within 10 min with 25 mM potassium phosphate (pH 7.5) containing 8% 1-propanol as running buffer. A 60 cm coated fused-silica capillary (52.6 cm effective length x 75 microm I.D.), with an electric field of 375 V/cm at 30 degrees C was used. The limit of detection ranged from 0.01 mM (3.0 microg/ml) to 0.03 mM (7.7 microg/ml). Linearity between peak areas and concentrations ranging from 0.05 mM to 1.75 mM were determined for each isoxazolinone. The correlation coefficient was 0.9954 or greater. Both relative migration time and peak area were reproducible. The RSD of relative migration time is between 0.44 and 1.94% and RSD of peak area is between 1.26 and 6.86%. The concentrations of isoxazolinones in Lathyrus odoratus and L. sativus seedlings obtained by CZE were in agreement with the previous results from HPLC.     

Optimization of the chiral resolution of baclofen by capillary electrophoresis using beta-cyclodextrin as the chiral selector

The chiral resolution of baclofen was achieved by capillary electrophoresis using a fused-silica capillary (60 cm x 75 microm ID). The background electrolyte (BGE) was phosphate buffer (pH 7.0, 50 mM)-acetonitrile (95:5 v/v) containing 10 mM beta-cyclodextrin. The applied voltage was 15 kV. The values of alpha and R(s) were 1.06 and 1.00, respectively. The electrophoretic conditions were optimized varying the pH and the ionic strength of the BGE, concentrations of beta-cyclodextrin and acetonitrile and the applied voltage.     

Analysis of metacycline by capillary electrophoresis

The development and validation of an optimized capillary electrophoresis method for the determination of metacycline in the presence of its related substances by capillary electrophoresis is shown. The influence of methanol as organic modifier, buffer pH, buffer concentration, capillary length, column temperature, Triton X-100 and methyl-beta-cyclodextrin was investigated. A central composite design was performed in order to optimize the method. The optimal separation conditions were: uncoated fused-silica capillary (39 cm total length, 31 cm effective length, 50 microm ID); as background electrolyte a solution of 160 mM sodium carbonate and 1 mM EDTA (pH 10.35)/methanol (89:13 v/v); temperature, 15 degrees C; voltage, 12 kV. The method showed good selectivity, repeatability, linearity, and sensitivity. The limits of detection and quantitation are 0.024% and 0.06%, respectively, relative to a 2.5 mg/mL solution. Six commercial samples were analyzed quantitatively.     

Simultaneous determination of polyamines and catecholamines in PC-12 tumor cell extracts by capillary electrophoresis with laser-induced fluorescence detection

A method to detect polyamines and catecholamines in PC-12 tumor cell extracts by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is described for the first time. Both derivatization conditions and buffer concentrations and pH were optimized. Under optimized conditions the polyamines (putresine, spermine, spermidine) and catecholamines (dopamine, norepinephrine, epinephrine, serotonin) were derivatized with fluorescein isothiocyanate and separated at 25 kV in a fused-silica capillary (50 microm ID x 40 cm) with 0.1 M borate, pH 9.0, in less than 18 min. The influence of running buffer conditions, such as buffer pH and concentrations, were also investigated. Linearity of the analytes ranged from 0.05 to 1.0 micromol/L, and the detection limit (S/N = 3 ) ranged from 0.03 to 2.50 nmol/L. The concentrations of polyamines and catecholamines in PC-12 tumor cell extracts were determined with this method.     

Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions

Separations of phiX-174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5-5 microg/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8,000,000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high-speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.     

Development and validation of a capillary electrophoresis method for the measurement of short-chain organic acids in natural rubber latex

Short-chain organic acid contents in serum of natural latex are interesting to measure and capillary electrophoresis (CE) has proved to be a good tool for their study. In the present work a method has been developed to identify the short-chain organic acids present in sera of natural rubber latex (oxalic, formic, fumaric, aconitic, succinic, malic, glutaric, citric, acetic, glycollic, propionic and quinic acids), the separation was optimised and the quantification method validated. The separation was performed on a CE system with UV detection at 200 nm. The separation was carried out with an uncoated fused-silica capillary (57 cm x 50 microm I.D.) and was operated at -10 kV potential. The separation buffers were prepared with 0.5 M H3PO4, 0.5 mM cetyltrimethylammonium bromide and pH adjusted by adding NaOH to 6.25 except for propionic acid which was better measured at pH 7.00. Validation parameters are adequate and limits of detection range from 0.005 mM to 1.6 mM. Short-chain organic acids were measured with this method in sera of three different types of latex.     

Post-column reactor of coaxial-gap mode for laser-induced fluorescence detection in capillary electrophoresis

A post-column reactor with coaxial-gap mode is developed for laser-induced fluorescence detection (LIF) in capillary electrophoresis (CE). The reactor can be assembled simply and conveniently, in which a thin polyimide sleeve of 10-mm length obtained from the capillary coating is used to align separation and reaction capillary with a 20 microm gap. Naphthalene-2,3-dicarboxaldehyde and 2-mercaptoethanol are used as derivatization reagents and delivered into the reaction capillary through the annulus between the separation capillary and polyimide sleeve and the gap of two capillaries by gravity. A reaction distance from the gap to detection point is 5mm. For the post-column reactor of CE-LIF, several configuration parameters are optimized, including liquid level difference between the derivatization solution and outlet buffer, annular dimension between the outer diameter of etched separation capillary and the inner diameter of polyimide sleeve, and reaction distance, etc. The detection limits in the range from 8.0x10(-8) to 1.0x10(-6) mol/L and linear calibration range more than two orders of magnitude are obtained for amino acids. The separation efficiency ranges from 1.35x10(5) to 1.67x10(5) theoretical plates.     

Determination of bisphenol A and 10 alkylphenols in serum using SDS micelle capillary electrophoresis with gamma-cyclodextrin.

A new micelle capillary electrophoresis based on cyclodextrin micellar electrokinetic chromatography (MEKC) for the determination of bisphenol A and 10 alkylphenols in rat serum is reported. Several surfactants and dextrins were studied. Bisphenol A and alkylphenols were separated using a 50 microm x 50 cm capillary with 20 mM borate phosphate buffer (pH 8.0) containing 20 mM sodium dodecylsulfate and 5 mM gamma-cyclodextrin as carrier. The method could determine 0.6-2000 microg/mL of phenols in 100 microL serum by photometric detection at 214 nm. Using 2.0 mL serum, 1.0 ng/mL of phenols could be determined. The relative standards deviations were 6.3-7.7% at 10 microg/mL in serum. The recoveries were 91.8-93.0% with 10 microg/mL serum samples.