On-line concentration and determination of tobacco-specific N-nitrosamines by cation-selective exhaustive injection-sweeping-micellar electrokinetic chromatography

In this paper, a micellar electrokinetic chromatography (MEKC) method combined with cation-selective exhaustive injection (CSEI) and sweeping was developed to separate and concentrate four tobacco-specific N-nitrosamines (TSNAs) including N′-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL). Experimental parameters affecting separation efficiency and enhancement factors were investigated in detail. Under the optimum MEKC condition, NAB, NNK, NNAL and iso-NNAL were baseline separated with high separation efficiencies and good peak shapes. Furthermore, with the preconcentration by CSEI-sweeping-MEKC, the sensitivity enhancement factors for NAB, NNK, NNAL and iso-NNAL in terms of peak areas ranged from 6.0 × 10³ to 1.5 × 10⁴, and the detection limits (LOD, S/N = 3) of four TSNAs were in the range of 0.004-0.016 μg/mL. In addition, this method had fairly good repeatability, and the RSDs of retention time and peak area were less than 1% and 5%, respectively. Finally, this method showed promising capabilities in the application of detecting and analyzing TSNAs in human urine samples.     

Analyses of gibberellins by capillary electrophoresis-mass spectrometry combined with solid-phase extraction

A new, simple and rapid capillary electrophoresis-mass spectrometry (CE-MS) method, using a cationic polymer-coated capillary to reverse electroosmotic flow, is proposed and validated for the separation and simultaneous quantification of 11 gibberellins (GAs). Under optimum conditions, a baseline separation of 11 GAs, including GA1, GA3, GA4, GA5, GA6, GA7, GA13, GA19, GA20, GA24 and GA53 was accomplished within 25 min using 70 mM ammonium formate/formic acid buffer (pH 3.8) and 2% (v/v) acetonitrile with -25 kV as the separation voltage. Satisfactory results were obtained in terms of linearity (R2 between 0.984 and 0.995), precision (RSD of migration time below 0.8%) and sensitivity (LOD between 0.31 and 1.02 microM). Furthermore, a novel solid-phase extraction (SPE) procedure was developed for the pre-concentration and purification of GAs using Oasis MAX cartridges. The combination of SPE and CE-MS approach was applied to screen for endogenous GAs present in coconut (Cocos nucifera L.) water sample. To illustrate the applicability of the method, GA1 and GA3 were successfully detected and quantified in coconut water. Finally, the GA1 and GA3 identities were further unequivocally confirmed by CE-tandem MS experiments operating in the multiple reaction monitoring mode.     

Separation of naturally occurring triterpenoidal saponins by capillary zone electrophoresis.

A high-performance capillary electrophoresis (HPCE) was successfully applied to the separation and quantitation of naturally occurring oleanene triterpenoidal saponins. The HPCE adapted to the separation of two pairs of disteriomeric saponins (1-2) or (3-4), obtained from Trifolium alexandrinum seeds, was based on capillary zone electrophoresis (CZE) in borate buffer with UV detection at 195 nm. An usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to determination of individual saponins by CZE. The separation parameters such as borate concentration, pH and applied voltage were varied in order to find the best compromise that complied with demands for high separation, short duration and sufficiently high detector response. The optimum running conditions were found to be 60 mM borate buffer, pH 10 and 12 kV. Under the alkaline borate electrolyte, no resolution was achieved for the saponins (1 and 3) or (2 and 4) in a single mixture, except when 20 mM beta-cyclodextrin was added to the running electrolyte. With the combined techniques of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring diasteriomeric saponins is now available.     

Study of the metabolism on tobacco-specific N-nitrosamines in the rabbit by solid-phase extraction and liquid chromatography–tandem mass spectrometry

Metabolism of four tobacco-specific N-nitrosamines (TSNAs), N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been studied by solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). 4-(Methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was used as internal standard. SPE and LC–MS–MS was found to be a rapid, simple, sensitive, and selective method for analysis of TSNAs in rabbit serum. The relative standard deviation (R.S.D., n = 6) for analysis of 5 ng mL⁻¹ and 0.5 ng mL⁻¹ standards and of serum sample spiked with 5 ng mL⁻¹ standards of five TSNAs was 2.1–11% and recovery of 5 ng mL⁻¹ standards from serum was 100.2–112.9%. A good linear relationship was obtained between peak area ratio and concentration in the range of 0.2–100 ng mL⁻¹ for NNAL and 0.5–100 ng mL⁻¹ for other four TSNAs, with correlation coefficients (R ²) >0.99 (both linear and log–log regression). Detection limits for standards in solvent were between 0.04 and 0.10 ng mL⁻¹. Doses of TSNAs administered to rabbits via the auricular vein were 4.67 μg kg⁻¹ and 11.67 μg kg⁻¹, in accordance with the different levels in cigarettes. Metabolic curves were obtained for the four TSNAs and for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK; on the basis of these curves we modeled metabolic kinetic equations for these TSNAs by nonlinear curve fitting.     

Application of capillary electrophoresis in the quality control of osmotically treated water

Summary This paper discusses the use of capillary zone electrophoresis (CZE) with indirect UV detection for the separation and detection of ions. By use of a 70 cm×75 μm i.d. capillary at −15 kV and an electrophoretic buffer containing sodium chromate, 1-butanol, and cetyltrimethylammonium bromide as electroosmotic-flow modifier (pH 8) nine inorganic and organic anions were separated in less than 10 min. By use of the same type of capillary at 20 kV and an electrophoretic buffer containing imidazole and 18-crown-6 (pH 5) eight cations were separated in less than 5 min. The different variables that affect the separation were studied and optimized; the compounds were detected at low mg L⁻¹ levels after hydrodynamic injection under pressure. The method was tested with osmotically treated waters, and the results compared with those obtained by ion chromatography for anion analysis and by atomic absorption spectroscopy for cation analysis.     

Separation of phospholipids by capillary zone electrophoresis with indirect ultraviolet detection

A simple method for separation of different anionic and zwitterionic phospholipid classes by capillary zone electrophoresis (CZE), using indirect UV detection with adenosine monophosphate (AMP) as background electrolyte and the UV-absorbing additive, was successfully developed in this study. The separation conditions including apparent pH (pH*) of running buffer, concentration of AMP, organic solvent, applied voltage and capillary temperature were systematically optimized. The application of this method to human blood sample was also briefly examined.     

Determination of furosine in food products by capillary zone electrophoresis-tandem mass spectrometry

A new method for qualitative and quantitative analysis of furosine in food products by capillary electrophoresis coupled to tandem mass spectrometry (CE-MS(2) ) has been optimized and validated. In analytical conditions, the pH value of the run buffer was similar to that of the hydrolyzed samples, which helps in avoiding interactions of the analyte with silanol groups of the inner wall of the fused silica capillary. In all previous CE methods proposed in literature, no SPE treatment has been used. The method has been applied to the analysis of food products, such as flour, having low amounts of furosine. Flour samples of different origin (wheat, chestnut, lupin, einkorn, chickpeas) have been investigated. Different food products such as pasta, milk, and tigelle bread (a typical Italian unleavened bread) were also analyzed. Some of the samples have also been analyzed by reversed-phase HPLC, and when compared to CE-MS, data obtained showed good agreement. CE-MS, in comparison to HPLC, showed advantages in terms of time of analysis and cost. The validation procedure provided good values for LOD and LOQ (respectively 0.07 and 0.25 mg L(-1) ), recovery (77 and 97%), and precision investigated in terms of intraday repeatability (RSD%: 4-6% for peak areas; 1-2% for migration times) and intermediate precision (below 16% for peak areas, and below 7% for migration times). Therefore, the technique reported can be proposed as a powerful analytical method and a valid alternative to common traditional analytical techniques.     

Applications of a sulfonated-polymer wall-modified open-tubular fused-silica capillary in capillary zone electrophoretic separations

A fused-silica capillary that is wall-modified via chemically bonding a sulfonated polymer to the capillary wall has a uniform negative charge density on its surface and produces an electroosmotic flow (EOF) greater than 4 x 10(-4) cm2 V(-1) s(-1) The EOF is nearly independent of buffer pH over the pH range of 2 to 10 and is lower than the EOF obtained for the bare fused-silica capillary at the more basic pH but is higher at the more acidic buffer pH. Optimization of buffer pH can be based on analyte pKa values to improve the overall quality of the capillary zone electrophoresis (CZE) separation of complex mixtures of weak acid and base analytes. Because of the high EOF in an acidic buffer, the capillary is useful for the separation of weak organic bases which are in their cation forms in the acidic buffer. EOF for the sulfonic acid bonded phase capillary can be adjusted via buffer additives such as organic solvent, tetraalkylammonium salts, multivalent cations and alkylsulfonic acids. The advantages of utilizing buffer pH and the EOF buffer modifiers to enhance migration time, selectivity, and resolution in CZE separations with this capillary are illustrated using a series of test analyte mixtures of inorganic anions, carboxylic acids, alkylsulfonic acids, benzenesulfonic acids, sulfas, pyridines, anilines or small-chain peptides.     

Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary

A capillary zone electrophoresis (CZE) method was developed for the rapid analysis of charge heterogeneity of immunoglobulin G (IgG) monoclonal antibodies (mAbs). The separation was carried out in a short, dynamically coated fused-silica capillary. A number of separation parameters were investigated and optimized, including pH, concentration of the separation buffer (ε-amino caproic acid), concentration of the triethylenetetramine (TETA) dynamic coating, the capillary internal diameter and the field strength used for the separation. The effects of between-run flushing of the capillary and the data acquisition rate were also evaluated. Under the optimized conditions, a fast (<5 min), selective and reproducible separation of mAb charge variants was achieved under a very high electric field strength (1000 V/cm). This method also requires only a short conditioning of the capillary, with between-run conditioning completed within 2 min. The method was evaluated for specificity, sensitivity, linearity, accuracy and precision. The same separation conditions were applied to the rapid separation (2-5 min) of charge variants of multiple monoclonal antibodies with pI in the range of 7.0-9.5. Compared with other existing methods for charge variants analysis, this method has several advantages including a short run time, rapid capillary conditioning and simple sample preparation.     

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms.     

Metabolic study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in vitro in human bronchial epithelial cells using chiral capillary electrophoresis

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) with one chiral center at the carbinol is a major metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As tobacco specific N-nitrosamines (TSNAs), NNK and NNAL are the most pulmonary carcinogens in tobacco products and smoke. In this paper, a chiral CE method modified with highly sulfated β-cyclodextrin (S-β-CD) was developed to investigate the stereoselective formation of NNAL from NNK in vitro in normal human bronchial epithelial (NHBE) cells. Combined with solid phase extraction (SPE) of the cell samples, NNK and NNAL enantiomers were baseline separated under the proposed CE conditions, with satisfactory recoveries (72.5-113% for NNK and (±)-NNAL) and low limits of detection (LOD, 2.5-3 μg/mL for NNK and (±)-NNAL). The cytotoxicity of NNK in NHBE cells was investigated through the cell counting kit (CCK) assay and proved to be highly dependent on the NNK's concentration. The metabolic results obtained from CE analysis demonstrated that NNK was preferentially metabolized to (+)-NNAL through carbonyl reduction. Meanwhile, the ratio of [(+)-NNAL]/[(-)-NNAL] was independent of NHBE cells' incubation time with NNK, but could be changed according to the original incubation concentration of NNK. This chiral CE method could be useful for the study on toxicology and metabolic transformations of related TSNAs.     

1-苯基-3-甲基-5-吡唑啉酮衍生中药寡糖的毛细管区带电泳分离

建立了一种可用于中药寡糖分离的毛细管区带电泳(CZE)分析方法。分离条件: 未涂层熔融石英毛细管柱(48 cm×50 μm,有效长度38 cm),紫外检测波长245 nm,采用1-苯基-3-甲基-5-吡唑啉酮(PMP)为寡糖衍生试剂,50 mmol/L磷酸盐溶液(pH 2.5)为运行缓冲液,电压15 kV,重力进样10 cm×2 s。针对中药寡糖实际样品的复杂性,通过添加多种常见单糖进行方法的实用性验证,并且将方法用于板蓝根多糖的控制降解产物的分离。结果表明,板蓝根寡糖组分可按相对分子质量从小到大的顺序分离,分离效果令人满意。该分离方法操作简单、高效,可用于中药寡糖实际样品分析。另外,还对单糖、寡糖PMP衍生物的电泳迁移行为进行了初步的理论探讨。     

Determination of cytokinins in coconut (Cocos nucifera L.) water using capillary zone electrophoresis-tandem mass spectrometry

The applicability of CZE in combination with MS and MS/MS methods for the simultaneous separation and determination of 12 cytokinins was investigated for the first time. Cytokinins were first completely separated by CZE within less than 20 min using a volatile buffer and then detected directly by MS or MS/MS. Satisfactory separation of the 12 cytokinin standards was achieved using a 25 mM ammonium formate/formic acid buffer (pH 3.4) and 3% ACN v/v with a separation voltage of 25 kV. On the basis of the resolution of the neighboring peaks, the various parameters for CZE-MS optimization, such as buffer pH value, concentration of buffer and organic modifier, applied voltage and sheath liquid, were evaluated systematically. MS/MS with multiple reaction monitoring detection was carried out to obtain sufficient selectivity and sensitivity. The combination of on-line sample stacking and CZE-MS/MS achieved a detection limit in the range of 0.05-0.18 microM for the 12 cytokinins at an S/N of 3. The optimized CZE-MS/MS method was simple, rapid, low cost, robust and highly selective. Furthermore, the developed method was successfully applied to screen for endogenous cytokinins in purified coconut water extract sample. Nine cytokinins were detected and quantified in coconut water after SPE.     

Separation and determination of enkephalin-related peptides using capillary electrophoresis

CZE with UV-absorption detection has been used for the separation and determination of enkephalin-related peptides. The experimental conditions, such as pH and concentration of running buffer, applied voltage, injection method, and time, were investigated in detail. Excellent separation efficiency could be obtained for ten enkephalin-related peptides with a 50 microm (ID) x 58 cm capillary using sodium dihydrogen phosphate as the running buffer (pH 3.11) when 20 kV of applied voltage was used. The concentration detection limits were found to be in the range of 0.31-1.94 microg/mL (defined as S/N = 3). The proposed method has been applied to analyze the spiked cerebrospinal fluid (CSF) sample, and the results showed that CZE is a powerful technique for separation and detection of the above biological peptides.     

Migration mechanism of bases and nucleosides in oil-in-water microemulsion capillary electrophoresis.

The electrophoretic behaviors of five bases and corresponding nucleosides in the oil in water (o/w) microemulsion capillary electrophoresis, microemulsion electrokinetic chromatography (MEEKC), were examined in comparison with those in normal capillary zone electrophoresis (CZE). The microemulsion systems were composed of heptane, sodium dodecyl sulfate (SDS), 1-butanol and 10 mM phosphate buffer (pH 7.0) or toluene, SDS, 1-butanol and 5 mM carbonate buffer (pH 10.0). CZE was carried out in the range of pH 9.7-10.9, and the dissociation constants, pKa, of the bases and nucleosides and the electrophoretic mobilities of the anionic forms were determined. The electrophoretic behaviors of the solutes in the microemulsion systems were analyzed from their pKa, the electrophoretic mobilities of the anions determined by CZE, and the distribution constants, K(D), of the neutral forms between the microemulsion droplets and the outer aqueous phase. The importance of adsorption mechanism in MEEKC system was suggested from the correlation between log K(D) and log P.     

Enantioseparation of warfarin and its metabolites by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method with direct ultraviolet (UV)-absorbance detection is presented for the simultaneous enantiomeric separation of warfarin and its main metabolites, including warfarin alcohols, 4'-, 6-, and 7-hydroxywarfarin, using highly sulfated beta-cyclodextrin (HS-beta-CD) as the chiral selector. This chiral separation method was optimized in terms of the electrophoretic parameters, which included the concentration of HS-beta-CD used, the type and composition of organic modifier added to the background electrolyte (BGE) buffer, and the BGE buffer pH. Chiral separation of warfarin and its major metabolites was achieved with high resolution, selectivity, efficiency, repeatability, and reproducibility. This optimized chiral analysis of warfarin along with its metabolites was completed within a satisfactory electrophoresis time of 20 min.     

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

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

Capillaries modified by noncovalent anionic polymer adsorption for capillary zone electrophoresis, micellar electrokinetic capillary chromatography and capillary electrophoresis mass spectrometry

A simple coating procedure for generation of a high and pH-independent electroosmotic flow in capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) is described. The bilayer coating was formed by noncovalent adsorption of the ionic polymers Polybrene and poly(vinylsulfonate) (PVS). A stable dynamic coating was formed when PVS was added to the background electrolyte. Thus, when the PVS concentration in the background electrolyte was optimized for CZE (0.01%), the EOF differed less than 0.3% after 54 runs. The electroosmotic mobility in the coated capillaries was (4.9+/-0.1) x 10(-4) cm2V(-1)s(-1) in a pH-range of 2-10 (ionic strength = 30 mM). When alkaline compounds were used as test substances intracapillary and intercapillary migration time variations (n = 6) were less than 1% relative standard deviation (RSD) and 2% RSD, respectively in the entire pH range. The coating was fairly stable in the presence of sodium dodecyl sulfate, and this made it possible to perform fast MEKC separations at low pH. When neutral compounds were used as test substances, the intracapillary migration time variations (n = 6) were less than 2% RSD in a pH range of 2-9. In addition to fast CZE and MEKC separations at low pH, analysis of the alkaline compounds by CE-MS was also possible.     

A capillary zone electrophoresis for determination of thiolic peptides in biological samples

A new method to improve the analyses of thiolic peptides (cysteine, γGlu-Cys, glutathione, phytochelatins and desglycyl-phytochelatins) derivatized with monobromobimane (mBrB) in complex biological samples by CZE is described. The method involves a SPE using Sep-Pak Light C18 Cartridges after derivatization and a later CZE analysis. Elution of mBrB-thiols was achieved with 10 mM HCl + 70% methanol v/v in deionised water. Electrophoretic parameters, such as BGE pH and concentration, different organic additives (methanol and trifluoroethanol), applied voltage and capillary length were studied in order to establish suitable analytical conditions. Optimum separation of the mBrB-thiolic peptides was obtained with 100 mM sodium borate buffer at pH 7.60. The electrophoretic conditions were +15 kV, capillary length of 90 cm from inlet to detector (98 cm total length, 50 μm ID), samples were loaded into the capillary by hydrodynamic injection (50 mbar, 20 s) and detection was performed at 390 nm. The improved method showed good reproducibility, linearity and sensitivity. The LODs and LOQs estimated using a standard of GSH were 1.41 and 4.69 μM respectively.     

Separation of human fibrinopeptides by capillary zone electrophoresis

We describe a method for the simultaneous determination of the five fibrinopeptide forms derived from the thrombin-promoted activation of human fibrinogen by capillary zone electrophoresis (CZE). The fibrinopeptide mixture was first desalted by a solid-phase extraction (SPE) step. The analysis was performed in reversed polarity in a highly cross-linked polyethylene glycol (PEG)-coated capillary with UV-light absorption detection at 200 nm. Several parameters including buffer concentration and pH, presence of an organic modifier, temperature, and applied voltage, have been tested. The best separations were obtained within 20 min, utilizing a 20 mM sodium phosphate buffer without organic modifier, in the narrow 6.1-6.2 pH range, at 25 degrees C, with an applied voltage of 20 kV. Quantitative analysis is made possible by the use of sheep fibrinopeptide A as an internal standard to correct for both extraction and injection errors.