Capillary electrochromatography of complex plasma matrix on a C18/SCX column using UV-vis and mass spectrometric detection

Summary Separations of compounds in plasma were performed on a Hypersil Duet C₁₈/SCX capillary electrochromatography (CEC) column, utilizing an automated injection system combined with a short in-house designed and fabricated micro-electrospray CEC mass spectrometer interface. Protein precipitation was used prior to the CEC separations. More than two hundred separations of the corticosteroids Dexamethasone and Betamethasone 17-valerate, and Fluticasone Propionate in complex plasma matrix were performed on a single column under isocratic conditions. The method demonstrated good reproducibility, selectivity, sensitivity and high efficiencies. Linear calibration with good correlation was typical. Estimated detection limits in the low micromolar and nanomolar range for all compounds were obtained using UV-Vis absorbance (UV) and Electrospray Mass Spectrometric (ES/MS) detection respectively. Efficiencies for all compounds were typically 87,500 plates on a 25 cm column (350,000 plates m⁻¹) and increased with the number of plasma samples injected, up to 250,000 plates per column (1,000,000 plates m⁻¹). These very high observed plate counts may be artificially enhanced by the inadequate scan possibilities of the MS over very narrow chromatographic peaks.     

Double‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis

Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined with various detection methods provided remarkable developments for high‐sensitivity glycan profiling. The coating of the capillary is needed for highly polar molecules from complex biosamples. A poly(vinyl alcohol)‐coated capillary is commonly utilized in the capillary electrophoresis separation of saccharides sample due to the high‐hydrophilicity properties. A modified facile coating workflow was carried out to acquire a novel multiple‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable analysis of glycans. The migration time fluctuation was used as index in the optimization of layers and a double layer was finally chosen, considering both the effects and simplicity in fabrication. With migration time relative standard deviation less than 1% and theoretical plates kept stable during 100 consecutive separations, the method was presented to be suitable for the analysis of glycosylation with wide linear dynamic range and good reproducibility. The glycan profiling of enzymatically released N‐glycans from human serum was obtained by the presented capillary electrophoresis method combined with mass spectrometry detection with acceptable results.     

Enhancing separation in short‐capillary electrophoresis via pressure‐driven backflow

We present a novel easy‐to‐operate and efficient method to improve the separation efficiency in short‐capillary electrophoresis by introducing steady backflow to counterbalance electro‐osmotic flow without the use of any external pressure. The backflow was easily generated by tapering the capillary end, which was achieved by heating a straight capillary and stretching it with a constant force. We investigated the net fluidic transport rate under different tip lengths and separation voltages. Good run‐to‐run repeatability and capillary‐to‐capillary reproducibility of the present method were obtained with RSD less than 1.5%, indicating the stability of the fluid transport rate in the tapered capillary, which ensures the quantification and repeatability of capillary zone electrophoresis (CZE) analysis. Enhanced separation of the tapered short capillary electrophoresis was demonstrated by CZE analyzing amino acids and positional isomers. Baseline separations were achieved in less than 60 s using a tapered capillary with the effective length of 5 cm, while no separation was achieved using a normal capillary without a tapered tip. The present study provides a promising method to use pressure‐driven backflow to enhance separation efficiency in short‐capillary electrophoresis, which would be of potential value in a wide application for fast analysis of complex samples.     

Gas chromatography of amino acids: Use of a fused-silica capillary column in the determination of plasma amino acids

A capillary chromatographic procedure using a fused silica column is described which can be used to quantitatively determine amino acids in plasma following the pre-chromatographic “clean-up” described in a recent paper [1]. In substituting this procedure for that involving a packed column, advantage has been taken of the greater resolving power to separate amino acids from background component peaks. In order to extend this advantage and provide a sound basis for quantitative analysis, the technique of cold on-column injection was employed. As a result, good precision of standard analysis was obtained with relative standard deviation (RSD) values for all amino acids of less than 4%. Application of the entire procedure to plasma samples yields RSD values of better than 10% for all amino acids with recoveries ranging from 72% to 104%. Simultaneous determination of plasma amino acid levels by gas chromatography (GC) using capillary columns and by classical ion exchange (CIE) showed reasonable agreement. Statistical evaluation showed no significant difference between twelve amino acids. Values for the remaining two, namely, phenylalanine and histidine are significantly different (p < 0.005). Comparison of the values obtained from GC capillary and packed columns reveals no significant difference between fourteen amino acids. Significant differences exist between results for phenylalanine and tyrosine (p < 0.001). It is concluded that there is good agreement between data obtained by GC capillary and CIE techniques and that differences between results for phenylalanine and histidine are method related.     

新型毛细管电泳化学发光系统应用于氨基酸分离测定

本研究建立一种以过氧草酸酯为化学发光体系的新型毛细管电泳化学发光系统,并将该系统应用于氨基酸的分离测定.该系统设计了新型的电极模式与反应混和模式以获得高的信号稳定性与检测灵敏度.以一端与高压电源负极相连的铂丝连接到不锈钢注射针头为接地电极,电泳毛细管穿过注射针头,且其末端到达注射针头的出口,电泳毛细宇航局末端与化学发光试剂在反应池中以相对的方向靠近.实验结果表明,该系统稳定性好、分离效率高、灵敏度高、精密度好,对天冬氨酸与亮氨酸的检出限分别为2.0和 1.1 nmol/L.峰高及迁移时间的相对标准偏差分别为2.3%～3.8%和1.2%～1.5%.     

Preparation of silica-based nanoparticle having surface-bound octanoyl-aminopropyl moieties and its applications for the capillary electrochromatography separation of charged and neutral compounds

A kind of novel amphiphilic silica-based nanoparticle having surface-bound octanoyl-aminopropyl moieties (OA-NP) with the diameter of ~250 nm was successfully prepared and characterized by elemental analysis, Fourier transform infrared spectrometry. The potential use of OA-NP as pseudostationary phase in capillary electrochromatography for the separation of aromatic acids, basic, and neutral compounds was investigated. Five aromatic acids were separated rapidly with high column efficiency as they migrate in the same direction with the EOF under optimum experimental conditions. Under a running buffer with the composition of 40% methanol, 10 mM phosphate buffer (pH 5.5) with 1.0 mg/mL OA-NPs added, basic compounds investigated were baseline resolved with relatively symmetrical peaks. Due to the existence of polar acyl amide group on the surface of OA-NPs, "silanol effect" that occurs between the positively charged basic analytes and the silanols of the capillary column was greatly suppressed. Furthermore, the newly synthesized OA-NPs were also tried for the separation of some neutral analytes, and satisfactory separations were obtained.     

Comparison of three modifications of fused‐silica capillaries and untreated capillaries for protein profiling of maize extracts by capillary electrophoresis

In this work, capillary electrophoresis was applied to protein profiling of fractionated extracts of maize. A comparative study on the application of uncoated fused‐silica capillaries and capillaries modified with hydroxypropylmethylcellulose, ω‐iodoalkylammonium salt and a commercially available neutral capillary covalently coated with polyacrylamide is presented. The coating stability, background electrolyte composition, and separation efficiency were investigated. It was found that for zeins separation, the most stable and efficient was the capillary coated with polyacrylamide. Finally, the usefulness of these methods was studied for the differentiation of zein fraction in transgenic and nontransgenic maize. Zeins extracted from maize standards containing 0 and 5% m/m genetic modification were successfully separated, but slight differences were observed in terms of the zein content. Albumin and globulin fractions were analyzed with the use of unmodified fused‐silica capillary with borate buffer pH 9 and the capillary coated with polyacrylamide with phosphate buffer pH 3. In the albumin fraction, additional peaks were found in genetically modified samples.     

Determination of total phosphorus in soya food samples by capillary isotachophoresis (cITP)

Capillary isotachophoresis (cITP) was applied for the determination of total phosphorus in soya food. The leading electrolyte was 8 mM HCl adjusted with β-alanine to pH=3.55 plus 3 mM bis-tris-propane and 0.2% hydroxyethylcellulose, whereas the terminating electrolyte 5 mM citric acid. Obtained results were compared with the spectrophotometric method. The correlation coefficients were 0.9996 for cITP and 0.9986 for standard method indicating the satisfactory precision of the calibration curves. The separation of anions was achieved within 25 min. Accuracy was determined using standard reference material (non-fat milk powder) and recovery assay based on standard additions method. Obtained results were discussed in respect to the accuracy and statistical parameters. Satisfactory values of recovery ranged between 99.22 and 99.85%, whereas R.S.D.<1% what demonstrate the advantage of cITP method in the routine analyses of phosphorus content in food samples.     

Highly selective and efficient separations of a wide range of acidic species in capillary electrophoresis employing non-aqueous media

Summary The novel application of CE using non-aqueous media has been studied for the separation of a range of acidic compound types. This enabled the first quantitative assay by CE employing non-aqueous media to be performed. Separation selectivity manipulation for closely related species was achieved through variation of organic solvent types and composition, ionic strength changes, alteration of pH^* values and the addition of cyclodextrin additives soluble in organic solvents. This offers a greater range of possibilities during method development than use of aqueous buffer. The generation of low operating currents permitted rapid, highly efficient and selective separations to be achieved by applying high field strengths across short capillaries. Optimised rinsing and capillary regenerating procedures were devised which allowed highly repeatable separations to be achieved with migration time repeatability below 1% RSD. Use of internal standards also allowed 1% RSD values to be obtained for injection precision. Routine operating effects were assessed and it was observed that stacking effects remain important in non-aqueous CE. Also, optimal separations are obtained when the samples are diluted in the pure solvent used to prepare the electrolyte. The application range of non-aqueous CE was appreciably extended to include acidic drugs, dyes, surfactants and preservatives.     

One-pot labeling-based capillary zone electrophoresis for separation of amino acid mixture and assay of biofluids

A fast, simple and cost-effective one-pot labeling strategy coupled with capillary zone electrophoresis was developed for the complete separation of amino acid mixture. The strategy includes two steps of reactions: Cyanuric chloride was made to react first with 7-amino-1,3-naphthalenedisulfonic acid monopotassium salt at 0 °C for 10 min, and then with amino acids at 55 °C for 6 min. The resulted products, after diluted with water, were injected into capillary zone electrophoresis system for separation. Using a running buffer of 20 mM sodium tetraborate decahydrate at pH 10.1, nineteen amino acids were efficiently separated in 25 min, with relative standard deviation of 0.36–1.6% and 0.96–2.1% (within and between days, respectively) for migration time and 0.030–1.6% and 0.22–2.4% (within and between days, respectively) for peak area. The proposed method has been successfully applied to the determination of free amino acids in biofluids, including human serum, urine, and saliva. The linearity of quantification was over two orders of magnitude for most amino acids, with a correlation coefficient larger than 0.999. The average recovery, determined by spiking a known amount of amino acid standards into real samples, was in a range from 91.6% to 105.9%. This method can be a noninvasive means since it could directly assay the urine and saliva samples.     

An ultrasonication-assisted extraction and derivatization protocol for GC/TOFMS-based metabolite profiling

Conventional chemical derivatization of metabolites in biological specimens is time-consuming, which limits the throughput and efficiency of metabolite profiling using a gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) platform. We report an ultrasonication-assisted protocol which reduces the derivatization time from hours to about 30 min and significantly enhances the derivatization efficiency prior to a GC/TOFMS analysis. The protocol was evaluated using 40 compounds representing different classes of human metabolites, and demonstrated good analytical precision and accuracy. In comparison with the conventional method, the new protocol was able to increase the intensity of most of the identified peaks (71.0%) in the GC/TOFMS chromatograms of human serum samples. The detected compounds with increased intensity include most amino acids, keto-containing organic acids, carbonyl-containing carbohydrates, and unsaturated fatty acids. We applied this protocol in a metabolomic study of human serum samples obtained from 34 patients diagnosed with hypertension and 29 age- and gender-matched healthy subjects. Metabolite markers associated with hypertension, including glucosamine, D-sorbitol, 1-stearoylglycerol, and homocysteine, were identified and validated by statistical methods and use of reference standards. Our work highlights the potential of this novel approach for the large-scale metabolite profiling of samples generated from plant, animal, and clinical and epidemiological studies.     

Capillary electrophoresis separation of aminoalkanol derivatives of 1,7‐dimethyl‐8,9‐diphenyl‐4‐azatricyclo[5.2.1.02,6]dec‐8‐ene‐3,5,10‐trione as potential anticancer drugs

The purpose of this study, the direct separation of aminoalkanol derivatives I and II of 1,7‐dimethyl‐8,9‐diphenyl‐4‐azatricyclo[5.2.1.0^2,6]dec‐8‐ene‐3,5,10‐trione, which was found in earlier studies as potential anticancer drugs, were performed. Capillary electrophoresis offers the possibility of fast, cheap, and reproducible separations for compounds I and II . In this paper, the simultaneous separation of I and II by capillary zone electrophoresis has been achieved within 8 min by use of 50 mM phosphate buffer of pH 2.5. Analysis of the two compounds in the serum plasma standards was conducted. Limits of detection of I and II by UV absorbance at 200 nm were achieved in the range of 156.3–156.6 ng/mL. The method was validated for linearity, accuracy, precision, limits of detection, and quantification. The calibration equation revealed a good linear relationship (r² = 0.998–0.999). Sufficient recovery was observed in the range of 96.3–99.5%. The method showed good reproducibility with intra‐ and interday precision of 0.97 and 1.76%, respectively. The quantification limits for the compounds were in the range of 477.0–479.8 ng/mL. The proposed method was applied to the analysis of real serum samples.     

Simultaneous determination of DTPA,EDTA, and NTA by capillary electrophoresis after complexation with copper

We present a method for simultaneous determination of the aminopolycarboxylic acids DTPA, EDTA and NTA in dishwashing detergents, paper mill waters, and natural waters by capillary electrophoresis (CE). The complexing agents were examined as their copper(II) complexes and separated by conventional CE with reversed polarity of the applied voltage. The optimum separation conditions were established by varying the pH and phosphate and tetradecyltrimethylammonium bromide (TTAB) concentrations in the run buffer. The separations were carried out in a fused-silica capillary (61 cm×75 m i.d.) filled with phosphate buffer (80 mmol L^–1, TTAB concentration 0.5 mmol L^–1, pH 7.1, voltage –20 kV) using direct UV detection at 191 and 254 nm. With this CE method all the peaks in the electropherograms were properly separated, the calibration plots gave good correlation coefficients and all three complexing agents could be detected in less than 4 min. Linear calibration plots were obtained for CuDTPA, CuEDTA and CuNTA; limits of detection were 0.03 mmol L^–1 for all complexing agents and recoveries for all tested samples were within the range 104±7%. Results obtained from dishwashing detergent samples were found to be reliable and comparable with those from HPLC (R²=0.989) and UV–Vis (R²=0.985) methods.     

High performance separations of nucleic acids using capillary electrophoresis and high-performance liquid chromatography

High resolution separations of nucleic acids have been performed using high performance capillary electrophoresis (HPCE) and high performance liquid chromatography (HPLC). Electropherograms showing HPCE separations of single and double stranded DNA are presented and compared with HPLC separations. Single base resolution of poly(dA) oligonucleotides in the size range of 12 to 60-mers was achieved in 35 min using HPCE. Plate numbers for HPCE are in the hundreds of thousands and reproducibility is about 1–2 % (RSD). In comparison with HPLC separations, the resolution of nucleic acids obtained using HPCE is much better than that using HPLC, while reproducibility of HPCE is comparable with that of HPLC.     

Elimination of the artefact peaks in capillary electrophoresis determination of glutamate by using organic solvents in sample preparation

Focusing on the demand from the food industry for fast and reliable alternative methods to control the quality of food products, we present in this paper a method for amino acid separation and glutamic acid quantification in complex matrices employing capillary electrophoresis with capacitively coupled contactless conductivity detection. We demonstrate by simulation and experimentally the use of organic solvents in sample preparation to prevent peak splitting and increase stacking in capillary electrophoretic separations of amino acids. Additionally, we obtained results for glutamic acid quantification comparable to those obtained via traditional methods used at industrial sites. We tested premium and low‐cost samples with large variations in their glutamic acid content, which demonstrated the wide range of applicability of the method presented herein. The results of the proposed capacitively coupled contactless conductivity detection based capillary electrophoresis method agreed with those obtained by an enzymatic detector and ultra high performance liquid chromatography coupled to tandem mass spectrometry, considering a confidence level of 95%.     

Separation of basic proteins and peptides by capillary electrophoresis using a cationic surfactant

Incorporation of a low concentration of cetyltrimethylammonium bromide (CTAB) in the running electrolyte is shown to dynamically coat the silica capillary and to reverse the direction of electroosmotic flow. The CTAB coating prevented interaction of proteins with the capillary surface and enabled sharp peaks to be obtained in the electropherograms. A systematic study of experimental parameters demonstrated the importance of selecting a suitable buffer electrolyte and an appropriate pH. Excellent separations were obtained for five proteins, three enkephalins, and six dipeptides with an efficiency of approximately 500,000 theoretical plates per meter. The method developed is very simple to perform and was found to give excellent reproducibility.     

Simultaneous determination of six toxic alkaloids in human plasma and urine using capillary zone electrophoresis coupled to time‐of‐flight mass spectrometry

A novel capillary zone electrophoresis separation coupled to electro spray ionization time‐of‐flight mass spectrometry method was developed for the simultaneous analysis of six toxic alkaloids: brucine, strychnine, atropine sulfate, anisodamine hydrobromide, scopolamine hydrobromide and anisodine hydrobromide in human plasma and urine. To obtain optimal sensitivity, a solid‐phase extraction method using Oasis MCX cartridges (1 mL, 30 mg; Waters, USA) for the pretreatment of samples was used. All compounds were separated by capillary zone electrophoresis at 25 kV within 12 min in an uncoated fused‐silica capillary of 75 μm id × 100 cm and were detected by time‐of‐flight mass spectrometry. This method was validated with regard to precision, accuracy, sensitivity, linear range, limit of detection (LOD), and limit of quantification (LOQ). In the plasma and urine samples, the linear calibration curves were obtained over the range of 0.50–100 ng/mL. The LOD and LOQ were 0.2–0.5 ng/mL and 0.5–1.0 ng/mL, respectively. The intra‐ and interday precision was better than 12% and 13%, respectively. Electrophoretic peaks could be identified by mass analysis.     

Generic capillary electrophoresis conditions for chiral assay in early pharmaceutical development

Generic capillary electrophoresis (CE) conditions have been implemented for chiral separations in early pharmaceutical development. The chiral CE separations of several pharmaceutical samples at different stages of development, i.e., discovery, process chemistry, and investigative new drug application, have been obtained using sulfated beta-cyclodextrin (CD). Several sulfated beta-CDs have been screened to select an appropriate enantioselective agent. The use of a generic CE method allows for a convenient and rapid chiral recognition of different weak bases, with minimal or no method development. CE using sulfated beta-CD for the chiral separation of N-benzoyl methyl piperazine has been validated for linearity, precision, accuracy, limits of detection and quantitation (LOD, LOQ). Although less sensitive than a specific liquid chromatography method using a Chiralpak AD column, the overall performance of the chiral CE method was found comparable. Validation data demonstrate that a LOD of 0.1%, sufficient to fulfill regulatory requirements, is achievable by chiral CE.     

Quantification of deformed peaks in capillary gas chromatography (CGC): application to simultaneous analysis of free fatty acids and less polar compounds in aqueous distillery effluent

The purpose of this study was to find a simple and rapid method allowing the simultaneous quantification of some alcoholic fermentation inhibitors present in aqueous distillery effluent in order to evaluate its recycling properties. A capillary gas chromatography (CGC) method was tested for the quantification of both short chain fatty acids (acetic to hexanoic) and neutral compounds (butane 2,3-diol, 2-furaldehyde, phenyl-2-ethane1-ol). A polyvalent column coated with ^®trifluoro-propyl-polysiloxane, allowing water injection, was tested and experiments were performed directly on untreated samples. During the development of the method, a deformation of acid peaks was observed; that could be explained by a secondary equilibrium, added to the chromatographic equilibrium. Although the acid peaks were deformed, calibration curves were produced and rigorously validated, proving that quantification is possible even when the best chromatographic conditions have not been achieved. Eventually, the method enabled the concentration of eight major fermentation inhibitors in distillery effluent to be measured.     

Assay for the simultaneous determination of guanidinoacetic acid, creatinine and creatine in plasma and urine by capillary electrophoresis UV-detection

Guanidinoacetic acid (GAA) measurement has recently become of great interest for the diagnosis of creatine (Cn) metabolism disorders, and research calls for rapid and inexpensive methods for its detection in plasma and urine in order to assess a large number of patients. We propose a new assay for the measurement of GAA by a simple CZE UV-detection without previous sample derivatization. Plasma samples were filtered by Microcon-10 microconcentrators and directly injected into the capillary, while for urine specimens a simple water dilution before injection was needed. A baseline separation was obtained in less than 8 min using a 60.2 cm x 75 microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer pH 2.25 at 15 degrees C. The performance of the developed method was assessed by measuring plasma creatinine and Cn in 32 normal subjects and comparing the data obtained by the new method with those found with the previous CE assay. Our new method seems to be an inexpensive, fast and specific tool to assess a large number of patients both in clinical and in research laboratories.