Capillary electrophoresis method for the analysis of inorganic anions, organic acids, amino acids, nucleotides, carbohydrates and other anionic compounds.

A previously developed capillary zone electrophoresis (CZE) method with indirect UV detection for the simultaneous determination of inorganic and organic anions, amino acids and carbohydrates using 20 mM 2,6-pyridinedicarboxylic acid (PDC) as the background electrolyte was extended to allow determination of 206 anions including those above--mentioned and physiological amino acids, nucleotides, aromatic acids, haloacetic acids, alcohols, phosphorylated saccharides, oxyhalides, metal oxoacids, metal-ethylenediaminetetraacetic acid (EDTA) complexes, forensic anions, Good's buffers and herbicides. Every compound could be analyzed and their electrophoretic mobility determined simply by selecting detection wavelength. This method is simple and universal for anion analysis, and could be readily applied to the simultaneous determination of anionic compounds. In this work, it was used to identify and quantify important anions in sea urchin and sake.     

Direct multicomponent analysis of beer samples constituents using micellar electrokinetic capillary chromatography

A capillary electrophoretic method was developed using micellar electrokinetic capillary chromatography (MEKC) with diode-array detection to analyze simultaneously 26 beer constituents in a single procedure, including alcohols, iso-alpha-acids, amino acids, flavonoids, isoflavonoids, a vitamin, purine and pyrimidine bases. After filtration, sample components were separated with an uncoated capillary and a 25 mM sodium borate and 110 mM SDS buffer at pH 10.5. Analyses were run at 14 kV and 8 s of hydrodynamic injection with UV detection at 210 nm and 270 nm. The proposed method was successfully applied to the direct determination of beer constituents without any sample cleanup procedures.     

Resolution of overlapped peaks of amino acid derivatives in capillary electrophoresis using multivariate curve resolution based on alternating least squares

The application of chemometric techniques to the resolution of overlapped peaks in capillary electrophoresis (CE) is described. When a physical separation can not be completely accomplished, chemometrics might still resolve the determination of the analytes mathematically. CE with diode array detection can provide a large amount of data consisting of spectra registered over time. In this study, the capillary electrophoretic separation of 1,2-naphthoquinone-4-sulfonate derivatives of amino acids is studied. Most of the common amino acid derivatives can be separated at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate + isopropanol (3:1 v/v) solution as background electrolyte. However, peaks of certain derivatives (Phe, His, Leu and Ile) still overlap. A multivariate curve resolution method based on an alternating least squares optimization procedure is used for the resolution of the overlapped electrophoretic peaks. The method takes advantage of spectral and electrophoretic differences of analytes to recover their pure electrophoretic and spectral profiles. In addition, each analyte in the mixture can be quantified using the corresponding standards.     

Continuous flow derivatization system coupled to capillary electrophoresis for the determination of amino acids

A derivatization system coupled to capillary electrophoresis for the determination of amino acids using 1,2-naphthoquinone-4-sulfonate as a labeling agent is described. In this system, amino acids are derivatized on-line in a three-channel flow manifold for sample, reagent and buffer solutions. The reaction takes place in a PTFE coil heated at 80 degrees C. The resulting solution, which contains the amino acid derivatives, is introduced into the electrophoretic system by means of an appropriate interface. Subsequently, amino acid derivatives are separated at 25 kV using a 40 mM sodium tetraborate aqueous solution with 30% (v/v) isopropanol solution as a running buffer. The electropherograms are monitored spectrophotometrically at 230 nm. The method has been applied to the determination of amino acids in feed samples and pharmaceutical preparations. A good concordance of the predicted values with those given by a standard amino acid analyzer is shown.     

Separation of twenty underivatized essential amino acids by capillary zone electrophoresis with contactless conductivity detection

Twenty underivatized essential amino acids were separated using capillary zone electrophoresis and consequently detected with contactless conductivity detection (CCD). A simple acidic background electrolyte (BGE) containing 2.3 M acetic acid and 0.1% w/w hydroxyethylcellulose (HEC) allowed the electrophoretic separation and sensitive detection of all 20 essential amino acids in their underivatized cationic form. The addition of HEC to the BGE suppressed both, electroosmotic flow and analyte adsorption on the capillary surface resulting in an excellent migration time reproducibility and a very good analyte peak symmetry. Additionally, the HEC addition significantly reduced the noise and long-term fluctuations of the CCD baseline. The optimized electrophoretic separation method together with the CCD was proved to be a powerful technique for determination of amino acid profiles in various natural samples, like beer, yeast, urine, saliva, and herb extracts.     

Determination of folic acid by capillary electrophoresis with chemiluminescence detection

A rapid and simple method is presented for the determination of folic acid (FA) by capillary electrophoresis (CE) with chemiluminescence (CL) detection. This method was based on enhance effect of FA on the CL reaction between luminol and BrO(-) in alkaline aqueous solution. Optimal separation and determination was obtained with an electrophoretic buffer of 35 mM sodium borate (pH 9.4) containing 0.8 mM luminol, and an oxidizer solution of 1.6 mM NaBrO in 100 mM NaCO(3) buffer solution (pH 12.0). Under the optimal conditions, the determination of FA was achieved in less than 20 min, and the detection limit was 2.0 x 10(-8) M (S/N=3). The relative standard deviations (RSDs) on peak area and migration time were in the 1.5 and 1.1%, respectively. The present CE-CL method was applied to the determination of FA in commercial pharmaceutical tablets, apple juices and human urine.     

Separation of phenolic acids in soil and plant tissue extracts by co-electroosmotic capillary electrophoresis with direct UV detection

Summary A capillary zone electrophoretic method for the analysis of phenolic acids in soil and plant extracts was developed with direct UV detection using a phosphate electrolyte solution. The electrophoretic separation required the phenolic acids to be charged at a pH above their pKa in order to achieve their migration towards the anode. Electroosmotic flow (EOF) was reversed in direction by adding tetradecyltrimethylammonium bromide (TTAB). Factors affecting the separation selectivity, including the buffer pH and EOF modifiers, were investigated systematically. Eight phenolic acids were separated and detected in 10 min using an electrolyte containing 25 mM phosphate, 0.5 mM TTAB and 15% acetonitrile (v/v) at pH of 7.20. Linear plots for the test phenolic acids were obtained in a concentration range of 0.01–1 mM with detection limits in the range of 1.0–7.0 μM. The recoveries ranged from 92.8 to 102.3% in soil and plant tissues samples spiked at 100 μM and the relative standard deviation based on the peak area were ranged 2.0 to 4.5%. The proposed method was used for the determination of phenolic acids in plant tissue and soil extracts with direct injection.     

Simultaneous determination of catecholamines and amino acids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A selective and sensitive method was developed for separation and simultaneous determination of catecholamines and amino acids by MEKC with LIF. Interestingly enough, such work has been firstly performed on catecholamines derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole and the detailed derivatization mechanism was discussed. After derivatization at 60 degrees C for 20 min, NBD-labeled catecholamines and amino acids were separated in a buffer system containing 10 mM sodium tetraborate-Na2HPO4, 20 mM SDS, and 10% v/v ACN at pH 9.75. SDS micelles were employed to improve the fluorescence intensity of catecholamine derivatives efficiently. Under optimum conditions, two catecholamines and 11 amino acids were separated in a short 13 min analysis time and the RSDs for migration time and peak area were less than 0.60 and 6.50%, respectively. The method was successfully applied for the quantification of catecholamines and amino acids in Portulaca oleracea L., human urine sample, and mixed injection sample.     

Enantioseparation of anionic analytes by non-aqueous capillary electrophoresis using quinine and quinidine derivatives as chiral counter-ions

A non-aqueous capillary electrophoretic method developed for the enantioseparation of N-protected amino acids has been applied to the investigation of five new quinine and quinidine derivatives as chiral selectors: 1-adamantyl carbamoylated quinine, 3,4-dichlorophenyl carbamoylated quinidine, allyl carbamoylated dihydroquinine, allyl carbamoylated dihydroquinidine and 1-methyl quininium iodide. The composition of the background electrolyte was 12.5 mM ammonia, 100 mM octanoic acid in an ethanol-methanol (60:40 v/v) mixture containing a 10 mM concentration of the chiral selector. Under these conditions, the enantioseparation of a series of various benzoyl, 3,5-dinitrobenzoyl and 3,5-dinitrobenzyloxycarbonyl amino acid derivatives was studied with respect to selectand-selector relationship and enantioselectivity.     

Analysis of amino acids by capillary zone electrophoresis with electrochemical detection

The precapillary derivatization of 20 amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) and CN(-) was investigated. All these derivatized amino acids could be oxidized on the carbon fiber microdisk bundle electrode except proline. Capillary zone electrophoresis with electrochemical detection was employed for the analysis of 19 amino acids. The optimum conditions of separation and detection were borate, pH 9.48, for the electrolyte, 18 kV for the separation voltage and 1.15 V versus a saturated calomel electrode for the detection potential. Limits of detection of concentration or mass for individual amino acids were between 1.7 x 10(-7) and 1.8 x 10(-6) mol/L or 84 and 893 amol (according to the signal-to-noise ratio of 3) for the injection voltage of 6 kV and injection time of 10 s. The relative standard deviations were between 0.80 and 2.3% for the migration times and 1.4 and 6.4% for the electrophoretic peak currents. From a mixture of 19 amino acids, 10 amino acids (Arg, Lys, Orn, Try, Ser, Ala, Gly, Cys, Glu, Asp) could be well separated. The other 9 amino acids appeared on three electrophoretic peaks. From the samples, in which the nine amino acids do not exist simultaneously, some of them could also be detected. The method was applied to the determination of amino acids in beer by the standard addition method. The recovery for the amino acids in beer was 91-109%.     

Determination of five priority haloacetic acids by capillary electrophoresis with contactless conductivity detection and solid phase extraction preconcentration

A sensitive capillary electrophoretic separation method with contactless conductivity detection (C4D) for analysis of five priority haloacetic acids (HAA5) is presented. The analytes were baseline separated in an electrolyte composed of 20 mM 2-(N-Morpholino) ethanesulfonic acid (MES), 20 mM L-histidine (HIS), and 30 μM cetyltrimethylammonium bromide (CTAB) at pH 6.0 in less than 4 min. A simplified solid-phase extraction (SPE) preconcentration procedure on highly cross-linked polystyrene-divinylbenzene (PS-DVB) type sorbent was developed and optimized with respect to short preconcentration time. HAA5 from a 25-mL sample aliquot of tap and swimming pool water could be preconcentrated in less than 5 min using an in-house made SPE column with recoveries ranging from 23 to 98%. Combining the SPE preconcentration procedure with capillary electrophoretic analysis, the attained limits of detection were between 6.1 and 12.2 μg/L with total analysis time of less than 10 min.     

Separation of dansylated amino acid enantiomers by chiral ligand-exchange CE with a zinc(II) L-arginine complex as the selecting system

A facile chiral ligand-exchange capillary electrophoretic method has been explored for the enantioseparation and UV detection of dansyl-amino acids with Zn(II) L-arginine complex as a chiral selecting system. Successful enantioseparation of 17 pairs of amino acid enantiomers has been achieved with a buffer of 100 mM boric acid, 5 mM ammonium acetate, 3 mM ZnSO4 and 6 mM L-Arg at pH 8.0, of which 10 pairs were fully resolved with resolution in between 1.59 and 4.21. This new method was shown to be applicable to the separation of some mixed pairs of amino acids and to the quantitative analysis of some real samples such as rice vinegars, with a linear range between 0.8 and 150 microg/mL, correlation coefficient above 0.99 and recovery in between 90.1 and 112.4%. It was found that amino acids with low resistance side chain(s), low tendency to form intramolecular hydrogen bond or high tendency to form intermolecular hydrogen bonds are more easily enantioseparated than those with extra carboxyl and/or phenyl groups. By the use of the suggested buffer, the running pH should be selected at 7.4-9.0 to compromise the resolution and elution speed.     

毛细管电泳-间接紫外检测法测定蜂蜜中的氨基酸

采用毛细管电泳-间接紫外检测法同时分离测定蜂蜜中的赖氨酸、色氨酸、谷氨酸等9种氨基酸。考察了磷酸浓度、进样方式和缓冲液pH对分离效率和重现性的影响。在分离电压为-15 kV、检测波长为220 nm条件下,以含有0.5 mmol/L十六烷基三甲基溴化铵、20 mmol/L烟酸、10%甲醇的10 mmol/L磷酸二氢钠缓冲溶液(pH 10.2)为运行缓冲液,9种组分在11 min内达到基线分离;检出限最低可达到0.3 mg/L;线性范围为1.0~1000 mg/L;日间及日内精密度为0.64%~5.83%。实际样品中除甲硫氨酸外的8种氨基酸的加标回收率为60.00%~118.37%。将该方法应用于不同蜜源植物和产地的蜂蜜样品的测定,在市售的5种蜂蜜中均检测到脯氨酸、丝氨酸和天冬氨酸,而只在荔枝蜜中检测到苏氨酸。该方法可以为蜂蜜的蜜源鉴别及质量评估提供借鉴方法。     

Determination of amino acids in urine by cyclodextrin-modified capillary electrophoresis-laser-induced fluorescence detection

Capillary electrophoresis (CE) combined with laser-induced fluorescence detection is applied to the determination of amino acids in urine samples. The urine samples are first ultrafiltered, to remove proteins and large peptides, and the filtrates are then directly labeled by reaction with fluorescein isothiocyanate (FITC). Cyclodextrin-modified CE using alpha-cyclodextrin is employed for the separation of the FITC-labeled amino acids. Seven amino acids are clearly separated from side reaction products produced during the labeling reaction, when an 80mM borate buffer containing 45mM alpha-cyclodextrin is used as the running buffer. For quantitative analysis, rhodamine B is added to the labeled urine samples as an internal standard. The calibration curves for phenylalanine, glutamine, proline, glycine, serine, alanine, and valine are linear in the range of 10microM to 100microM. The concentration limits of detection for all of the amino acids are estimated to be 160~330nM. Conversely, the limit of quantitation (LOQ) was ~10microM and the limitations are due to the labeling efficiency rather than the sensitivity of the detector. Three amino acids in urine samples, glutamine, glycine, and alanine, are readily quantitated, while the concentrations of the others are below the LOQ. The present method would permit the determination of seven amino acids in urine successfully.     

Use of nucleic acids in the mobile phase for the determination of ascorbic acid in foods by high-performance liquid chromatography with electrochemical detection

The sodium salts of amino acids, nucleic acids and organic acids were examined in a new mobile phase for the determination of ascorbic acid (AA) in foods. It was possible to use disodium guanosine-5'-monophosphate (GMP) (20 mM GMP, pH 2.1) in a new mobile phase after comparison of five mobile phases. The proposed method is simple, rapid (analysis time: ca. 6 min), sensitive (detection limit: ca. 0.1 ng per injection (5 microl) at a signal-to-noise ratio of 3), highly selective and reproducible [relative standard deviation: ca. 2.7% (n=7)]. The calibration graph of AA was linear in the range of 0.1 to 50 ng per injection (5 microl). Recovery of AA was over 90% by the standard addition method.     

乘子法在多组分光度分析中的应用

报道了将乘子法用于样品中多组分的分光光度法同时测定;介绍了乘子法的原理和计算步骤。对复方新诺明模拟样中磺胺甲恶唑、甲氧苄氨嘧啶测定的平均回收率分别为９９．７９％,１００．６７％,ＲＳＤ分别为０．４５％．０．９５％（ｎ＝９）,测定了５个批号复方新诺明片剂和针剂的含量,结果满意。     

Simultaneous Determination of Imidazole Amino Acids, Aromatic Amino Acids, and Creatinine by Dual-Mode Gradient HPLC Using a Low-Capacity Sulfo-Functionalized Poly(Ethylstyrene-Divinylbenzene) Resin Column

This paper presents a low-capacity cation-exchange chromatography method for the analysis of UV-absorbing dipeptides and amino acids. A newly marketed low-capacity cation-exchange column packed with sulfo-functionalized highly cross-linked macroreticular poly(ethylstyrene-divinylbenzene) copolymer was used for the simultaneous determination of imidazole amino acids, aromatic amino acids, and creatinine in urine samples. A dual-mode binary gradient chromatography method was established using two solvents, A: 15 mM H₃PO₄/5 mM ethylenediamine and B: 15 mM H₃PO₄/5 mM ethylenediamine/40 (v/v) % CH₃CN at 40 °C, with an optimized time program for changing the delivery ratio of A/B and the flow rate. Good chromatograms were obtained within an acceptable cycle time of 25 min. The quantification data were satisfactory for all analytes, showing the relative standard deviations (RSD) of retention times between 0.08 and 1.68 %; RSDs of area intensities between 0.23 and 2.60 %; and linear regression lines with r ² more than 0.9994. The method could determine the creatinine ratios of the diagnostic markers on the single chromatographic run, which enabled to discriminate disease from health. For example, the creatinine ratios for phenylketonuria were significantly higher than those for controls. The method can provide highly cost-efficient information or useful knowledge for clinical and pharmaceutical studies.     

Capillary electrophoresis of some free fatty acids using partially aqueous electrolyte systems and indirect UV detection. Application to the analysis of oleic and linoleic acids in peanut breeding lines

This study has shown for the first time the suitability of CE with a partially aqueous electrolyte system for the analysis of free fatty acids (FFAs) in small portions of single peanut seeds. The partially aqueous electrolyte system consisted of 40 mM Tris, 2.5 mM adenosine-5'-monophosphate (AMP) and 7 mM alpha-CD in (N-methylformamide) NMF/dioxane/water (5:3:2 by volume) mixture, pH 8-9. While AMP served as the background UV absorber for indirect UV detection of the FFAs, the alpha-CD functioned as the selectivity modulator by affecting the relative effective electrophoretic mobilities of the various FFAs due to their differential association with alpha-CD. This CE method allowed the screening of peanut seeds for their content of oleic and linoleic acids, which is essential in breeding of peanuts of high-oleic acid content. The extraction method of FFAs from peanut seeds is very reproducible with a high recovery approaching quantitative yield (approximately 97% recovery).     

Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method for the simultaneous determination of l‐valine,l‐leucine,l‐isoleucine,l‐phenylalanine,and l‐tyrosine in human serum

l ‐Valine, l ‐leucine, l ‐isoleucine, l ‐phenylalanine, and l ‐tyrosine are important proposed biomarkers for the early detection and diagnosis of type 2 diabetes. A simple and selective hydrophilic interaction chromatography with tandem mass spectrometry method was developed for the simultaneous determination of these amino acids in human serum, using stable isotope‐labeled amino acids as internal standards. Chromatographic separation was carried out on a Syncronis HILIC column (150 mm × 2.1 mm, 5 μm) with the column temperature of 35°C and a mobile phase consisted of acetonitrile/120 mM ammonium acetate (89:11, v/v), and the run time was 11.0 min. The mass spectrometric analysis was performed using a QTRAP 5500 mass spectrometer coupled with an electrospray ionization source in positive ion mode. As these five amino acids are endogenous compounds in serum, we used the corresponding stable isotope‐labeled amino acids to evaluate the matrix effect and recovery in serum. The matrix effect was 98.7–107.3%, and the recovery was 92.7–102.3%. Calibration curves spiked unlabeled amino acids in water were linear over the range of 0.200–100 μg/mL. The accuracy, inter‐, and intraday precision were below 10.2%. Analytes were stable during the study. This assay method has been validated and applied to the early diagnosis research of type 2 diabetes.     

Characterization of paromomycin sulfate by capillary electrophoresis with UV detection after pre-capillary derivatization

Summary A capillary electrophoretic method has been developed for determination of the components of the paromomycin mixture salt. Paromomycin was detected at 330 nm after pre-capillary derivatization witho-phthaldialdehyde and thioglycolic acid. The electrophoretic separation was performed in a fused-silica capillary at 25°C and 18kV with a background electrolyte comprising 40mm sodium tetraborate, 3mm β-cyclodextrin, and 12.5% (v/v) methanol. Although the analysis time was reduced to 10min, five peaks could be separated to baseline. The relative standard deviation of the ratios (peak area/internal standard peak area) was <5% for all peaks.