Improved method for the simultaneous determination of whey proteins, caseins and para-kappa-casein in milk and dairy products by capillary electrophoresis

A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-kappa-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated capillary, in combination with electrophoresis buffer 0.48 M citric acid-13.6 mM citrate-4.8 M urea at pH 2.3, and a separation voltage of 25 kV, a complete separation of beta-lactoglobulin and para-kappa-casein was achieved, permitting the quantification of both components.     

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

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

Séparation niobium-tantale contribution à l'étude de l'électrophorése sur papier

A study has been made of the separation of Nb and Ta on a micro-scale by paper electrophoresis, using oxalic and citric complexes and different buffer solutions.A fairly good separation is obtained by using oxalic complexes and, as buffer solution, a mixture of citric acid and potassium citrate of pH 3.42 and ionic strength 0.4.Different factors influencing the displacement of ions by paper electrophorcsis have been examined. It is shown that, if saturation currents are minimized, the displacement is practically proportional to the time of electrophoresis, and more difficult separations become possible using longer strips.     

Feasibility of nonvolatile buffers in capillary electrophoresis-electrospray ionization-mass spectrometry of proteins

The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5-10 microM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.     

Efficient applications of capillary electrophoresis-tandem mass spectrometry to the analysis of adrenoreceptor antagonist enantiomers using a partial filling technique

Throughout the separation of chiral basic drugs by capillary electrophoresis (CE) with neutral hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral selector, the sensitivity of detection can be improved by using tandem mass spectrometric (MS-MS) detection with a partial filling technique rather than with UV spectrometric detection. Prior to sample injection. the capillary was partly filled with HP-beta-CD dissolved in volatile ammonium formate buffer (pH 4, ionic strength 50 mM). The effects of modifying the HP-beta-CD concentration in the selector zone and the length of the separation zone on the enantioresolution and the signal-to-noise ratio of the pseudo-molecular MH+ ion were investigated. For a given selector zone length, as the concentration of the neutral cyclodextrin increases, the resolution between enantiomers becomes higher (the opposite of the behavior of the signal-to-noise ratio) and then reaches an optimum value. The decrease of the selector zone length lowered the resolution between the enantiomers but increased peak efficiencies and signal-to-noise ratio values. Accordingly, partial capillary filling at 80% (v/v) and 10 mM concentration of HP-beta-CD was selected as a suitable compromise between resolution and sensitivity of MS detection. Limits of detection for each adrenoreceptor antagonist enantiomer were 5 ng/ml (0.02 microM) in CE-MS-MS instead of 150 ng/ml (0.60 microM) in CE-UV, which enhances sensitivity by a factor of 30.     

Nucleoside monophosphates recognition using macrocyclic polyamine bonded phase in capillary electrochromatography

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

Chiral CE of aromatic amino acids by ligand-exchange with zinc(II)-L-lysine complex

A novel method of chiral ligand-exchange CE was developed with either L- or D-lysine (Lys) as a chiral ligand and zinc(II) as a central ion. This type of chiral complexes was explored for the first time to efficiently separate either individual pairs of or mixed aromatic amino acid enantiomers. Using a running buffer of 5 mM ammonium acetate, 100 mM boric acid, 3 mM ZnSO(4) x 7H(2)O and 6 mM L-Lys at pH 7.6, unlabeled D,L-tryptophan, D,L-phenylalanine, and D,L-tyrosine were well separated, giving a chiral resolution of up to 7.09. The best separation was obtained at a Lys-to-zinc ratio of 2:1, zinc concentration of 2-4 mM and running buffer pH 7.6. The buffer pH was determined to have a strong influence on resolution, while buffer composition and concentration impacted on both the resolution and peak shape. Boric acid with some ammonium acetate was an adoptable buffer system, and some additives like ethylene diamine tetraacetic acid capable of destroying the complex should be avoided. Fine-tuning of the chiral resolution and elution order was achieved by regulating the ratio of L-Lys to D-Lys; i.e. the resolution increased from zero to its highest value as the ratio ascended from 1:0 to 1:infinitive, and L-isomers eluted before or after D-isomers in excessive D- or L-Lys, respectively.     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

Determination of trace cationic impurities in butylmethylimidazolium-based ionic liquids: from transient to comprehensive single-capillary counterflow isotachophoresis-zone electrophoresis

Determination of impurities in ionic liquids (ILs) remains a difficult task. In this work, the hyphenation of isotachophoretic (ITP) preconcentration to zone electrophoresis (ZE) has been explored for the trace analysis of the cationic impurities Na(+), Li(+), and methylimidazolium (MI(+)) in butylmethylimidazolium (BMI(+))-based ILs. Simultaneous detection of UV-transparent and UV-absorbing impurities was ensured by a BGE composed of creatinine-acetate buffer. To induce ITP, three different strategies were evaluated: (i) Sample self-stacking ensured by the addition of ammonium acetate (NH(4)Ac) to 25-50-fold diluted IL solution (transient ITP). (ii) Complete ITP-ZE separation performed in a single capillary: ITP was realized in discontinuous electrolytes comprising an 80 mM NH(4)Ac, 40 mM acetic acid, 30 mM alpha-CD, pH 5.05, leading electrolyte (LE) and a 10 mM creatinine, 10 mM acetic acid, pH 4.9, terminating electrolyte (TE). To create the ZE stage, the ITP stack of analytes was moved back toward the capillary inlet by pressure and simultaneously the capillary was filled with the BGE. This protocol made it possible to accommodate a 2.5-times diluted IL sample. (iii) Complete counterflow ITP-ZE with continuous electrokinetic sample supply: the ITP stage was performed in a capillary filled with a 150 mM NH(4)Ac, 75 mM acetic acid, 30 mM alpha-CD, pH 5.0 LE, with 40-times diluted IL at the capillary inlet. BMI(+) from IL acts as the terminating ion. The LODs reached in this latter case were at the 10 and 1 ppb levels for MI(+) and Li(+) in diluted IL matrix, respectively.     

Discontinuous electrolyte systems for improved detection of biologically active amines and acids by capillary electrophoresis with laser-induced native fluorescence detection

On-line concentration and separation of biologically active amines and acids by capillary electrophoresis (CE) in conjunction with laser-induced fluorescence using an Nd:YAG laser at 266 nm under discontinuous conditions is presented. The suitable conditions for simultaneous analysis of amines and acids were: samples were prepared in a solution (pH* 3.1) consisting of 10 mM citric acid, 89% acetonitrile (ACN), and water; a capillary was filled with 1.5 M Tris-borate (TB) buffer (pH 10.0); and the anodic vial contained PTG10 buffer (pH* 9.0) that consists of 50 mM propanoic acid, Tris, 10% glycerol, and water. After injecting a large-volume sample, amines and acids were separately stacked at the front (cathodic side) and back (anodic side) of the acidic sample zone, mainly because of changes in their electrophoretic mobilities as a result of changes in pH, viscosity, and electric field when high voltage was applied. When the sample was injected at 15 kV for 360 s, the concentration limits of detection (LODs) for 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) were 0.27 and 0.31 nM, respectively, which are about 400- and 800-fold sensitivity improvements when compared to those injected at 1 kV for 10 s. For the analysis of amines, samples were prepared in 100 mM citric acid (pH* 1.8) containing 89% ACN and both the capillary and anodic vial were filled with 400 mM PTG20 (propanoic acid, Tris, 20% glycerol, and water) at pH* 4.5. Using a large injection volume (15 kV for 360 s), we achieved concentration LODs of 17 pM and 0.3 nM for tryptamine and epinephrine, which are about 5200- and 14,000-fold sensitivity improvements, respectively, in comparison with those injected at 1 kV for 10 s. The features of simplicity (no sample pretreatment), rapidity (12 min), and sensitivity for identification of amines and acids of interest in urine samples show diagnostic potential of the two approaches developed in this study.     

Determination of adduct forms of antitumor ruthenium(III) complex with cytosolic components by capillary electrophoresis with mass spectrometry

Using capillary electrophoresis with inductively coupled plasma mass spectrometry, the transformation of adduct forms of indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] with apo-transferrin and albumin under the effect of active intracellular reducing agents, glutathione and ascorbic acid, and citric acid as a complexant was studied under conditions simulating a cytosolic environment. These adducts of ruthenium with transport proteins are forms in which the anticancer drug exists after intravenous administration. Two modes of interaction of adducts with glutathione, ascorbic acid, and citric acid were studied: in a capillary using a background electrolyte containing a cytosolic active ingredient and upon incubation of the reaction components in the off-line mode, followed by their separation and determination by capillary electrophoresis. Incubation with intracellular components and separation were carried out in a 10 mM phosphate buffer solution (pH 6.0) containing 4 mM NaCl. The effect 1–10 mM of glutathione, 10 mM of ascorbic acid, and 50 mM of citric acid on the adducts was studied. It is shown that under the selected model conditions, new forms of ruthenium do not emerge.     

Determination of ammonium and metal ions by capillary electrophoresis-potential gradient detection using ionic liquid as background electrolyte and covalent coating reagent

A capillary zone electrophoresis (CZE)-potential gradient detection (PGD) method coupled with field-amplified sample injection was developed to determine alkali metal, alkaline-earth metal, nickel, lead and ammonium ions. The capillary surface was coated with dialkylimidazolium-based ionic liquid and thus the electroosmotic flow (EOF) of the capillary was reversed. The buffer composed of 7.5 mM lactic acid, 0.6 mM 18-crown-6, 12 mM alpha-cyclodextrin (alpha-CD); it was adjusted to pH 4.0 by 1-hexyl-3-methylimidazolium hydroxide. The 11 cations were baseline separated within 14 min with 5.1-18.9 x 10(4) plates (for 40-cm-long capillary) in separation efficiency, and the detection limits were in the range of 0.27-7.3 ng/ml. The method showed good reproducibility in terms of migration time with RSD < or = 0.90% for run-to-run and < or = 1.65 for day-to-day assessment.     

Separation and determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation by capillary zone electrophoresis

A capillary zone electrophoresis method was developed for the separation and determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation. The factors that could affect the separation were studied, such as the types and concentrations of electrolytes, pH, ionic strength and organic modifier. The optimum running buffer was 20 mmol/L of ammonium acetate containing 0.2 mol/L of glacial acetic acid (pH 3.64). The applied voltage was 25 kV and the wavelength of the UV detector was set at 214 nm. The established method with dopamine hydrochloride as internal standard was linear in the range of 5-100 microg/mL for both strychnine and brucine. The recovery was 102.96% for strychnine and 98.56% for brucine. The extracts of Strychnos nux-vomica and its preparation could be directly injected for analysis.     

Capillary electrophoretic method for nucleotide analysis in cells: application on inherited metabolic disorders

Purine and pyrimidine nucleotides influence many metabolic pathways and their analogs have been widely used in medicine. A capillary electrophoretic method was developed for measuring intracellular nucleotides. The final BGE consisted of 40 mM citric acid with addition of 0.8 mM CTAB titrated by gamma-aminobutyric acid to pH 4.4. The electrophoretic separations were carried out in an uncoated silica capillary (id/od - 75/375 microm; effective/total length - 90/97 cm). The method allows a complete separation of 21 nucleotides and deoxynucleotides within 15 min with separation efficiencies up to 400,000 theoretical plates per meter. Due to the use of an acidic separation medium, the method offers a high selectivity toward the studied analytes versus possible interferences from matrices. Sample preparation was optimized in order to shorten work-time and prevent analyte degradation. The method was applied for analyzing nucleotides in human erythrocytes and Chinese hamster ovary cells. Diagnostic potential for inherited metabolic disorders of nucleotide metabolism is presented.     

Modeling the adsorption of Cd(II) onto goethite in the presence of citric acid

The adsorption of cadmium onto goethite in the presence of citric acid was measured as a function of pH and cadmium concentration at 25 degrees C. Potentiometric titrations were also performed on the system. Cadmium adsorption onto goethite was enhanced above pH 4 in the presence of 50 microM, 100 microM and 1 mM citric acid. While there was little difference between the enhancements caused by 50 and 100 microM citric acid below pH 6, above pH 6 further enhancement is seen in the presence of 100 microM citric acid. When 1 mM citric acid was present, the enhancement of cadmium adsorption was greater below pH 6, with increased Cd(II) adsorption down to pH 3.5. However, above pH 6, 1 mM of citric acid caused slightly less enhancement than the lower citric acid concentrations. ATR-FTIR spectra of soluble and adsorbed citrate-cadmium species were measured as a function of pH. At pH 4.6 there was very little difference between the ternary Cd(II)-citric acid-goethite spectrum and the binary citric acid-goethite spectrum. However, spectra of the ternary system at pH 7.0 and 8.7 indicated the presence of additional surface species. Further analysis of the spectra suggested that these were metal-ligand outer-sphere complexes. Data from the adsorption experiments and potentiometric titrations of the ternary Cd(II)-citric acid-goethite system were fitted by an extended constant-capacitance surface complexation model. The spectroscopic data were used to inform the choice of surface species. Three reactions in addition to those for the binary Cd(II)-goethite and citric acid-goethite systems were required to describe all of the data. They were [formula in text], [formula in text], and [formula in text]. Neither the spectroscopy nor the modeling suggested the formation of a ternary inner-sphere complex or a surface precipitate under the conditions used in this study.     

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

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

Two fluoroalcohols as components of basic buffers for liquid chromatography electrospray ionization mass spectrometric determination of antibiotic residues

Two fluoroalcohols--1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFTB)--were evaluated for the first time as volatile buffer acids in the basic mobile phase for reversed-phase chromatography with electrospray ionization-mass spectrometric (LC-ESI-MS) detection of five antibiotics. Chromatographic separation as well as positive and negative ion ESI-MS intensities using these novel buffer components were compared to traditional buffer systems. Overall, the highest signal intensities and best chromatographic separation for the five antibiotics (ciprofloxacin, norfloxacin, ofloxacin, sulfadimethoxine and sulfamethoxazole) were achieved using 5 mM HFIP as the buffer acid to methanol : water mobile phase (pH of the aqueous component adjusted to 9.0 with ammonium hydroxide). Comparable results were achieved using 5 mM HFTB (pH adjusted to 9.0 with ammonium hydroxide). The suitability of HFIP for analysis of antibiotic residues in lettuce is demonstrated.     

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.     

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.     

Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction

A capillary zone electrophoresis (CZE) method for separation of adenosine and N⁶-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20 min with high theoretical plate number (up to 400 000 for isopentenyladenosine triphosphate) was achieved using a 100 mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69–1.27 μmol L⁻¹). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R² > 0.999) was achieved over the concentration range 5–1000 μmol L⁻¹. The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE–ESI-MS using a volatile buffer (30 mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products – isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction.