Hydrophilic interaction liquid chromatography tandem mass spectrometry analysis of malonyl‐coenzyme A in breast cancer cell cultures applying online solid‐phase extraction

Cofactors such as coenzyme A and its derivatives acetyl‐coenzyme A and malonyl‐coenzyme A are involved in many metabolic pathways. Due to trace level concentrations in biological samples and the high reactivity of cofactors, a fast, sensitive, and selective method for quantification is mandatory. In this study, online solid‐phase extraction was coupled successfully to hydrophilic interaction liquid chromatography with tandem mass spectrometry for isolation of analytes in complex matrix and quantification by external calibration. Online solid‐phase extraction was carried out by application of a weak anion‐exchange column, whereas hydrophilic interaction liquid chromatography separation was performed on an amide modified stationary phase. Sample preparation of the extracts before the analysis was reduced to a centrifugation and dilution step. Moreover, the applied online solid‐phase extraction significantly reduced matrix effects and increased the signal‐to‐noise ratio. The limit of detection and the limit of quantification were in the lower nanomolar range. Finally, the applicability of this method was demonstrated on MCF‐7 breast cancer cell cultures, a commonly used model system, where acetyl‐coenzyme A and malonyl‐coenzyme A were determined using standard addition procedure in concentrations of 1.98 μM and 41 nM, respectively.     

Novel adsorptive materials by adenosine 5ʹ‐triphosphate imprinted‐polymer over the surface of polystyrene nanospheres for selective separation of adenosine 5ʹ‐triphosphate biomarker from urine

In this study, we have developed a method to assess adenosine 5?‐triphosphate by adsorptive extraction using surface adenosine 5′‐triphosphate‐imprinted polymer over polystyrene nanoparticles (412 ± 16 nm) for selective recognition/separation from urine. Molecularly imprinted polymer was synthesized by emulsion copolymerization reaction using adenosine 5′‐triphosphate as a template, functional monomers (methacrylic acid, N‐isopropyl acrylamide, and dimethylamino ethylmethacrylate) and a crosslinker, methylenebisacrylamide. The binding capacities of imprinted and non‐imprinted polymers were measured using high‐performance liquid chromatography with UV detection with a detection limit of 1.6 ± 0.02 µM of adenosine 5′‐triphosphate in the urine. High binding affinity (Q_MIP, 42.65 µmol/g), and high selectivity and specificity to adenosine 5′‐triphosphate compared to other competitive nucleotides including adenosine 5?‐diphosphate, adenosine 5?‐monophosphate, and analogs such as adenosine, adenine, uridine, uric acid, and creatinine were observed. The imprinting efficiency of imprinted polymer is 2.11 for urine (Q_MIP, 100.3 µmol/g) and 2.51 for synthetic urine (Q_MIP, 48.5 µmol/g). The extraction protocol was successfully applied to the direct extraction of adenosine 5′‐triphosphate from spiked human urine indicating that this synthesized molecularly imprinted polymer allowed adenosine 5′‐triphosphate to be preconcentrated while simultaneously interfering compounds were removed from the matrix. These submicron imprinted polymers over nano polystyrene spheres have a potential in the pharmaceutical industries and clinical analysis applications.     

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

CE was used for the first time to study the two‐substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine‐5′‐triphosphate were in‐capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine‐5′‐triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero‐potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine‐5′‐diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose‐1,6‐bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in‐capillary mixed using an electrophoretically mediated microanalysis approach.     

Electrophoretic measurement of water charge density and ion hydration

Water exchange between bulk water and water-ion complexes will be at equilibrium when the charge density of the complex surface equals the charge density of bulk water, producing a constant radius water-ion complex. This complex will migrate in an electric field at a velocity proportional to the complex radius. CE velocity is the sum of the complex charge-dependent velocity and the buffer electro-osmotic flow. Simultaneous use of both a base (1.07 mM imidazole) and an acid (1.5 mM MOPS) buffer negates EOF at pH 7.4. Electric fields below 300 V/cm (potassium, calcium) and 400 V/cm (magnesium) yield migration velocities with no dehydration of the water-ion complexes. The number of waters per complex increase with the ion charge density: K⁺ 1.90, Ca⁺⁺ 5.90, Mg⁺⁺ 6.59 waters/ion. The charge densities of the complexes are similar: K⁺ 1.24, Ca⁺⁺ 1.43, Mg⁺⁺ 1.21 e/nm², for an average bulk water charge density of 1.29 ± 0.11 (SD) e/nm². The addition of 0.1% Triton increases the number of waters for Mg⁺⁺ to 25.33 and lowers the charge density to 0.497 e/nm². High electric field dehydration shows that calcium will be fully dehydrated at 638.3 V/cm and magnesium fully dehydrated at 925.5 V/cm, which occur at 6.15 and 5.78 nm from the membrane. Dehydrated magnesium will then bind to calcium channels leading to decreased smooth muscle activation.     

Development of a silica monolith modified with Fe3O4 nano‐particles in centrifugal spin column format for the extraction of phosphorylated compounds

In this study, citrate‐stabilised iron oxide nano‐particles (～16 nm) have been immobilised on commercial silica monolithic centrifugal spin columns (MonoSpin) for the extraction of phosphorylated compounds. Two alternative strategies were adopted involving either direct electrostatic attachment to an aminated MonoSpin (single‐layer method) in the first instance, or the use of a layer‐by‐layer method with poly(diallyldimethylammonium) chloride. Field‐emission scanning electron spectroscopy and energy‐dispersive X‐ray spectroscopy was used for confirming notably higher coverage of nano‐particles using the layer‐by‐layer method (2.49 ± 0.53 wt%) compared with the single‐layer method (0.43 ± 0.30 wt%). The modified monolith was used for the selective separation/extraction of adenosine monophosphate, adenosine diphosphate and adenosine triphosphate with elution using a phosphate buffer. A reversed‐phase liquid chromatographic assay was used for confirming that adenosine, as a non‐phosphorylated control was not retained on the modified MonoSpin devices, whereas recovery of 80% for adenosine monophosphate, 86% for adenosine diphosphate and 82% for adenosine triphosphate was achieved.     

Capillary ion electrophoresis-capacitively coupled contactless conductivity detection of inorganic cations in human saliva on a polyvinyl alcohol-coated capillary

Capillary ion electrophoresis–capacitively coupled contactless conductivity detection (CIE-C4D) with a polyvinyl alcohol chemically coated capillary (PVA capillary) was used to analyze inorganic cations (Na⁺, K⁺, NH₄⁺, Mg²⁺, and Ca²⁺) commonly found in human saliva. The PVA capillary, which was made by our laboratory, minimized electro-osmotic flow in the wide pH range of the background electrolyte (BGE), and the PVA layer adsorbed to capillary wall did not affect the conductimetric background level. In this study, we determined an optimized BGE of 30 mM lactic acid/histidine plus 3 mM 18-crown-6 for the CIE-C4D system using the PVA capillary, which could simultaneously improve the separation of Mg²⁺ and Ca²⁺ from Na⁺ and that of K⁺ from NH₄⁺. This system obtained highly reproducible separation of cations in human saliva samples within 8 min at 20 kV without deprotonation. The quantifiability of cations in human saliva samples on the CIE-C4D system was demonstrated through identification by ion chromatography with satisfactory results.     

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.     

Ultra‐fast adenosine 5′‐triphosphate,adenosine 5′‐diphosphate and adenosine 5′‐monophosphate detection by pressure‐assisted capillary electrophoresis UV detection

Herein, we report a new CE method to measure adenine nucleotides adenosine 5′‐triphosphate, adenosine 5′‐diphosphate, and adenosine 5′‐monophosphate in red blood cells. For this purpose, 20 mmol/L sodium acetate buffer at pH 3.80 was used as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of 25 kV and an overimposed pressure of 0.2 psi from inlet to outlet. A rapid separation of these analytes in less than 1.5 min was obtained with a good reproducibility for intra‐ and inter‐assay (CV<4 and 8%, respectively) and an excellent analytical recovery (from 98.3 to 99%). The applicability of our method was proved by measuring adenine nucleotides in red blood cells.     

Rapid and cost‐effective method for the simultaneous quantification of seven alkaloids in Corydalis decumbens by microwave‐assisted extraction and capillary electrophoresis

A rapid and cost‐effective method based on microwave‐assisted extraction followed by capillary electrophoresis was developed for simultaneous quantification of seven alkaloids in Corydalis decumbens for the first time. The main parameters affecting microwave‐assisted extraction and capillary electrophoresis separation were investigated and optimized. The optimal microwave‐assisted extraction was performed at 40°C for 5 min using methanol/water (90:10, v/v) as the extracting solvent. Electrophoretic separation was achieved within 15 min using an uncoated fused‐silica capillary (50 μm internal diameter and 27.7 cm effective length) and a 500 mM Tris buffer containing 45% v/v methanol (titrated to pH^* 2.86 with H₃PO₄). The developed method was successfully applied to the quantification of seven alkaloids in Corydalis decumbens obtained from different regions of China. The combination of microwave‐assisted extraction with capillary electrophoresis was an effective method for the rapid analysis of the alkaloids in Corydalis decumbens .     

Determination of creatine and phosphocreatine in muscle biopsy samples by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis method with contactless conductivity detection was evaluated as a new approach for quantification of creatine and phosphocreatine in human quadriceps femoris biopsy samples. The running buffers employed consisted of 1 M acetic acid at a pH of 2.3 for the determination of creatine and 50 mM 3-(N-morpholino)propanesulfonic acid/30 mM histidine at a pH of 6.4 for the determination of phosphocreatine in the centrifuged muscle extracts. The limits of detection for creatine and phosphocreatine were found to be 2.5 and 1.0 μM, respectively. Creatine and phosphocreatine were determined in six human muscle biopsy samples and the results were found comparable to those of a standard enzymatic assay. The procedures developed for creatine and phosphocreatine also allow the determination of creatinine as well as adenosine diphosphate and adenosine triphosphate.     

Nuclear magnetic resonance to study the interactions acting in the enantiomeric separation of homocysteine by capillary electrophoresis with a dual system of γ‐cyclodextrin and the chiral ionic liquid EtCholNTf2

The enantiomeric separation of 9‐fluorenylmethoxycarbonyl chloride (FMOC)‐homocysteine (Hcy) by CE was investigated using γ‐CD and the chiral ionic liquid (R)‐(1‐hydroxybutan‐2‐yl)(trimethyl)azanium‐bis(trifluoromethanesulfon)imidate (also called (R)‐N,N,N‐trimethyl‐2‐aminobutanol‐bis(trifluoromethane‐sulfon)imidate) (EtCholNTf₂) as chiral selectors. Using 2 mM γ‐CD and 5 mM EtCholNTf₂ in 50 mM borate buffer (pH 9), FMOC‐Hcy enantiomers were separated with a resolution value of 3.8. A reversal in the enantiomer migration order in comparison with the single use of γ‐CD in the separation buffer was obtained. Then, NMR experiments were carried out to elucidate the interactions taking place in the enantiomeric separation of FMOC‐Hcy. NMR analyses highlighted the formation of an inclusion complex since the hydrophobic group of FMOC‐Hcy was inserted into the γ‐CD cavity. Moreover, interactions between EtCholNTf₂ and γ‐CD were also observed, suggesting that the chiral ionic liquid would also enter the cavity of the γ‐CD.     

Use of Ionic liquids as additives in ion exchange chromatography for the analysis of cations

The behavior of acids (citric acid, nitric acid, oxalic acid, tartaric acid) as a mobile phase and imidazolium ionic liquids (the bromides, tetrafluoroborates and hexafluorophosphates of 1‐ethyl, 1‐butyl, and 1‐hexyl‐3‐methylimidazolium) as additives in ion exchange chromatography for cations (Na⁺, K⁺, Mg²⁺, Ca²⁺) separation were studied. The results showed that nitric acid and 1‐hexyl‐3‐methyl‐imidazolium hexafluorophosphate offered the most interesting features in the separation of cations, such as lower retention time and better resolution. The selected optimal conditions were achieved by adding 0.10 mM 1‐hexyl‐3‐methyl‐imidazolium hexafluorophosphate in 4.0 mM HNO₃ mobile phase for the separation of four cations with the flow rate of 0.9 mL/min at room temperature (25°C). The linear regression equations of Na⁺, K⁺, Mg²⁺, Ca²⁺ were S  = 4.4763c  + 0.0209, S  = 3.8903c  – 0.0087, S  = 6.3974c  – 0.0173, and S  = 7.601c  – 0.0339 and the limits of detection of Na⁺, K⁺, Mg²⁺, Ca²⁺ were 0.296, 4.98, 0.0970, and 1.22 μg/L, respectively. In this work, four cations in samples were successfully detected.     

Affinity capillary electrophoresis employed for determination of stability constants of antamanide complexes with univalent and divalent cations in methanol

Affinity capillary electrophoresis (ACE) and pressure‐assisted ACE were employed to study the noncovalent molecular interactions of antamanide (AA), cyclic decapeptide from the deadly poisonous fungus Amanita phalloides, with univalent (Li⁺, Na⁺, K⁺, and NH₄⁺) and divalent (Mg²⁺ and Ca²⁺) cations in methanol. The strength of these interactions was quantified by the apparent stability constants of the appropriate AA‐cation complexes. The stability constants were calculated using the nonlinear regression analysis of the dependence of the effective electrophoretic mobility of AA on the concentration of the above ions in the BGE (methanolic solution of 20 mM chloroacetic acid, 10 mM Tris, pH_MeOH 7.8, containing 0–50 mM concentrations of the above ions added in the form of chlorides). Prior to stability constant calculation, the AA effective mobilities measured at actual temperature inside the capillary and at variable ionic strength of the BGEs were corrected to the values corresponding to the reference temperature of 25°C and to the constant ionic strength of 10 mM. From the above ions, sodium cation interacted with AA moderately strong with the stability constant 362 ± 16 L/mol. K⁺, Mg²⁺, and Ca²⁺ cations formed with AA weak complexes with stability constants in the range 37–31 L/mol decreasing in the order K⁺ > Ca²⁺ > Mg²⁺. No interactions were observed between AA and small Li⁺ and large NH₄⁺ cations.     

Development of a microbioreactor with ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) immobilized on a polyacrylamide-coated capillary at the outlet

A simple and fast method of immobilization of cell membrane suspension containing human ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) on a polyacrylamide-coated capillary was developed. The enzyme microbioreactor was prepared by hydrodynamic injection of a small plug of the polycationic electrolyte hexadimethrine bromide (HDB) followed by a suspension of an enzyme-containing membrane preparation. In order to shorten the enzyme assay time and to increase the throughput of the assay, the capillary was coated from the outlet end and all injections were performed from the outlet end of the capillary. For the monitoring of the enzymatic reaction, the substrate ATP dissolved in reaction buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM Hepes, pH 7.4, internal standard: 10 μM UMP) in the absence or presence of inhibitor was injected electrokinetically and incubated in the microbioreactor for 1 min with 1 kV of applied voltage. Then, the electrophoretic separation of the reaction products was initiated by applying a constant current of 60 μA. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by UV absorbance at 260 nm. The new method was compared with an at-capillary-inlet method without immobilization of the enzyme. The results (K_m values, K_i values for inhibitor) obtained with both methods were similar and comparable with literature data. The developed outlet immobilized enzyme microreactor using a coated capillary is very fast, simple and most economic allowing multiple use of the enzyme.     

Chiral separation of 12 pairs of enantiomers by capillary electrophoresis using heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector and the elucidation of the chiral recognition mechanism by computational methods

Chiral separation of 12 pairs of basic analyte enantiomers including oxybutynin, bambuterol, tradinterol, clenbuterol, clorprenaline, terbutaline, tulobuterol, citalopram, phencynonate, fexofenadine, salbutamol, and penehyclidine was conducted by capillary electrophoresis using a single‐isomer anionic β‐cyclodextrin derivative, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin as the chiral selector. Parameters influencing separation were studied, including background electrolyte pH, heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin concentration, buffer concentration, and separation voltage. A background electrolyte consisting of 50 mM Tris‐H₃PO₄ and 6 mM heptakis‐(2,3‐diacetyl‐6‐sulfato)‐β‐cyclodextrin at pH 2.5 was found to be highly efficient for the separation of most enantiomers, with other conditions of normal polarity mode at 10 kV, detection wavelength of 210 nm using hydrodynamic injection for 3 s. Under the optimal conditions, baseline resolution (>1.50) for 11 pairs of enantiomers and somewhat lower resolution for penehyclidine enantiomers (1.17) were generated. Moreover, the possible mechanism of separation of clenbuterol, oxybutynin, salbutamol, and penehyclidine was investigated using a computational modeling method.     

Separation of Anthraquinones by Capillary Electrophoresis and High‐Performance Liquid Chromatography

The separation and determination of twelve anthraquinones, viz. anthraquinone 1, chrysphanol 2, aloe‐emodin 3, alizarin 4, anthraquinone‐2‐carboxylic acid 5, purpurin 6, sennoside B 7, sennoside A 8, emodin 9, quinalizarin 10, rhein 11, and anthraflavic acid 12, were achieved by capillary electrophoresis (CE) and high‐performance liquid chromatography (HPLC). Detection at 260 nm with a buffer solution containing 30 mM sodium borate (adjusted to pH = 10.56 with 0.05N NaOH) and acetonitrile (9 : 1) in CE or with a linear gradient elution containing 20 mM KH₂PO₄ with 0.05% phosphoric acid (pH = 2.91) and methanol in HPLC was found to be the most suitable approach for this separation. Contents of six components (2, 3, 7, 8, 9, 11) in crude Rhei Rhizoma extract could easily be determined within 39 min by CE or 63 min by HPLC. The effects of buffers on this separation and the validation of the two methods were studied.     

Chiral separation of lansoprazole and rabeprazole by capillary electrophoresis using dual cyclodextrin systems

Novel capillary electrophoresis methods using CDs as chiral selectors were developed and validated for the chiral separation of lansoprazole and rabeprazole, two proton pump inhibitors. Fourteen different neutral and anionic CDs were screened at pH 4 and 7 in the preliminary analysis. Sulfobutyl‐ether‐β‐CD with a degree of substitution of 6.5 and 10 at neutral pH proved to be the most suitable chiral selector for both compounds. Various dual CD systems were also compared, and the possible mechanisms of enantiomer separation were investigated. A dual selector system containing sulfobutyl‐ether‐β‐CD degree of substitution 6.5 and native γ‐CD proved to be the most adequate system for the separations. Method optimization was carried out using an experimental design approach, performing an initial fractional factorial screening design, followed by a central composite design to establish the optimal analytical conditions. The optimized methods (25 mM phosphate buffer, pH 7, 10 mM sulfobutyl‐ether‐β‐CD/20 mM γ‐CD, +20 kV voltage; 17°C temperature; 50 mbar/3 s injection, detection at 210 nm for lansoprazole; 25 mM phosphate buffer, pH 7, 15 mM sulfobutyl‐ether‐β‐CD/30 mM γ‐CD, +20 kV voltage; 18°C temperature; 50 mbar/3 s injection, detection at 210 nm for rabeprazole) provided baseline separation for lansoprazole (R_s = 2.91) and rabeprazole (R_s = 2.53) enantiomers with favorable migration order (in both cases the S‐enantiomers migrates first). The optimized methods were validated according to current guidelines and proved to be reliable, linear, precise, and accurate for the determination of 0.15% distomer as chiral impurity in dexlansoprazole and dexrabeprazole samples.     

Simultaneous determination of deoxycytidine diphosphate and deoxycytidine triphosphate by capillary electrophoresis with transient isotachophoretic stacking: a sensitive monitoring method for ribonucleotide reductase activity

A simple and rapid capillary electrophoretic method was developed for simultaneous determination of sub‐micromolar 2′‐deoxycytidine 5′‐diphosphate (dCDP) and 2′‐deoxycytidine 5′‐triphosphate (dCTP) levels in enzyme assays without using radioactively labeled substrates. The separation was performed at 25°C using MES in the BGE as the terminating ion, the chloride ions in the sample buffer as the leading ion, and PEG 4000 in the BGE as the EOF suppressor for sample stacking by transient isotachophoresis (tITP). Several parameters affecting the separation were investigated, including the pH of the BGE, the concentration of sodium chloride in the sample buffer, and the concentrations of MES and PEG 4000 in the running buffer. Good separation with high separation efficiency was achieved within 6 min under optimal conditions. In comparison with the simple CZE method, the present tITP‐CZE method enabled a 150‐fold increase in the injection time without any decrease in resolution and the sensitivity was enhanced up to two orders of magnitude with the new method. The linear range of the method was 0.1–10 μM for dCDP and dCTP. The limits of detection of dCDP and dCTP were 85 and 73 nM, respectively. The proposed method was successfully applied for the activity assay of ribonucleotide reductase from Hep G2 and Sf9 cells.     

Efficient sample clean‐up and online preconcentration for sensitive determination of melamine in milk samples by capillary electrophoresis with contactless conductivity detection

Based on an efficient sample clean‐up and field‐amplified sample injection online preconcentration technique in capillary electrophoresis with contactless conductivity detection, a new analytical method for the sensitive determination of melamine in milk samples was established. In order to remove the complex matrix interference, which resulted in a serious problem during field‐amplified sample injection, liquid–liquid extraction was utilized. As a result, liquid–liquid extraction provides excellent sample clean‐up efficiency when ethyl acetate was used as organic extraction by adjusting the pH of the sample solution to 9.5. Both inorganic salts and biological macromolecules are effectively removed by liquid–liquid extraction. The sample clean‐up procedure, capillary electrophoresis separation parameters and field‐amplified sample injection conditions are discussed in detail. The capillary electrophoresis separation was achieved within 5 min under the following conditions: an uncoated fused‐silica capillary, 12 mM HAc + 10 mM NaAc (pH = 4.6) as running buffer, separation voltage of +13 kV, electrokinetic injection of +12 kV × 10 s. Preliminary validation of the method performance with spiked melamine provided recoveries >90%, with limits of detection and quantification of 0.015 and 0.050 mg/kg, respectively. The relative standard deviations of intra‐ and inter‐day were below 6%. This newly developed method is sensitive and cost effective, therefore, suitable for screening of melamine contamination in milk products.     

Simultaneous quantification of energetically important metabolites in various cell types by CZE

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field‐enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on‐line pre‐concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.