Field-amplified sample injection and in-capillary derivatization for sensitivity improvement of the electrophoretic determination of histamine

The feasibility of the combination of field-amplified sample injection (FASI) and in-capillary derivatization was explored for improving sensitivity of histamine in capillary electrophoresis (CE). Naphthalene-2,3-dicarboxaldehyde (NDA) was used as derivatization reagent. The reagent and sample was introduced by tandem mode. The derivatization was accomplished by at-inlet mode with standing time of 1.5 min. The combination of FASI and in-capillary derivatization was successfully achieved with about 400-fold concentration sensitivity enhancement compared to pre-capillary derivatization at the same set-up. The detection limit of concentration for histamine reached 1.25 x 10(-11) M by CE and fluorescence detection with S/N = 3. Parameters affecting FASI and in-capillary derivatization process including sample matrix, buffer concentration and reagent injection amount, were investigated.     

Field-amplified sample injection and in-capillary derivatization for capillary electrophoretic analysis of metal ions in local wines

In-capillary derivatization and field-amplified sample injection (FASI) coupled to capillary zone electrophoresis (CZE) was evaluated for the analysis of metals (Co(II), Cu(II), Ni(II), and Fe(II)) using 2-(5-Nitro-2-Pyridylazo)-5-(N-Propyl-N-Sulfopropylamino)Phenol (Nitro-PAPS) as the derivatizing agent. For FASI, the optimum conditions were water as sample solvent, 1 s hydrodynamic injection (0.1 psi) of a water plug, 5 s of electrokinetic introduction (10 kV) of the sample. The in-capillary derivatization was successfully achieved with zone-passing strategy in order tandem injection of Nitro-PAPS reagent (0.5 psi, 7 s), a small water plug (0.1 psi, 1 s), and metal ion introduction (10 kV, 5 s). The solution of 45 mmol L^− 1 borate pH 9.7 and 1.0 × 10^− 5 mol L^− 1 Nitro-PAPS containing 20% acetonitrile was used as the running buffer. The limit of detection obtained by the proposed method was lower than those from pre-capillary derivatization about 3–28 times. The recovery of the method was comparable to pre-capillary derivatization method. In-capillary derivatization-FASI-CZE was applied to analysis of metals in wine samples. The results were compared with those obtained by CZE with pre-capillary derivatization method and atomic absorption spectrometry (AAS).     

Constant pressure-assisted head-column field-amplified sample injection in combination with in-capillary derivatization for enhancing the sensitivity of capillary electrophoresis

In this work, a novel method combining constant pressure-assisted head-column field-amplified sample injection (PA-HC-FASI) with in-capillary derivatization was developed for enhancing the sensitivity of capillary electrophoresis. PA-HC-FASI uses an appropriate positive pressure to counterbalance the electroosmotic flow in the capillary column during electrokinetic injection, while taking advantage of the field amplification in the sample matrix and the water of the “head column”. Accordingly, the analytes were stacked at the stationary boundary between water and background electrolyte. After 600 s PA-HC-FASI, 4-fluoro-7-nitro-2,1,3-benzoxadiazole as derivatization reagent was injected, followed by an electrokinetic step (5 kV, 45 s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 10 min for derivatization reaction under 35 °C, then the capillary temperature was cooled to 25 °C and the derivatives were immediately separated and determined under 25 °C. By investigating the variables of the presented approach in detail, on-line preconcentration, derivatization and separation could be automatically operated in one run and required no modification of current CE commercial instrument. Moreover, the sensitivity enhancement factor of 520 and 800 together with the detection limits of 16.32 and 6.34 pg/mL was achieved for model compounds: glufosinate and aminomethylphosphonic acid, demonstrating the high detection sensitivity of the presented method.     

A dual preconcentration method by combining micro matrix solid-phase dispersion extraction with field-enhanced sample injection and micelle to cyclodextrin stacking for sensitive analysis of neutral coumarins

A rapid, sensitive, environmental friendly dual preconcentration method by combining micro matrix solid-phase dispersion extraction with field-enhanced sample injection and micelle to cyclodextrin stacking has been developed for the determination of furocoumarins. Molecular sieve, KIT-6, was used as an adsorbent in micro matrix solid-phase dispersion process. The important parameters affecting off-line and online CE preconcentration efficiency were optimized. Under the optimal experimental conditions, all analytes showed good linearity (R² > 0.999). The LODs of notopterol, isoimperatorin, and imperatorin were 0.1 μg/mL, 1.2 mg/kg, and 1.0 mg/kg, respectively. Compared with the normal CE method, the enrichment times were up to 300. Moreover, Angelicae Dahuricae Radix was used as the mode of complex solid sample matrix to demonstrate the prospect of application of this methodology. The results showed the proposed strategy is promising for determining trace furocoumarins in complex matrix samples, which might be applied as a powerful and economic tool in monitoring illegal cosmetic adding.     

Capillary electrophoresis determination of biogenic amines by field-amplified sample stacking and in-capillary derivatization

A sensitive CE method for determining biogenic amines in wines based on in-capillary derivatization with 1,2-naphthoquinone-4-sulfonate is presented. In this method, reagent and buffer solutions are introduced hydrodynamically into the capillary whereas the sample is injected electrokinetically, thus, allowing a selective preconcentration of the analytes by field-amplified sample stacking. Amines are labeled inside the capillary using a zone-passing derivatization approach in mixed tandem mode. The most relevant variables influencing on the derivatization and separation as well as significant interactions have been evaluated using experimental design. Multi-criteria decision making is utilized for the simultaneous optimization of interacting variables through overall desirability response surfaces. The validation of the method has proven an excellent separation performance and accuracy for the determination of biogenic amines such as histamine, tryptamine, phenylethylamine, tyramine, agmatine, ethanolamine, serotonin, cadaverine, and putrescine in red wines. Detection limits range from 0.02 mg/L for ethanolamine to 0.91 mg/L for serotonin. The RSDs for migration time and peak area are around 1.2 and 6.2%, respectively. Red wines from different Spanish regions have been analyzed using the proposed method.     

Capillary electrophoresis with field-enhanced stacking for rapid and sensitive determination of strychnine and brucine

A new capillary electrophoresis procedure with field-enhanced stacking concentration for the analysis of strychnine and brucine is established. After optimization of the separation and concentration conditions, the two alkaloids can be separated within 5 min and quantified with high sensitivity (The detection limits were 1.0 ng mL(-1) for strychnine and 1.4 ng mL(-1) for brucine). The method was useful for qualitative and quantitative analysis of strychnine and brucine in Strychnos nux-vomica L with recovery of 105.1% for strychnine and 98.4% for brucine.     

Enzymatic in-capillary derivatization for glucose determination by electrophoresis with spectrophotometric detection

The following paper compares several procedures of in-capillary bienzymatic derivatization with regard to glucose determination with the use of glucose oxidase and horseradish peroxidase. The procedures discussed below include continuous contact in the capillary, plug-plug injection, and sequential injection with incubation in the capillary inlet. The reaction of hydrogen peroxide catalyzed by peroxidase was performed using two different substrates. The best results were achieved for nicotinamide adenine dinucleotide, reduced disodium salt (NADH) acting both as a chromogenic reagent and a substrate for peroxidase, while the method employed was sequential injection and incubation at the capillary inlet. The LOD was estimated to be 25 nM with a linear response up to 0.1 microM.     

Determination of kanamycin by electrophoretically mediated microanalysis with in-capillary derivatization and UV detection

An electrophoretically mediated microanalysis (EMMA) approach, used to perform on-line chemistry between two small molecules, has been characterized and optimized. The plug-plug type EMMA method involved electrophoretic mixing and subsequent reaction of nanoliter plugs of kanamycin-containing samples and 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid within the confines of the capillary column, which acts as a microreactor. Analyses were performed by pressure-injecting a plug of kanamycin sandwiched in two reagent plugs. A potential of 375 Vcm(-1) was then applied to electrophoretically mix the two reactants, and an incubation time of up to 5 min allowed the reaction to proceed prior to the application of a separation potential of 588 Vcm(-1). UV detection was at 335 nm. The background electrolyte was 30 mM sodium tetraborate at pH 10.0, containing 16% of methanol. The method was validated in terms of linearity, limits of quantitation and detection, and precision. The method allows determination of kanamycin in bulk samples as a fully automated procedure.     

Analysis of anabolic androgenic steroids in urine by full-capillary sample injection combined with a sweeping CE stacking method

This study describes an on-line stacking CE approach by sweeping with whole capillary sample filling for analyzing five anabolic androgenic steroids in urine samples. The five anabolic steroids for detection were androstenedione, testosterone, epitestosterone, boldenone, and clostebol. Anabolic androgenic steroids are abused in sport doping because they can promote muscle growth. Therefore, a sensitive detection method is imperatively required for monitoring the urine samples of athletes. In this research, an interesting and reliable stacking capillary electrophoresis method was established for analysis of anabolic steroids in urine. After liquid–liquid extraction by n-hexane, the supernatant was dried and reconstituted with 30 mM phosphate buffer (pH 5.00) and loaded into the capillary by hydrodynamic injection (10 psi, 99.9 s). The stacking and separation were simultaneously accomplished at ?20 kV in phosphate buffer (30 mM, pH 5.0) containing 100 mM sodium dodecyl sulfate and 40 % methanol. During the method validation, calibration curves were linear (r?≥?0.990) over a range of 50–1,000 ng/mL for the five analytes. In the evaluation of precision and accuracy for this method, the absolute values of the RSD and the RE in the intra-day (n?=?3) and inter-day (n?=?5) analyses were all less than 6.6 %. The limit of detection for the five analytes was 30 ng/mL (S/N?=?5, sampling 99.9 s at 10 psi). Compared with simple MECK, this stacking method possessed a 108- to 175-fold increase in sensitivity. This simple and sensitive stacking method could be used as a powerful tool for monitoring the illegal use of doping.     

A simple, rapid and sensitive method for determination of aldehydes in human blood by gas chromatography/mass spectrometry and solid-phase microextraction with on-fiber derivatization

Aldehydes are considered potential markers for enhanced oxidative stress and have been proposed as a diagnostic measure of cancer status. Do to their volatility and activity, it is very difficult to accurately measure aldehydes in human blood. In the present work, gas chromatography/mass spectrometry (GC/MS) and solid-phase microextraction (SPME) with on-fiber derivatization was developed for determination of aldehydes in human blood. O-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) in aqueous solution was first adsorbed by a SPME fiber, and then the aldehydes in blood samples were headspace extracted by the SPME fiber and rapidly derivatized with PFBHA on the SPME fiber. Finally, the oximes formed were desorbed and detected by GC/MS in electron ionization (EI) mode. Validation of the present method was carried out, and the method was applied to quantitative analysis of the aldehydes in lung cancer blood. The results demonstrated that GC/MS and SPME with on-fiber derivatization is a simple, rapid, sensitive and solvent-free method for the determination of aldehydes in human blood.     

Analysis of selenomethylselenocysteine and selenomethionine by LC-ESI-MS/MS with diethyl ethoxymethylenemalonate derivatization

A simple method to identify and determine Selenomethionine (SeMet) and Selenomethylselenocysteine (Se-MeSeCys) with diethyl ethoxymethylenemalonate (DEEMM) derivatization and LC-ESI-MS/MS determination was developed. Separation of SeMet and Se-MeSeCys was achieved in 15.3 minutes. The calibration graph was linear in the range of 0.32 pmol to 49 pmol for SeMet and 0.34 pmol to 40 pmol for Se-MeSeCys. To prevent oxidation of SeMet, 2-mercaptoethanol was introduced to the calibration solutions. Detection limits were 0.1 pmol, which are comparable to LC-ICP-MS analysis. The developed method therefore offers an alternative to LC-ICP-MS offering similar sensitivity and additionally allows identification. The method was used to determine Se-MeSeCys and SeMet in onion samples.     

A sensitive spectrophotometric method for lead determination by flow injection analysis with on-line preconcentration

A new flow injection (FI) system for the determination of Pb(II) at trace level with a preconcentration step and spectrophotometric detection is proposed. It is based on preconcentration of lead ions on chitosan and dithizone-lead complex formation in aqueous medium (pH 9). The chemicals and FIA variables influencing the performance of the system were optimized and applied to the determination of lead in natural, well, and drinking water samples. It is a simple, highly sensitive, and low cost alternative methodology. The method provided a linear rage between 25 and 250 μg l⁻¹, a detection limit of 5.0 ng ml⁻¹ and a sample throughput of 15 h⁻¹. The obtained results of spiked samples are in good agreement between the proposed method and ICP-AES.     

A sensitive and rapid method for determination of pyrophosphatase activity

A new, rapid and sensitive method of measuring the rate and amount of PPi hydrolysis is described. The method is based on registration of small pH changes, caused by decrease of hydrogen-ion concentration during the pyrophosphatase reaction. With this detected value of hydrogen-ion concentration change and the buffering capacity of each reaction mixture determined by titration, the rate and amount of PPi hydrolysis can be calculated. The results obtained by pH method are in good agreement with values determined by direct phosphate analysis. The described method can be applied to the continuous determination of pyrophosphatase activity in chromatophores, mitochondria, crude extracts or purified enzymes.     

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.     

Sensitivity enhancement by on-line preconcentration and in-capillary derivatization for the electrophoretic determination of amino acids

This study describes an application of on-line preconcentration by large-volume stacking in combination with in-capillary derivatization for enhancing spectrophotometric detection sensitivity in capillary electrophoresis. The method is illustrated by an example dealing with the determination of amino acids with 1,2-naphthoquinone-4-sulfonate as a labelling agent. Samples are dissolved in water in order to create a stacking process based on differences in the conductivity between this medium and a concentrated running buffer. The in-capillary derivatization is accomplished following a sandwich procedure in which the sample is inserted between two segments of reagent. Amino acid derivatives are obtained and separated in a fused-silica capillary with a sodium borate electrolyte buffer using 2-propanol as an organic modifier. The method is applied to the analysis of amino acids in pharmaceutical and feed samples. A good concordance between the predicted values and those obtained with the standard method is observed, with overall quantification error below 5%. The proposed procedure allows the detection limits sensitivity to be enhanced in 1000-fold with respect to conventional precapillary derivatization.     

A rapid assay method for catechol-O-methyltransferase activity by flow injection analysis

A rapid assay employing flow injection analysis (FIA) to determine the activity of purified catechol-O-methyltransferase (COMT) from porcine liver is described. The method was based on the determination of normetanephrine, the 3-O-methyl metabolite of the substrate norepinephrine. Excess norepinephrine was removed from the incubation mixture by alumina extraction twice to allow normetanephrine to be subjected to flow injection analysis, coulometrical oxidation, fluorogenic reaction with ethylenediamine and fluorescence detection. K(m) and V(max) values for COMT obtained with the system were 503 microM and 4.51 nmol/min/mg protein, respectively. The method is suitable for screening of COMT inhibitors or activators, as a large number of samples, up to 200, can be processed in one working day.     

Determination of histamine and some other amines by high-performance capillary electrophoresis with on-line mode in-capillary derivatization

We have developed a method for the determination of histamine (His), tyramine (Tyr) and cadaverine (Cad) using high-performance capillary electrophoresis with fluorescence detection and an on-line mode in-capillary derivatization with o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as derivatization reagent. HPCE separation of His, Tyr, Cad and Spermidine (Spd) was influenced by sodium dodecyl sulfate (SDS) and phosphate–borate buffer (pH 10) concentration. After optimization of the method, a 4-component amine solution containing His, Tyr, Cad and Spd could be separated and detected by using 2 mM OPA/NAC–20 mM SDS–20 mM phosphate–borate buffer (pH 10) as a run buffer at an applied voltage of 25 kV, with detection at 340 nm. Although a practical sensitivity level can be obtained by using fluorescence detection (λ_ex=340 nm, λ_em=450 nm) instead of ultraviolet–visible detection, Spd was not detected at all. The precision (relative standard deviation; n=15) of this method for within- and between-days is less than 2.9% (peak area) and 1.3% (migration time), respectively. Linearity for these analytes, except for Spd, was established over a concentration range of 0.02 to 1.00 μmol/ml and detection limits (S/N=3) range from 1 nmol/ml for His and Tyr to 5 nmol/ml for Cad. The determination of His and some amines in aging raw fish meat samples (room temperature, 48 h) was carried out using the described method with fluorescence detection.     

A rapid and sensitive method for quantitation of nucleosides in human urine using liquid chromatography/mass spectrometry with direct urine injection

Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) with single-parameter detectors like electron-capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley et al., Rapid Commun Mass Spectrom. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI-MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI-MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive-ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2-400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03-14.92% (inter-day) and 0.54-14.39% (intra-day), respectively.     

A sensitive method for simultaneous determination of four macrolides by CE with electrochemiluminescence detection and its applications in human urine and tablets

A sensitive method for simultaneous determination of azithromycin (AZI), acetylspiramycin (ACE), erythromycin (ERY), and josamycin (JOS) was developed by CE coupled with electrochemiluminescence detection with Ru(bpy)₃²⁺. The parameters related to separation and detection were investigated in detail. The four macrolides were well separated and detected within 6 min under the optimized conditions. The LOD (S/N=3) of AZI, ACE, ERY, and JOS were 1.2×10^?9, 7.1×10^?9, 3.9×10^?8 and 9.5×10^?8 mol/L, respectively. The LOQ (S/N=10) of AZI, ACE, ERY, and JOS in human urine were 8.2×10^?8, 2.5×10^?7, 8.9×10^?7 and 1.2×10^?6 mol/L, respectively. The recoveries of the four macrolides in human urine and pharmaceutical tablet samples were 85.0–104.0% at different concentration levels.