Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.
A rapid procedure for the determination of 2-aminoisobutyric acid in enzalutamide bulk drug substance based on hydrophilic interaction chromatography with fluorescence detection was developed. Fluorescence detection after postcolumn derivatization with o-phthaldialdehyde/2-mercaptoethanol was carried out at excitation and emission wavelength of 345 nm and 450 nm, respectively. The postcolumn reaction conditions such as reaction temperature, mobile phase and derivatization reagent flow rate and the reagents concentrations were studied and optimized due to steric hindrance of amino group of 2-aminoisobutyric acid. The derivatization reaction was applied for the hydrophilic interaction chromatography method which was based on COSMOSIL HILIC column with a mobile phase consisting of a mixture of 25 mmol/L acetic acid adjusted to pH 5.5 (using 1 mol/L potassium hydroxide) and acetonitrile using an isocratic elution (28:72, ν/ν). The benefit of the reported approach consists in a simple sample pretreatment and a quick and sensitive hydrophilic interaction chromatography method. The developed method was validated in terms of linearity, limit of detection, limit of quantification, accuracy, precision and selectivity according to the International Conference on Harmonisation guidelines. The developed method was demonstrated to be applied for the analysis of 2-AIBA in routine quality control evaluation of commercial samples of enzalutamide bulk drug substance.
- MeSH
- acetonitrily chemie MeSH
- antagonisté androgenních receptorů analýza chemie MeSH
- fenylthiohydantoin analogy a deriváty analýza chemie MeSH
- fluorescenční spektrometrie přístrojové vybavení metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- kontaminace léku prevence a kontrola MeSH
- kyseliny aminoisomáselné analýza MeSH
- limita detekce MeSH
- merkaptoethanol chemie MeSH
- o-ftalaldehyd chemie MeSH
- reprodukovatelnost výsledků MeSH
- řízení kvality * MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
A completely new voltammetric method has been developed for quantitative determination of food additive Taurine (Tau) in energy drinks. This electroanalytical method is based on voltammetric oxidation of o-phthalaldehyde-ethanthiol derivative of Tau at glassy carbon electrode in 95% methanol containing 0.1 mol L-1 lithium perchlorate. Working conditions necessary for quantitative Tau derivatization reaction and electrochemical detection using square wave voltammetry were optimized. Linear range from 1.0 × 10-5 to 1.0 × 10-4 mol L-1 characterized by coefficient of determination 0.9998, limits of quantification 6.8 × 10-6 mol L-1 and detection 2.1 × 10-6 mol L-1 were obtained at pulse amplitude 50 mV and frequency 80 Hz. Analytical method of calibration curve was used for evaluation of Tau content in several commercially available energy drinks. The procedure was validated using standard reference high performance liquid chromatography (HPLC) method. Both methods showed nearly identical Tau content, around 0.35% (w/w). Besides its reliability to the Tau determination, that is totally comparable to reference method used, present voltammetric approach is more advantageous on the economic and simplicity basis. Finally, developed voltammetric method could find employment in food quality control.
A sensitive and rapid high-performance liquid chromatography (HPLC) method was developed to enantioseparation of N-acetyl-dl-cysteine after precolumn derivatization using o-phthaldialdehyde and primary aliphatic amines. Seven polysaccharide-based chiral columns were tested in a reversed phase mode. Under the optimal chromatographic conditions, N-acetyl-dl-cysteine derivatives were completely enantioseparated on Chiralcel OZ-3R column with the resolution more than 2.5. The impact of various primary aliphatic amine additives as co-reagents (ethyl-, 1-propyl-, 1-butyl-, 1-pentylamine, (R)-sec-butylamine, tert-butylamine, isobutylamine, cyclopropyl-, cyclobutyl-, cyclopentyl and cyclohexylamine) used in precolumn derivatization step on the retention behavior (retention factor, selectivity and column efficiency) of N-acetyl-dl-cysteine derivatives was investigated. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH, salt concentration in the mobile phase and column temperature on the retention and selectivity was investigated. The developed method was properly validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy, precision, intermediate precision and selectivity according to International Council for Harmonisation (ICH) of Technical Requirements for Pharmaceuticals for Human Use guidelines using internal normalization procedure. Proposed HPLC method was successfully applied to the determination of optical purity in commercially available N-acetyl-L-cysteine samples.
A rapid, precise and specific hydrophilic interaction chromatography (HILIC) combined with postcolumn derivatization using o-phthaldialdehyde and fluorescence detection was developed and validated for the determination of selected pharmaceutically active thiols. The analysis was carried out on a Diol HILIC column using a mobile phase consisting of acetonitrile and solution of 10 mmol/L citric acid adjusted with 1-propylamine to pH 5.5 in ratio 75:25 (v/v) for separation of cysteine and homocysteine and in ratio 85:15 (v/v) for separation of N-acetyl-l-cysteine and captopril. The postcolumn derivatization reaction was performed at room temperature using reagent (5 mmol/L OPA in 0.05 mol/L 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid at pH 7) delivered at the flow rate of 0.3 mL/min. Fluorescence detection was carried out at excitation and emission wavelength of 345 nm and 450 nm, respectively. The effect of chromatographic conditions including acetonitrile content, salt concentration in the mobile phase and mobile phase pH on the retention of tested thiols was investigated. The postcolumn reaction conditions such as reaction temperature, derivatization reagent flow rate, o-phthaldialdehyde concentration and derivatization reagent pH were deeply studied. The developed method was validated in terms of linearity, accuracy, precision and selectivity according to the International Conference on Harmonisation guidelines. The HILIC method was successfully applied for the analysis of commercially available samples of pharmaceutically active thiols such as captopril, N-acetyl-l-cysteine (NAC) and cysteine.
- MeSH
- acetonitrily chemie MeSH
- acetylcystein analýza MeSH
- cystein analýza MeSH
- fluorescenční spektrometrie MeSH
- hydrofobní a hydrofilní interakce MeSH
- kaptopril analýza MeSH
- o-ftalaldehyd chemie MeSH
- propylaminy MeSH
- sulfhydrylové sloučeniny analýza chemie MeSH
- teplota MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Fluorometric glutathione assays have been generally preferred for their high specificity and sensitivity. An additional advantage offered by fluorescent bimane dyes is their ability to penetrate inside the cell. Their ability to react with glutathione within intact cells is frequently useful in flow cytometry and microscopy. Hence, the aims of our study were to use monochlorobimane for optimizing a spectrofluorometric glutathione assay in cells and then to compare that assay with the frequently used ortho-phthalaldehyde assay. We used glutathione-depleting agents (e.g., cisplatin and diethylmalonate) to induce cell impairment. For glutathione assessment, monochlorobimane (40μM) was added to cells and fluorescence was detected at 394/490nm. In addition to the regularly used calculation of glutathione levels from fluorescence change after 60min, we used an optimized calculation from the linear part of the fluorescence curve after 10min of measurement. We found that 10min treatment of cells with monochlorobimane is sufficient for evaluating cellular glutathione concentration and provides results entirely comparable with those from the standard ortho-phthalaldehyde assay. In contrast, the results obtained by the standardly used evaluation after 60min of monochlorobimane treatment provided higher glutathione values. We conclude that measuring glutathione using monochlorobimane with the here-described optimized evaluation of fluorescence signal could be a simple and useful method for routine and rapid assessment of glutathione within intact cells in large numbers of samples.
- MeSH
- biotest ekonomika metody MeSH
- buněčné linie MeSH
- cisplatina toxicita MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie ekonomika metody MeSH
- glutathion analýza metabolismus MeSH
- lidé MeSH
- malonáty toxicita MeSH
- o-ftalaldehyd chemie MeSH
- průtoková cytometrie MeSH
- pyrazoly chemie MeSH
- senzitivita a specificita MeSH
- studie proveditelnosti MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A rapid procedure for the determination of memantine based on hydrophilic interaction chromatography with fluorescence detection was developed. Fluorescence detection after postcolumn derivatization with o-phtaldialdehyde/2-mercaptoethanol was performed at excitation and emission wavelengths of 345 and 450 nm, respectively. The postcolumn reaction conditions such as reaction temperature, derivatization reagent flow rate, and reagents concentration were studied due to steric hindrance of amino group of memantine. The derivatization reaction was applied for the hydrophilic interaction liquid chromatography method which was based on Cogent Silica-C stationary phase with a mobile phase consisting of a mixture of 10 mmol/L citric acid and 10 mmol/L o-phosphoric acid (pH 6.0) with acetonitrile using an isocratic composition of 2:8 v/v. The benefit of the reported approach consists in a simple sample pretreatment and a quick and sensitive hydrophilic interaction chromatography method. The developed method was validated in terms of linearity, accuracy, precision, and selectivity according to the International Conference on Harmonisation guidelines. The developed method was successfully applied for the analysis of commercial memantine tablets.
- MeSH
- antiparkinsonika analýza MeSH
- hydrofobní a hydrofilní interakce MeSH
- memantin analýza MeSH
- merkaptoethanol chemie MeSH
- o-ftalaldehyd chemie MeSH
- silikáty chemie MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
Glutathione is an important tripeptide involved in a variety of cellular processes. Thus, precise knowledge of its levels is essential. Glutathione exists in two free forms-reduced and oxidized-and a number of methods exist to measure its levels. The aim of our work was to optimize a spectrofluorometric assay for reduced glutathione based on the reaction between glutathione and o-phthalaldehyde. We found that a change of excitation wavelength to 340 nm and modification of pH to 6.0 enhance sensitivity and specificity of the method (intraassay coefficient of variation CV < 3%, interassay CV = 5.1%, recovery = 98-102%, linearity = 0-1000 μM GSH, calibration R2 = 1.00). We also anticipated possible effect of various amino acids on the fluorescence signal, but no interference was found. We compared the optimized fluorometric method with a popular enzymatic recycling glutathione assay and found very strong correlation of results (r = 0.99, n = 45). We introduce here an optimized fluorometric method possessing sufficient sensitivity and specificity that is comparable to the enzymatic glutathione assay. Because the fluorometric assay procedure is faster and lower in cost, it could be ideal for routine analysis of reduced glutathione levels in a large number of samples.
A rapid procedure based on direct extraction and HILIC separation of aliskiren (ALI) degradation product - 3-amino-2,2-dimethylpropanamide (ADPA) with fluorescence detection has been developed. The formation of ADPA from ALI under different conditions was studied. The evaluation of HILIC method robustness was performed using multifactorial experiments with fixed factors (one-level Plackett-Burman design). XBridge HILIC column with isocratic elution using mobile phase 10 mM K(2)HPO(4) pH 7.2-acetonitrile (26:74; v/v) was employed. Fluorescence detection after post column derivatization using o-phthaldialdehyde (OPA) reagent was performed at excitation and emission wavelength of 345 nm and 450 nm, respectively. The reported method has an advantage of a simple sample pre-treatment and quick and very sensitive measurement. The method was successfully applied for the analysis of commercially available ALI samples.
- MeSH
- amidy analýza chemie MeSH
- aminokyseliny analýza MeSH
- antihypertenziva analýza chemie MeSH
- časové faktory MeSH
- chromatografie kapalinová metody MeSH
- fluorescenční spektrometrie metody MeSH
- fumaráty analýza chemie MeSH
- hmotnostní spektrometrie metody MeSH
- o-ftalaldehyd chemie MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
A rapid procedure for the determination of lysine based on hydrophilic interaction chromatography (HILIC) separation of arginine and lysine with fluorescence detection has been developed. The separation conditions and parameters of lysine postcolumn derivatization with o-phtaldialdehyde (OPA)/2-mercaptoethanol were studied. The various HILIC columns were employed using isocratic elution. Fluorescence detection was performed at excitation and emission wavelength of 345 nm and 450 nm, respectively. An advantage of the reported method is a simple sample pre-treatment and a quick and very sensitive HPLC method. The developed method was successfully applied for analysis of commercial samples of Ibalgin Fast tablets (Zentiva, Czech Republic).
- MeSH
- arginin analýza MeSH
- časové faktory MeSH
- fluorescenční spektrometrie MeSH
- hydrofobní a hydrofilní interakce MeSH
- indikátory a reagencie MeSH
- léčivé přípravky analýza MeSH
- limita detekce MeSH
- lysin izolace a purifikace MeSH
- merkaptoethanol chemie MeSH
- o-ftalaldehyd chemie MeSH
- referenční standardy MeSH
- reprodukovatelnost výsledků MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
