Current Analytical Chemistry - Volume 10, Issue 3, 2014

In the present study we report the first automated flow-based derivatization method for the determination of the anti-spastic active pharmaceutical ingredient baclofen. The analyte reacts on-line with o-phthalaldehyde in the presence of N-acetylcysteine in a sequential injection manifold to form a highly fluorescent derivative (340 / 455 nm). The various instrumental (flow rate, volumes of sample and reagents, length of reaction coil) and chemical (pH, amount concentrations of the reagents) variables were carefully investigated. The analytical performance of the proposed method included linearity (5 – 150 % or 1 - 30 mg L-1 baclofen), limits of detection (60 µg L-1) and quantification (200 µg L-1), accuracy, precision and selectivity. Analytical applications involved assay, content uniformity and dissolution studies of baclofencontaining formulations at a sampling throughput of 35-40 h-1.

In this work, an overview of analytical methods employing derivatization reactions in chemiluminescence detection is presented. Method development, instrumental configurations and hyphenated manifolds, specifically designed or implemented to accommodate CL detection, along with their analytical applications for the determination of a wide variety of organic compounds such as biochemically active substances, pharmaceutical active compounds, environmental pollutants and food constituents, are summarized. The analytical merits of derivatization reactions in chemiluminescence analysis and their potential for the development of sensitive and selective methods are elucidated.

We have developed a sensitive, simple and cheap method for determination of lipoic acid in urine and dietary supplement tablets. The method is based on conversion of lipoic acid to its thiol counterpart – dihydrolipoic acid – by reductive cleavage with tris(2-carboxyethyl)phosphine hydrochloride prior to precolumn derivatization with 1-benzyl-2- chloropyridinium bromide, followed by ion-pairing reversed-phase liquid chromatography separation and ultraviolet detection of its 2-S-pyridinium derivative at 321 nm. Bathochromic shift from the reagent absorption maximum at 275 nm to that of derivative maximum at 321 nm, taking place during reaction, is analytically advantageous. In developing this method the following parameters were investigated and optimized: the time, pH and reagent excess in the derivatization step and mobile phase buffer concentration, pH, organic modifier and column temperature in the separation step. Calibration plot, understood as the relationship between instrument response and known concentration of the analyte, demonstrated linearity of results when applied to normal urine spiked with growing amounts of lipoic acid within the tested range 0.2 – 50 µmol/L. The method was validated for urine samples received from healthy donors and urine concentration of lipoic acid was monitored after oral administration of 600 mg of lipoic acid.

A new, simple HPLC method is described for the isocratic separation of biogenic monoamines, in milk samples, in the presence of hexylamine as the internal standard, after pre-column derivatization with naphthalene-2,3- dicarboxaldehyde /cyanide ions. The derivatization reaction variables including buffer pH and concentration, reaction time, naphthalene-2,3-dicarboxaldehyde (NDA) and cyanide concentrations were optimized. The isoindoles formed were eluted by means of methanol/water (80:20, v/v), at a flow rate of 1 mL min-1, on an Inertsil ODS-3 column (250 x 4 mm i.d., 5 µm), in less than 30 min. Fluorometric detection was used, at excitation and emission wavelengths of 424 and 494 nm, respectively. The limits of detection were in the pg level (7 – 80 pg) using a 10 µL-loop. The whole procedure was evaluated and fully validated for the determination of biogenic amines in milk samples, after ultrasound-assisted liquidliquid extraction. The intra-assay precision and accuracy were calculated in samples spiked with different levels of biogenic amines and yielded RSD and recovery values in the range 0.3 – 4.2% and 82.5 – 103.8% respectively.

Flow Injection analysis (FIA) has been established as a useful tool for the automation of chemical analysis. In principle, analytes are injected as a discrete zone into a flowing stream followed by physical/chemical processing that is governed by the dispersion mechanism. The products/complexes/ derivatives or detectable species are measured in a continuous mode. However, in many instances the reactions products need to be extracted into organic solvents or vice versa or signals from complexes do not satisfy the required sensitivity or selectivity. An interesting and viable alternative is to use micellar solutions that are aggregated structures of surfactant molecules. Many reports are found in the literature utilizing micellar solutions in flow injection analysis for the determination of organic analytes through derivatization. Spectrophotometry and luminescence spectroscopy are major techniques employed as detection systems. Sensitivity, selectivity, simplicity and high sample throughput in green working environment is evidenced from the literature. Emerging trends and future potentials of micellar solutions in conjunction with FIA are reviewed. Solvolysis chemistry of micellar solutions and interaction mechanisms are also discussed.

A determination method for amino acids in mouse plasma was developed by HPLC with fluorescence detection. The developed methodology is based on a pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) carried out in the sample injection loop. The optimized derivatization time and temperature were 10 min and 60 °C, respectively. The separation of 21 amino acid derivatives was achieved on a monolithic silica column with gradient elution. The amino acid derivatives were detected with excitation and emission at 470 nm and 530 nm, respectively. The lower limit of detection for the individual amino acids was 1.52 to 47.0 fmol. The calibration curves for all the amino acids were linear over the tested range of 0.03 to 30 pmol. The validation data showed that the assay is sensitive, selective and reproducible for determination of amino acids. The analytical method was successfully applied to determine the amino acids in a mouse plasma sample.

The problems and risks associated with mycotoxin contamination of foods and feedstuffs have led to the development of a variety of confirmatory analytical methods for their determination, based on chromatographic techniques and capillary electrophoresis. In this paper, an overview of the analytical methods developed in the last decade and based on post-column derivatization coupled with high-performance liquid chromatography and fluorescence detection is reported. A particular focus on the determination of aflatoxins (B1, B2, G1 and G2), fumonisins (B1 and B2) and trichotecenes deoxynivalenol (DON) and nivalenol (NIV) in cereals products for human and animal consumption is given. Important aspects as the optimization of the experimental conditions and validation of the analytical methods are also described.

A new UHPLC protocol for the determination of amino acids after pre-column derivatization with 6- aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was presented. Applying a simple and easy to prepare binary eluent system, the 20 major proteinogenic amino acids were separated in about 7 min with a total runtime of 10.5 min until the next injection. Since AQC amino acid derivatives were primarily designed to be used with fluorescence detection but facilitate UV detection as well, both approaches were evaluated for their applicability with the established UHPLC method. 6-aminoquinoline, the hydrolysis by-product of derivatization which implies similar absorbance as amino acid derivatives, was effectively separated prior to the polar amino acids, hence no interferences in UV detection were observed. For UV detection at 254 nm, all amino acids exhibited a quite similar response, whereas the respective fluorescence yield at 395 nm emission (excitation at 245 nm) indicated significant dependencies from the applied conditions mainly affected by aqueous quenching. Regarding the possible sensitivity compared to UV, fluorescence detection proved to be superior with detection limits ranging down to the low fmol level. Additionally, the established method was successfully applied to analyze amino acid levels intrinsic to commercial hydrolysates/peptones, that were primarily destined for microbiological use. The found proportion of amino nitrogen ranged from 8% for soy protein acid hydrolysate to 2% for fish peptone, while both detection approaches proved to be equally suitable for the given application.

An accurate, simple and sensitive method for determination of tiopronin (TP, N-(2-mercaptopropionyl)- glycine, Thiola) in human urine has been devised. Tiopronin is an important compound and is mainly used as a drug to prevent kidney stones. It works by removing the extra cystine from the body. Therefore, the reliable assay of tiopronin in biological matrices and pharmaceutical preparations is highly desirable. The proposed analytical procedure consists of reduction of tiopronin dimmer and its unsymmetrical disulfides with endogenous thiols, derivatization with 2-chloro-1- methylquinolinium tetrafluoroborate (CMQT) followed by acetonitrile stacking in capillary electrophoresis separation and ultraviolet-absorbance detection of TP-CMQT derivative at 350 nm. Baseline electrophoretic separation was achieved using standard fused-silica capillary (effective length 47.5 cm, 50 µm i.d.) and 0.25 mol L-1, Tris/HCl buffer adjusted to pH 2.0. The lower limit of quantitation for derivatized TP in urine was 5 µmol/L. The calibration curve showed linearity in the range 5-160 µmol L-1 urine with a regression coefficient corresponding to 0.9987 and the relative standard deviation of the points of the calibration curve being lower than 7%. The method can be used for a pharmacokinetic study with human subjects.

High-performance liquid chromatography coupling with atmospheric pressure ionization mass spectrometry (LC–API–MS) has been widely used in the pharmaceutical industry, clinical research, environmental science, metabolomics studies and so on. However, LC-API-MS often suffers from the matrix effect in its applications in complex samples. Matrix effects can heavily affect reproducibility, linearity, and accuracy of the method leading to erroneous quantitation. The use of isotope-labeled analyte as internal standards provides an efficient solution to overcome matrix effects in LCAPI- MS analysis. However, the isotope-labeled standards are expensive and not always available, especially in the analysis of large numbers of analytes. In recent years, the stable isotope-coded derivatization reagents, as an alternative way, have been used to quantify small molecules in LC-API-MS analysis of complex samples. In this review, an overview of differential isotope-coded derivatization reagents is presented, focusing on the applications to low molecular weight compounds by LC-API-MS. The recently reported isotope-coded derivatization reagents were summarized in category of their target analytes, specifically peptides, amines, phenols, carboxylic acids, aldehyde or ketone, saccharides and thiols. It indicates that isotope-labeled derivatization reagent would have an increasingly wide range of applications in the LC-APIMS analysis.

The Schiff bases are formed by condensation of different β-diketones, salicylaldehydes or 2-hydroxyacetophenes with various amines. These bases react with metal and oxo-metal cations to form stable metal chelates which absorb/fluoresce in UV or visible region of light. The Schiff bases have been used in the determination of metal ions by spectrophotometry, spectrofluorimetry, gas chromatography, liquid chromatography and capillary electrophoresis. Ion-selective electrodes using Schiff’s bases is an emerging area for metal ion analysis. This review covers brief synthesis, chelation properties and analytical applications of Schiff bases.

The absorption of the diatomic molecule AlF in the C2H2/N2O flame at 227.66 nm reveals an interesting feature. The calibration curve of the AlF absorption plotted against a rising concentration of hydrofluoric acid in solutions of constant aluminum content consists of two subsequent linear sections of different slopes. The bend position is reproducibly found at a molar fluorine-to-aluminum ratio of 3, calculated from the composition of the studied solutions. To explain this behavior, the most prominent aluminum flame species Al, AlF, and AlO were recorded as a function of the burner gas composition and flame observation height, using a high-resolution continuum source flame absorption spectrometer. As a result, the two-sectioned calibration curve is explained by two different pathways of AlF molecule formation: At a molar fluorine-to-aluminum ratio of below 3, aluminum is transported into the flame by two parallel pathways. One is the common pathway in absence of fluorine via the reduction of oxidic and/or carbidic species by the flame gases. The second pathway comprises the formation of gaseous AlF3 and its decomposition into AlF molecules and, subsequently, Al atoms. The fractionation of AlF3 releases Al atoms much faster than through the reduction of the oxidic and/or carbidic species. At molar fluorine-to-aluminum ratios of above 3, all aluminum is introduced to the flame via gaseous AlF3. A further increase of the hydrofluoric acid concentration increases the fluorine atom concentration in the flame, so that the AlF formation is determined by the recombination of aluminum and fluorine atoms.

The molecular absorption of the diatomic AlF molecule in the C2H2/N2O flame was studied using a highresolution continuum source flame atomic absorption spectrometer. AlF has a structured absorption spectrum in the range of 227.30 nm and 227.80 nm. From this band system, the remarkably narrow absorption band at 227.66 nm proved to be the optimum for analytical purposes. The signal intensity was studied as a function of the C2H2 : N2O ratio, the aspiration flow, and the aluminum concentration added to the analytical solution to generate the AlF molecules in the flame. The AlF molecule formation is significantly affected by the bonding state of the fluorine source used. Compared to ionic bound fluorine, organic bound fluorine leads to a markedly less sensitive molecular absorbance of AlF. Furthermore, several ions, such as Na+, K+ and NH4+ +, and acids, such as HCl, CH3COOH, and HNO3, affect the AlF signal intensity severely. It has to be concluded that the determination of fluorine by AlF F MAS only leads to reliable analytical results in simple matrices.

In this report we aimed to examine the effect of metal / metal oxide nano / micro particles on the electrochemical responds of composite, carbon paste electrode (CPE) and solid glassy carbon electrode (GCE). Therefore, three different configurations of CPE modified with titanium (IV) oxide micro particles (TiO2-µp) and gold nanoparticles (Au-np) and three different configurations of GCE including TiO2-µp, Au-np and manganese (IV) oxide nanoparticles were prepared. Their electrochemical performances were examined towards ferricyanide. As a result, better electrochemical results were obtained with TiO2-µp incorporated CPE where Au-np was attached with chitosan onto the electrode surface. Also for GCE, again TiO2-µp -p modified GCE gave the best results.

An innovative microbiological assay was developed for the determination of colistin sulphate at various concentrations (192-469 IU/mL); using Bordetella bronchiseptica ATCC 4617 as microorganism test. This assay revealed subtle changes not demonstrated by chemical methods, such as chemical degradation and low of potency, but by evaluating the potency of colistin, which is very important for the analysis of antibiotics. The method was validated according to ICH guidelines, proving to be linear (r2 = 0.9985), precise (CV = 1.32%) and accurate (100.9 ± 1.33%). It was concluded that the microbiological assay is satisfactory for in vitro quantitation of the antibacterial activity of colistin sulphate.