Direct UV Detection Research Articles

Development of capillary electrophoresis methods for the detection of microbial metabolites on potential future spaceflight missions.

The search for chemical indicators of life is a fundamental component of potential future spaceflight missions to ocean worlds. Capillary electrophoresis (CE) is a useful separation method for the determination of the small organic molecules, such as amino acids and nucleobases, that could be used to help determine whether or not life is present in a sample collected during such missions. CE is under development for spaceflight applications using multiple detection systems, such as laser induced fluorescence (LIF) and mass spectrometry (MS). Here we report CE-based methods for separation and detection of major polar metabolites in cells, such as amino acids, nucleobases/sides, and oxidized and reduced glutathione using detectors that are less expensive alternatives to LIF and MS. Direct UV detection, indirect UV detection, and capacitvely coupled contactless conductivity detection (C4D) were tested with CE, and a combination of direct UV and C4D allowed the detection of the widest variety of metabolites. The optimized method was used to profile metabolites found in samples of Escherichia coli and Pseudoalteromonas haloplanktis and showed distinct differences between the species.

Potential application of a chromatographic method in the accurate simultaneous determination of amikacin sulfate and its preservation additives in different pharmaceutical dosage forms

Aminoglycosides are antibiotics with high clinical importance that face a great challenge of detection in the UV range. This study aimed to develop an accurate, simple, and sensitive validated high-performance liquid chromatographic (HPLC) method for the determination of amikacin sulfate, one of the widely used aminoglycoside antibiotics in the pharmaceutical formulations, as the target analyte without interference from its preservation additive sodium metabisulfite in the pharmaceutical dosage forms without the usage of any derivatization reactions. The developed method is based on direct UV detection at 205 nm using a gradient mobile phase consisting of acetonitrile as an organic solvent and buffered solution; pH 3.0. This method was developed on a reversed-phase ZORBAX ECLIPSE XDB-C8 (4.6 × 250 mm, 5-μm) analytical column with a flow rate of 1.5 mL min−1 at a column temperature of 35 °C. The separation of amikacin sulfate and sodium metabisulfite was achieved at 3.7, and 1.5 min, respectively. The developed method validation was performed as prescribed by the International Conference on Harmonization (ICH) guidelines to confirm its linearity, precision, and accuracy. Finally, using simple, commonly available analytical tools, safe buffer concentration on the HPLC system, and without any complicated equipment or challenging derivatization steps, our validated HPLC method was applied for the simultaneous determination of amikacin and sodium metabisulfite in commercially available pharmaceutical formulations in a single method of analysis.

Detection techniques for carbohydrates in capillary electrophoresis – a comparative study

Capillary electrophoresis methods for the separation of carbohydrates with four different detection techniques, namely direct UV, indirect UV, capacitively coupled conductivity, and laser-induced fluorescence detection, were tested and their performance was evaluated and compared in terms of linearity, limits of detection and quantitation, repeatability, recovery, analysis time, and sample treatment. The test set of analytes comprised sucrose, glucose, and fructose. The effect of using lactose as an internal standard on the individual methods was investigated, too. The results showed that laser-induced fluorescence detection is a technique of choice for applications requiring the detection of very low amounts of reducing carbohydrates. Contactless conductivity detection is favorable when detection sensitivity is not a crucial parameter but fast and reliable analysis is required. When only a UV detector is available as a standard part of capillary electrophoresis instruments, direct UV detection can be used when analysis time is not a critical parameter. For fast analysis with UV detectors, indirect UV detection is the technique of choice. Finally, to verify the applicability of the tested methods, samples of cola beverage, honey, and orange juice were analyzed and the results obtained by all four methods were compared.Graphical abstract

Alternative method for microenzymatic inhibition activities monitoring from Baccharis trimera extracts by CZE-UV.

Capillary zone electrophoresis with direct UV detection (CZE-UV) was used to investigate the hypothesis about the extract of Baccharis trimera enzymatic activities as an analytical approach to monitoring the phenomenon. The aim of this work was to investigate enzymatic bioactivities of the hydroalcoholic and infusion extracts of B. trimera through screening evaluation of the inhibition of the enzymes acetylcholinesterase (AChE) and α-glycosidase (α-GLY). An alternative approach using CZE-UV to hydroalcoholic and infusion extracts of B. trimera monitoring was applied to evaluate the inhibition ability of the enzymes AChE and α-GLY. The result of the reaction of acetylthiocholine (AThCh) with AChE was thiocholine (TCh) and acetic acid, and from the amount of TCh generated, the AChE inhibition was calculated. For the inhibition study of the two enzymes, the reactions of the extracts were optimised to be performed in situ, inside the capillary column, and the introduction of the solutions was performed through ordered sequential plug injections. Samples extracted with 70% ethanol presented 7.80% inhibition for AChE and 0.51% for α-GLY, while samples extracted with 96% ethanol resulted in 6.89% inhibition for AChE and no inhibition activity for α-GLY. In the present work, the potentialities of CZE-UV for the study of hydroalcoholic and infusion extracts of B. trimera were demonstrated. The experimental results were useful for the calculation of the percentage of the inhibition activities of the AChE and α-GLY enzymes.

Concepts for New Rapid Simple HPLC Method for Quantification of Fosfomycin Trometamol in Pharmaceutical Dosage Forms with Direct UV Detection

Two different concepts for developing direct HPLC-UV methods for quantifying fosfomycin trometamol were developed without any derivatization and modification of the analyte. In the first concept, without the use of alkylamines as ion-pairs in the mobile phase, by using cyanopropyl CN and a strong anion-exchanger column, we investigated the possibility of their highly polar and anion-exchanging forces and mechanisms to retain, separate and detect trometamol without the help of additional agents or modifiers. In the second concept, the most frequent reversed-phase C18 columns with different characteristics and vendors were tested in combination with different length-based alkylamines with 3–10 C atoms in their chains. In our research, we found that the ion-pairing of fosfomycin with 6–10 C-atom-based alkyl-length of aliphatic chains manifested the most appropriate strength of interactions between alkyl-paired trometamol molecules and octadecylsilane or C18 bonded RP column to achieve optimal retention, selectivity and peak shape on chromatograms, with the possibility for the fine-tuning of elution time. The simplicity of our method concept omits the need for expensive and sophisticated columns like HILIC, C30 graphite carbon, and mixed-mode-based columns for easier retaining, separation, and determination of fosfomycin, and for its quantification purposes, especially in high-throughput analyses in regular quality-control laboratories.

Determination of purity and anionic exchange efficiency of amino acid ionic liquids synthesis by multiple-injection capillary zone electrophoresis

The purity of ionic liquids (IL) is important for its performance, so the methods able to quantify low concentration impurities are necessaries. Therefore, this paper aims to determine the purity and the anionic exchange efficiency for the preparation of amino acids ionic liquids (AAIL). For this, a Multiple-Injection Capillary Zone Electrophoresis (MICZE) method with direct UV detection was developed and optimized for iodide (I−) determination as impurity of AAIL synthesis, comparing this method with traditional injection modes. Furthermore, this paper aims to demonstrate the use of factorial design methods for the optimization of MICZE method. The method optimization allowed five consecutives I− injections of sample in a single run, with analysis time of less than 3 min, showing a larger analytical frequency, counting with 31 injections per hour. It was possible to determine the high purity of the prepared and analyzed AAIL, ranging from 89% to 95% and its overall ionic exchange yield varying from 55% to 87%.

Separation and Determination of d-Allose in Presence of Process-Related Impurities by Capillary Electrophoresis

A simple and effective capillary electrophoresis (CE) method for the separation and quantification of d-allose in the presence of its process impurities, namely sucrose, d-glucose, d-fructose, d-psicose, and d-altrose, has been developed and validated. The optimum conditions for the separation with an acceptable resolution are found to be as follows: background electrolyte (BGE): 36 mM of Na2HPO4 and 130 mM NaOH; pH: 12.6; and voltage: 18 kV with direct UV detection at 265 nm. The present method overcomes the limitations of existing analytical method like sample preparation, poor resolution, and sensitivity. The method is optimized with the established standard curves for d-allose and its related impurities in the concentration range 0.25 to 3.0 mM with coefficient of determinations > 0.99 and the limit of detection (LOD) in the range of 0.11 to 0.20 mM. Separation of d-allose and its impurities present in the mixture is achieved using the current method with good resolution, linearity, and reproducibility. The present method is suitable for the analysis of the final product in the enzymatically synthesized d-allose and also for reaction monitoring of in-process samples.

Study of the Oxidative Forced Degradation of Glutathione in Its Nutraceutical Formulations Using Zone Fluidics and Green Liquid Chromatography

In the present study, we report the results of our investigation of the oxidative forced degradation of glutathione in its nutraceutical formulations by two validated analytical methods. The first is based on the reaction of glutathione with o-phthalaldehyde through an automated zone fluidics flow platform and fluorimetric detection (λex/λem = 340/425 nm). The second is based on the separation of glutathione and its oxidation product by a green reversed-phase HPLC method coupled to direct UV detection, at 210 nm. A solution of 3% w/v H2O2 provided fast oxidation of more than 95% of glutathione to yield oxidized glutathione in a time period of 180 min. The mechanism of the oxidation was proved to follow pseudo-first order kinetics. The k, t90 and t1/2 values were calculated.

Characterization of hydrosoluble fraction and oligomers in poly(vinylidene chloride) latexes by capillary electrophoresis using electrophoretic mobility modeling

The characterization of hydrosoluble oligomers in latexes is an important topic in emulsion polymerization since oligomers are suspected to be responsible for latex destabilization. In this work, the hydrosoluble fraction of poly(vinylidene chloride) latexes synthesized by emulsion polymerization of three monomers (acrylic acid, methyl acrylate, vinylidene chloride) was characterized by capillary electrophoresis (CE). CE using direct UV detection permitted to estimate residual monomers and surfactants concentrations contained in the latexes serums. Water soluble oligomers, polymerization initiator (persulfate) and other inorganic anions were detected by indirect UV detection. The oligomers content in the dry extract of serum was estimated to be about 6% (% m/m) represented mainly by 9 different compounds belonging to 3 different families. Using a semi-empirical electrophoretic mobility modeling, the charge number of these oligomers was estimated to be −2 and the molar masses were estimated in the range of ∼300–550 g.mol−1. Oligomer samples obtained by surfactant-free polymerization, with different initial monomers proportions, provided qualitatively 14 different oligomers, including the 9 oligomers previously detected in the serums. Finally, the latex was characterized (electrophoretic mobility and zeta potential) using its serum as a background electrolyte. This approach could be very useful to study the behavior of the latexes, and possibly destabilization effect, in analytical conditions very close to its real environment / applications.

Identification of non-protein nitrogen compounds separated by CZE without derivatization from TCA extract from salted herring meat

Capillary zone electrophoresis (CZE) is a good method for separation of small charged molecules like amino acids and small peptides. However, their identification in biological samples is quite difficult. Proteolysis products in salted fish are usually determined by chemical methods and there is no suitable simple and quick direct instrumental method. In this work the identification of compounds in trichloroacetic extracts from salted herring meat separated by CZE with direct UV detection was performed. There were determined theoretical migration time of amino acids and peptides based on the Offord’s model and experimental migration time of 67 standards. Standards were separated individually, in the mixture and with samples (increasing concentration). On the electrophoregram of salted herring meat extract there were noticed 36 peaks, divided into 4 fields A–D. Among them there were identified in sequence: (A) creatinine, Lys, Arg and His, (B) creatine, (C) Trp, Met, Tyr and Phe and (D) cystine. Described method may be suitable for fast monitoring of the salted herring ripening process.

Flow Injection Analysis with Direct UV Detection Following Electric Field Driven Membrane Extraction.

A method for on-line matrix elimination to enable selective quantification of ultraviolet absorbing analytes by a flow-injection analysis procedure is described. Selectivity is achieved by electric field driven extraction across a polymer inclusion membrane. The method was demonstrated on the example of the determination of naproxen from spiked human urine. Membranes of 10 μm thickness were employed which consisted of 7.5 mg cellulose triacetate as base polymer, 5 mg of o-nitrophenyl octyl ether as plasticizer and 7.5 mg of Aliquat 336 as cationic carrier. Ten μL of sample was introduced into a continuous stream of background solution consisting of 100 µM aqueous NaClO4 with a flow rate of 2 μL/min while applying a voltage of 150 V to the extraction cell. The target ion was electrokinetically transported across the membrane and enriched in 1.5 μL of a stagnant acceptor solution. This was subsequently pumped past a flow-through UV detector for quantification. The method showed a linear range from 5 to 200 µM with a correlation coefficient of 0.9978 and a reproducibility of typically 7% (n = 8). The detection limit of the method for naproxen was 2 µM.

Determination of gibberellic acid in a commercial seaweed extract by capillary electrophoresis

Gibberellic acid (GA 3 ) is an important plant growth regulator used in agriculture. This work has developed and validated a simple method for the quantification of GA 3 in a seaweed extract product using capillary electrophoresis with direct UV detection. A fused-silica capillary of 50 µm inner diameter and 30 cm of effective length with extended light path, 50 mmol.L -1 of borate buffer as background electrolyte solution (pH 9.11), voltage of +30kV applying hydrodynamic injection of 50 mbar for 5 s and length detection of 200 nm were employed. The method was validated according to the International Conference Harmonisation guidelines. Satisfactory results in the range of 10 – 100 mg.L -1 were obtained for precision (RSD below 3.62%), linearity (R 2 : 0.9976, lack of fit test p > 0.05), specificity and accuracy for which the limit of detection and quantitation were 3.07 and 9.29 mg.L -1 , respectively. The results indicate that the method is suitable for implementation in agroindustry in order to assure the quality control of GA 3 of seaweed products.

SIMULTANEOUS DETERMINATION OF BENZOIC ACID AND SORBIC ACID IN FOOD PRODUCTS BY CAPILLARY ELECTROPHORESIS

In this work, a rapid and easy capillary electrophoretic method for simultaneous determination of benzoic acid and sorbic acid in food samples using direct UV detection was reported. The separation of acids was achieved in fused silica capillary at 28 kV. 20 mM borate buffer at pH 9.3 was used as running buffer; cinnamic acid was chosen as internal standard for quantification. Under the optimal separation conditions benzoic acid and sorbic acid were detected in 3.1 min. The linear ranges were between 0.005-0.4 mM. The correlation coefficients for each calibration curve were calculated as 0.999. The reproducibility of peak area and migration time for each acid were less than 3% (R.S.D.). The limits of detection were found 0.405 m g/mL for benzoic acid and 0.415 m g/mL for sorbic acid. The limits of quantification were 1.35 μg/mL and 1.38 μg/mL respectively.

Simultaneous Determination of Tizanidine, Nimesulide, Aceclofenac and Paracetamol in Tablets and Biological Fluids Using Micellar Liquid Chromatography.

A simple, sensitive and rapid micellar liquid chromatographic method was developed and validated for simultaneous determination of four drugs, namely, paracetamol (PAR), tizanidine (TZD), aceclofenac (ACF) and nimesulide (NMD). Good chromatographic separation was achieved using Cyano column and micellar mobile phase consisting of 120 mM sodium dodecyl sulfate, 25 mM phosphate buffer and 10% (V/V) butanol. The pH was adjusted to three using phosphoric acid. The total retention time was below 10 min. The analysis was performed at a flow rate of 1 mL/min and a column temperature of 40°C with direct UV detection at 230 nm. Diclofenac sodium was used as the internal standard. The proposed method was validated according to the ICH guidelines and was successfully applied to the analysis of these drugs in their tablet dosage forms with high accuracy. Limits of detection were found to be 0.03, 0.07, 0.033 and 0.11 μg/mL for PAR, ACF, TZD and NMD, respectively. The high sensitivity of developed method permitted its application to the in-vitro determination of the cited drugs in spiked human plasma and urine samples, and the obtained results were satisfactory. However, PAR could not be determined in spiked human urine because its peak overlapped with that of the urine peak.

Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection.

The branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) play a pivotal role in the human body. Herein, we developed capillary electrophoresis (CE) coupled with conventional UV detector to quantify underivatized BCAAs in two kinds of sport nutritional supplements. For direct UV detection at 195 nm, the BCAAs (Leu, two enantiomers of Ile and Val) were separated in a background electrolyte (BGE) consisting of 40.0 mmol/L sodium tetraborate, and 40.0 mmol/L β-cyclodextrin (β-CD) at pH 10.2. In addition, the indirect UV detection at 264 nm was achieved in a BGE of 2.0 mmol/L Na2HPO4, 10.0 mmol/L p-aminosalicylic acid (PAS) as UV absorbing probe, and 40.0 mmol/L β-CD at pH 12.2. The β-CD significantly benefited the isomeric separation of Leu, L- and D-Ile. The optimal conditions allowed the LODs (limit of detections) of direct and indirect UV absorption detection to be tens μmol/L level, which was comparable to the reported CE inline derivatization method. The RSDs (relative standard deviations) of migration time and peak area were less than 0.91% and 3.66% (n = 6). Finally, CE with indirect UV detection method was applied for the quantitation of BCAAs in two commercial sport nutritional supplements, and good recovery and precision were obtained. Such simple CE method without tedious derivatization process is feasible of quality control and efficacy evaluation of the supplemental proteins.

Capillary electrophoresis method for the analysis of organic acids and amino acids in the presence of strongly alternating concentrations of aqueous lactic acid.

During the production of bio-based bulk chemicals, such as lactic acid (LA), organic impurities have to be removed to produce a ready-to-market product. A capillary electrophoresis method for the simultaneous detection of LA and organic impurities in less than 10min was developed. LA and organic impurities were detected using a direct UV detection method with micellar background electrolyte, which consisted of borate and sodium dodecyl sulfate. We investigated the effects of electrolyte composition and temperature on the speed, sensitivity, and robustness of the separation. A few validation parameters, such as linearity, limit of detection, and internal and external standards, were evaluated under optimized conditions. The method was applied for the detection of LA and organic impurities, including tyrosine, phenylalanine, and pyroglutamic acid, in samples from a continuous LA fermentation process from post-extraction tapioca starch and yeast extract.

Simultaneous determination of aliphatic, aromatic and heterocyclic biogenic amines without derivatization by capillary electrophoresis and application in beer analysis

Biogenic amines (BAs) play significant roles in indicating human health or food quality. Aiming to simultaneously determine three structures (aliphatic, aromatic and heterocyclic) of underivatized BAs, we explored a simple and rapid capillary electrophoresis (CE) method only coupled with conventional UV detector for the separation of thirteen key BAs. The strategy is to choose a UV absorbing probe as co-ion in the background electrolyte (BGE), and different BAs could be characterized by positive or negative peaks according to the fact that their UV absorptivity coefficients at a certain wavelength are better or worse than that of the UV absorbing probe. After the detailed investigation of critical parameters as pH, the concentration of Imidazole (Im) and α-cyclodextrin (α-CD), the optimized BGE consisted of 12.0mmol/L Im as the UV probe and 10.0mmol/L α-CD as the additive (at pH 4.50 adjusted with acetic acid). With such condition, the targets of thirteen BAs were baseline separated in 9.0min and appeared at nine positive peaks and four negative peaks at 200nm. The obtained LODs and LOQs (S/N=3 or 10) were in the range of 0.36–3.67 and 1.2–12.2μmol/L, respectively. The interday RSDs of migration time and peak area were less than 0.7% and 4.7% (n=6), respectively. To the best of our knowledge, this is the first report on separating diverse structures of BAs by using Im as UV absorbing probe. The thirteen BAs were simultaneously detected by direct and indirect UV detection in a CE process. To verify the applicability, this method was used to analyze BAs in commercial beer samples. The recoveries of all BAs except carnosine (not identified by the interference) ranged from 70.4 to 119.6%, and four aliphatic and aromatic amines were satisfactorily identified and quantified.

Development of a fast method for simultaneous determination of hippuric acid, mandelic acid, and creatinine in urine by capillary zone electrophoresis using polymer multilayer-coated capillary.

This paper reports the development of a fast separation method employing capillary zone electrophoresis for the simultaneous determination of hippuric acid, mandelic acid, and creatinine in samples of urine using a coated capillary. The background electrolyte was composed of 10mmolL-1 tris(hydroxymethyl)aminomethane and 30mmolL-1 2-hydroxy-isobutyric acid at pH3.6. The internal standard was 3,5-dihydroxybenzoic acid. Separations were performed in a fused silica capillary (32cm total length, 8.5cm effective length, and 50μm internal diameter) coated with crosslinked hydroxypropyltrimethyl ammonium chloride chitosan and κ-carrageenan. Direct UV detection was performed at a wavelength of 200nm. Samples and standards were injected hydrodynamically (-50mbar, 3s) using the short-end injection procedure. The electrophoretic system was operated under constant voltage of 30kV with positive polarity on the injection side. The separation time for hippuric acid, mandelic acid, and creatinine was less than 70s. The evaluation of some analytical parameters of the method for the three analytes showed good linearity (R 2 > 0.99), limit of detections of 0.21 to 0.63mgL-1, inter-day precision better than 3.0% (peak area), and recovery in the range of 98 to 106%. The method developed was applied in the analysis of the three analytes in urine samples. Graphical Abstract New method using capillary zone electrophoresis for analysis of creatinine, hippuric acid and mandelic acid in urine.

Assay Development for Aminoglycosides by HPLC with Direct UV Detection.

The absence of UV chromophores in the structure of most aminoglycosides impedes an easy and straightforward development of analytical methods. It was demonstrated that addition of borate to the mobile phase allowed direct detection of aminoglycosides at the lower end of the UV spectrum. Although the borate ion complexes preferably with compounds possessing vicinal diols, the aim of this study was to develop an assay for tobramycin that does not contain vicinal diols in its structure. Amikacin, an aminoglycoside with a broad spectrum, was added to the study for its high therapeutic importance. An assay for both has been developed and validated. This method was developed on an XBridge C18 column (4.6 mm x 250 mm; 5 µm) and the mobile phase consisting of methanol-disodium tetraborate decahydrate buffer (0.1 M; pH=9.0)-water (20:20:60) supplemented with 1 g/L sodium octanesulfonate was run isocratically at a column temperature of 40°C. After validation according to International Conference on Harmonization guidelines, this method was tested on two commercially available pharmaceutical formulations, Amukin® and Tobrex®. The developed method was fast, accurate, easy to use and cheap as it did not require sophisticated equipment or tedious derivatization steps.

Investigation and application of photochemically induced direct UV detection of low or non-UV absorbing compounds by capillary electrophoresis

Some low or non-UV absorbing compounds like amino acids might be accessible to direct UV detection by capillary electrophoresis (CE), due to the photochemical reaction in the detection window of the separation capillary under extremely strong alkaline conditions. However, with regards to the photochemical reaction procedure and the influencing factors in CE, no comprehensive studies have been done. Herein, two strategies were applied to investigate the photochemical reaction mechanism including the introduction of an additional UV lamp and the utilization of driving pressure. The former confirmed the occurrence of photolysis, while the latter solved the interference of electroosmotic flow (EOF). Furthermore, the online photochemical reaction and online preconcentration technique were combined to develop a rapid, simple and sensitive method for determination of seven essential amino acids (valine, leucine, phenylalanine, methionine, tryptophan, threonine and lysine). Eventually, the developed method was successfully applied to the analysis of real samples with good reproducibility and reliability. This novel and simple method, based on the photochemical reactions occurring in the detection window and coupling with online preconcentration techniques, shows a great potential for the rapid and sensitive detection of low or non-UV absorbing compounds.