Analytical studies on some pesticides with antifungal effects: Simultaneous determination by HPLC, investigation of interactions with DNA and DNA damages.

A simple, and fast method was developed for the simultaneous determination of five fungicides, namely thiram (THR), epoxiconazole (EPO), hexaconazole (HEX), tebuconazole (TEB), and diethofencarb (DIE), in different matrices by HPLC-UV. Parameters influencing the peak shape and resolution, such as the composition of mobile phase, pH and concentration of buffer solution, and column temperature, were examined and optimized. The proposed method was validated in terms of linearity, sensitivity, precision, and accuracy. Forced degradation studies were carried out for all analytes to demonstrate the specificity of the method and to evaluate the stability of analytes under different conditions. DNA interaction and DNA damage studies were conducted by HPLC and comet assay, respectively. All fungicides were found to bind DNA, except for DIE. While the binding coefficients for EPO, HEX, and TEB were of the order of 104, THR was found to interact more strongly with DNA with a binding coefficient of higher than 106. DIE did not induce DNA damage at any concentration tested. On the other hand, TEB, HEX, and EPO induced DNA damage up to 30 µg/mL. THR showed cytotoxic effects at 20 and 30 µg/mL and caused significant DNA damage at lower concentrations.

Lab-in-syringe automated protein precipitation and salting-out homogenous liquid-liquid extraction coupled online to UHPLC-MS/MS for the determination of beta-blockers in serum.

A sample preparation method involving tandem implementation of protein precipitation and salting-out homogenous liquid-liquid extraction was developed for the determination of beta-blockers in serum. The entire procedure was automated using a computer-controlled syringe pump following the Lab-In-Syringe approach. It is based on the denaturation of serum proteins with acetonitrile followed by salt-induced phase separation upon which the proteins accumulate as a compact layer at the interphase of the solutions. The extract is then separated and diluted in-syringe before being submitted to online coupled UHPLC-MS/MS. A 1 mL glass syringe containing a small stir bar for solution mixing at up to 3000 rpm, was used to deal with sample volumes as small as 100 µL. A sample throughput of 7 h-1 was achieved by performing the chromatographic run and sample preparation procedure in parallel. Linear working ranges were obtained for all analytes between 5 and 100 ng mL-1, with LOD values ranging from 0.4 to 1.5 ng mL-1. Accuracy values in the range of 88.2-106% and high precision of <11% RSD suggest applicability for routine analysis that can be further improved using deuterated standards.

Automated centrifugation-less milk deproteinization and homogenous liquid-liquid extraction of sulfonamides for online liquid chromatography.

A novel approach to the determination of sulfonamides in milk based on the Lab-In-Syringe technique is presented. The method involves automated salting-out liquid-liquid extraction of the analytes, allowing simultaneous sample deproteination without requiring centrifugation or manual sample handling. The procedural parameters, including salt type, solvent-to-sample ratio, and salt solution volume, were studied. The extracts obtained were diluted in situ and transferred to online coupled liquid chromatography for analyte separation carried out in parallel to the subsequent extraction. In this way, a sample throughput of approximately 6 h-1 was achieved. The detection limits ranged from 25 to 32 µg L-1 using a 500 µL milk sample and spectrophotometric detection. The applicability of the developed method to sample analysis was proven by recovery values ranging from 73.2% to 94.1% (86.3% on average) for milk samples of different fat content spiked at 3 µg mL-1 level. Straightforward automation of one of the most laborious preparation steps in food analysis, i.e., deproteination, was demonstrated.

Lab-In-Syringe automation of deep eutectic solvent-based direct immersion single drop microextraction coupled online to high-performance liquid chromatography for the determination of fluoroquinolones.

Lab-In-Syringe direct immersion single drop microextraction is proposed as an automated sample pretreatment methodology and coupled online to HPLC with fluorescence detection for the determination of fluoroquinolones in environmental waters. For the first time, a drop of a natural deep eutectic solvent (NADES), synthesized from hexanoic acid and thymol, has been used as an extractant in automated single-drop microextraction. The extraction procedure was carried out within the 5 mL void of an automatic syringe pump. A 9-position head valve served the aspiration of all required solutions, air, waste disposal, and hyphenation with the HPLC instrument. Sample mixing during extraction was done by a magnetic stirring bar placed inside the syringe. Only 60 µL of NADES were required omitting toxic classical solvents and improving the greenness of the proposed methodology. By direct injection, linear working ranges between 0.1 and 5 µg L-1 were achieved for all fluoroquinolones. The limit of quantification values and enrichment factors ranged from 20 ng L-1 to 30 ng L-1 and 35 to 45, respectively. Accuracies obtained from the analysis of spiked surface water and wastewater treatment plant effluent analysis at two concentration levels (0.5 and 4 µg L-1) ranged from 84.6% to 119.7%, with RSD values typically <3%.

Novel Trends in Analytical Methods for ß-Blockers: An Overview of Applications in the Last Decade.

ß-blockers are among the most prescribed drugs and frequently recommended in patients with hypertension, angina, myocardial infarction, arrhythmias, and heart failure. Additionally, since ß-blockers cause low anxiety, they are abused by athletes in branches where aiming is important. ß-blocker residues may be present in environmental samples due to high consumption. At this point, the development of sensitive, rapid, and reliable analytical methods is very important for determination of ß-blockers in different matrices. Among all the analytical methods, chromatographic methods are used in a wide range for the determination of ß-blockers as they enable simultaneous determination of many analytes and removal of interfering components. Electrochemical methods, which usually do not require any pretreatment process, have become important due to their reliability, high specificity, and fast response. Simple and rapid optical methods can be used in laboratories where modern and expensive instruments such as high-performance liquid chromatography or gas chromatography are not available. The objective of this review is to provide an overview of the analytical methods for determination of ß-blockers in pharmaceuticals, biological fluids, and environmental samples. Some selected studies for the determination of ß-blockers published in the last decade are discussed. Furthermore, current trends and future perspectives on ß-blocker analyses are also suggested.

Analytical Methods for Determination of Antiviral Drugs in Different Matrices: Recent Advances and Trends.

Viruses are the main pathogenic substances that cause severe diseases in humans and other living things. They are among the most common microorganisms, and consequently, antiviral drugs have emerged to prevent and treat viral infections. Antiviral drugs are an essential drug group considering their prescription and consumption rates for different diseases and indications. Therefore, it is crucial to develop accurate and precise analytical methods to detect antiviral drugs in various matrices. Chromatographic techniques are used frequently for the quantification purpose since they allow simultaneous determination of antivirals. Electrochemical methods have also gained importance since the analysis can be performed quickly without the need for pretreatment. Spectrophotometric and spectrofluorimetric methods are used because they are simple, inexpensive, and less time-consuming methods. The purpose of this review is to present an overview of the analysis of currently used antiviral drugs from 2010 to 2021. Since studies on antiviral drugs are numerous, selected publications were reviewed in this article. The analysis of antiviral drugs was divided into three main groups: chromatographic, spectrometric, and electrochemical methods which were applied to different matrices, including pharmaceutical, biological, and environmental samples.

Vortex assisted liquid-liquid microextraction based on in situ formation of a natural deep eutectic solvent by microwave irradiation for the determination of beta-blockers in water samples.

In this study, a simple, green, and reliable method combining vortex-assisted liquid-liquid microextraction based on in situ formation of a novel hydrophobic natural deep eutectic solvent (NADES-VA-LLME) and high-performance liquid chromatography (HPLC) was developed for the determination of metoprolol and propranolol in water samples. The novel NADES was synthesized in situ within only 20 s by subjecting the water sample containing azelaic acid and thymol to microwave irradiation at 50 ˚C. Initial studies indicated that a 17:1 ratio of thymol to azelaic acid yielded the highest response for analytes. The influence of 7 parameters, including NADES volume, salt amount, sample pH, vortex time, centrifugation time, microwave time, and temperature, were screened using a 27-3 fractional factorial design. The obtained significant parameters were optimized by response surface methodology employing a Box-Behnken design. The method displayed satisfactory linearity (r=0.9996) for metoprolol and propranolol with limits of detection of 0.2 and 0.1 µg/L, respectively. The relative standard deviation at 2.5, 40, and 80 µg/L levels was lower than 6%, with accuracy in the range of 90.8-100.2%. Enrichment factors were 147.0 and 144.4 for metoprolol and propranolol, respectively. This study demonstrates that the developed in situ NADES-VA-LLME-HPLC technique can be considered as a fast and environmentally friendly alternative for isolation/preconcentration of ß-blockers from water samples.

Interaction of citalopram and escitalopram with calf Thymus DNA: A spectrofluorometric, voltammetric, and liquid chromatographic approach.

Citalopram (CIT) and its S-enantiomer, escitalopram (ESC), are antidepressants belonging to the class called selective serotonin reuptake inhibitors and have many different pharmacological and biological properties. Understanding the interaction mechanism of small drug molecules with DNA both helps in the development of new DNA-targeted drugs and provides more in-depth knowledge for controlling gene expression. In this study, the interaction of CIT and ESC with double-stranded calf thymus DNA (ct-dsDNA) was investigated for the first time. Spectrofluorometric, liquid chromatographic, and voltammetric response profiles of drugs and ct-dsDNA at different concentrations showed DNA-drug complex formation. Calculated binding constants were greater with all three techniques for ESC compared to CIT and were of the order of 103-104, which is in accordance with those of well-known groove binders. The results also showed the significant effect of chirality on complex formation. The thermodynamic parameters, including free energy change (ΔG < 0) and enthalpy change (ΔH < 0) obtained at different temperatures, indicated that complex formation was mainly driven by hydrogen bonding and van der Waals forces for both drugs. The results of this study may enhance the understanding of the interaction between CIT or ESC and ct-dsDNA and can be considered as the pioneer for future studies to uncover possible hidden phenotypes of these compounds.

Simple and Fast Determination of Terbinafine in Human Urine by Dilute and Shoot HPLC-DAD Using a Core-Shell Column.

BACKGROUND: Terbinafine is an allylamine antifungal that is effective against many fungi, dermatophytes and moulds. Analytical methods are required for the determination of terbinafine in biological fluids to perform therapeutic drug monitoring and pharmacokinetic studies. OBJECTIVE: The aim of this study was to develop and validate a novel and fast method combining dilute and shoot approach and high-performance liquid chromatography coupled with photodiode array detection for the determination of terbinafine in human urine. METHODS: Chromatographic parameters including mobile phase composition, pH, flow rate and injection volume were assessed and optimized. The separation of terbinafine and naproxen (internal standard) was achieved within 3 min using a C18 core-shell column (Raptor ARC-18, 100 x 4.6 mm, 2.7 µm) under isocratic conditions. Samples were eluted from the column at the flow rate of 1.4 mL/min using a mobile phase containing 0.2% triethylamine in water (pH 3.4 with formic acid): acetonitrile (45:55, v/v). RESULTS: The presented technique was linear in the range of 25-2000 ng/mL. Intra- and inter-day reproducibility at four quality control levels (25, 200, 750 and 1500 ng/mL) were less than 7%, with relative errors ranging from -5.40% to 5.91%. The limit of detection was 12.60 ng/mL. The developed method has three main advantages compared to existing methods: simplicity and greenness of sample preparation, use of core-shell column and short analysis time. CONCLUSION: The results of this study indicate that the combination of dilute and shoot approach and core-shell column can be regarded as an advantageous application for the fast determination of terbinafine in the urine.

Determination of aluminum concentrations of parenteral nutrition solutions by HPLC.

Aluminum (Al) contamination of parenteral nutrition (PN) solutions has been known for over 30 years. In particular, vascular intake of Al leads to its accumulation in tissues. In this study, 8 all-in-one PN solutions the aluminum concentration was analyzed by high-performance liquid chromatography. The mean Al concentration of the glucose solutions of the PN solutions combinations was 16.36 ± 8.31 µg/L, the mean Al concentration of the amino acid solutions was 4.96 ± 3.73 µg/L, and the mean Al concentration of the lipid solutions was 9.09 ± 11.23 µg/L. The Al concentration of the PN5 glucose and PN2 lipid solutions were above 25 µg/L, which is the limit set by the Food and Drug Administration (FDA). No studies in the literature have examined the Al concentrations of all-in-one PN solutions via HPLC. In two of the analyzed solutions, the Al concentration was found to be higher than the limit set by the FDA.

Determination of levofloxacin, ciprofloxacin, moxifloxacin and gemifloxacin in urine and plasma by HPLC-FLD-DAD using pentafluorophenyl core-shell column: Application to drug monitoring.

Concentrations of fluoroquinolones, which are used in the treatment of many bacterial infections, should be monitored in biological fluids as they exhibit concentration-dependent bactericidal activity. In this study, a liquid chromatography method for the determination of levofloxacin, ciprofloxacin, moxifloxacin and gemifloxacin in human urine and plasma was developed for the first time. The efficiency of five different columns for the separation of these fluoroquinolones was compared. Experimental parameters that affect the separation, such as percentage of organic solvent, pH, temperature, gradient shape and detector wavelength, were optimized by a step-by-step approach. Using a pentafluorophenyl core-shell column (100 × 4.6 mm, 2.7 µm), the separation of four analytes was accomplished in <7.5 min. The developed method was validated for the determination of analytes in both urine and plasma with respect to sensitivity, specificity, linearity (r ≥ 0.9989), recovery (79.46-102.69%), accuracy, precision and stability (85.79-111.07%). The intra- and inter-day accuracies were within 89.55-111.94% with relative standard deviations of 0.35-8.05%. The feasibility of method was demonstrated by analyzing urine and plasma samples of patients orally receiving levofloxacin, ciprofloxacin or moxifloxacin. The developed method is suitable for therapeutic drug monitoring of these fluoroquinolones and can be applied to pharmacokinetic and toxicological studies.

Effect of cytokinins on in vitro multiplication, volatiles composition and rosmarinic acid content of Thymus leucotrichus Hal. shoots.

An efficient in vitro multiplication protocol was designed to Thymus leucotrichus, a subshrub and perennial herb growing naturally in the Northwest of Turkey. Of all basal media studied, Murashige and Skoog medium was found to be superior to the others, providing higher shoot formation and the maximum shoot length. Varying concentrations of cytokinins, i.e., 6-benzyladenine, thidiazuron, 2-isopentenyladenine and kinetin were supplemented in the nutrient media to observe their effects on shoot development and biomass. Rosmarinic acid content and volatile compositions of both naturally growing plants and in vitro multiplied plantlets were also evaluated. 6-benzyladenine (1.0 mg/L) and kinetin (0.5 mg/L) were found to be optimum for shoot number and shoot elongation, respectively. Thidiazuron (1.0 mg/L) was superior for biomass production. Rosmarinic acid content of in vitro multiplied plants was found to be higher than that of wild plants, reaching a maximum with 0.5 mg/L 2-isopentenyladenine, which yielded 10.15 mg/g dry weight. The highest thymol content was obtained with 1.0 mg/L kinetin (55.82%), while thidiazuron (0.1 mg/L) increased carvacrol production (12.53%). Overall, Murashige and Skoog medium supplemented with 1.0 mg/L kinetin was determined to be the most favorable medium studied.

Fast determination of anthocyanins and free pelargonidin in fruits, fruit juices, and fruit wines by high-performance liquid chromatography using a core-shell column.

Anthocyanins are water-soluble pigments that are liable for colors ranging from red to blue of most fruits, vegetables, and flowers. A novel and fast method was developed for the determination of five anthocyanins and free pelargonidin by high-performance liquid chromatography coupled to photodiode array detection. A 10% formic acid and acetonitrile mixture was employed as mobile phase in the gradient elution mode. Mobile phase composition, column temperature, flow rate, injection volume, and column conditioning time were optimized by employing a stepwise strategy. Using a C18 core-shell column (100 × 4.6 mm, 2.7 µm), the separation of six analytes was accomplished in less than 9.5 min with a run-to-run analysis time of 19 min. The developed method was validated with respect to linearity (r > 0.9999), limit of detection, limit of quantification, intra-/interday precision (<2%), accuracy (98.6-104.4%), and specificity. Afterwards, the method was applied to the determination of anthocyanins present in 15 different samples including fruits, fruit juices, and fruit wines.