Development and validation of a simple and affordable LC-UV method for identification and assay of selected antimicrobial medicines.

Antimicrobials, particularly antibiotics, are among the most common classes of drugs reported as substandard and falsified (SF) in developing countries. Therefore, it is important to develop simple and affordable analytical methods for the quality control of antimicrobial medicines. In this study, a liquid chromatographic method with ultraviolet detection (LC-UV) was developed and validated for the screening and quantification of 13 antimicrobial medicines and one beta-lactamase inhibitor in pharmaceutical formulations. LC separation was carried out on a Kinetex C18 column (150 mm × 4.6 mm, 2.6 µm) with gradient elution. The mobile phase consisted of mixtures of acetonitrile-water-10 mM phosphate buffer pH 3.5 at ratios of 3:92:5, v/v/v for mobile phase A and 50:45:5, v/v/v for mobile phase B with a flow rate of 0.5 mL/min. The screening method was intended for confirmation of the identity of the actives and validated for specificity and robustness, whereas the quantification method (using only a different detection wavelength) was further validated in terms of linearity, accuracy, sensitivity and precision (repeatability, intermediate precision). For all compounds, the method was found to be linear (r2 > 0.999), precise (%RSD < 1%), accurate (% recovery of 98-102%), sensitive, specific and robust. The developed LC method was successfully applied for the identification and assay of 12 antimicrobial samples from Ethiopia. Among the 12 samples analyzed, one (8.3%) product was confirmed to be falsified.

A fast and efficient method for screening and evaluation of hypoglycemic ingredients of Traditional Chinese Medicine acting on PTP1B by capillary electrophoresis.

As a pivotal enzyme that regulates dephosphorylation in cell activities and participates in the insulin signaling pathway, protein tyrosine phosphatase 1B (PTP1B) is considered to be an important target for the therapy of diabetes. In this work, a rapid and efficient inhibitor screening method of PTP1B was established based on capillary electrophoresis (CE), and used for screening and evaluating the inhibition effect of Traditional Chinese Medicine on PTP1B. Response Surface Methodology was used for optimizing the conditions of analysis. After method validation, the enzyme kinetic study and inhibition test were performed. As a result, the IC50 of PTP1B inhibitors Ⅳ and ⅩⅧ were consistent with reported values measured by a conventional method. It was found that the extracts of Astragalus membranaceus (Fisch) Bunge and Morus alba L. showed prominent inhibition on the activity of PTP1B, which were stronger than the positive controls. Meanwhile, on top of the excellent advantages of CE, the whole analysis time is less than 2 min. Thus, the results demonstrated that a fast and efficient screening method was successfully developed. This method could be a powerful tool for screening inhibitors from complex systems. It can also provide an effective basis for lead compound development in drug discovery.

Impact of diluents on the calibration of direct dynamic thermal desorption prior to gas chromatography.

A thermal desorber (TD) can be used in different ways to introduce samples in a gas chromatographic (GC) system. Besides its conventional use where the collected analytes are released from the sorbent in the sample tube, direct dynamic desorption (DDD) is an interesting option where a solid sample is put directly in the TD tube. However, since no sorbent is used for the sample, proper calibration is not straightforward. This issue was investigated in the present work using offline liquid calibration (OLC) and inline liquid calibration (ILC). Unexpectedly, ILC yielded a lower response than OLC. This could be related to the adsorption kinetics of the analytes and water on the cold trap of the TD. More insight was gained performing double injection ILC experiments with toluene as diluent for the analytes and injecting water before or after the toluene solution. This revealed a clear influence of the diluent. The influence of water was further explored applying two cold trap temperatures (4 °C and -30 °C). Inserting a LiCl trap in the TD tube to capture the water was found to be an effective solution for the problem. Finally, quantitative aspects of this approach were demonstrated.

Determination of inorganic ions and carbohydrates in cardioplegia and nephroplegia solutions by ion chromatography.

In this study, methods for analyzing inorganic ions and carbohydrates in cardioplegia and nephroplegia solutions were developed and validated using ion chromatography with both conductivity and pulsed amperometric detection. The inorganic ions such as sodium, potassium, and calcium were separated by a cation-exchange column with 27 mM methanesulfonic acid as mobile phase at 0.5 mL/min. The anion (chloride) and carbohydrates (mannitol and glucose) were analyzed by an anion-exchange column using a mobile phase of 20 mM sodium hydroxide at 1.0 mL/min. The methods showed a high sensitivity for all analytes, with quantification limits from 0.0002 to 0.06 mg/L. Good linearities between the peak areas and concentrations were found for all analytes within the selected concentration range (R2  > 0.999). Relative standard deviation values for repeatability and interday precision were 0.1%-1.0% and 0.7%-1.6%, respectively. The accuracy was validated by determining the percentage recovery, which was between 98.0% and 101.3% for all analytes, indicating good accuracy of the methods. The robustness was verified by using an experimental design. Finally, real samples were analyzed to determine the content of the analytes. All assay values were between 96.8% and 102.5%.

A comprehensive review of capillary electrophoresis-based techniques for erythropoietin isoforms analysis.

Different CE techniques have been used to analyze erythropoietin. These techniques have been shown to be effective in differentiating and quantifying erythropoietin isoforms, including natural and recombinant origins. This review provides a comprehensive overview of various capillary electrophoresis-based techniques used for the analysis of erythropoietin isoforms. The importance of erythropoietin in clinical practice and the necessity for the accurate analysis of its isoforms are first discussed. Various techniques that have been used for erythropoietin isoform analysis are then described. The main body of the review focuses on the different capillary electrophoresis-based methods that have been developed for erythropoietin isoform analysis, including capillary zone electrophoresis and capillary isoelectric focusing. The advantages and drawbacks of each method as well as their applications are discussed. Suggestions into the future directions of the area are also described.

Overview of chromatographic and electrophoretic methods for the determination of branched-chain amino acids.

The significance of branched-chain amino acids in diseases was clearly shown over the years. This review aims to describe the available techniques for their analytical determination. The article provides examples of the use of various analytical methods. The methods are divided into two categories: derivatization and non-derivatization approaches. Separation is achieved through different chromatography or capillary electrophoresis techniques and can be combined with different detectors such as flame ionization, ultraviolet, fluorescence, and mass spectrometry. It compares the application of various derivatization reagents or detection as such for different detectors.

Simultaneous determination of ceftazidime and pyridine in human plasma by LC-UV.

A sensitive, accurate and precise liquid chromatography (LC) method for the simultaneous determination of ceftazidime and pyridine in human plasma has been developed and validated. Acetonitrile (ACN) was employed to precipitate the proteins in the plasma samples. Chromatographic separation was performed with a Kinetex® C18 (150 mm × 3 mm, 2.6 µm) column with gradient elution. Ammonium formate 20 mM and ACN were mixed in a ratio of 98:2 (v/v) for mobile phase A and 85:15 (v/v) for mobile phase B. Both were adjusted to pH 4.5 with formic acid. The flow rate was 0.4 mL/min. UV detection was performed at 254 nm. Calibration curves were linear in the range from 0.3 to 225 µg/mL for ceftazidime and from 0.2 to 10 µg/mL for pyridine with correlation coefficients ≥ 0.999. Within- and between-run precision and accuracy were satisfactory with coefficients of variation (CV) ≤ 8.0% and deviations ≤ 7.0%, respectively. The method fulfilled all validation criteria prescribed by the European Medicines Agency guidelines. Next, it has been used successfully to analyze plasma samples of patients who received ceftazidime under intermittent and continuous administration. With intermittent administration, the concentration of the antibiotics reached a peak and then dropped quickly, which may be below the minimal inhibitory concentration (MIC). With continuous administration, the concentration of the antibiotics remained stable over 24 h, certainly above the MIC. Although the same tendency in ceftazidime concentration changes over time was observed, a difference in concentration amongst the patients was noticeable. The concentration of pyridine in plasma was negligible.

Indoleamine 2,3 dioxygenase 1 immobilization on magnetic nanoparticles for screening inhibitors from coffee.

In this study, a ligand fishing method was developed to screen potential indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors from coffee extracts by immobilization of IDO1 enzyme on amino-modified magnetic nanoparticles combined with UHPLC-Q-TOF-MS/MS analysis. Parameters including enzyme concentration, immobilization time, the pH of glutaraldehyde and the amount of magnetic nanoparticles were optimized. The results indicated that immobilized IDO1 could be reused 5 times and was stable during storage for 7 days. Several IDO1 ligands were captured by incubating immobilized IDO1 with coffee extract, of which 10 showed an obvious difference comparing to non-conjugated bare nanoparticles. In vitro inhibitory activity was further performed by CE analysis, in which ferulic acid and chlorogenic acid had better IDO1 inhibitory activity, with IC50 value of 113.7 µM and 307.5 µM. These results demonstrate that this method provides an effective platform for identifying and screening IDO1 inhibitors from natural products.

Differentiation of tetracyclines and their 4-epimers by mass spectrometry of the alkali metal adduct ions.

Tetracyclines (TCs) are a family of broad-spectrum antibiotics. During the manufacturing process or storage, epimerization of tetracyclines could occur, leading to 4-epimers which are nearly inactive. From an analytical point of view, isomers are often difficult to distinguish. Previously, four pairs of TCs (oxytetracycline, tetracycline, doxycycline, chlortetracycline and their respective 4-epimers) were differentiated by mass spectrometry (MS) through protonated ions. However, they do not follow common rules and so it is still quite difficult to differentiate between them. In order to solve this, the four pairs were differentiated in the current study by collision induced dissociation (CID) spectra of the alkali adduct ions, including lithium, sodium and potassium. In the spectra of the sodium adducts, all studied tetracyclines showed a tendency to form [M+Na-NH3]+ ions, while the 4-epimers liked to form [M+Na-NH3-H2O]+ ions. Meanwhile, energy resolved mass spectrometry (ERMS) showed that all four 4-epimers' sodium adducts had the tendency to fragment at higher energy points. In the CID spectra of lithium adducts of TCs, a similar trend was observed for three pairs, except for doxycycline. For potassium adducts, the fragmentation was found to be less discriminative. As was derived from the 3D model, the four pairs all interact with the alkali metal through the dimethyl amino group at the C-4 position. The lithium adduct species also bound through the hydroxyl group at the C-5 position. If the TCs did not have a hydroxyl group at the C-5 position, they bound with the hydroxyl group at the C-6 position. For the same TC, with an increase of the diameter of the metal ion, the loss of H2O decreased gradually. As sodium adduct ions are common during the ionization process, TCs and their 4-epimers could be differentiated rapidly by ERMS of the sodium adduct ions.

Screening assays for tyrosine kinase inhibitors: A review.

Tyrosine kinases have been intensively investigated as drug targets for several decades, since they regulate many cellular processes including cell growth, differentiation, and proliferation. Indeed, the deregulation of tyrosine kinases has been confirmed to play a vital role in the pathophysiology of many diseases. During the last few years, varieties of techniques have been developed to search for new tyrosine kinase inhibitors for cancer therapy, such as traditional filtration binding assay, scintillation proximity assay and some high-throughput screening methods. In this review, we describe the basic rules, merits and demerits, and application of a number of general and advanced technologies. The purpose of this review is to provide an insight into the numerous assays to achieve the exploration of new tyrosine kinase inhibitors.

Off-line and on-line liquid chromatography-mass spectrometry methods with immobilized bio-macromolecules for drug screening from natural sources.

Natural products are an important source of major compounds in drug discovery. Currently, rapid screening and identification of bioactive compounds is a challenge due to the complicated chemical composition of natural products. Affinity screening methods based on liquid chromatography coupled with mass spectrometry have seen increasing interest in the past few years. In this review, the various strategies are classified into off-line and on-line modes. The principles and applications of these screening methods such as magnetic nanoparticles, affinity solid-phase extraction, immobilized liposome chromatography and cell membrane chromatography are introduced. This review covers the research advances of LC-MS based screening methods from 2019 to mid 2022, discussing their advantages and disadvantages, and providing an outlook for the future of this field.

Determination of Inorganic Ions in Parenteral Nutrition Solutions by Ion Chromatography.

A new, simple and sensitive ion chromatography (IC) method for the determination of sodium, potassium, magnesium, calcium and chloride in a parenteral nutrition (PN) solution was developed and validated. Before sample analysis, a sample pretreatment by calcination was applied which could totally remove interference from other constituents of the PN solution. Methanesulfonic acid (MSA) and sodium hydroxide were used as the mobile phase for the determination of cations and anions, respectively. The calibration curves showed good correlation between analyte peak area and concentration (r2 > 0.999). Detection limits ranged from 0.0001 to 0.02 mg/L and quantification limits from 0.0002 to 0.06 mg/L. Relative standard deviation (RSD) values for repeatability and inter-day precision did not exceed 1.0% and the recoveries for all analytes were between 99.1−101.1%. The robustness was verified by using an experimental design.

Migration study of phenolic endocrine disruptors from pacifiers to saliva simulant by solid phase microextraction with amino-functionalized microporous organic network coated fiber.

Parabens, bisphenols, and triclosan are used in many baby products, including pacifiers. However, the migration through oral saliva will result in a potential health risk. The present study proposes a sensitive and simple method for the analysis of these chemicals in saliva simulants by solid phase microextraction (SPME) with amino-functionalized microporous organic network (MON-NH2) coated fiber. The MON-NH2 showed an excellent adsorption ability for phenolic compounds. The adsorption isotherm fitted the Langmuir isotherm model and the adsorption kinetics followed the pseudo second-order model. The developed SPME method exhibited wide linear ranges (0.005-500 µg/L), good linearity, low limits of quantitation (0.005 µg/L), great recoveries (87.0-112.5 %), and excellent precision (RSD < 8.3 % for intra-day and RSD < 13.7 % for inter-day). Mathematical models based on Fick's second law were applied to predict migration from pacifiers into saliva simulants and a good fit between theoretical and experimental migration results was found. The daily exposure assessment results indicated that these chemicals in pacifiers do not pose unacceptable health risks to infants. However, exposure risks still should be monitored and appropriate precautions are still needed to protect infants from exposure to these chemicals.

A disease-associated missense mutation in CYP4F3 affects the metabolism of leukotriene B4 via disruption of electron transfer.

BACKGROUND: Cytochrome P450 4F3 (CYP4F3) is an ω-hydroxylase that oxidizes leukotriene B4 (LTB4), prostaglandins, and fatty acid epoxides. LTB4 is synthesized by leukocytes and acts as a chemoattractant for neutrophils, making it an essential component of the innate immune system. Recently, involvement of the LTB4 pathway was reported in various immunological disorders such as asthma, arthritis, and inflammatory bowel disease. We report a 26-year-old female with a complex immune phenotype, mainly marked by exhaustion, muscle weakness, and inflammation-related conditions. The molecular cause is unknown, and symptoms have been aggravating over the years. METHODS: Whole exome sequencing was performed and validated; flow cytometry and enzyme-linked immunosorbent assay were used to describe patient's phenotype. Function and impact of the mutation were investigated using molecular analysis: co-immunoprecipitation, western blot, and enzyme-linked immunosorbent assay. Capillary electrophoresis with ultraviolet detection was used to detect LTB4 and its metabolite and in silico modelling provided structural information. RESULTS: We present the first report of a patient with a heterozygous de novo missense mutation c.C1123 > G;p.L375V in CYP4F3 that severely impairs its activity by 50% (P < 0.0001), leading to reduced metabolization of the pro-inflammatory LTB4. Systemic LTB4 levels (1034.0 ± 75.9 pg/mL) are significantly increased compared with healthy subjects (305.6 ± 57.0 pg/mL, P < 0.001), and immune phenotyping shows increased total CD19+ CD27- naive B cells (25%) and decreased total CD19+ CD27+ IgD- switched memory B cells (19%). The mutant CYP4F3 protein is stable and binding with its electron donors POR and Cytb5 is unaffected (P > 0.9 for both co-immunoprecipitation with POR and Cytb5). In silico modelling of CYP4F3 in complex with POR and Cytb5 suggests that the loss of catalytic activity of the mutant CYP4F3 is explained by a disruption of an α-helix that is crucial for the electron shuffling between the electron carriers and CYP4F3. Interestingly, zileuton still inhibits ex vivo LTB4 production in patient's whole blood to 2% of control (P < 0.0001), while montelukast and fluticasone do not (99% and 114% of control, respectively). CONCLUSIONS: A point mutation in the catalytic domain of CYP4F3 is associated with high leukotriene B4 plasma levels and features of a more naive adaptive immune response. Our data provide evidence for the pathogenicity of the CYP4F3 variant as a cause for the observed clinical features in the patient. Inhibitors of the LTB4 pathway such as zileuton show promising effects in blocking LTB4 production and might be used as a future treatment strategy.

Hollow fiber membrane-protected amino/hydroxyl bifunctional microporous organic network fiber for solid-phase microextraction of bisphenols A, F, S, and triclosan in breast milk and infant formula.

Bisphenols and triclosan have been used in various products, and exposure to these chemicals may affect human health. The present study proposes a sensitive method for the determination of bisphenols A, F, S, and triclosan. The fiber was coated by amino/hydroxyl bifunctional microporous organic network and protected by polyvinylidene fluoride hollow fiber membrane for direct immersion solid phase microextraction. The limit of detection was 0.005 µg/L (µg/kg), and the recoveries were in the range of 76.7% to 107.5% (87.4% to 107.6%) for breast milk (infant formula), with intra-day and inter-day precisions <10.5% (7.3%) and 13.6% (8.4%), respectively. Fiber-to-fiber reproducibility of < 9.5% and a lifespan of >100 cycles were obtained. The 95th percentile estimated daily intake of total bisphenols was close to temporary tolerable daily intake for infants fed by human milk, which highlighted the needs for further attention on human exposure to BPA and its substitutes.

Determination of pesticide residues in chilli and Sichuan pepper by high performance liquid chromatography quadrupole time-of-flight mass spectrometry.

In this study, a multi-component analytical method for the detection of pesticide residues in chilli and Sichuan pepper by high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC-Q-TOF/MS) was developed and validated. The sample preparation is based on an extraction step with acetonitrile followed by a cleanup step using primary secondary amine, C18, graphitized carbon black and anhydrous magnesium sulfate. Values of matrix effects ranged from -55.8 to 26.0 % for chilli, and -69.1 to 24.0 % for Sichuan pepper. The LOQ of ≤ 5 µg kg-1 was achieved for all the target pesticides. Applying the method to real samples, some pesticides were found at high concentrations, which were beyond the MRL set by the EU. The results showed that the developed method could be used for the quantitative analysis of target pesticides and non-target screening for potential metabolites in chilli and Sichuan pepper.

The effect of Gamma and Ethylene Oxide Sterilization on a Selection of Active Pharmaceutical Ingredients for Ophthalmics.

Ensuring the sterility of life science products plays a pivotal role in the healthcare sector. Gamma irradiation and ethylene oxide sterilization are two commonly applied methods for the sterilization of medical devices, packaging components and Active Pharmaceutical Ingredients (API) for medicinal products. Focussed studies on the effects of sterilization processes on APIs remain limited. In this research study, five APIs, frequently used in sterile ophthalmic preparations were subjected to both gamma irradiation and ethylene oxide under different process conditions. The following APIs of GMP quality were selected: dexamethasone, aciclovir, tetracycline hydrochloride, triamcinolone and methylprednisolone. Analyses were performed using High Performance Liquid Chromatography equipped with UV detection and the effect of sterilization conditions on the APIs was evaluated by the assay and related substances test prescribed by the European Pharmacopoeia (Ph. Eur.). It was concluded that exposure to ethylene oxide resulted in compliance with Ph. Eur. for all APIs. While dexamethasone and methylprednisolone did not meet the requirement for the Ph. Eur. after exposure to gamma irradiation, the other three APIs did meet the requirement under the specified irradiation conditions. Subsequent optimization of sterilization parameters positively influenced the compliance to the Ph. Eur. requirements.

Quantification of allantoin and other metabolites of the purine degradation pathway in human plasma samples using a newly developed HILIC-LC-MS/MS method.

The development of a simple HILIC-LC-MS/MS method to quantify the plasma levels of allantoin, inosine, hypoxanthine, and adenosine, using stripped plasma for the bioanalytical method validation, was the purpose of this study. Chromatographic separation conducted using an XBridge BEH Amide column (2.1 × 150 mm, 3.5 µm) was achieved under gradient elution with two mobile phases: 0.1% formic acid-ACN (5:95) and 0.1% formic acid-ACN (50:50). Multiple reaction monitoring MS detection was performed using a triple quadrupole. The method validation experiments were performed according to the European Medicines Agency and the U.S. Food and Drug Administration guidelines. The lower LOQ was 50 nM, 5 nM, 20 nM, and 2 nM for allantoin, inosine, hypoxanthine, and adenosine, respectively. The recovery was repeatable and stable. The intraday precision ranged from 1.6% to 6.5%, while the interday precision ranged from 3.4% to 58.7%. Therefore, it is necessary to make a matrix-matched calibration curve each day to overcome this issue. Since the quality control samples' stability did not always comply with the guidelines, the samples need to be analyzed soon after collection.

Evaluation of aloins, pH and moisture in aloe leaf gel-based personal care products.

OBJECTIVE: Some easily applicable analytical methods were explored to evaluate the quality of personal care products containing aloe leaf gel. Aloins should be absent in these products in view of their side effects. To check this, liquid chromatography (LC) was applied. METHODS: The LC method used a C18 monolithic column combined with gradient elution and ultraviolet (UV) detection. The mobile phase consisted of a mixture of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The method was validated with respect to specificity, linearity, precision and accuracy. Next, it was practically applied for the analysis of commercial samples. In addition, the pH and moisture content were determined. RESULTS: The LC results indicated that aloins were detected in 25% of the analysed commercial samples. Further, it turned out that 42% of the test samples were found to be in the basic pH range and 33% of them contained excessive moisture. CONCLUSION: Proper quality control and adequate labelling of aloe leaf gel-based cosmetics are mandatory to avoid side effects.

OBJECTIF: Certaines méthodes analytiques facilement applicables ont été explorée pour évaluer la qualité des produits de soins personnels contenants du gel de feuille d'aloe. Les aloïnes doivent être absentes de ces produits en raison de leurs effets secondaires. Pour vérifier cela, la chromatographie liquide (CL) a été appliquée. MÉTHODES: La méthode CL a utilisé une colonne monolithique C18 combinée à une élution par gradient et une détection ultraviolette (UV). La phase mobile était constituée d'un mélange de 0,1 % d'acide formique dans l'eau (A) et de 0,1 % d'acide formique dans l'acétonitrile (B). La méthode a été validée en ce qui concerne la spécificité, la linéarité, la précision et l'exactitude. Ensuite, elle a été appliquée pour l'analyse d'échantillons commerciaux. De plus, le pH et la teneur en humidité ont été déterminés. RÉSULTATS: Les résultats CL ont indiqué que des aloïnes ont été détectées dans 25 % des échantillons commerciaux analysés. Il s'est avéré que 42 % des échantillons se trouvaient dans la plage de pH basique et que 33 % d'entre eux contenaient une humidité excessive. CONCLUSION: Un contrôle de qualité approprié et un étiquetage adéquat des cosmétiques à base de gel de feuille d'aloe sont obligatoires pour éviter des effets secondaires.