Search Results (1,188)

The article presents the first method based on high-performance liquid chromatography and ultraviolet detection (HPLC-UV) for the determination of timonacic (thioproline, 1,3-thiazolidine-4-carboxylic acid, tPro) in pharmaceutical tablets and face care products (creams, sera, foundations, suncreams). Sample preparation primarily involves solid-liquid extraction (SLE) of tPro with 0.2 mol/L phosphate buffer pH 6, derivatization with 0.25 mol/L 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT), followed by polytetrafluoroethylene (PTFE) membrane filtration. The chromatographic separation of the stable UV-absorbing 2-S-quinolinium derivative is achieved within 14 min at 25 °C on a Zorbax SB-C18 (150 × 4.6 mm, 5 µm) column using gradient elution. The eluent consists of 0.1 mol/L trichloroacetic acid (TCA), pH 1.7, in a mixture with acetonitrile (ACN) delivered at a flow rate of 1 mL/min. The analyte is quantified by monitoring at 348 nm. The assay linearity was observed within 0.5–125 μmol/L. The limit of quantification (LOQ) was found to be 0.5 μmol/L. The accuracy ranged from 93.22% to 104.31% and 97.38% to 103.48%, while precision varied from 0.30% to 11.23% and 1.13% to 9.64% for intra- and inter-assay measurements, respectively. The method was successfully applied to commercially available on the Polish market pharmaceutical and cosmetic products. Full article

Background/Objectives: The emergence of cathinone-based psychostimulants necessitates ongoing research and analysis of the characteristics and properties of novel derivatives. The metabolic fate of five novel cathinone-derived substances (ASProp, MASProp, MASPent, PySProp, and PySPent) containing a selenophene moiety was investigated in vitro and in vivo. Methods: All compounds were incubated individually with pooled human liver S9 fraction. A monooxygenase activity screening investigating the metabolic contribution of eleven recombinant phase I isoenzymes was conducted. Rat urine after oral administration was prepared by urine precipitation. Liquid chromatography–high-resolution tandem mass spectrometry was used for the analysis of all samples. Reversed-phase liquid chromatography (RPLC) and zwitterionic hydrophilic interaction liquid chromatography (HILIC) were used to evaluate and compare the metabolites’ chromatographic resolution. Results: Phase I reactions of ASProp, MASProp, MASPent, PySProp, and PySPent included N-dealkylation, hydroxylation, reduction, and combinations thereof. The monooxygenase activity screening revealed the contribution of various isozymes. Phase II reactions detected in vivo included N-acetylation and glucuronidation. Both chromatographic columns complemented each other. Conclusions: All substances revealed metabolic reactions comparable to those observed for other synthetic cathinones. Contributions from isozymes to their metabolism minimized the risk of drug–drug interactions. The identified metabolites should be considered as targets in human biosamples, especially in urine screening procedures. RPLC and HILIC can both be recommended for this purpose. Full article

This study presents an advanced analytical method for the simultaneous quantification of malondialdehyde (MDA), a biomarker of oxidative stress, and diphenyl phosphate (DPhP), a metabolite of the organophosphate flame retardant triphenyl phosphate (TPhP), in human urine. The method integrates hydrophilic interaction liquid chromatography (HILIC), a type of liquid chromatography suitable for polar compounds, for MDA separation, and an online restricted access material (RAM), a preconcentration column, for DPhP isolation, achieving high specificity and sensitivity. Validation with certified urine samples confirmed its robustness across diverse analyte concentrations and complex biological matrices. The optimized clean-up steps effectively minimized carryover, allowing for high-throughput analysis. Application to 72 urine samples revealed a significant positive correlation (ρ = 0.702, p-value = 1.9 × 10⁻⁷) between MDA and DPhP levels, supporting a potential link between oxidative stress and TPhP exposure. The subset analysis demonstrated a statistically significant moderate positive correlation in women (ρ = 0.622, p-value = 0.020), although this result should be interpreted with caution because of the limited sample size (N = 14). This method provides a powerful tool for biomonitoring oxidative stress and environmental contaminants, offering valuable insights into exposure-related health risks. Full article

Background/Objectives: Selinexor is a selective nuclear-export inhibitor approved for hematologic malignancies, characterized by extensive plasma protein binding (>95%). However, a validated analytical method to accurately measure the clinically relevant unbound fraction of selinexor in human plasma has not yet been established. This study aimed to develop a fully validated bioanalytical assay for simultaneous quantification of total and unbound selinexor concentrations in human plasma. Methods: We established and fully validated an analytical method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) capable of quantifying total and unbound selinexor concentrations in human plasma. Unbound selinexor was separated using ultrafiltration, and selinexor was efficiently extracted from 50 μL of plasma by liquid–liquid extraction. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase (0.1% formic acid:methanol, 12:88 v/v) with a relatively short runtime of 2.5 min. Results: Calibration curves showed excellent linearity over a range of 5–2000 ng/mL for total selinexor (r² ≥ 0.998) and 0.05–20 ng/mL for unbound selinexor (r² ≥ 0.995). The precision (%CV ≤ 10.35%) and accuracy (92.5–104.3%) for both analytes met the regulatory criteria. This method successfully quantified selinexor in plasma samples from renally impaired patients with multiple myeloma, demonstrating potential inter-individual differences in unbound drug concentrations. Conclusions: This validated bioanalytical assay enables precise clinical pharmacokinetic assessments in a short runtime using a small plasma volume and, thus, assists in individualized dosing of selinexor, particularly for renally impaired patients with altered protein binding. Full article

This paper presents a numerical investigation into the generation of 2D ordered pillar array columns for liquid chromatography columns, focusing on the development of an algorithm for the automatic creation of unit-cell morphologies and their subsequent computational fluid dynamics (CFD) simulation. The algorithm is developed to incorporate functional and operational constraints, which ensure that the generated structures are permeable and suitable for chromatographic separations. The functional constraints include the principal pathway and no dry void constraints, while the operational constraints involve symmetry and porosity thresholds. The algorithm’s efficacy is demonstrated with a reduction rate of 97.8% for order 5 matrices. CFD simulations of the generated morphologies reveal that the homogeneity of the fluid velocity profile within the unit cell is a key determinant of separation performance, suggesting that refining the resolution of discrete unit cells could enhance separation efficiency. Future work will explore the inclusion of more complex morphologies and the impact of particle shape and size on separation efficiency. Full article

The application of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) in the analysis of peptide therapeutics demonstrates its capacity to achieve high sensitivity and selectivity, which are essential qualities for the expanding peptide therapeutic industry. Given the challenges posed by hydrophilic peptides in reversed-phase chromatography, we investigated the necessity of a derivatization procedure to improve chromatographic separation and quasimolecular ion fragmentation during MS/MS detection. We investigated how eight different derivatizing agents react with a hydrophilic lysine- and arginine-containing human ezrin peptide-1 (HEP-1) to identify the most suitable one. The results showed that the reaction of HEP-1 with propionic anhydride proceeds most rapidly and completely, providing a high and reproducible yield of the product, which has sufficient retention on the RP column. The 4-propionylated derivative of HEP-1, compared to the other derivatives considered, demonstrates the most pronounced MS/MS fragmentation. The retention time of 2.42 min allows the separation of the substance from the interfering components of the blood plasma matrix and provides a limit of quantification of 5.00 ng/mL, which allows the use of this derivatizing agent for subsequent applications in pharmacokinetic studies, and this approach can improve the analytical parameters of similar peptides in other HPLC-MS/MS studies. Full article

A sensitive method was developed for detecting diazepam residues in aquatic products using magnetic dispersive solid-phase extraction (MDSPE) coupled with ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). Samples extracted with 1% ammonia–acetonitrile were purified using synthesized Fe₃O₄@SiO₂-PSA nanoparticles via MDSPE before UPLC-MS/MS analysis. Separation was performed on a C₁₈ column with gradient elution using 0.1% formic acid–2 mM ammonium acetate/methanol. Detection employed positive electrospray ionization (ESI⁺) in multiple reaction monitoring (MRM) mode. Characterization confirmed Fe₃O₄@SiO₂-PSA’s mesoporous structure with excellent adsorption capacity and magnetic properties. The method showed good linearity (0.1–10 μg/L, r > 0.99) with an LOD and LOQ of 0.20 μg/kg and 0.50 μg/kg, respectively. Recoveries at 0.5–15.0 µg/kg spiking levels were 74.9–109% (RSDs 1.24–11.6%). This approach provides rapid, accurate, and high-precision analysis of diazepam in aquatic products, meeting regulatory requirements. Full article

Edaravone is used to treat motor neurone disease (MND) by slowing disease progression and prolonging survival time. Currently, it is available as an IV infusion (Radicava^®, Jersey City, NJ, USA) and an oral liquid suspension (Radicava ORS^®, Jersey City, NJ, USA). Development of novel edaravone formulations is still an active field of research that requires a validated stability-indicating assay capable of providing specific, precise, and accurate quantification of edaravone content. In this study, we developed and validated a stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method for edaravone quantification. Ten RP-HPLC methods based on the previously published literature were evaluated during method development. The optimal method employed a gradient method on an Agilent ZORBAX Extend-C18 column (150 × 4.6 mm, 5 µm) and produced a sharp and symmetrical drug peak. The method was further validated according to ICH Q2(R2) guidelines for specificity, linearity, sensitivity, accuracy, and precision. Successful separation of edaravone from void signals and degradant products was achieved. The method was precise and accurate at the concentration range of 6.8–68.6 µg/mL and was recommended to use without methyl hydroxybenzoate (MHB) as an internal standard. Full article

Estrogens are potent hormones involved in numerous physiological and pathological processes. Their typically low concentrations in biological samples necessitate highly sensitive analytical methods for accurate quantification. This study presents a high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for quantifying estradiol and its metabolites in blood serum and saliva. Analytes were extracted using solid-phase microextraction with a divinylbenzene sorbent and methanol as the desorption agent. FLD was performed after the derivatization of the analytes with dansyl chloride. Separation was achieved on a Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 µm) at 50 °C using water with 0.1% formic acid and methanol as the mobile phase at 0.5 mL/min. A gradient elution increased the methanol concentration from 76% to 100% over 0–8 min, then it returned to 76% at 8.1 min and was held until 11 min had passed. Detection was at λ_EX 350 nm and λ_EM 530 nm. Good linearity was observed for estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol (10–300 ng/mL; R² = 0.9893–0.9995). The LOQ for all analytes was 10 ng/mL. Solid-phase microextraction (SPME) offered advantages over liquid–liquid extraction. The method is suitable for quantifying estrogens in the 10 ng/mL–1 µg/mL range. Full article

Heterozygous mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), are major risk factors for Parkinson’s Disease (PD). Ambroxol, a small chaperone originally used as a mucolytic agent, has been shown to cross the blood–brain barrier, enhance GCase activity, and reduce α-synuclein levels, making it a promising therapeutic candidate for disease-modifying effects in GBA1-associated PD (GBA1-PD). This study aimed to develop a method to quantify ambroxol levels in human plasma and cerebrospinal fluid (CSF) using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Ambroxol was determined by online solid-phase extraction (SPE), coupled with LC-MS/MS, by gradient elution on a monolithic column. Detection employed a 3200 QTRAP tandem mass spectrometer in the positive electrospray ionization mode. Calibration curves exhibited linearity across the analyzed ranges in both plasma and CSF. The recovery rate ranged from 106.7% to 113.5% in plasma and from 99.0% to 103.0% in CSF. No significant matrix effect was observed. Intra-day and inter-day precisions were below 11.8% in both matrices, and accuracy ranged from 89.9% to 103.1% in plasma and from 96.3% to 107.8% in CSF. We evaluated and confirmed the stability of the analyte in plasma and CSF across various storage conditions. The method was successfully validated according to European Medicine Agency (EMA) guidelines and its applicability was confirmed in the context of a multicenter, randomized, double-blind, placebo-controlled, phase II study, designed to monitor the ambroxol levels in the plasma and CSF of GBA1-PD. Full article

The increasing environmental concerns and regulatory restrictions on toxic conventional solvents have driven the search for sustainable alternatives. Dimethyl isosorbide (DMI), a bio-renewable solvent, has shown potential as a replacement for short-chain glycol ethers, although its use as solvent in liquid chromatography (LC) is underexplored. This study presents a physicochemical characterization of DMI with a particular focus on its application as an innovative solvent in LC analyses. The partition coefficient (log P = −0.44) was determined using the OECD 107 method, and viscosity measurements for DMI and its mixtures with water and ethanol were conducted at 25 °C, 40 °C, and 60 °C. Viscosity ranged from 1.28 mPa·s at 60 °C to 2.62 mPa·s at 40 °C. The Central Composite Face 2³ experimental design for studying the chromatographic behavior of DMI confirmed that 50% (v/v) DMI can be effectively utilized in the mobile phases, at a column temperature of 40 °C, with backpressures ranging from 160 to 300 bar and a UV cut-off at 240 nm. Its effectiveness as an eluent in LC was demonstrated for the quantification of methylparaben and propylparaben in pharmaceutical formulations. This study highlights DMI’s promise as a sustainable bio-renewable alternative to conventional organic solvents used as eluents in LC, supporting eco-friendly practices in pharmaceutical analysis. Full article

Among grapevine trunk diseases, Eutypa dieback, caused by the fungus Eutypa lata, is one of the most critical ones, due to its widespread infection in vineyards and the lack of effective treatments. This fungus is a vascular pathogen that enters grapevines through pruning wounds. The infection process is associated with phytotoxic metabolites produced by the fungus, and as such, the identification of new metabolites from different culture conditions and broths could provide valuable insights into the fungus’s enzymatic system and help its control. For the purposes of this study, the OSMAC (one strain, many compounds) approach was applied to investigate the secondary metabolism of E. lata strain 311 isolated from Vitis vinifera plants in Spain. A total of twenty metabolites were isolated, including five reported for the first time from E. lata and four that are newly identified compounds in the literature: eulatagalactoside A, (R)-2-(4′-hydroxy-3′-methylbut-1′-yn-1′-yl)-4-(hydroxymethyl)phenol, (S)-7-hydroxymethyl-3-methyl-2,3-dihydro-1-benzoxepin-3-ol, and (3aR,4S,5R,7aS)-4,5-dihydroxy-6-((R)-3′-methylbuta-1′,3′-dien-1′-ylidene)hexahydrobenzo[d][1,3]dioxol-2-one. These compounds were extracted from fermentation broths using silica gel column chromatography and high-performance liquid chromatography (HPLC). Their structures were elucidated through extensive 1D and 2D NMR spectroscopy, along with high-resolution electrospray ionization mass spectrometry (HRESIMS). Compounds were evaluated for phytotoxicity against Phaseolus vulgaris, with only eulatagalactoside A producing white spots after 48 h. Additionally, the antibacterial activity against Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae of selected compounds was tested. The compounds (R)-2-(4′-hydroxy-3′-methylbut-1′-yn-1′-yl)-4-(hydroxymethyl)phenol and (S)-7-(hydroxymethyl)-3-methyl-2,3-dihydrobenzo[b]oxepin-3-ol showed the most significant antimicrobial activity against Gram-positive bacteria, inhibiting S. aureus by over 75%, with IC₅₀ values of 511.4 µg/mL and 617.9 µg/mL, respectively. Full article

A rapid, traceable, and highly sensitive method was developed for the simultaneous separation and quantification of 24 water-soluble synthetic colorants in premade cocktails, utilizing ultra-performance liquid chromatography coupled with diode array detection (UPLC-DAD). The purity of each colorant was individually confirmed through multi-wavelength analysis. Chromatographic conditions, including mobile phase composition and gradient elution, were meticulously optimized, achieving the separation of the 24 colorants on a BEH C18 column using a linear gradient elution within 16 min. The mobile phase consisted of an ammonium acetate solution (100 mmol/L, pH 6.25) and a mixed organic solvent of methanol and acetonitrile (2:8, v/v). The method exhibited excellent linearity across the concentration range of 0.005–10 μg/mL, with limits of detection (LODs) ranging from 0.66 to 27.78 μg/L for all 24 colorants. The method also demonstrated good precision (0.1–4.9%) at various concentration levels and recoveries ranging from 87.8% to 104.5% at spiked concentrations of 0.1, 0.5, and 1.0 μg/mL. A comparison with other published methods for colorant determination in food samples using HPLC-DAD and LC-MS (2014–2024) revealed that the proposed method offers superior performance in terms of the number of analytes detected, lower limits of detection, and reduced analytical time. Finally, the method was successfully applied to the analysis of colorants in premade cocktails from different sources. Full article

The global increase in chicken meat production and consumption has heightened concerns regarding the safety of chicken meat and its derived products. This study aimed to investigate the presence of Penicillium and Aspergillus mycotoxins in 50 samples of chicken breast muscle and liver collected from the Croatian market. Eight mycotoxins commonly produced by Aspergillus and Penicillium species were analyzed: aflatoxins B₁ (AFB₁), G₁ (AFG₁), B₂ (AFB₂), and G₂ (AFG₂); sterigmatocystin (STC); ochratoxin A (OTA); cyclopiazonic acid (CPA); and citrinin (CIT). Mycotoxin concentrations were determined using liquid chromatography–tandem mass spectrometry (LC-MS/MS) following sample cleanup with immunoaffinity columns while a QuEChERS-based method was applied for CPA. Mycotoxin occurrence was higher in liver samples, indicating the liver as primary site of mycotoxin accumulation compared to muscle tissue, where only CPA was detected. CPA was present in 20% of all samples, with the highest concentration (6.50 µg/kg) found in breast muscle, detected for the first time in fresh meat. AFB₁ and OTA were each detected in 10% of samples, and CIT was found in 4%—all exclusively in liver tissue. Notably, 4 out of the 17 contaminated samples contained more than one mycotoxin. Although the detected concentrations can be considered too low to pose an immediate health risk, the contamination rate suggests further research into these mycotoxins in chicken and other poultry species is needed. Full article

Structural characterization of natural products in complex herbal extracts remains a major challenge in phytochemical analysis. In this study, we present a novel post-acquisition data-processing strategy—key ion diagnostics–neutral loss filtering (KID-NLF)—combined with ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) for systematic profiling of the medicinal plant Terminalia chebula. The strategy consists of four main steps. First, untargeted data are acquired in negative electrospray ionization (ESI⁻) mode. Second, a genus-specific diagnostic ion database is constructed by leveraging characteristic fragment ions (e.g., gallic acid, chebuloyl, and HHDP groups) and conserved substructures. Third, MS/MS data are high-resolution filtered using key ion diagnostics and neutral loss patterns (302 Da for HHDP; 320 Da for chebuloyl). Finally, structures are elucidated via detailed spectral analysis. The methanol extract of T. chebula was separated on a C18 column using a gradient of acetonitrile and 0.1% aqueous formic acid within 33 min. This separation enabled detection of 164 compounds, of which 47 were reported for the first time. Based on fragmentation pathways and diagnostic ions (e.g., m/z 169 for gallic acid, m/z 301 for ellagic acid, and neutral losses of 152, 302, and 320 Da), the compounds were classified into three major groups: gallic acid derivatives, ellagitannins (containing HHDP, chebuloyl, or neochebuloyl moieties), and triterpenoid glycosides. KID-NLF overcomes key limitations of conventional workflows—namely, isomer discrimination and detection of low-abundance compounds—by exploiting genus-specific structural signatures. This strategy demonstrates high efficiency in resolving complex polyphenolic and triterpenoid profiles and enables rapid annotation of both known and novel metabolites. This study highlights KID-NLF as a robust framework for phytochemical analysis in species with high chemical complexity. It also paves the way for applications in quality control, drug discovery, and mechanistic studies of medicinal plants. Full article