Elution Research Articles

Strong Anion Exchange On-Line Solid Phase Extraction and Liquid Chromatographic Determination of Cefuroxime in Spiked Human Plasma Samples

ABSTRACT On-line solid phase extraction and liquid chromatography (SPE-LC) allows direct determination of drugs from all biological fluids without any sample pre-treatment. Ion exchange is one of the SPE types used for the extraction of drug molecules, especially those containing carboxyl or amino groups. In this study, a novel sample pre-treatment free, fully automated on-line SPE-LC method was developed for the determination of cefuroxime, a broad-spectrum second-generation cephalosporin antibiotic, in spiked human plasma samples. A quaternary amine functionlized hydrophilic modified styrene polymer strong anion exchange (SAX) sorbent was utilized as the solid phase, which was in-house packed using a tailor-made SPE hardware. A C18 analytical column (150 × 4.6 mm, 5 μm) was connected to SPE by a 10-port switching valve. A mobile phase composed of acetonitrile and 10 mm aqueous ortho-phosphoric acid solution was used with a dual gradient elution (1.0 mL/min). Cefuroxime was detected at 276 nm. Total analysis time was 13 min including equilibration-fractionation-separation-washing steps. Linear range was 5.00–200.00 µg/mL. LOD and LOQ were 1.03 and 3.11 µg/mL, respectively. Developed on-line SPE-LC method was novel and practical with recovery values higher than 90% and RSDs ≤2.82%, indicating that the SAX sorbent was effective for the extraction of cefuroxime from spiked plasma samples.

Stability indicating eco-friendly HPLC method development and validation for the estimation of bisoprolol fumarate and telmisartan

BackgroundTelmisartan and bisoprolol fumarate together are two medications that diminish arterial pressure. The current study comprises an evaluation of the proposed methodology's greenness regarding the HPLC method used to govern the medication mixture regardless of dose form A novel stability suggesting HPLC method's environmental effect was evaluated using the greenness metrics. Stress conditions comprising acidic, alkaline, oxidative, thermal, and photolytic degradation were applied for both of the medications.ResultsThe RP—HPLC method employing a reversed-phase C18 column with a gradient approach, the HPLC chromatography was carried out. The mobile phase consisted of acetonitrile, methanol, and phosphate buffer (60:35:5, %v/v/v), with the stationary phase being the Unisphere C18 column Agela Tech. The RP-HPLC method uses UV detection at 224 nm with chromatographic purification spanning linearities of 2.5–12.5 μg/mL for bisoprolol fumarate and 40.0–200.0 μg/mL for telmisartan, correspondingly. The procedure is accurate and precise, as demonstrated by an outcome that % RSD inside the permissible range. Additionally, various stressors were introduced to the medications. The approach's green credentials with respect to solvent utilization, chemical substances, expenditure of energy, and waste formation have been verified by the greenness data collected during the evaluation. No chromatographic or spectrum impediments caused by formulation additives have been observed.ConclusionBisoprolol fumarate and telmisartan could be measured simultaneously using the devised RP-HPLC method, which was simple, quick, sensitive, accurate, precise, linear, and stability indicating. The proposed approach showed ecological friendliness, robustness, sensitivity, and ease of use. As a result, the devised method could be applied to the regular quality checking of tablets and bulk medications.

Research on detection methods of related substances and degradation products of the antitumor drug selpercatinib

BackgroundSelpercatinib, a selective RET kinase inhibitor, is approved for treating various cancers with RET gene mutations such as RET-rearranged thyroid cancer and non-small cell lung cancer. The presence of process-related and degradation impurities in its active pharmaceutical ingredient (API) can significantly affect its safety and effectiveness. However, research on detecting these impurities is limited.MethodsThis study developed and systematically validated a High-Performance Liquid Chromatography (HPLC) method for identifying selpercatinib and its related impurities. The method utilized a 4.6 mm × 250 mm chromatographic column with 5 μm particles, employing a flow rate of 1.0 mL/min, a detection wavelength of 235 nm, an injection volume of 10 μL, and a column temperature of 35°C. Mobile phase A was composed of a 9:1 ratio of water to acetonitrile, with the aqueous component adjusted to pH 2.5 and containing 2 mM potassium dihydrogen phosphate (KH2PO4) and 0.4% triethylamine. Mobile phase B was pure acetonitrile. The gradient elution program was as follows: 0–2 min, 5%B; 2–15 min, 5% to 15%B; 15–30 min, 15% to 35%B; 30–35 min, 35% to 45%B; 35–36 min, 45% to 5%B; 36–45 min, 5%B.ResultsThe chromatographic method established in this study was validated according to the ICH Q2 (R1) guidelines. The developed HPLC method demonstrated excellent specificity, sensitivity, stability, linearity, precision, accuracy, and robustness. It efficiently separated the impurities present in selpercatinib, thereby confirming the method’s efficacy in ensuring the purity and quality of the drug.ConclusionThe chromatographic method established in this study can be used for the detection of selpercatinib and its impurities, providing significant reference value for the quality research of selpercatinib bulk drug and its preparations, and ensuring the safety of medication for patients.

A Novel High-Conjugated Probe of 4-(Acridone-10-yl)-Phenylethyl Chloroformate (APE-Cl) for Rapid Detection of Amino Compounds Using HPLC with Fluorescence Detection.

The fluorescence detection of amino compounds and the evaluation of their content in environmental samples are vital, not only for assessing food quality but also for studying soil organic matter. Here, we present the synthesis and application of a novel fluorescent probe, 4-(9-acridone)benzylmethyl carbonochloride (APE-Cl), for detecting amino compounds via a chloroformate reaction with fluorescence detection. The complete derivatization reaction of APE-Cl with amino compounds can be accomplished in aqueous acetonitrile within 5min at room temperature, using 0.2M borate buffer (pH = 9.0). APE-amine derivatives exhibited intense fluorescence with an excitation maximum at λex 254nm and an emission maximum at λem 418nm. All derivatives demonstrated high stability, strong fluorescence, and elevated ionization potential under atmospheric pressure chemical ionization (APCI-MS) in positive ion detection mode. The method, combined with gradient elution, provides baseline resolution of common amine derivatives on a reversed-phase C18 column. The LC separation for the derivatized amines shows good reproducibility with aqueous acetonitrile as the mobile phase. The relative standard deviations (n = 6) for each amine derivative are < 3.99%. The detection limits (at a signal-to-noise ratio of 3) per injection ranged from 1.68 to 11.2 femtomole. The established pre-column derivatization method for determining amino compounds in practical samples proved to be satisfactory.

Numerical solution of a nonlinear model of chromatography considering different isotherms and gradient profiles

In gradient chromatography, the mobile phase composition is progressively altered to regulate the propagation rates of the injected mixture components. Resultantly, improved separation of mixture components can be attained, the recycling time that must pass between two consecutive injections can be reduced, and, hence, the column’s efficiency and selectivity can be enhanced. A coupled modeling framework, integrating a lumped kinetic model and the convection–diffusion equation for solvent volume fraction, is employed to approximate the process. The influence of different isotherm models on the process dynamics is examined through numerical simulations. A semi-discrete second-order finite volume scheme is suggested to approximate the model equations. Impacts of positive and negative gradients, gradient shapes, gradient starting and ending times, axial dispersion, Henry’s constants, nonlinearity coefficients, mass transfer coefficients, and different adsorption isotherms are investigated. The theoretical findings presented in this study may facilitate further advancements in the field of gradient elution chromatography.

Development and comprehensive greenness assessment of HPLC method for quality control of β-sitosterol in pharmaceutical ointments with trio-color coded evaluation

A simple, rapid, and reproducible high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of β-sitosterol in the pharmaceutical dosage form of moist exposed burn ointment (MEBO). This method involved an effective sample procedure for extraction of β-sitosterol from MEBO using an alkali saponification agent composed of 0.8 N ethanolic NaOH and diethyl ether. The chromatographic separation was achieved on a C18 column (50 × 3.0 mm, 2.5 μm), using a mobile phase composed of methanol and acetonitrile (70:30 v/v) pumped in an isocratic mode at a flow rate of 0.7 mL/min. The column temperature was maintained at 40 °C, the injection volume was 10 µL, and the detection wavelength was 203 nm. Employing these conditions, the retention time was found to be 2.10 min. The developed method was validated for its specificity, linearity, accuracy, precision, the limit of detection, the limit of quantification, robustness, and solution stability based on International Council for Harmonisation (ICH) guidelines Q2 (R1). Our proposed method demonstrated superior performance compared to other reported methods. It exhibited a linearity range of 30 to 500 µg/mL and improved detectability with a limit of detection (LOD) of 4.65 µg/mL, highlighting its high sensitivity. Additionally, the separation was achieved in a remarkably short analysis time of just 2.1 min, which not only enhanced throughput but also significantly minimized waste and solvent consumption, thereby making it a more sustainable and effective alternative for β-sitosterol extraction. Moreover, in the light of green and white analytical chemistry, a comprehensive ecological and sustainable tri-color coded assessment protocol was established. The proposed method has been successfully applied to quantify β-sitosterol in commercial products (MEBO®, Avomeb® and BISTROl®) demonstrating its suitability for routine quality control analysis of β-sitosterol in pharmaceutical ointment dosage forms.

Quantitative analysis of quercetin in tea mistletoe (Scurrula oortiana Dans.)

Indonesia has a variety of plants that are useful as medicine. One is the Tea Mistletoe plant (Scurrula oortiana Dans.). This plant contains flavonoid compounds that are effective as antioxidants, especially in the form of quercetin. Quercetin in the tea mistletoe plant can prevent cell damage caused by free radicals, where cells need this antioxidant to avoid the harmful effects of excessive reactive oxygen species (ROS) production and prevent damage to immune cells. This study aims to analyze the levels of quercetin in Tea Mistletoe extract using quantitative analysis methods such as thin-layer chromatography and UV-Vis spectrophotometry. Tea Mistletoe powder was extracted by maceration with 96% ethanol solvent and then concentrated using a rotary evaporator and water bath. The thick extract obtained was then tested for phytochemical screening and specific and non-specific parameters. Analysis of flavonoid quercetin compounds was carried out using a thin layer chromatography method using a mobile phase of a mixture of chloroform, ethyl acetate, and n-butanol with a ratio of 5:4:1 and a stationary phase of Silica Gel GF₂₅₄. Determination of flavonoid levels was carried out using UV-Vis spectrophotometry at a wavelength of 428 nm. The results of this study indicate that the Mistletoe Tea plant contains flavonoids, alkaloids, steroids, tannins, saponins, phenolics, and glycosides. All test parameters meet the specified requirements. The thin layer chromatography test showed the Rf value of the Mistletoe Tea extract of 0.78. The linearity test showed a correlation value (r) = 0.9988 with a total flavonoid compound content of 372.250 mg QE/g extract, or a percentage of 37.225%.

Development and Validation of a Robust LC-MS/MS Method for Quantitation of a Novel Kinase Inhibitor, Ritlecitinib, in Rat Plasma: Application in Pharmacokinetic Study

Background Ritlecitinib (RIT) is a novel kinase inhibitor with promising therapeutic applications in the treatment of autoimmune disease alopecia areata. Accurate quantification of RIT in biological matrices is essential for pharmacokinetic studies and drug development. Objectives This study aims to develop and validate a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of RIT in rat plasma and to apply this method in a pharmacokinetic study. Materials and Methods A sensitive and selective LC-MS/MS method was developed in a AB Sciex QTRAP 5500 coupled with Shimadzu Prominence LC-20AD UFLC (ultra-fast liquid chromatography) System using an C18 column (Xselect HSS T3 2.5 µm, 2.1x150 mm) with a mobile phase consisting of 0.1% formic acid in high-performance liquid chromatography (HPLC) grade water and 0.1% formic acid in acetonitrile (70:30, v:v). RIT ( m/z 286.1 ↓ 105.7) and [ 13 C4]-ritlecitinib (internal standard, IS) ( m/z 290.1 ↓ 109.7) were detected in a positive mode by electrospray ionization (ESI) using multiple reaction monitoring (MRM) and extracted from rat plasma samples through protein precipitation We validated the method according to United States Food and Drug Administration (U.S. FDA) guidelines, assessing parameters such as linearity, accuracy, precision, selectivity, sensitivity, recovery and stability. The validated method was then applied to a pharmacokinetic study in rats following the administration of RIT. Results On rat plasma, the LC-MS/MS method has shown exceptional linearity across the concentration range of 5–100 ng/mL. The FDA’s approved standards of ±15% matched the accuracy and precision measurements. The matrix effect was negligible, and the mean recovery was about 93%, indicating that RIT was fully extracted from plasma. Different experimental conditions found RIT to be stable. RIT is quickly absorbed, with a peak plasma concentration (Cmax) of 43.82 ng/mL occurring 0.5–0.75 hours after the dose. It then drops in concentration in a way that is biexponential, with a half-life at the end of 1.5 hours. Conclusion A highly sensitive and specific LC-MS/MS method for quantitation of RIT in rat plasma was successfully developed and validated. The method was effectively applied to a pharmacokinetic study, providing critical data on the absorption and elimination characteristics of RIT in rats. This established approach may support additional preclinical pharmacokinetic investigations of RIT.

Exploratory multivariate analysis using R Language for method development in liquid chromatography.

The visual evaluation of data derived from screening and optimization experiments in the development of new analytical methods poses a considerable time investment and introduces the risk of subjectivity. This study presents a novel approach to processing such data, based on factor analysis of mixed data and hierarchical clustering - multivariate techniques implemented in the R programming language. The methodology is demonstrated in the early-stage screening and optimization of the chromatographic separation of 15 structurally diverse drugs that affect the central nervous system, using a custom R Language script. The presented explorative approach enabled the identification of key parameters affecting the separation and significantly reduced the time required to evaluate the comprehensive dataset from the screening experiments. Based on the data analysis results, the optimal combination of stationary phase and mobile phase composition was selected, considering retention, overall resolution, and peak shape of compounds. Additionally, compounds vulnerable to changes in selected chromatographic conditions were identified. As a complement to the presented R Language script, a web-based application ChromaFAMDeX has been developed to offer an intuitive interface that enhances the accessibility of the used statistical methods. Accompanying the publication, the R script and the link to the standalone application are provided, enabling replication and adaptation of the methodology.

Isolation of a Unique Monoterpene Diperoxy Dimer From Ziziphora clinopodioides subsp. bungeana Together With Triterpenes With Antidiabetic Properties.

Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f. is used in traditional medicine for various purposes. Previous phytochemical studies focused on phenolic compounds, but triterpenoids were almost overlooked. The study focused on the isolation of compounds with dual antidiabetic activity from the aerial parts of Z. clinopodioides subsp. bungeana. Separation of CHCl3-soluble fraction by silica gel column chromatography using different mobile phases and purification of compounds by semi-preparative HPLC or preparative TLC. The structures of pure compounds were elucidated by 1D and 2D NMR experiments along with HRMS. Compound 1 was additionally identified by the single crystal X-ray diffraction method. α-Glucosidase inhibitory assay and GLUT4 expression and translocation in C2C12 myotubes were conducted to evaluate antidiabetic potential of isolated compounds. This phytochemical study led to the isolation of 20 compounds, including a unique monoterpene diperoxy dimer (1). Compounds 7 and 9-11 displayed more potent α-glucosidase inhibitory activity (IC50 45.3-135.3 μM) than acarbose used as a positive control (IC50 264.7 μM), while only pomolic acid (5) increased GLUT4 translocation in C2C12 myotubes in a significant manner. Extensive chromatographic separation led to the isolation and identification of a unique monoterpene diperoxy dimer (1) from aerial parts of Z. clinopodioides subsp. bungeana. Some triterpenes inhibited α-glucosidase, another increased GLUT4 translocation. Although none of the isolated compounds demonstrated dual antidiabetic activity, selected triterpenes proved to be potent antidiabetic agents in vitro.

Development of a new method using dispersive liquid-liquid microextraction with hydrophobic natural deep eutectic solvent for the analysis of multiclass emerging contaminants in surface water by liquid chromatography-mass spectrometry.

A new analytical method was developed for the determination of 14 multiclass emerging organic contaminants in surface waters using LC-MS, and Dispersive Liquid-Liquid Microextraction (DLLME) for extraction. Different Natural Deep Eutectic Solvents (NADESs) composed of terpenes and organic acids were tested as extraction solvents and characterized by Fourier Transform Infrared Spectroscopy (FTIR), Hydrogen Nuclear Magnetic Resonance Spectroscopy (1H-NMR), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), density, and viscosity, eliminating the need to use traditional chlorinated solvents. NADES produced with butyric acid and thymol showed the best results and was selected for application for the first time in the extraction of emerging organic contaminants of different classes in water samples. Vortex was used as the dispersion mode, eliminating the use of the dispersion solvent. Chromatographic conditions and sample preparation were optimized using multivariate experimental designs. The optimized chromatographic conditions included the column oven temperature, mobile phase modifiers, and stationary phase type. The optimized conditions for sample preparation included the extraction temperature and pH, salting out effect, and extraction solvent volume. The analytical performance was evaluated through repeatability and intermediate precision tests, with RSD values below 20%, and recoveries between 70 and 120%. The coefficient of determination was greater than 0.98 for all analytes. LOQs varied between 1.5 and 35 μg L-1. DLLME is a simple technique, it does not require expensive and specific equipment. Furthermore, replacing traditional chlorinated solvents with NADES makes the procedure more environmentally friendly. The method presented here can be applied to a wide range of analytes for the analysis of fresh, brackish, and salt waters. Up to the present moment, this is the first study using NADES based thymol and butyric acid for the determination of multiclass emerging contaminants in surface waters samples.

Determination of oxyphenisatine and its total ester derivatives content in fermented green plum by ultra performance liquid chromatography-tandem mass spectrometry

Illegal additives such as oxyphenisatine and its esters are prevalent in the slimming food industry, necessitating a robust analytical method for their detection. This study presents a novel UPLC-MS/MS method for the rapid and accurate quantification of total oxyphenisatine levels in fermented green plum, following hydrolysis of its esters. An efficient ultrasonic extraction with a methanol and 0.1 mol/L NaOH mixture (5:5, v/v) was optimised to hydrolyse esters to oxyphenisatine within 18 min. Chromatographic separation was conducted on a C18 column (Waters Acquity UPLC BEH, 2.1 × 100 mm, 1.7 μm) with a mobile phase of 5 mmol/L ammonium acetate and acetonitrile under gradient elution at a flow rate of 0.3 mL/min. The method demonstrated linearity (r 2 > 0.999) over 0.1–500 µg/L, with a LOD of 10 µg/kg and LOQ of 30 µg/kg. Quantitative analysis employed positive ion multi-response monitoring and external standardisation, achieving recoveries of 92.4–97.0% and RSDs of 2.9–4.1%. Application to ten real samples gave a 90% detection rate, with measured values closely aligning with theoretical predictions (−11.3 to 13.2% relative difference) and oxyphenisatine content ranging from 159 µg/kg to 452 mg/kg. This UPLC-MS/MS method provides a reliable and efficient tool for monitoring the presence of oxyphenisatine and its derivatives in the context of food safety.

Capsaicin content in chillies (Capsicum annuum L., Capsicum frutescens L., Capsicum chinense Jacq.) of Northeast India by high performance thin layer chromatography (HPTLC)

Capsaicin is the primary bioactive constituent in chillies, responsible for its incomparable pungent taste and many health advantages. In the current study, 32 samples of three different species of Capsicum (Capsicum annuum L., Capsicum frutescens L., Capsicum chinense Jacq.) from various geographical regions in Northeast India were evaluated for their total capsaicin content by high performance thin layer chromatography (HPTLC). An excellent separation of capsaicin (Rf = 0.268 ± 0.02) was obtained using toluene and ethyl acetate as mobile phase at a 7:3 (v/v) ratio. The calibration curve was described by the equation Y = 5.661 × 10−9X + 1.225 × 10−4 with a correlation coefficient (r) of 0.99894. The percentage of capsaicin was found to range from 1.99 to 24.48% w/w. The linearity range for the curve for capsaicin was found 200–1000 ng per spot. The limit of detection and quantification were determined to be 45.98 and 97.70 ng, respectively. The proposed validated HPTLC method may be used for routine quality testing and quantification of capsaicin in chilli samples.

Development of a Multianalyte Method for the Determination of Phenolic Compounds in Residues from Beverage Production Using a Cyclofructan-Based Column in HILIC-MS/MS

This study expands and shows the versatility of applications in hydrophilic interaction chromatography coupled with triple quadrupole mass spectrometry (HILIC-MS/MS) using a FRULIC-N column to determine 31 phenolic compounds in beverage industry residues (coffee, apple juice, beer, and wine). Optimization, via Doehlert design, determined the acetonitrile proportion (50-90%) and pH (3.0-7.0) in the mobile phase (MP). A 22 factorial design assessed the concentration of ammonium acetate (10-110 mmol L-1) in the MP and column temperature (20-60 °C). Injection volume (5-45 µL) was univariately optimized. Separations at 60 °C reduced chromatographic run time to less than 11 min. Gradient elution (500 µL min-1) employed an MP comprising acetonitrile and ammonium acetate (110 mmol L-1, pH 7). Validation demonstrated average coefficient of determination (R2 ) 0.9707, limit of detection 0.001-0.503 mg L-1 and quantification 0.004-1.524 mg L-1, precision &lt; 12.2%, recovery 92.4-110.8%. The method was applied to five residues, indicating its viability for determining phenolics in beverage production residues.

Development and validation of a RP-HPLC method for simultaneous determination of cimetidine, metoprolol tartrate and phenol red for intestinal perfusion studies.

A new reversed phase high-performance liquid chromatography (RP-HPLC) method, with a short analysis time and easy to apply, was developed for the simultaneous detection of cimetidine (CIM), metoprolol tartrate (MT) and phenol red (PR) for use in intestinal perfusion studies. The analysis was performed with phosphate buffer (pH 5.0, 12.5mM)-acetonitrile mixture as mobile phase and C18 column (Inertsil ODS-3; 5µm, 4.6×250 mm) as stationary phase. Gradient analysis conditions were used and the acetonitrile ratio in the mobile phase varied from 10 to 50% in 10min. Total run time for analysis was 10min and the injection volume was 20µL. Detection of compounds was performed at 207nm. Under optimum HPLC conditions, retention times were 4.03min for CIM, 6.99min for MT and 8.49min for PR. The method was validated according to ICH Q2 (R1) guideline for specificity, linearity, sensitivity, precision, accuracy, stability and robustness. Developed method was linear and determination coefficients of the calibration curves were 0.9993, 0.9991 and 1.0 for CIM, MT and PR, respectively. The limits of quantification were 6.20, 2.78 and 0.45μg/mL for CIM, MT and PR, respectively. The precision and accuracy values of the developed analytical method met the ICH Q2 (R1) limits. The applicability of the method was demonstrated by preliminary in-situ intestinal perfusion studies. In conclusion, samples obtained from in-situ intestinal perfusion studies performed to examine the absorption/permeability of CIM, MT, and PR can be analyzed with the developed HPLC method.

Development and Validation of the RP-HPLC Method for Dexamethasone Sodium Phosphate Determination in Nasal Chitosan Microsphere Preparations

The purpose of this work was to provide a robust, sensitive, accurate, and straightforward analytical method for measuring dexamethasone sodium phosphate (DSP) in chitosan microspheres preparedn using the spray drying method. DSP was quantitatively analyzed using RP-HPLC with an ultraviolet detector at 254 nm, a mobile phase that contained a mixture of acetonitrile and 0.1% sodium dihydrogen phosphate monohydrate (50:50) operating isocratically at a flow rate of 1.0 mL/min, and a stationary phase that was a C18 PrincetonSPHER-100 C18-QB 100A HPLC Column (250 × 4.6 mm, 5 um). The ICH recommendations were followed in the validation of the analytical method. DSP had a retention duration of 2.899 minutes and a tailing factor of 0.827. The RP-HPLC method was linear (R = 0.9992) in the 15-60 ug/mL concentration range. The limits of quantitation (LOQ) and detection (LOD) were 4.425 ug/mL and 1.327 ug/mL, respectively. The relative standard deviations for the intra-day and inter-day precisions were 0.057-0.876% and 0.780-0.949%, respectively. The recovery percentages at 50, 100, and 200% concentration levels were within the 99.269-100.980% range. The validated method has been successfully applied to determine DSP entrapment efficiency in chitosan microspheres. A linear, sensitive, accurate, precise, and robust technique of determining DSP in chitosan microsphere preparations is offered by the established RP-HPLC method.

Determination of 12 halogenated organic pollutants in edible fish by ultra performance liquid chromatography-high resolution mass spectrometry combined with ultrasound-assisted extraction and gel permeation chromatography purification

Halogenated organic pollutants (HOPs) have attracted considerable attention owing to their persistence, bioaccumulation, and toxicity. The development of methods to detect HOPs in fish is challenging owing to the compositional complexity of fish matrices, which contain high levels of lipids and relatively low concentrations of HOPs. In addition, the lipophilicity of most HOPs renders their extraction difficult. Moreover, the simultaneous determination of multiple HOPs to achieve the high-throughput screening of these analytes is complex. In this study, a reliable and efficient pretreatment method based on ultrasound-assisted extraction, gel permeation chromatography purification, and ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was developed for the determination of 12 HOPs in edible fish. The procedures of sample extraction and purification and LC-HRMS detection parameters were optimized to improve the performance of the method. Fresh fish samples were thoroughly rinsed with water, and non-edible parts, including the skin, bones, and phosphorus, were removed. The fish were weighed, cut into small pieces, and vacuum freeze-dried for 48 h. Subsequently, a freeze grinder was used to grind the dried fish into a fine powder. Exactly 2 g of the fish powder was weighed, fortified with isotope-labeled internal standards of the HOPs, and allowed to stand for 5 min. Methanol-acetonitrile (1∶1, v/v) was then added, followed by vortex mixing and ultrasonication. After centrifugation, the supernatant was transferred to a fresh tube. The extraction process was repeated twice and all extracts were combined. The extract was evaporated under a gentle nitrogen flow and redissolved in a mixture of ethyl acetate-cyclohexane (1∶1, v/v). The sample mixture was cleaned using gel permeation chromatography, and the eluate was collected and concentrated under a nitrogen flow. Sample residuals were reconstituted with water-methanol (1∶1, v/v) prior to instrumental analysis. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm). Water containing 2 mmol/L NH4Ac and acetonitrile were used as the mobile phases, and an optimized gradient elution program was applied. Isotope dilution and an internal standard method were used to quantify the HOPs. An electrospray ionization source operated in negative mode was applied to ionize the HOPs, and a full scan together with data-dependent acquisition (DDA) was applied for HRMS. Excellent linearities (R2>0.99) were obtained for all HOPs in the quantification range of 1.0-1000.0 ng/mL. The limits of quantification were 0.5 ng/g. The analytical method was validated using pooled fish samples fortified with HOP standards (4, 40, and 400 ng/g). The recoveries of the HOPs were in the range of 67.6%-133.8%, and the corresponding RSDs were 0.5%-15.6%. A total of 27 commercially available fish samples were analyzed using the developed method, and the results revealed the presence of HOPs in the fish, indicating the practicability of the method for real-world samples. The developed method is rapid, accurate, precise, and suitable for detecting HOPs in fish. This study provides a useful approach for environmental monitoring and food safety assurance by enabling the accurate and efficient analysis of HOPs in commonly consumed fish. Given increasing global concerns over HOPs, the method developed in this study will provide practical technical support for consumers aiming to reduce their exposure to and the adverse impacts of HOPs via fish.

Solid-phase extraction coupled with high performance liquid chromatography-triple quadrupole mass spectrometry for simultaneous determination of seven coumarins in water samples from drinking water treatment plants

Chlorinated coumarins, which are as cytotoxic as highly toxic halobenzoquinones toward CHO-K1 cells, have recently been identified as disinfection byproducts in drinking water disinfection processes. Therefore, detecting coumarins in water samples collected at various stages from drinking water treatment plants helps assess the formation of chlorinated coumarins in drinking water. Hence, a simple, rapid, accurate, and sensitive method for quantifying coumarins in water samples is required. In this study, a method was developed based on solid-phase extraction coupled with high performance liquid chromatography-triple-quadrupole mass spectrometry for analyzing seven coumarins in water samples from drinking water treatment plants, including 6,7-dihydroxycoumarin, 7-hydroxycoumarin, 6-hydroxy-4-methylcoumarin, 8-chloro-7-hydroxycoumarin, coumarin, 7-chloro-6-hydroxy-4-methylcoumarin, and 3,8-dichloro-7-hydroxycoumarin. Sample pretreatment involved solid-phase extraction using HLB columns, followed by elution with water and methanol, each containing 0.25% formic acid. The extracted solution was separated via gradient elution using a Phenomenex Luna C18 column (100 mm×2.0 mm, 3 mm) with 0.1% formic acid aqueous solution and methanol as the mobile phases, with analytes detected by triple-quadrupole mass spectrometry equipped with electrospray ionization source in multiple reaction monitoring mode. The matrix effect, precision, and accuracy of the developed method were investigated using raw and treated water as matrices. Matrix effects of 0.84-1.12 were recorded for the detection of 6,7-dihydroxycoumarin, 7-hydroxycoumarin, 6-hydroxy-4-methylcoumarin, and coumarin in raw water, while values of 0.67-0.70 were recorded for 8-chloro-7-hydroxycoumarin, 7-chloro-6-hydroxy-4-methylcoumarin, and 3,8-dichloro-7-hydroxycoumarin in finished water. The three chlorinated coumarins exhibited matrix effects above 0.80 after the finished water matrix had been diluted four times. These results suggest that only simple solid-phase extraction or sample dilution is required to accurately determine the seven coumarins in drinking water from treatment plants. In addition, these coumarins exhibited good linear relationships at their respective mass concentrations. The precision and accuracy of the method were evaluated using raw and treated water as matrices. The seven coumarins exhibited good linearities by triple-quadrupole mass spectrometry in a certain range, with correlation coefficients (r) greater than 0.99 and method detection limits (MDLs) of 0.67-1.12 ng/L. The seven coumarins exhibited recoveries of 61.4%-91.5% at three spiked levels (20, 50, 100 ng/L) with relative standard deviations (RSDs, n=6)≤11.2%. The developed method can be used to analyze water samples from various treatment stages of a drinking-water treatment plant. 7-Hydroxycoumarin, 6,7-dihydroxycoumarin, and coumarin were detected at levels of 0.21-27.9 ng/L in 100% of the samples, while 6-hydroxy-4-methylcoumarin was not detected in raw water, post-coagulated water, and post-carbon-filtered water, but was found in sand-filtered water and finished water, with higher levels recorded for the latter (4.69 ng/L) than the former (1.79 ng/L). 8-Chloro-7-hydroxycoumarin was only detected in treated water (0.07 ng/L). This method is highly precise and accurate, provides results in short analysis times, and can be used to effectively monitor coumarins in real water samples and assess their removal during drinking-water treatment.

Stationary phase effects in hydrophilic interaction liquid chromatographic separation of oligonucleotides.

The use of liquid chromatography coupled with mass spectrometry (LC-MS) for the characterization of oligonucleotides and nucleic acids is a powerful analytical method. Recently, hydrophilic interaction chromatography (HILIC) has been proposed as a reasonable alternative to ion-pair reversed phase separations of oligonucleotides prior to MS. A wide variety of HILIC stationary phase surface chemistries are currently available. Although their selectivity can be considerably different, few studies have compared these chemistries for LC-MS analysis of oligonucleotides. We evaluated ten different HILIC column chemistries to understand their capabilities for separating a variety of oligonucleotides. In general, we found that most columns were ineffective at separating larger (n > 15-mer) oligonucleotides under the mobile phase and gradient conditions evaluated here. However, several stationary phases were found to be effective for separating smaller oligonucleotides such as endonuclease digestion products. Given that early eluting oligonucleotides were found to be compatible with standard electrospray ionization conditions, several different HILIC stationary phase options are available for LC-MS studies of smaller oligonucleotides including those generated in RNA modification mapping experiments.

Characterization of main degradation products from dendrobine under stress conditions by multistage cleavage of UPLC-ESI-IT-TOF.

Dendrobine is a sesquiterpene alkaloid primarily used in the treatment of inflammatory diseases, immune system disorders, and conditions related to oxidative stress. To understand the possible degradation pathways of dendrobine for its quality control, we conducted an in-depth investigation of its degradation products using forced degradation methods. The separation of dendrobine and its degradation products was achieved on a Shim-pack XR-ODS III (75 mm × 2 mm, 1.6 µm) column with a methanol-water mixture as the mobile phase under isocratic conditions, the isolated compounds were examined in positive ion mode with an ion trap-time of flight mass spectrometer (IT-TOF). In order to obtain in-depth structural information about the degradation products, mass spectrometry was performed using a five-stage fragmentation approach. This method allowed for thorough structural clarification via several rounds of selective fragmentation and high-resolution detection. System control and data acquisition were managed using LCMSsolution 3.81 software. The results showed that dendrobine undergoes significant degradation under oxidative, acidic, hydrolytic and thermal conditions, resulting in the formation of several degradation products with notable structural changes. Under oxidative conditions, dendrobine primarily generates two degradation products with mass increases of 16 Da and 32 Da, indicating mono-oxidation and di-oxidation reactions. Acidic degradation led to the identification of three degradation products, including a novel compound with an 18 Da mass increase, suggesting potential hydrolysis or dehydration reactions. Hydrolytic and thermal conditions resulted in the formation of two and three degradation products, respectively, with structural changes indicating possible molecular cleavage and reorganization mechanisms. In contrast, dendrobine exhibited strong stability under alkaline and photolytic conditions, with no significant degradation products detected. Detailed characterization of the degradation products via multi-stage mass spectrometry revealed key reaction pathways and mechanisms involved in dendrobine's degradation, providing critical insights for assessing its chemical stability and optimizing storage conditions.