Positive Electrospray Research Articles

Insights into the Chemical Exposome during Pregnancy: A Non-Targeted Analysis of Preterm and Term Births.

Human-made chemicals are ubiquitous, leading to chronic exposure to complex mixtures of potentially harmful substances. We investigated chemical exposures in pregnant women in New York City by applying a non-targeted analysis (NTA) workflow to 95 paired prenatal urine and serum samples (35 pairs of preterm birth) collected as part of the New York University Children's Health and Environment Study. We analyzed all samples using liquid chromatography coupled with Orbitrap high-resolution mass spectrometry in both positive and negative electrospray ionization modes, employing full scan and data-dependent MS/MS fragmentation scans. We detected a total of 1524 chemical features for annotation, with 12 chemicals confirmed by authentic standards. Two confirmed chemicals dodecyltrimethylammonium and N,N-dimethyldecylamine N-oxide appear to not have been previously reported in human blood samples. We observed a statistically significant differential enrichment between urine and serum samples, as well as between preterm and term birth (p < 0.0001) in serum samples. When comparing between preterm and term births, an exogenous contaminant, 1,4-cyclohexanedicarboxylic acid (tentative), showed a statistical significance difference (p = 0.003) with more abundance in preterm birth in serum. An example of chemical associations (12 associations in total) observed was between surfactants (tertiary amines) and endogenous metabolites (fatty acid amides).

Intrinsic Chemical Drivers of Organic Aerosol Volatility: From Experimental Insights to Model Predictions

AbstractAccurately predicting the volatilities of molecules in aerosols is challenging but crucial for understanding the atmospheric effects of aerosols. We used negative and positive ion electrospray ionization Fourier‐transform ion cyclotron resonance mass spectrometry (FT‐ICR MS) to identify differences in the molecular compositions of gas and particle phase samples from urban atmosphere. We aimed to identify intrinsic chemical parameters that determine and predict the organic aerosol volatility. We found higher average molecular weights, carbon mass percentages, and double bond equivalents (DBE) but lower average O/C ratios and oxygen mass percentages in the particle phase than the gas phase. We identified DBE, which display a significant negative correlation with volatility, as a key parameter. We proposed to improve the previous model for predicting organic aerosol volatility by incorporating DBE as a new variant; and the result showed that this subsequently improved the accuracy of the model, particularly for compounds with minimal or no heteroatoms (0–2) such as hydrocarbons (CH). The revised model offers insights into the contributions of DBE, carbon, nitrogen, oxygen, and sulfur atoms to the volatilities of diverse organic molecules in aerosols and could be applied to improve our understanding of the phase distributions of volatile organic compounds in the ambient air.

Application of Human Plasma Targeted Lipidomics and Analysis of Toxic Elements to Capture the Metabolic Complexities of Hypothyroidism.

Hypothyroidism (HT) affects millions worldwide and can lead to various lipid disorders. The metabolic complexity and the influence of toxic elements in autoimmune and non-autoimmune HT subtypes are not fully understood. This study aimed to investigate the relationships between plasma lipidome, toxic elements, and clinical classifications of HT in unexposed individuals. Samples were collected from 120 adults assigned to a study group with Hashimoto's disease and non-autoimmune HT, and a healthy control group. Quantification of 145 pre-defined lipids was performed by using triple quadrupole tandem mass spectrometry (TQ MS/MS) in multiple reactions monitoring (MRM) mode via positive electrospray ionization (ESI). Levels of toxic elements were determined using inductively coupled plasma mass spectrometry (ICP-MS). Significant associations between altered levels of several components of the plasma lipidome and Al, Cd, Ni, As, and Pb with HT were found. We show metabolic differences in lysophosphatidylcholines (LPC) and phosphatidylcholines (PC) between HT and controls, with distinct predicted activation patterns for lysolecithin acyltransferase and phospholipase A2. There are significant changes in the lipidome profiles of healthy subjects compared to euthyroid HT patients treated with L-thyroxine, which are related to the type of hypothyroidism and non-occupational exposure to toxic elements.

Simultaneous analysis of certain tree-nut allergens in cookies by LC-MS/MS

In this study, four tree-nut allergens were identified using liquid chromatography-mass spectrometry (LC-MS/MS) with a reverse-phase XSelect HSS C18 SB column (100 mm x 2.1 mm, 3.5 &amp;micro;m) and corresponding guard column. The gradient solvent program employed 0.1% formic acid in water and 0.1% formic acid in acetonitrile, with a flow rate of 0.5 mL/min, a positive ion electrospray ionization (ESI+) source, and a multiple reaction monitoring (MRM) mode. The method was validated following the guidelines of AOAC International. Results showed that the method had good specificity, with calibration curves constructed in the concentration ranges of 30.5 - 1220 &amp;micro;g/g for almonds, 102 - 4080 &amp;micro;g/g for cashews, 128 - 5120 &amp;micro;g/g for hazelnuts, and 122 - 4880 &amp;micro;g/g for pistachios. Limits of detection were 9 &amp;micro;g/g for almonds, 30.6 &amp;micro;g/g for cashews, 38.4 &amp;micro;g/g for hazelnuts, and 36.6 &amp;micro;g/g for pistachios, while limits of quantification were 30.5 &amp;micro;g/g for almonds, 102 &amp;micro;g/g for cashews, 128 &amp;micro;g/g for hazelnuts, and 122 &amp;micro;g/g for pistachios. The repeatability and accuracy of the method met AOAC requirements. The method was used to analyze the content of four groups of nut allergens (almond, cashew, hazelnut, and pistachio) in ten cookie samples from the Hanoi area. Results indicated that three samples contained undeclared allergens on the food labels.

Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100×3mm; 1.7μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8min including column equilibration, which enables the analysis of 160 samples per day.

Accurate determination of three new antihypertensive drugs illegally added to food using ultra-high performance liquid chromatography-tandem mass spectrometry

Effective strategies are required to address food safety issues related to the illegal addition of antihypertensive drugs to food and claims of antihypertensive function. In this study, a novel ultra-high performance liquid chromatography-triple-quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of three antihypertensive drugs (azilsartan, candesartan cilexetil, and lacidipine) in 12 food matrices (pressed candies, solid beverages, alternative teas, tea drinks, biscuits, jellies, mixed liquors, oral liquids, medicinal teas, tablets, hard capsules, and soft capsules). Initially, mass spectrometry parameters, such as the collision energies of the three antihypertensive drugs, were optimized. Subsequently, the response intensities and chromatographic separation conditions of the three drugs in different mobile phases were compared. In addition, to enhance the recoveries, various extraction solvents and purification methods, including solid-phase extraction (SPE) columns and the QuEChERS technique, were investigated. In the developed method, sample determination involved three steps. First, the sample was extracted using 0.2% (v/v) formic acid in acetonitrile and then filtered using high-speed centrifugation, in addition, the extracted solution of alternative tea and medicinal tea was purified using the QuEChERS technique. Second, the supernatant was diluted with water, and filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane. Finally, the analytes were separated on an Agilent Eclipse Plus RRHD C18 column (50 mm×2.1 mm, 1.8 μm) using a 5 mmol/L ammonium formate aqueous solution and acetonitrile as the mobile phases under gradient elution conditions and then detected using UHPLC-MS/MS with positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Quantitative analysis was performed using a matrix-matched external standard method. Methodological validation showed good linear relationships for all three antihypertensive drugs in the investigated concentration ranges, with correlation coefficients (r2) greater than 0.996. The limit of detection (LOD) and limit of quantification (LOQ) of lacidipine were 0.02 mg/kg and 0.04 mg/kg, respectively, whereas those of the other two drugs were 0.01 mg/kg and 0.02 mg/kg, respectively. In the 12 food matrices, the average recoveries of the drugs ranged from 86.6% to 107.5% with relative standard deviations (RSDs) of 1.1%-10.9% (n=6) at low, medium, and high spiked levels. Furthermore, this method was successfully applied to the analysis of real food samples. Overall, the newly developed method is simple, rapid, sensitive, accurate, and suitable for the qualitative and quantitative determination of antihypertensive drugs in different food matrices. This work could provide technical support for food safety agencies in implementing measures against the illegal addition of antihypertensive drugs to food.

Molecular characterization of atmospheric organic aerosols in typical megacities in China

Atmospheric aerosols in megacities impact air quality and public health. However, limited information exists on the detailed molecular composition of organic aerosols in urban areas. This study characterized the molecular composition of organic aerosols (OA) in Shanghai, Beijing and Guangzhou, China, during summer and winter of 2021. Liquid chromatography-orbitrap mass spectrometry detected 4536−5560 and 2067− 3489 organic molecular formulas in positive (ESI+) and negative (ESI−) electrospray ionization modes, respectively. CHO and CHON compounds accounted for over 80% and 60% of total abundance in ESI+ and ESI−, respectively, suggesting their significant contribution to urban OA. The number and abundance percentages of CHO showed obvious seasonal variation, with more CHO in summer than in winter, while CHON exhibited the opposite trend in Beijing and Shanghai. Compared with winter, a lower unsaturation degree, reduced aromaticity, and higher oxidation state of OA in summer were observed in Beijing and Shanghai, while these seasonal variations were not as obvious in Guangzhou, likely due to regional climate differences. The number percentage of common compounds between Beijing and Shanghai was higher than that between Guangzhou and Beijing (or Shanghai). Nitroaromatic compounds were more prevalent in winter than in summer. Further analysis of atmospheric formation relevance and precursor-product pairs suggested that CHON compounds are derived from the oxidization or hydrolyzation processes, revealing potential chemical transformations of these aerosols. This study characterized the chemical makeup of organic aerosols, providing insight into their sources and characteristics in these cities.

Pharmacokinetic Analysis of Gatifloxacin and Dexamethasone in Rabbit Ocular Biofluid Using a Sensitive and Selective LC-MS/MS Method.

Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL-1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.

Determination of deoxynivalenol and metabolites in maize samples by LC-MS/MS

In Vietnam, the regulation QCVN 8-1:2011/BYT has been issued to control mycotoxin content in food products. However, there are currently no regulations or studies on their derivatives and metabolites. Deoxynivalenol-3-glucoside (D3G) and two acetylated derivatives of deoxynivalenol (DON), namely 15-acetyldeoxynivalenol (15-AcDON) and 3- acetyldeoxynivalenol (3-AcDON), have been frequently detected in naturally contaminated grains such as wheat, barley, and corn. This study developed and applied a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine deoxynivalenol (DON) and its metabolites, including deoxynivalenol-3-glucoside (D3G), 3- acetyldeoxynivalenol (3-AcDON), and 15-acetyldeoxynivalenol (15-AcDON) in maize. The instrumental conditions comprised a C18 HSS SB (150 mm &amp;times; 2.1 mm &amp;times; 1.7 &amp;micro;m) column for separation and an MS/MS detector in positive and negative electrospray ionization (ESI) source, employing multi-reactive ion monitoring mode, with an injection volume of 2 &amp;micro;L. The method has been fully validated and meets the performance requirements of AOAC, demonstrating high specificity. The linearity range was established from 100 &amp;micro;g/kg to 1000 &amp;micro;g/kg, with a method detection limit (MDL) of 30 &amp;micro;g/kg. The repeatability and recovery rates were determined to be between 1.45% to 4.31% and 90% to 110%, respectively. The validated method was applied to analyze 50 maize samples. Deoxynivalenol (DON) was detected in 10 samples (20%), while deoxynivalenol-3-glucoside (D3G), 3- acetyldeoxynivalenol (3-AcDON), and 15-acetyldeoxynivalenol (15-AcDON) were not detected in any of the samples.

Development of an advanced analytical technique for detecting multiple pesticide residues in vegetables through liquid chromatography tandem mass spectroscopy (LC-MS/MS)

A comprehensive LC-MS/MS method, which employs Positive Electrospray Ionization (PEI) and Multiple Reaction Monitoring (MRM) was developed for the simultaneous determination of 35 pesticides belonging to various chemical classes in tomato, brinjal, chili, and okra samples. Extraction was facilitated using a modified QuEChERS method, which allows efficient sample analysis in a single run. Calibration curves for each pesticide exhibited linearity within the concentration range of 0.0025 to 0.1 µg mL−1, with correlation coefficients ranging from 0.993 to 0.999. Mean recoveries at five fortification levels (0.01 to 0.5 µg kg−1) ranged from 80 to 90%, demonstrating satisfactory precision (RSD < 20%). The matrix effects, mitigated through an optimized cleanup process, were observed within the range of 6.42% to 19.52%. The developed method having the limit of quantification of 0.01 mg kg−1 for all 35 pesticides, proved to be highly sensitive and rapid for multi-residue estimation in diverse vegetable samples. Subsequently, the method was used to analyze the market samples from Varanasi, India, which revealed the presence of pesticides like Chlorpyrifos, Chlorantraniliproleand Indoxacarb in tomato, brinjal, chili and okra. Therefore, the method could be considered as a robust tool for monitoring pesticide residues in vegetables, aiding in quality assessment and regulatory compliance in the agriculture sector.

Impact of Protonation Sites on Collision-Induced Dissociation-MS/MS Using CIDMD Quantum Chemistry Modeling.

Protonation is the most frequent adduct found in positive electrospray ionization collision-induced mass spectra (CID-MS/MS). In a parallel report Lee, J. J. Chem. Inf. Model. 2024, 10.1021/acs.jcim.4c00760, we developed a quantum chemistry framework to predict mass spectra by collision-induced dissociation molecular dynamics (CIDMD). As different protonation sites affect fragmentation pathways of a given molecule, the accuracy of predicting tandem mass spectra by CIDMD ultimately depends on the choice of its protomers. To investigate the impact of molecular protonation sites on MS/MS spectra, we compared CIDMD-predicted spectra to all available experimental MS/MS spectra by similarity matching. We probed 10 molecules with a total of 43 protomers, the largest study to date, including organic acids (sorbic acid, citramalic acid, itaconic acid, mesaconic acid, citraconic acid, and taurine) as well as aromatic amines including uracil, aniline, bufotenine, and psilocin. We demonstrated how different protomers can converge different fragmentation pathways to the same fragment ions but also may explain the presence of different fragment ions in experimental MS/MS spectra. For the first time, we used in silico MS/MS predictions to test the impact of solvents on proton affinities, comparing the gas phase and a mixture of acetonitrile/water (1:1). We also extended applications of in silico MS/MS predictions to investigate the impact of protonation sites on the energy barriers of isomerization between protomers via proton transfer. Despite our initial hypothesis that the thermodynamically most stable protomer should give the best match to the experiment, we found only weak inverse relationships between the calculated proton affinities and corresponding entropy similarities of experimental and CIDMD-predicted MS/MS spectra. CIDMD-predicted mechanistic details of fragmentation reaction pathways revealed a clear preference for specific protomer forms for several molecules. Overall, however, proton affinity was not a good predictor corresponding to the predicted CIDMD spectra. For example, for uracil, only one protomer predicted all experimental MS/MS fragment ions, but this protomer had neither the highest proton affinity nor the best MS/MS match score. Instead of proton affinity, the transfer of protons during the electrospray process from the initial protonation site (i.e., mobile proton model) better explains the differences between the thermodynamic rationale and experimental data. Protomers that undergo fragmentation with lower energy barriers have greater contributions to experimental MS/MS spectra than their thermodynamic Boltzmann populations would suggest. Hence, in silico predictions still need to calculate MS/MS spectra for multiple protomers, as the extent of distributions cannot be readily predicted.

Identification and quantitation of carbon–carbon double bond isomers of fatty acid in livestock and poultry meat

Livestock and poultry meat is one of the main consumption sources of fatty acids (FAs) in China. FAs are the main component of lipids and essential nutrients for the human body. The location of carbon–carbon double bond (CC) in unsaturated fatty acids (UFAs) significantly affects the nutrition and flavor quality of meat products. This study established an analytical method for identifying the CC positions of UFAs based on the Paternò-Büchi (PB) reaction using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF). The FA extract was derivatized with 2-acetylpyridine under ultraviolet light at 254 nm. The derivatized FA was then analyzed using targeted MS/MS mode in positive electrospray ionization. Limits of detection (LOD) and limits of quantitation (LOQ) of 21 UFAs were 0.004–0.680 and 0.013–2.267 μg/L, respectively. Furthermore, for the diagnostic ions at the same functional group terminal fragmented from derivatives of FA CC isomers, the abundance ratio was linear with that of the corresponding content, which could be applied to non-targeted screening of CC positions in FAs. Based on this method, a total of 28 UFAs were screened in five types of livestock and poultry meats in this study. Among them, four isomers of C17:1, four of C18:1, two of C18:2, two of C20:3 and two of C22:5 were identified. The precise identification and quantitation of the double bond positions of fatty acids will be of great significance for the exploration of the nutritional components in livestock and poultry meat.

Development and validation of a highly sensitive UPLC-MS/MS method for the determination of Huperzine A in rat plasma.

Huperzine A is a reversible and selective cholinesterase inhibitor and has been approved for the treatment of Alzheimer's diseases. In this study, we developed a highly sensitive and specific ulta-high-performance liquid chromatography-tandem mass spectrometry method for the determination of Huperzine A in rat plasma. An aliquot of 50 μL of rat plasma sample was pretreated with 200 μL of acetonitrile-methanol (v/v; 1:1) containing 0.2% formic acid followed by solid phase extraction. The resulting sample was separated on a Waters ACQUITY BEH C18 column using acetonitrile and water containing 0.2% formic acid as mobile phase, at a flow rate of 0.3mL/min. Multiple-reaction monitoring (MRM) mode was used for quantitative analysis of Huperzine A in positive electrospray ionization. In the concentration range of 0.01-10ng/mL, Huperzine A showed excellent linearity with correlation coefficient > 0.998. The intra- and inter-day RSD% were less than 9.7%, while the RE% ranged from -6.7% to 10.0%. The mean recovery was >84.5%. The validated method was demonstrated to be selective, sensitive, and reliable, which has been successfully applied to pharmacokinetic study of Huperzine A in rat plasma. Huperzine A displayed a long half-life in rat plasma and high oral bioavailability.

Critical evaluation of high-resolution and low-resolution mass spectrometry for biomonitoring of human environmental exposure to pesticides

Despite increasing debates about their potential side effects on human health, data concerning the risks and the impacts associated with pesticides remains scarce. Analytical tools allowing the measurement of most pesticides and/or their metabolites to which the population can be exposed are also of need. In the present study, the limits of detection (LODs) of 3 different screening procedures based on either Low-Resolution and High-Resolution Mass Spectrometry (LR-MS and HR-MS) for the determination of pesticides in serum (among which carbamates, dithiocarbamates, neonicotinoids, organochlorines, organophosphates and pyrethroids) were explored. For HR-MS, a quadrupole time-of-flight was used in positive and negative electrospray ionization modes and data were obtained from either a targeted scheduled MSMS acquisition (HR-MSMS) or a data-independent acquisition (HR-DIA). For LR-MS, a triple quadrupole was used and data were acquired with a classical multiple-reaction monitoring (MRM) mode. Depending on the approach, the LOD values varied from 0.05 to 10 ng/mL. For the lowest concentrations, the proportion of molecules detected was systematically greater for the LR-MS approach, while those of HR-MSMS were better than those of HR-DIA. These differences in the LOD values were confirmed in a sample of 174 serums in which LR-MRM detected 89 compounds while HR-MSMS and HR-DIA detected 79 and 75 compounds, respectively. Nevertheless, for environmental and occupational purposes, HRMS approach could probably be efficient to detect most of pesticides and their metabolites in human serum and could be suitable for human biomonitoring studies or fundamental research exploring the impact of exposure to pesticides on human health.

Pharmaceutical metabolite identification in lettuce (Lactuca sativa) and earthworms (Eisenia fetida) using liquid chromatography coupled to high-resolution mass spectrometry and in silico spectral library.

Pharmaceuticals released into the aquatic and soil environments can be absorbed by plants and soil organisms, potentially leading to the formation of unknown metabolites that may negatively affect these organisms or contaminate the food chain. The aim of this study was to identify pharmaceutical metabolites through a triplet approach for metabolite structure prediction (software-based predictions, literature review, and known common metabolic pathways), followed by generating in silico mass spectral libraries and applying various mass spectrometry modes for untargeted LC-qTOF analysis. Therefore, Eisenia fetida and Lactuca sativa were exposed to a pharmaceutical mixture (atenolol, enrofloxacin, erythromycin, ketoprofen, sulfametoxazole, tetracycline) under hydroponic and soil conditions at environmentally relevant concentrations. Samples collected at different time points were extracted using QuEChERS and analyzed with LC-qTOF in data-dependent (DDA) and data-independent (DIA) acquisition modes, applying both positive and negative electrospray ionization. The triplet approach for metabolite structure prediction yielded a total of 3762 pharmaceutical metabolites, and anin silico mass spectral library was created based on these predicted metabolites. This approach resulted in the identification of 26 statistically significant metabolites (p < 0.05), with DDA + and DDA - outperforming DIA modes by successfully detecting 56/67 sample type:metabolite combinations. Lettuce roots had the highest metabolite count (26), followed by leaves (6) and earthworms (2). Despite the lower metabolite count, earthworms showed the highest peak intensities, closely followed by roots, with leaves displaying the lowest intensities. Common metabolic reactions observed included hydroxylation, decarboxylation, acetylation, and glucosidation, with ketoprofen-related metabolites being the most prevalent, totaling 12 distinct metabolites. In conclusion, we developed a high-throughput workflow combining open-source software with LC-HRMS for identifying unknown metabolites across various sample types.

Determination of ADB-BUTINACA in rat plasma by UPLC-MS/MS and its application

AbstractA fast, accurate, sturdy, and sensitive UPLC-MS/MS method was developed to measure the levels of ADB-BUTINACA in rat plasma for a pharmacokinetic study. The ADB-BUTINACA and midazolam (internal standard) were separated on a UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using a gradient elution of acetonitrile and water (0.1% formic acid) as the mobile phase. ADB-BUTINACA and midazolam were detected in multiple reaction monitoring (MRM) mode for quantitative analysis, the mass detection was performed on a positive electrospray ionization (ESI) source. The calibration curve displayed excellent linearity from 1 to 1,000 ng mL−1, with a lower limit of quantification of 1.0 ng mL−1 and a limit of detection of 0.3 ng mL−1. The precision for both inter-day and intra-day assays was below 14%, while accuracy ranged from 92% to 111%. The method also showed an average recovery between 87% and 90%, with a matrix effect ranging from 104% to 111%. The data was acceptable according to the guidelines of the US Food and Drug Administration (FDA). This newly validated quantitative method successfully supported the pharmacokinetic study of ADB-BUTINACA.

Evaluating a novel method for vitamin A analysis in an observational study of the UAE's obese population

Vitamin A plays a critical role in various biological functions, including vision, cellular differentiation, and immune regulation. However, accurately assessing its status, particularly in obese individuals, presents challenges due to potential alterations in metabolism and distribution. This study utilized Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS) methodology to precisely measure serum vitamin A concentrations in population of UAE. The methodology's reliability and precision, as demonstrated through validation procedures, underscore its potential utility in clinical settings. Employing the Multiple Reaction Monitoring mode of positive ion electrospray ionization, the LC–MS/MS system achieves a limit of detection (LOD) of 0.48 ng/mL in serum, while adhering to FDA-US regulations for accuracy and compliance. A key aspect of this study was the application of LC–MS/MS to assess vitamin A status in an obese population within UAE. By employing a diverse cohort of 452 Emirati participants, including 277 individuals from a randomized controlled trial who were assessed at baseline and at 6th month, and 175 healthy individuals aged 18–82 assessed at baseline, this study explores the relationship between obesity and vitamin A levels, shedding light on potential implications for health and well-being. It was an observational study based on a new vitamin A method and participants were asked to eat vitamin A rich foods. The robust performance of the LC–MS/MS methodology positions it as a valuable tool for clinical research. By accurately quantifying vitamin A levels in human serum, this methodology opens avenues for advancing our understanding of vitamin A physiology and its implications for health, particularly in obese populations. In summary, this LC–MS/MS methodology presents a potent tool for clinical studies, providing reliable, specific, and robust detection of vitamin A in human serum, thus, opening a new frontier for advancing our understanding of vitamin A related physiology and health in the obese population.

Pharmacokinetics of Oleandrin Following Administration of a Nerium oleander Extract in Mice

Background. Nerium oleander Linn is native of Mediterranean regions of Africa and Europe, where it is used in folk medical practices. The whole plant is known to be toxic. Various studies have shown its antimicrobial and anticancer properties. Oleandrin, the main toxic component in Nerium oleander is an inhibitor of P-glycoprotein. Cardiac glycosides, including oleandrin, are substrates of P-glycoprotein. The presence of other inhibitors in the alcoholic extract may potentially modify the pharmacokinetics of oleandrin. Given the therapeutic potential and the associated toxicity of the alcoholic extract, it is important to investigate the kinetics of oleandrin within this extract. Purpose. The objective of this study was to determine the pharmacokinetic parameters in mice following oral and I.V. administrations of a hydroalcoholic extract of Nerium oleander. Methods. Pharmacokinetic investigations of oleandrin, a cardiotonic glycoside and main active compound in Nerium oleander, were conducted in mice following intravenous administration (30 mg/kg) and oral administration (150 mg/kg) of the hydrolcoholic extract of Nerium oleander. For the oral route, seven times were chosen for the mice sampling. Four times were chosen for the intravenous route. Three mice per group were used at each time point. In the excretion study, seven mice were housed in a mouse urine collection device and a dose of 150 mg/g was administered. A urine sample was collected after 48 hours. Oleandrin was measured by LC-MS/MS validated method. MS data were acquired in the positive ion electrospray ionization (ESI) mode. Two deuterated internal standards, cocaine-d3 and digoxin-d3, were used. The retention times were 8.47 min for oleandrin, 4.78 min for deuterated internal standard cocaine d3 and 5,37 min for deuterated internal standard digoxin d3. Results. The equivalent doses of oleandrin administred to mice were 1710 ug/kg for the oral route and 342 ug/kg for the intravenous route. Oleandrin was rapidly absorbed after oral administration (Cmax at 10 min). The AUC0-inf (ug/L*min) values obtained after intravenous and oral dosing were 34797.7 and 107222 respectively, resulting in an oral bioavailability of 61.6 %. The apparent volume of distribution (Vss) was 0.55 L/kg and Clearance ClT was 0.01 kg* L/min.

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Development and clinical implementation of an LC-HRMS method for ivacaftor, lumacaftor, tezacaftor and elexacaftor in human plasma and breast milk.

The four cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, ivacaftor, lumacaftor, tezacaftor, and elexacaftor, have revolutionised the treatment of CF by direct action on the protein target behind the disease's development. The aim was to develop and validate a quantification method for these CFTR modulators in plasma and breast milk to better understand inter-patient variability in pharmacokinetics and treatment outcome, including the risk of adverse drug reactions. The ability to monitor CFTR modulators in breast milk enables the estimation of the exposure of breastfed infant, with a potential concern for CFTR modulator-induced liver injury. The analysis was performed on a Thermo Vanquish Flex Binary UHPLC system coupled to a high-resolution mass spectrometer (HRMS), Thermo Q Exactive. The analytes were detected using positive electrospray ionisation in full scan mode. After sample preparation by protein precipitation, the supernatant was injected onto the LC system and the analytes were separated using a Zorbax SB-C18 Rapid Res HPLC column (3.5µm, 4.6 × 75mm). This is the first published method for CFTR modulators in breast milk. The validated quantification range for ivacaftor is 0.0050-10µg/mL with a coefficient of variation < 6% and a mean accuracy of 97-106%; for lumacaftor, tezacaftor, and elexacaftor, the validated quantification range is 0.050-100µg/mL with a coefficient of variation < 8% and a mean accuracy 93-106%. A simple and sensitive quantification method for CFTR modulators has been developed and used for routine analysis of human plasma and breast milk samples since 2022.