Positive Mode Electrospray Research Articles

Untargeted secondary metabolite profiling and putative pathway deduction of a marine Ochrophyte – Iyengaria stellata (Børgesen) using liquid chromatography-mass spectrometry

This study divulges biochemical traits, mineral makeup, and the secondary metabolite outline of the less explored Ochrophyte, Iyengaria stellata (Børgesen) using untargeted metabolite profiling along the Pingleshwar coast, Kachchh, India. Macro mineral Na (4921.33 ± 0.577 mg/100 g Dry Weight) and micro mineral Fe (859.76 ± 0.98 mg/100 g DW) were found to be in the highest quantities. A total of 108 putative metabolites were detected using both, the positive electrospray ionization mode (+ESI) and negative electrospray ionization mode (–ESI of the Liquid Chromatography-Mass Spectrometry-Time of Flight (LCMS-TOF). The identified metabolites included steroids, terpenoids, ketones, esters, polyphenols, anthraquinones, tocopherol, etc. The metabolomics data obtained from the +ESI mode led to the inference of a putative glycerophospholipid metabolism pathway that shall provide valuable insights for future metabolic engineering studies. This study highlights the first attempt to decipher the secondary metabolite composition of I. stellata thereby further accounting for its potential as a natural source in pharmaceutical and nutraceutical compositions.

Novel method for determination of methoxphenidine and its main metabolite in biological samples

Methoxphenidine (MXP) is new psychoactive substance belonging to the group of dissociative anesthetics. It appeared on the black market in 2013, and MXP intoxications have been reported with at least three deaths since then. To understanding the mechanism of toxicity and effects of MXP, it is important to know its pharmacological properties (e.g. toxicity, activity on receptors and ADME) and metabolism which may be further exploited to study its mechanism of action in vivo. Metabolism of such compound may be useful not only in toxicological screenings, but together with pharmacokinetic and pharmacodynamic data it enhances the knowledge how these substances may affect the living organism. It is still very little information on the MXP and a method for analysis of biological samples (e.g. brain, liver, lungs and serum) remain to be developed. Standards of MXP, main metabolite norMXP and internal standard of deuterated MXP (MXP-d3) was prepared in-house and used as external calibrators. For determination of MXP and norMXP in biological samples, UHPLC-MS was used. Quantitative analyses were performed using LC system Dionex Ultimate 3000 (Thermo-Fisher, MA, USA) and the chromatographic separation was performed using Poroshell 120, Phenyl Hexyl (2.1 × 100 mm, 2.7 μm) column (Agilent, CA, USA). Mobile phases consisted of 0.1% formic acid and 10mM ammonium formate in 5% methanol in water (v/v) (A) and in 5% acetonitrile in methanol (v/v) (B) and were used at a flow rate of 0.4mL/min. The analytes were detected in multiple reaction monitoring (MRM) mode using QTRAP 6500+ (AB Sciex, MA, USA) with a Turbo V Ion source operating in positive electrospray (ESI + ) mode. Wistar rats given 20 mg/kg MXP were used as real samples. At 0; 0.5; 1; 2; 4; 8 and 24 hours after administration, samples of livers, lungs, brain and 2 mL of blood serum were collected from rats. There were 8 samples from each tissue at each time point. To extract analytes from these complex biological matrices, protein precipitation and salting-out liquid-liquid extraction (SALLE) were used. Due to the complex matrix, it was important to develop an extraction method with sufficient yield and low matrix effect. Liquid-liquid extraction, SALLE and protein precipitation were tested for each type of biological sample separately. These methods were selected on the basis of a previous study of structurally similar substances. Protein precipitation was chosen as the best extraction method for processing blood serum samples and SALLE was chosen for preparation of three other tissues. All validation parameters met the guidelines from the US Food and Drug Administration (FDA). 8 samples of 4 biological samples were analyzed in 7-time intervals. Based on these data, MXP and norMXP concentration curves were generated. A quantification and extraction method for the determination of MXP and the major metabolite norMXP in blood serum, brain, liver and lung samples from Wistar rats was developed and optimized. Pharmacokinetic profiles for MXP and norMXP in all 4 types of biological material used, were established. This work was a part of the research project supported by the Ministry of Health of the Czech Republic (grant No. NU21-04-00307) and also it was supported from the grants of Specific university research–grant No. A2_FCHI_2022_003 and A2_FPBT_2022_023.

Vitamin and Mineral Supplementation and Rate of Weight Gain during the First Trimester of Gestation in Beef Heifers Alters the Fetal Liver Amino Acid, Carbohydrate, and Energy Profile at Day 83 of Gestation.

The objective of this study was to evaluate the effects of feeding heifers a vitamin and mineral supplement and targeting divergent rates of weight gain during early gestation on the fetal liver amino acid, carbohydrate, and energy profile at d 83 of gestation. Seventy-two crossbred Angus heifers were randomly assigned in a 2 × 2 factorial arrangement to one of four treatments comprising the main effects of vitamin and mineral supplementation (VTM or NOVTM) and feeding to achieve different rates of weight gain (low gain [LG] 0.28 kg/day vs. moderate gain [MG] 0.79 kg/day). Thirty-five gestating heifers with female fetuses were ovariohysterectomized on d 83 of gestation and fetal liver was collected and analyzed by reverse phase UPLC-tandem mass spectrometry with positive and negative ion mode electrospray ionization, as well as by hydrophilic interaction liquid chromatography UPLC-MS/MS with negative ion mode ESI for compounds of known identity. The Glycine, Serine, and Threonine metabolism pathway and the Leucine, Isoleucine, and Valine metabolism pathway had a greater total metabolite abundance in the liver of the NOVTM-LG group and least in the VTM-LG group (p < 0.01). Finally, both the TCA Cycle and Oxidative Phosphorylation pathways within the Energy Metabolism superpathway were differentially affected by the main effect of VTM, where the TCA cycle metabolites were greater (p = 0.04) in the NOVTM fetal livers and the Oxidative Phosphorylation biochemicals were greater (p = 0.02) in the fetal livers of the VTM supplemented heifers. These data demonstrate that the majority of metabolites that are affected by rate of weight gain or vitamin/mineral supplementation are decreased in heifers on a greater rate of weight gain or vitamin/mineral supplementation.

Glutathione reactivity with aliphatic polyisocyanates.

Isocyanate chemicals known to cause adverse health effects when inhaled are essential to making important products and are used in multiple industries. Glutathione (GSH), a major antioxidant of the lower airways with a well described role in xenobiotic metabolism, is a primary reaction target for di-isocyantes. However, GSHs reactivity with poly-isocyanates which have largely replaced diisocyanates (particularly aliphatic) in most end-user settings remains uncertain. We hypothesized aliphatic polyisocyanates would readily react with glutathione under physiologic conditions and the products could be identified using liquid chromatography (LC) coupled-mass spectrometry (MS) and tandem MS/MS. The data identified (tris)GSH-isocyanate adducts as the major reaction product of GSH with the most commonly used contemporary polymeric (tri-isocyanate) formulations of hexamethylene diisocyanate (HDI), the isocyanurate and biuret, as [M+H]+ ions of 1426.53 and 1400.55 m/z respectively in reverse phase LC-MS using electrospray in positive ion mode. The uretdione form of HDI, a stabilized dimer, formed two reaction products with GSH, a tris(GSH)-isocyanate reaction product recognized as a 1258.44 m/z [M+H]+ ion, and a bis(GSH)-isocyanate product identified as a 951.36 m/z [M+H]+ ion. Predicted structures for the newly described GSH-polyisocyanate reaction products, modeled based on collision induced dissociation (CID) fragmentation patterns in tandem MS/MS, support S-linkage of the GSH to N = C = O groups. In summary, industrially-used aliphatic polyisocyanates readily react with GSH to form primarily S-linked tris(GSH)-conjugates, a process that may play an important role in response to respiratory tract exposure.

Validated LC/MS/MS Method for the Determination of Rivastigmine in Human Plasma: Application to a Pharmacokinetic Study in Egyptian Volunteers to Determine the Effect of Gender and Body Mass Index.

The effect of gender and body mass index (BMI) on the pharmacokinetics of rivastigmine was studied in Egyptian human subjects using new bio-analytical validated LC/MS/MS method. In this study, Rivastigmine was estimated in human plasma using Escitalopram as an internal standard (IS). Rivastigmine and Escitalopram were extracted from human plasma samples by liquid-liquid extraction using diethyl ether (DEE)-dichloromethane (DCM) (70:30, v/v). Chromatographic separation was performed on a reversed phase C18 INERTSIL ODS column using 0.05% aqueous formic acid, acetonitrile in the ratio (50:50, v/v) as a mobile phase. Multiple reaction monitoring (MRM) was applied and operated by positive mode electrospray ionization. A significant difference between male and female Cmax (maximum plasma concentration) (P= 0.0205; CL = 95.4) was found using Mann-Whitney U test. Also, a moderate negative correlation was found between BMI and Tmax (time to peak plasma concentration) using spearman rho test. The calculated results confirm the difference of Rivastigmine pharmacokinetics between male and female subjects. Furthermore, it indicates that Rivastigmine dose adjustment may be necessary. The method was applied for the estimation of pharmacokinetic parameters in volunteers (n= 26, 17 male and 9 female) and the effects of gender and BMI were investigated.

Rapid Determination of 9 Tyrosine Kinase Inhibitors for the Treatment of Hepatocellular Carcinoma in Human Plasma by QuEChERS-UPLC-MS/MS.

A reliable and rapid method employing QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) pretreatment coupled with ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was successfully developed and validated for the analysis of nine tyrosine kinase inhibitors (TKIs) in human plasma. Biological samples were extracted with acetonitrile and salted out with 350 mg of anhydrous magnesium sulfate (MgSO4), followed by purification with 40 mg of ethyl enediamine-N-propylsilane (PSA) adsorbents. All analytes and internal standards (IS) were separated on the Hypersil GOLD VANQUISH C18 (2.1 mm × 100 mm, 1.9 μM) column using the mobile phases composed of acetonitrile (phase A) and 0.1% formic acid in water (phase B) for 8.0 min. Detection was performed by selection reaction monitoring (SRM) in the positive ion electrospray mode. Lenvatinib, sorafenib, cabozantinib, apatinib, gefitinib, regorafenib, and anlotinib rendered good linearity over the range of 0.1–10 ng/ml, and 1–100 ng/ml for tivantinib and galunisertib. All linear correlation coefficients for all standard curves were ≥ 0.9966. The limits of detection (LOD) and the limits of quantitation (LOQ) ranged from 0.003 to 0.11 ng/ml and 0.01–0.37 ng/ml, respectively. The method was deemed satisfactory with an accuracy of -7.34–6.64%, selectivity, matrix effect (ME) of 90.48–107.77%, recovery, and stability. The proposed method is simple, efficient, reliable, and applicable for the detection of TKIs in human plasma samples as well as for providing a reference for the clinical adjustment of drug administration regimen by monitoring the drug concentrations in the plasma of patients.

Evaluation of 4 quantification methods for monitoring 16 antibiotics and 1 beta-lactamase inhibitor in human serum by high-performance liquid chromatography with tandem mass spectrometry detection

Antibiotic (ATB) prescription in an intensive care unit (ICU) requires continuous monitoring of serum dosages due to the patient’s pathophysiological condition. Dosing adjustment is necessary to achieve effective targeted concentrations. Since ICUs routinely use a large number of ATBs, global monitoring needs to be developed. In the present study, we developed a global analytical method for extracting, separating and quantifying the most widely used ATBs in ICUs: amoxicillin, piperacillin, cefazolin, cefepime, cefotaxime, ceftazidime, ceftolozane, ceftriaxone, ertapenem, meropenem, ciprofloxacin, moxifloxacin, levofloxacin, daptomycin, dalbavancin, linezolid and a beta-lactamase inhibitor: tazobactam. To guarantee the robustness of the quantification, we differentiated the 16 ATBs and the beta lactamase inhibitor into 4 pools (ATB1 to ATB4), taking into account prescription frequency in the ICU, the physicochemical properties and the calibration ranges of the ATBs selected. The whole ATB was then separated with two LC columns in reversed phase: Kinetex Polar-C18 100 Å and Polar-RP-80 synergy, in less than 6.5 min. Detection was carried out by electrospray in positive ion mode, by tandem mass spectrometry (LC-MS/MS. The four quantification methods were validated according to the European guidelines on bioanalytical method validation (EMEA guide), after determining the extraction yields, matrix effects, recovery, precision, accuracy, within-run precision and between-run precision. For all analyses, bias is < 15% and is comparable to the literature and LOQs vary from 0.05 mg.L−1 for ciprofloxacin to 1.00 mg.L−1 for ceftriaxone and dalbavancin. The stability time of cefepime and piperacillin is 3 hrs and for the other ATBs 6 hrs in serum at room temperature. For long-term stability, freezing at − 80 °C guarantees 3 months of stability for ceftriaxone and dalbavancin and more than 6 months for the other ATBs.

LC‐MS based urine metabolomic profiling indicate altered gut microbiome and host lipid metabolites in Parkinson’s disease

Evidence indicates that host and gut microbial metabolism are dysregulated prior to the onset of motor symptoms, in the prodromal phase of Parkinson’s disease (PD). There is currently no definitive test for the diagnosis of Parkinson’s disease; this represents an urgent, unmet medical need to enable the effectiveness of current and future therapeutic interventions for PD. The aim of this study was to understand changes in host and microbial metabolism in PD patient biofluids which could reflect a transition from the healthy to diseased state. We performed a comprehensive untargeted metabolomics analysis of urinary lipid metabolites together with altered metabolites associated with the gut microbiota. A total of 64 urine samples consisting of 31 from age matched healthy controls and 33 from PD patients were used in this study. Samples were deproteinized and analysed by reverse phase (RP)/UPLC‐MS/MS methods with positive and negative ion mode electrospray ionization (ESI) and HILIC/UPLC‐MS/MS with negative ion mode ESI. 14 lipid metabolites were significantly altered in PD patients. Five metabolites were associated with bile acid metabolism and nine were from the cholesterol pathway. Changes in secondary bile acid metabolism, particularly secondary bile acids indicative of microbial metabolism changes, were evident in PD patients, with 11 altered metabolites associated with the gut microbiota being significantly altered. Interestingly, several lipid metabolites that were altered in PD patients correlated with specific gut microbiome metabolites. Our results provide new insights into altered lipid metabolism and gut dysbiosis in PD, however, confirmation in a larger cohort would be needed.

Spontaneous Water Radical Cation Oxidation at Double Bonds in Microdroplets.

Spontaneous oxidation of compounds containing diverse X=Y moieties (e.g., sulfonamides, ketones, esters, sulfones) occurs readily in organic-solvent microdroplets. This surprising phenomenon is proposed to be driven by the generation of an intermediate species [M+H2O]+·: a covalent adduct of water radical cation (H2O +· ) with the reactant molecule (M). The adduct is observed in the positive ion mass spectrum while its formation in the interfacial region of the microdroplet (i.e., at the air-droplet interface) is indicated by the strong dependence of the oxidation product formation on the spray distance (which reflects the droplet size and consequently the surface-to-volume ratio) and the solvent composition. Importantly, based on the screening of a ca. 21,000-compound library and the detailed consideration of six functional groups, the formation of a molecular adduct with the water radical cation is a significant route to ionization in positive ion mode electrospray, where it is favored in those compounds with X=Y moieties which lack basic groups. A set of model monofunctional systems was studied and in one case, benzyl benzoate, evidence was found for oxidation driven by hydroxyl radical adduct formation followed by protonation in addition to the dominant water radical cation addition process. Significant implications of molecular ionization by water radical cations for oxidation processes in atmospheric aerosols, analytical mass spectrometry and small-scale synthesis are noted.

Simultaneous quantification of trimethoprim metabolites in pediatric plasma

The dual agent antibiotic, trimethoprim/sulfamethoxazole (TMP-SMX), has been prescribed to treat or prevent infections for over 50 years. However, there are no published validated analytical methods for the measurement of TMP metabolites in humans. We developed methodology enabling reliable quantification of TMP and 5 metabolites in human plasma. Chromatographic separation was achieved in less than 8 min using a biphenyl column. Analytes were detected in positive electrospray mode using a tandem Waters Xevo-TQ-XS mass spectrometer. Precision and accuracy values for all analytes were within 15% of nominal values during assay validation.

Sweeping-micellar electrokinetic chromatography with tandem mass spectrometry as an alternative methodology to determine neonicotinoid and boscalid residues in pollen and honeybee samples

In this work, it is proposed for the first time an electrophoretic approach based on micellar electrokinetic chromatography coupled with tandem mass spectrometry (MEKC-MS/MS) for the simultaneous determination of nine neonicotinoids (NNIs) together with the fungicide boscalid in pollen and honeybee samples. The separation was performed using ammonium perfluorooctanoate (50 mM, pH 9) as both volatile surfactant and electrophoretic buffer compatible with MS detection. A stacking strategy for accomplishing the on-line pre-concentration of the target compounds, known as sweeping, was carried out in order to improve separation efficiency and sensitivity. Furthermore, a scaled-down QuEChERS was developed as sample treatment, involving a lower organic solvent consumption and using Z-Sep+ as dispersive sorbent in the clean-up step. Regarding the detection mode, a triple quadrupole mass spectrometer was operating in positive ion electrospray mode (ESI+) under multiple reaction monitoring (MRM). The main parameters affecting MS/MS detection as well as the composition of the sheath-liquid (ethanol/ultrapure water/formic acid, 50:49.5:0.5 v/v/v) and other electrospray variables were optimized in order to achieve satisfactory sensitivity and repeatability. Procedural calibration curves were established in pollen and honeybee samples with LOQs below 11.6 µg kg−1 and 12.5 µg kg−1, respectively. Precision, expressed as RSD, lower than 15.2% and recoveries higher than 70% were obtained in both samples. Two positive samples of pollen were found, containing imidacloprid and thiamethoxam. Imidacloprid was also found in a sample of honeybees. The obtained results highlight the applicability of the proposed method, being an environmentally friendly, efficient, sensitive and useful alternative for the determination of NNIs and boscalid in pollen and honeybee samples.

Development of a Method of Liquid Chromatography Coupled with Tandem Mass Spectrometry for Simultaneous Determination of Linezolid and Tedizolid in Human Plasma.

It is important to select appropriate antibiotics for infection control. Linezolid and tedizolid are newly developed and synthesized oxazolidinone antibacterial agents. It has been pointed out that there is a relationship between a high plasma concentration of the target drug and incidence of adverse effects, although it has been reported that neither linezolid nor tedizolid requires dose adjustment according to renal function. Due to the high incidence of adverse effects, both are often switched. Precise plasma concentration control by therapeutic drug monitoring (TDM) is desirable for reducing the adverse effects of both drugs and obtaining a better therapeutic effect. In this study, we aimed to establish a method for simultaneous quantification of linezolid and tedizolid in human plasma using LC coupled with tandem mass spectrometry. Sample preparation was performed by a simple operation with acetonitrile. Linezolid and tedizolid were separated by an octadecylsilyl column using a gradient elution of acetonitrile in aqueous 0.1% formic acid solution and were detected in the positive ion electrospray mode with multiple reaction monitoring. Quantification of linezolid and tedizolid ranged from 0.5 to 50 and 0.5 to 20 µg/mL, respectively. The intra-day and inter-day precision and accuracy of data were assessed and found to be acceptable. The developed method was successfully applied to measurement of the concentrations of linezolid and tedizolid. This simple method, which can simultaneously quantify both drug concentrations for daily TDM, could contribute to safer treatment of patients.

Isotonitazene, a novel psychoactive substance opioid, detected in two cases following a local surge in opioid overdoses.

Isotonitazene is a novel opioid that was first reported in Europe in 2019. There have been no reports of the detection of isotonitazene in patients presenting to the emergency department with acute drug toxicity. There was an increase in presentations to our emergency department with acute opioid toxicity in August 2021. We aim to describe this outbreak and provide detail on two cases in which isotonitazene was quantified in serum samples. Serum samples were available for comprehensive toxicological analysis for two presentations. Written consent was obtained and the samples were analysed through a Thermo XRS ultrahigh-performance liquid chromatography system, interfaced to a Thermo Q Exactive high-resolution accurate mass spectrometer, operating in heated positive ion electrospray mode. Acquired data were processed using Toxfinder software (Thermo) against a regularly updated in-house database. There was an increase in acute opioid presentations to our emergency department from a median of 10 per month to 36 in August 2021. Twenty were treated with naloxone, and 23 were admitted to the hospital for observation and treatment. Serum sample analysis from two patients with acute opioid toxicity responsive to naloxone detected the presence of isotonitazene (0.18 and 0.81 ng/ml). We report a cluster of acute opioid toxicity presentations to our Emergency Department with detection of isotonitazene in two cases. Analytical screening is important in determining the presence of novel psychoactive substances (NPS) and to help inform the public health of the implications of NPS use, particularly during clusters of acute recreational drug toxicity presentations.

Selective Plate-Based Assay for Trace EDTA Analysis via Boron Trifluoride-methanol Derivatization UHPLC-QqQ-MS/MS Enabling Biologic and Vaccine Processes.

The employment of ethylenediaminetetraacetic acid (EDTA) across several fields in chemistry and biology has required the creation of a high number of quantitative assays. Nonetheless, the determination of trace EDTA, especially in biologics and vaccines, remains challenging. Herein, we introduce an automated high-throughput approach based on EDTA esterification in 96-well plates using boron trifluoride-methanol combined with rapid analysis by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Derivatization of EDTA to its methyl ester (Me-EDTA) serves to significantly improve chromatographic performance (retention, peak shape, and selectivity), while also delivering a tremendous enhancement of sensitivity in the positive ion mode electrospray ionization (ESI+). This procedure, in contrast to previous EDTA methods based on complexation with metal ions, is not affected by high concentration of other metals, buffers, and related salts abundantly present in biopharmaceutical processes (e.g., iron, copper, citrate, etc.). Validation of this assay for the determination of ng·mL-1 level EDTA in monoclonal antibody and vaccine products demonstrated excellent performance (repeatability, precision, and linear range) with high recovery from small sample volumes while also providing an advantageous automation-friendly workflow for high-throughput analysis.

Study on molecular structure characteristics of natural dissolved organic nitrogen by use of negative and positive ion mode electrospray ionization Orbitrap mass spectrometry and collision-induced dissociation

Dissolved organic nitrogen (DON) in aquatic systems is an important component of the global nitrogen cycle. However, the molecular structural information of DON in natural water is still unknown. In this study, the molecular structural characteristics of DON molecules in three natural waters were studied by using negative and positive ion mode electrospray ionization (ESI) Orbitrap mass spectrometry and collision-induced dissociation (CID). The DON compounds in these natural water samples could be selectively ionized by a positive ESI source with formic acid as the ionization promoter. A fraction of DON may exist as amphoteric substance. Then, possible chemical structures were assigned for some of these DON molecules by CID. Possible O-containing functional groups could be assigned as carboxyl, hydroxyl, carbonyl and methoxyl in negative/positive ESI tandem mass spectra, and neutral loss of NH3 corresponding to amino groups was observed for the first time in a positive ESI CID MSMS analysis, which demonstrated that a fraction of DON in natural water may exist as amino acid-like compounds. The results demonstrate that the positive/negative ESI CID Orbitrap MSMS method could provide valuable molecular structure information on DON in natural water.

Integrated laser ablation-dropletProbe-mass spectrometry for absolute drug quantitation, metabolite detection, and distribution in tissue.

Spatially resolved and accurate quantitation of drug-related compounds in tissue is a much-needed capability in drug discovery research. Here, application of an integrated laser ablation-dropletProbe-mass spectrometry surface sampling system (LADP-MS) is reported, which achieved absolute quantitation of propranolol measured from <500 × 500 μm thin tissue samples. Mouse liver and kidney thin tissue sections were coated with parylene C and analyzed for propranolol by a laser ablation/liquid extraction workflow. Non-coated adjacent sections were microdissected for validation and processed using standard bulk tissue extraction protocols. High-performance liquid chromatography with positive ion mode electrospray ionization tandem mass spectrometry was applied to detect the drug and its metabolites. Absolute propranolol concentration in ~500 × 500 μm tissue regions measured by the two methods agreed within ±8% and had a relative standard deviation within ±17%. Quantitation down to ~400 × 400 μm tissue regions was shown, and this resolution was also used for automated mapping of propranolol and phase II hydroxypropranolol glucuronide metabolites in kidney tissue. This study exemplifies the capabilities of integrated laser ablation-dropletProbe-mass spectrometry (LADP-MS) for high resolution absolute drug quantitation analysis of thin tissue sections. This capability will be valuable for applications needing to quantitatively understand the spatial distribution of small molecules in tissue.

A Non-Covalent Dimer Formation of Quaternary Ammonium Cation with Unusual Charge Neutralization in Electrospray-Ionization Mass Spectrometry

Specific and nonspecific non-covalent molecular association of biomolecules is characteristic for electrospray-ionization mass spectrometry analysis of biomolecules. Understanding the interaction between two associated molecules is of significance not only from the biological point of view but also gas phase analysis by mass spectrometry. Here we reported a formation of non-covalent dimer of quaternary ammonium denatonium cation with +1 charge detected in the positive ion mode electrospray ionization mass spectrometry analysis of denatonium benzoate. Hydrogen deuterium exchange of amide and carbon-bonded hydrogens revealed that charge neutralization of one denatonium cation is the consequence of amide hydrogen dissociation. DFT (Density Functional Theory) calculations proved high thermodynamic stable of formed dimer stabilized by the short and strong N..H-N hydrogen bond. The signal intensity of the peak characterizing non-covalent dimer is low intensity and does not depend on the sample concentration. Additionally, dimer observation was found to be instrument-dependent. The current investigation is the first experimental and theoretical study on the quaternary ammonium ions dimer. Thus the present study has great significance for understanding the structures of the biomolecules as well as materials.

Detection and confirmation of zilpaterol in equine hair using liquid chromatography-mass spectrometry.

Zilpaterol is a β2 -adrenergic agonist and a repartitioning agent that has a high potential for abuse in equine performance athletes. Analysis of zilpaterol in hair is an alternative sampling matrix that extends detection time periods beyond those found in urine or blood samples. Our laboratory has been screening for zilpaterol in hair for many years and recently detected and confirmed its presence in official samples. Accordingly, a liquid chromatography-mass spectrometry method was developed and validated to detect and confirm zilpaterol in equine hair. Briefly, equine hair was decontaminated, cut, and pulverized prior to disruption and liquid-liquid extraction in basic conditions. Following extraction, the sample was introduced to an Agilent 1260 HPLC and zilpaterol was separated using a reverse phase gradient with a total run time of 12.5min. Following chromatographic separation, zilpaterol and its corresponding stable isotope labeled internal standard were introduced via positive mode electrospray ionization to a Thermo Q-Exactive Plus mass spectrometer and spectra collected using parallel reaction monitoring. The methodology was validated using in-house criteria including characterization of accuracy, precision, recovery, linear range, matrix effects, limit of detection, and limit of quantitation, and the method was found to be fit-for-purpose to confirm the presence of zilpaterol in equine hair. This methodology has been used to detect and confirm the presence of zilpaterol from out-of-competition hair samples submitted by regional racing authorities.

LC-MS/MS Analysis Elucidates a Daily Rhythm in Orexin A Concentration in the Rat Vitreous Body.

Orexins are two neuropeptides synthesised mainly in the brain lateral hypothalamic area. The orexinergic system provides arousal-dependent cues for a plethora of brain centres, playing a vital role in feeding behaviour, regulation of the sleep–wake cycle and circadian rhythms. Recently, orexins were found to be produced in the retina of an eye; however, their content in the vitreous body and possible daily pattern of expression have not yet been explored. In this manuscript, we describe the development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method designed for quantitative bioanalysis of orexin in the rat vitreous body. Orexin was extracted from vitreous body samples with a water:acetonitrile:formic acid (80:20:0.1; v/v/v) mixture followed by vortexing and centrifuging. Separation was performed on a reverse-phase HPLC column under gradient conditions. Orexin was analysed via multiple-reaction monitoring (MRM) in the positive electrospray mode. The total analysis time for each sample was less than 5.0 min. Once the method was fully optimised, it was then validated, following the 2018 FDA guidance on bioanalytical method validations. The calibration curves for orexin (1–500 ng/mL) were constructed using a linear regression with a 1/x2 weighting. The lower limit of quantitation for orexin was 1.0 pg/mL for the vitreous body. Intra-day and inter-day estimates of accuracy and precision were within 10% of their nominal values, indicating that the method is reliable for quantitation of orexin in the rat vitreous body. From the physiological perspective, our results are the first to show daily rhythm of orexin synthesis by the retina with possible implications on the circadian regulation of vision.

Changes in lipidomic profile by anti-retroviral treatment regimen: An ACTG 5257 ancillary study.

High cardiovascular disease risk in people living with HIV is partly attributed to antiretroviral therapy (ART). Lipid response to ART has been extensively studied, yet, little is known how small molecule lipids respond to Integrase inhibitor-based (INSTI-based) compared to Protease inhibitor-based (PI-based) ART regimens.Ancillary study to a phase 3, randomized, open-label trial [AIDS Clinical Trial Group A5257 Study] in treatment-naive HIV-infected patients randomized in a 1:1:1 ratio to receive ritonavir-boosted atazanavir (ATV/r), ritonavir-boosted darunavir (DRV/r) (both PI-based), or raltegravir with Tenofovir Disoproxil Fumarate-TDF plus emtricitabine (RAL, INSTI-based).We examined small molecule lipid response in a subcohort of 75 participants. Lipidomic assays of plasma samples collected pre- and post-ART treatment (48 weeks) were conducted using ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry. The effect of ART regimens was regressed on lipid species response adjusting for the baseline covariates (lipids, age, sex, race, CD4 level, BMI, and smoking). Results were validated in the Centers for AIDS Research Network of Integrated Clinical Systems study (N = 16).Out of 417 annotated lipids, glycerophospholipids (P = .007) and sphingolipids (P = .028) had a higher response to ATV/r and DRV/r compared to RAL. The lysophosphatidylcholine (LPCs(16:1),(17:1),(20:3)) and phosphophatidylcholine species (PCs(40:7),(38:4)) had an opposite response to RAL versus ATV/r in the discovery and validation cohort. The INSTI-based regimen had an opposite response of ceramide species ((d38:1), (d42:2)), PCs((35:2), (38:4)), phosphatidylethanolamines (PEs(38:4), (38:6)), and sphingomyelin(SMd38:1) species compared with the PI-based regimens. There were no differences observed between 2 PI-based regimens.We observed differences in response of small molecule lipid species by ART regimens in treatment-naive people living with HIV.