Electrospray Ionization Source Research Articles

Unravelling the structure of one-electron oxidation products of model peptide backbones containing methionine by IRMPD spectroscopy: the effect of the neighbouring groups.

A series of Methionine (Met) derivatives, where either the amino group and/or the carboxylic acid group is blocked by acetyl and/or methyl ester functionalities, has been investigated by Collision Induced Dissociation-tandem mass spectrometry (CID-MS2) and Infrared Multiple Photon Dissociation (IRMPD) spectroscopy. The CID-MS2 experiments were performed using a Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometer equipped with an electrospray ionization (ESI) source. The IRMPD spectra were recorded employing a Paul type ion-trap coupled with the free-electron laser (FEL) FELIX in the fingerprint region from 600 to 2000 cm-1. We show that the oxidation of the methionine residue with protected terminal groups induces the formation of a sulfoxide group. However, compared to the IRMPD spectrum of protonated methionine and methionine sulfoxide, significant spectral differences are observed in the spectra of model peptide backbones containing methionine. DFT calculations show that protonation occurs on the sulfoxide group in the gas phase for these model peptide backbones containing methionine, shifting the diagnostic signature of the sulfoxide group from 1000 to 862 cm-1.

Determination of three methylimidazole compounds in cosmetics by high performance liquid chromatography-tandem mass spectrometry

Methylimidazole compounds are byproducts formed during the caramel-coloring process and are used in various cosmetics. In addition metronidazole is an antibacterial and anti-inflammatory drug commonly used in modern medicine and is used in cosmetics to treat acne in the short-term. The illegal addition of metronidazole during cosmetics production can result in residual 2-methylimidazole (2-MEI), which, along with 4-methylimidazole (4-MEI), is a class 2B carcinogen. Therefore, establishing efficient, accurate, and sensitive analytical techniques for analyzing methylimidazole compounds in cosmetics is an urgent objective. In this study, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining 1-methylimidazole (1-MEI), 2-MEI, and 4-MEI in cosmetics was developed. Cosmetics samples were extracted via ultrasonication in acetonitrile and purified using a mixed cation-exchange (MCX) solid-phase extraction (SPE) column, with subsequent drying under a stream of nitrogen and redissolution in acetonitrile. The resulting solution was then filtered through a 0.22 μm organic filter membrane for further testing. The analytes were separated using an XBridge® shield RP18 chromatographic column (150 mm×4.6 mm, 3.5 μm) and isocratically eluted with 20 mmol/L ammonium formate solution (containing 0.1% formic acid)-acetonitrile (98∶2, v/v). The target compounds were ionized by electrospray ionization (ESI) source, analyzed in multi-reaction monitoring (MRM) mode, and quantified using the external standard method, with the peak area of the quantitative ion and the mass concentration of the compound taken as the longitudinal and transverse coordinates, respectively. Matrix-matching working curves were also constructed. 1-MEI exhibited good linear relationships in the range of 5-200 μg/L, with correlation coefficients (r2)≥0.9994, while 2-MEI and 4-MEI showed good linearities in the range of 2-100 μg/L with r2≥0.9984. The three methylimidazole compounds exhibited limits of detection (LODs) and quantification (LOQs) of 10-30 μg/kg and 25-100 μg/kg, respectively. Under three spiked levels (LOQ, 2LOQ, 10LOQ), the recoveries of three methylimidazole compounds were 80.9%-107.9%, with relative standard deviations (RSDs, n=6) of 1.2%-12.8%. The practicability of the method was examined using 48 cosmetic samples; 4-MEI was detected in nine samples at contents of 26-1000 μg/kg, while two samples contained 240 and 267 μg/kg of 2-MEI, respectively. 1-MEI was not detected in any of the 48 samples tested. The developed method is simple, fast, and highly sensitive, and provides methodological support for assessing risks and monitoring the three methylimidazole compounds in cosmetics through screening.

Online LC-Orbitrap MS method for the rapid molecular characterization of dissolved organic matter.

High-resolution mass spectrometry (HRMS) combined with electrospray ionization (ESI) has been the most useful technique for molecular characterization of dissolved organic matter (DOM) derived from diverse sources. However, the comprehensive detection of DOM composition was hindered by ionization suppression observed in ESI sources. HRMS coupled with liquid chromatography (LC) is a potential tool for comprehensive molecular characterization of DOM. The Suwannee River fulvic acids (SRFAs) and two DOM samples from seawater and refinery wastewater extracted by solid phase extraction (SPE) were analyzed by LC-Orbitrap MS coupled with ESI. The mobile phases, solvent composition, and gradient in the LC-Orbitrap MS analysis were optimized. The number of detected molecular formulae of SRFAs by online LC-Orbitrap MS was significantly increased by approximately 40% compared to direct injection. These additional detected compounds are mainly protein and lignin-like compounds, with a low O/C ratio and high H/C ratio. For the SRFAs, the relative standard deviations (%) of reproducibility are 5.51, 2.33, 7.97, and 1.80 for average O/C, H/C, double bond equivalent, and modified aromaticity index, respectively. This study proposed a simple and rapid method based on LC-Orbitrap MS for an in-depth analysis of the molecular composition of DOM, achieving a remarkable analysis time of only 5 min per sample. The rapid method provides a dependable and efficient approach for the molecular characterization of DOM, thereby advancing our comprehension and investigation of DOM across diverse ecosystems.

In vitro antifungal activity of extracts and alkaloid compounds from Piper arboreum against dermatophytes

Piper is widely distributed in subtropical regions and species of this genus are known for their potent pharmacological activities. Piper arboreum Aubl. is a traditional medicinal plant popularly known as "pau-de-angola", "jaborandi", and chili pepper, demonstrating antifungal, trypanocidal, antibacterial, and antioxidant activities. The leaves of P. arboreum were extracted using Soxhlet and dichloromethane to obtain the extract, which was then fractionated using solvents of different polarities. Samples were analyzed using ultra-high-performance liquid chromatography coupled with mass spectrometry equipped with an electrospray ionization source. Antifungal microdilution assays were performed, and scanning and transmission electron microscopy were used to assess the invasion of the pretreated nail. The minimum inhibitory concentration (MIC) values of the extract and a dichloromethane fraction were, respectively, 62.5 μg/ml and 16.0 μg/ml against Trichophyton rubrum, and 125 μg/ml and 62.5 μg/ml, and 500 μg/ml and 500 μg/ml against T. mentagrophytes, and Microsporum gypseum, respectively. No growth was observed on nail fragments exposed to the extract (at concentrations > 64 µg/ml and then inoculated with spore suspension. Transmission electron microscopy revealed strong inhibition of hyphal growth and an irregular growth pattern following treatment with the extract and the dichloromethane fraction. Results demonstrated the antifungal properties of the P. arboreum extract and its dichloromethane fraction against dermatophytes, with the identification of three different alkaloid compounds. The cytotoxicity was specific towards the fungal cells, and morphological and ultrastructural analyses indicated damage to the cell wall and cytoplasmic membrane as the potential mechanism of action. The leaf material used to generate the extract can be taken from the plant without any detrimental effect thus enabling strategies to be implemented for the exploitation of this species.

An Assessment of Surface Contamination and Dermal Exposure to 5-Fluorouracil in Healthcare Settings by UPLC-MS/MS Using a New Atmospheric Pressure Ionization Source

5-Fluorouracil (5-FU) is a well-known cytostatic drug, which is often used in cancer treatments. Yet, it is also a very dangerous compound for people who are occupationally exposed to it for a long time, such as pharmacy employees, nurses and cleaning staff. We aimed to improve and implement a LC-MS/MS method for 5-FU quantification on surface contamination samples collected with swabs in a pharmacy department and outpatient nursing station of a university hospital. To improve the existing methods to quantify 5-FU, we compared a LC-MS/MS method using the frequently applied electrospray ionization source (ESI) with a UniSpray ionization source (USI). To determine the contamination of 5-FU in a pharmacy department preparing 5-FU infusion bags, which are then given to patients in the outpatient nursing stations, we collected multiple surface swabs of the laminar flow cabinets and frequently touched objects, before the preparation and administration of 5-FU and afterwards. Furthermore, we sampled the protective gloves and the bare hands of employees of the pharmacy department, involved in the preparation of the infusion bags. Using the USI source, we were able to reach the lowest limit of quantification (LOQ). With this technique, we were able to detect 5-FU contamination on the laminar flow cabinets and frequently used objects in the pharmacy department and the outpatient nursing station in the very low ng/cm2 range. This contamination was mostly higher after preparation or administration than before. While we also found 5-FU on the protective gloves, we almost found no 5-FU on the skin of the pharmacy technicians preparing the 5-FU infusion bags. In conclusion, our method was able to detect very low concentrations of 5-FU contamination, but the contamination we found is very unlikely to result in any issues for the personnel working in these areas.

Detection of Veterinary Drugs in Food Using a Portable Mass Spectrometer Coupled with Solid-Phase Microextraction Arrow.

A portable mass spectrometer (PMS) was combined with a mesoporous silica material (SBA-15) coated solid-phase microextraction (SPME) Arrow to develop a rapid, easy-to-operate and sensitive method for detecting five veterinary drugs-amantadine, thiabendazole, sulfamethazine, clenbuterol, and ractopamine-in milk and chicken samples. Equipped with a pulsed direct current electrospray ionization source and a hyperboloid linear ion trap, the PMS can simultaneously detect all five analytes in approximately 30 s using a one-microliter sample. Unlike traditional large-scale instruments, this method shows great potential for on-site detection with no need for chromatographic pre-separation and minimal sample preparation. The SBA-15-SPME Arrow, fabricated via electrospinning, demonstrated superior extraction efficiency compared to commercially available SPME Arrows. Optimization of the coating preparation conditions and SPME procedures was conducted to enhance the extraction efficiency of the SBA-15-SPME Arrow. The extraction and desorption processes were optimized to require only 15 and 30 min, respectively. The SBA-15-SPME Arrow-PMS method showed high precision and sensitivity, with detection limits and quantitation limits of 2.8-9.3 µg kg-1 and 10-28 µg kg-1, respectively, in milk. The LOD and LOQ ranged from 3.5 to 11.7 µg kg-1 and 12 to 35 µg kg-1, respectively, in chicken. The method sensitivity meets the requirements of domestic and international regulations. This method was successfully applied to detect the five analytes in milk and chicken samples, with recoveries ranging from 85% to 116%. This approach represents a significant advancement in food safety by facilitating rapid, in-field monitoring of veterinary drug residues.

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 µm) 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 µg/g for almonds, 102 - 4080 µg/g for cashews, 128 - 5120 µg/g for hazelnuts, and 122 - 4880 µg/g for pistachios. Limits of detection were 9 µg/g for almonds, 30.6 µg/g for cashews, 38.4 µg/g for hazelnuts, and 36.6 µg/g for pistachios, while limits of quantification were 30.5 µg/g for almonds, 102 µg/g for cashews, 128 µg/g for hazelnuts, and 122 µg/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.

Development of a rapid and robust hydrop interaction liquid chromatography tandem mass spectrometry method for the detection of 13 endogenous amino acids as well as trimethylamine oxide in serum and tissues of the mice.

This work aimed to establish an HILIC-MS/MS method to simultaneously determine the levels of 13 endogenous amino acids and trimethylamine oxide in the biological samples from the mice. Electrospray ion source was used for the analysis of mass spectrometry. The 20 min separation was applied in a Dikma Inspire Hilic column (2.1 × 100.0mm, 3μM). Positive ion mode under an MRM model gave a satisfying response value. The limits of quantitation were evaluated by accuracy from -12.59% to 7.89% and precision from 1.77% to 14.00% as well as acceptable interday and intraday precision, matrix effect, recovery, and stability. Later, the assay was successfully used to measure the concentrations of the determinands in the biological samples. Individual and tissue distribution differences for these metabolites were observable. The amino acids had a consistent highest content in the spleens, while the lowest levels were found in the livers. Alanine was the most abundant amino acid in the serum, and taurine kept the highest content in all of the tissues. Trimethylamine oxide remained low level, especially in the liver samples.

B-177 Development of a Contrived Fecal Matrix for Clinical Validation of a LC-MS/MS Assay to Quantify Microbiota-derived Metabolites in Stool Specimens

Abstract Background The gut microbiota plays a critical role in health and its disruption can impact clinical outcomes in a variety of circumstances, including graft-versus-host disease following hematopoietic stem cell transplantation, Clostridioides difficile infections, and liver disease. Metagenomic and metabolomic analyses of fecal material is frequently performed in the research setting; however, evaluation of the gut microbiota is not yet incorporated into routine patient care. One barrier to its implementation is the lack of matrix-appropriate materials for validation of clinical assays. While surrogate fecal matrices are being developed for metagenomic sequencing, matrices specific for targeted metabolomics are currently unavailable. In this study, we designed a contrived fecal matrix to validate a new LC-MS/MS method for measurement of microbiota-derived metabolites from stool specimens. Methods Common food products and mucin were individually analyzed by LC-MS/MS for quantities of three microbiota-derived metabolites - butyrate, deoxycholic acid (DCA) and taurocholic acid (TCA). Food items with the lowest endogenous metabolite concentrations were selected to create a contrived fecal matrix with a consistency similar to human stool specimens. Calibrators and quality controls (QC) were then created by spiking butyrate, DCA, and TCA into either the contrived fecal matrix or 50% methanol (MeOH). The internal standards (ISTD) used were d7-butyrate, d4-DCA, and d4-TCA, respectively. Three human stool specimens were selected and spiked at a range of metabolite concentrations to assess matrix effects. All samples, calibrators and controls were homogenized in 80% MeOH extraction solvent containing the ISTDs using beadruptor tubes. The suspension was centrifuged and 25 µL of supernatant was derivatized using 12.5 µL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 12.5 µL 3-nitrophenylhydrazine at 40°C for 30 minutes. For optimal separation of isomers, 2 µL of sample was injected and separated on a CORTECS T3 column (1.6 µm, 2.1 x 100 mm, Waters) at 45°C with a flow rate of 0.350 mL/min and run-time of 20 min using the Exion LC AD (Sciex). Analytes were detected using a Sciex QTRAP 6500 mass spectrometer with an electrospray ionization source. Results A contrived fecal matrix containing a mix of avocado, black beans, gluten-free flour, and cornstarch blended in water and mucin was created. The contrived matrix was confirmed to contain minimal to no endogenous levels of the 3 metabolites of interest: butyrate, DCA, and TCA. Validation of the LC-MS/MS assay using the contrived fecal matrix showed a linear analytical measurement range from 25-2000 µM for butyrate, 1-60 µM for DCA, and 0.5-640 µM for TCA. Intra-assay precision at 7 QC levels for all analytes was <10%. The matrix effects from use of the contrived fecal matrix compared to patient fecal samples were 140-148% for d7-butyrate and 101-112% for d4-DCA across multiple spiked analyte concentrations. Conclusions Our LC-MS/MS method quantifies butyrate, deoxycholic acid, and taurocholic acid, metabolites which serve as indicators of microbiota health in liver disease patients. The contrived fecal matrix provides an additional tool for the development and validation of clinical assays to assess the gut microbiota.

B-182 Development and validation of an LC-MS/MS assay for simultaneous measurement of serum androstenedione and 17-hydroxyprogesterone in serum

Abstract Background Androstenedione and 17-hydroxyprogesterone are measured via immunoassays or high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories. Immunoassays have limited sensitivity and specificity for certain steroid hormones testing. We aimed to develop and validate a sensitive, accurate, and specific in-house LC-MS/MS assay to simultaneously measure both 17-hydroxyprogesterone and androstenedione in serum. Methods 13C-labeled androstenedione and 17-hydroxyprogesterone internal standards were added to calibrator, control, and patient samples. A hexane/ethyl acetate solution was used in a liquid-liquid extraction procedure. The organic layer was then removed and dried followed by reconstituting the extracts in 2.5 mM ammonium formate in 50% methanol. The HPLC mobile phase gradient began with 47%, 2.5 mM ammonium formate in water and 53%, 2.5 mM ammonium formate in methanol. This progressed to 56% of the methanol phase over three minutes. The mixtures were separated though an Agilent 1260 Infinity HPLC system with a Poroshell 120 EC-C18 analytical column (2.1 × 50 mm × 2.7 µm). The separated HPLC eluents then entered an Agilent 6460C QQQ mass spectrometer in positive ion mode through an electrospray ionization source where protonated androstenedione (m/z = 287.2) with the protonated androstenedione-[2, 3, 4-13C3] internal standard (m/z = 290.2) and protonated 17-hydroxyprogesterone (m/z = 331.2) with the protonated 17-hydroxyprogesterone-[2, 3, 4-13C3] (m/z = 334.2) were mass-selected for collision-induced dissociation. For androstenedione, respective quantifier and qualifier m/z values of 287.2 > 97.1 and 287.2 > 109.1 were used while respective quantifier and qualifier m/z values of 331.2 > 97.1 and 331.2 > 109.1 were used for 17-hydroxyprogesterone. Validation studies were performed including imprecision, lower limit of quantitation (LLOQ), analytical measurement range (AMR), interference, specificity, carryover, extraction recovery, matrix effect, and comparison with a reference laboratory LC-MS/MS method. The total allowable error limits used in this study were 23.5% for androstenedione and 30.2% for 17-hydroxyprogesterone. Results Total imprecision results across three concentrations yielded a maximum CV of 4.6 % for androstenedione and 6.5% for 17-hydroxyprogesteron. The AMRs of androstenedione and 17-hydroxyprogesterone were 0.2 to 13.4 and 0.1 to 22.0 ng/mL without significant carryovers. No significant interferences were observed from 11-deoxycorticosterone (700 ng/dL), testosterone (2500 ng/dL), DHEA (1000 ng/mL), bilirubin (30.1 mg/dL), hemoglobin (1032 mg/dL), or triglycerides (1858 mg/dL). The extraction recoveries of androstenedione and 17-hydroxyprogesterone were 98% and 81%, respectively. No significant matrix effects were identified. In comparison to the reference lab LC-MS/MS method, the in-house LC-MS/MS assay demonstrated a 0.523% bias for androstenedione and a -3.77% bias for 17-hydroxyprogesterone with both exhibiting excellent correlations with the reference lab measurements. More specifically, our results (as a function of values obtained from the reference lab) followed the relationship of y = 1.039x - 0.092 (R = 0.9951) for androstenedione and y = 0.957x + 0.01 (R = 0.9974) for 17-hydroxyprogesterone. Conclusions An LC-MS/MS assay is developed for simultaneous detection of serum androstenedione and 17-hydroxyprogesterone. The assay demonstrates acceptable imprecision, AMR, and accuracy. No significant carryover and interference from other structurally similar steroid hormones and other common interferents are identified.

Pharmacokinetic and tissue distribution study of pectolinarigenin in rats using UPLC-MS/MS

Pectolinarigenin (PEC), a natural flavonoid isolated from Cirsium japonicum, exhibits promising therapeutic potential for multiple cancers. In present study, a simple and sensitive UPLC-MS/MS method was established for the quantification of PEC in rat plasma and tissues. The assay procedure involved a one-step protein precipitation with tadalafil as the internal standard, and separation on a Welch Xtimate UHPLC C18 column by gradient elution of acetonitrile/aqueous formic acid (0.1 %, v/v) at a flow rate of 0.2 m L·min−1. The detection was conducted using multiple-reaction monitoring via an electrospray ionization source in positive ionization mode. The established method was proved to be highly sensitive with a good linearity (R2 > 0.99) in respective concentration range (0.1–100 ng·mL−1 in plasma and 1–10000 ng·mL−1 in tissues) and acceptable extraction recovery (≥71.17 %), matrix effect and stability, which was applied to study the pharmacokinetics and tissue distribution of PEC after intravenous (100 μg·kg−1) and oral administration (10, 20 and 40 mg·kg−1). PEC was promptly absorbed (Tmax ≤ 0.222 h) and maintained at a low level with slow elimination (t1/2 z ≥ 14.47 h) in rats after oral administration, resulting in extremely low bioavailability (0.56–0.68 %). However, PEC is widely distributed in rat tissues with high exposure in GI tract, liver and kidney. The bioavailability and tissue affinity were firstly revealed, which will guide directions for further development of PEC as an anti-tumor drug candidate.

Investigating the ions emitted by multiply charged ionic liquids from a porous electrospray ion source

A porous electrospray ion source was tested with three ionic liquids in order to investigate the effects of ionic liquid properties on the sizes of ion clusters emitted by purely ionic electrospray sources. Two of the ionic liquids, bis(1-ethyl-3-methylimidazolium) tetrathiocyanatocobaltate and 1,6-bis(3-methylimidazolium-1-yl)hexane bis(trifluoromethylsulfonyl)amide, were selected due to them having a dication or dianion, which were termed multiply charged ionic liquids due to them containing anions or cations with more than one charge within them. These were selected in order to investigate ionic clustering within electrospray ion emission and were compared against one of the most common ionic liquids, EMI-BF4. The current–voltage data showed that EMI-BF4 emitted similar levels of current to bis(1-ethyl-3-methylimidazolium) tetrathiocyanatocobaltate, even though the latter liquid had significantly lower conductivity and higher viscosity, suggesting an improvement in current speculated to be due to the extra charges contained by the ions. Time-of-flight and retarding potential analysis data are provided, showing all three liquids emitting ions comprising of monomers, dimers, trimers, and quadramers, with some of the 1,6-bis(3-methylimidazolium-1-yl)hexane bis(trifluoromethylsulfonyl)amide data showing heavier species emission. The data also suggested that the multiply charged ionic liquids produced ions that have two anions or cations attached, termed “double ions,” with these ions not been previously reported using porous electrospray sources. Furthermore, it was found that the dimers emitted by both of the multiply charged ionic liquids seemed to be more stable than EMI-BF4 dimers, providing insight into ion cluster formation.

Determination of Ibuprofen Enantiomers in Mouse Blood Using Liquid Chromatography-Tandem Mass Spectrometry and Its Application to a Pharmacokinetic Study.

The aim of this study was to establish a simple, fast, and sensitive method with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneously determining ibuprofen enantiomers using mouse blood in very small volumes. LC-MS/MS equipped with an electrospray ionization (ESI) source was used in negative ion mode and multiple-reaction monitoring mode. Enantiomer chromatographic separation was carried out on a Lux® 5 μm Cellulose-3 (250 × 4.6 mm, 5 μm) column at a flow rate of 0.6 mL/min. Samples were pretreated by extracting only 5 μL of blood with 40 μL of acetonitrile (containing 1.3% formic acid) so that a concentration-time profile could be completed using a single mouse. 2-(4-Propylphenyl) propanoic acid was used as an internal standard. Standard curves for each enantiomer were linear from 0.04 to 80.00 μg/mL, demonstrating a lower limit of quantitation (LLOQ) than all previously reported methods. This method was completely validated and successfully executed to investigate the pharmacokinetics of ibuprofen enantiomers after intravenous administration of racemic ibuprofen, (S)-(+)-ibuprofen, and (R)-(-)-ibuprofen in Kunming mice, respectively. The results showed that the pharmacokinetic profiles of the (R)-(-)-ibuprofen and (S)-(+)-ibuprofen were significantly different, indicating the unidirectional inversion of R-(-)-ibuprofen to (S)-(+)-ibuprofen.

UPLC-MS/MS analysis of axitinib and pharmacokinetic application in beagle dogs

ObjectiveTo develop a method for determining the concentration of axitinib in beagle dog plasma and utilize this method to investigate the pharmacokinetics of orally administered axitinib in beagle dogs. MethodsPlasma samples were processed using acetonitrile precipitation and analyzed by UPLC-MS/MS with sunitinib as an internal standard (IS). Chromatographic separation was achieved on a Waters Acquisition UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm) with a gradient elution of acetonitrile and 0.1 % formic acid. Mass spectrometry uses an electrospray ion source for positive ion detection in a multiple reaction monitoring mode. The monitored ion transitions for axitinib and sunitinib were m/z 387 → 355.96 and m/z 399.3 → 282.96, respectively. Six beagle dogs were administered 0.33 mg/kg of axitinib orally, and venous blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h post-dose for pharmacokinetic analysis. ResultsThe assay demonstrated a linear range of 0.5–100 ng/mL (r2 = 0.9992), and the lower limit of quantification was up to 0.5 ng/mL. Precision, as assessed by relative standard deviation (RSD), was within 8.64 % for both intraday and interday variability. The relative error (RE) for precision from −2.77 %–1.20 %. The recovery rate of the analytes exceeded 85.28 % and the matrix effect was approximately 100 %. Plasma samples maintained stability under various conditions, including room temperature storage for 12 h, processed on an automatic sampler at 4 °C for 6 h, three freeze-thaw cycles, and long-term storage at −80 °C for 60 days. Pharmacokinetic parameters were determined using DAS 2.0 software, revealing a half-life (T1/2) of 6.05 h and an area under the curve (AUC (0 →∞)) of 97.13 ng h/mL for axitinib. ConclusionsThe UPLC-MS/MS method developed in this study offers high specificity, rapid analysis, high recovery, excellent linearity, and minimal plasma volume requirements, making it well-suited for pharmacokinetic and drug interaction studies in beagles dogs.

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 × 2.1 mm × 1.7 µm) 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 µL. The method has been fully validated and meets the performance requirements of AOAC, demonstrating high specificity. The linearity range was established from 100 µg/kg to 1000 µg/kg, with a method detection limit (MDL) of 30 µg/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.

Metabolomics-based biomarkers of fermented dairy and red meat intake: a randomized controlled trial in healthy adults.

Dietary assessment is usually performed through imprecise tools, leading to error-prone associations between diet and health-related outcomes. Metabolomics has been applied in recent years to develop biomarkers of food intake (BFIs) and to study metabolites in the diet-microbiome crosstalk. Candidate BFIs exist to detect intake of meat and to a lesser extent dairy, but validation and further development of BFIs are needed. Here, we aim to identify biomarkers that differentiate between intakes of red meat and dairy, to validate previously reported BFIs for these foods, and to explore the effect of protein-matched meals on selected microbial metabolites. We conducted a randomized, controlled, cross-over single-meal study comparing a meal with highly fermented yogurt and cheese, and a meal with beef and pork meatballs. Postprandial urine samples from 17 subjects were collected sequentially after each meal up to 24h and analyzed by untargeted metabolomics through ultra-high-performance-liquid chromatography (UHPLC) coupled via electrospray (ESI) source to a qTOF mass spectrometer. Univariate (repeated measures ANOVA) and multivariate (PLSDA, ML-PLSDA) data analyses were used to select BFIs differentiating the two meals. 3-Indoxyl sulfate, p-cresol sulfate, and several other microbial amino acid catabolites were additionally explored within the urine profiles. Thirty-eight markers of meat and dairy intake were selected and are presented along with their excretion kinetics. Carnosine, taurine, and creatine, as well as hydroxyproline-based dipeptides are confirmed as meat BFIs. For dairy, previously reported metabolites such as acyl-glycines are confirmed, while proline-based dipeptides are reported as novel putative BFIs. Microbial metabolites showed only marginal evidence of differential formation after the two meals. This study allowed us to validate the postprandial kinetics of previously suggested biomarkers of meat and dairy intake and to identify new potential biomarkers. The excretion kinetics are useful to ensure that the collection of urine covers the correct time window in future dietary studies. The BFIs add to the existing body of biomarkers and may further be used in combination to provide a more reliable assessment of meat and dairy intake. Proteolytic microbial metabolites should be further investigated to assess the effect of different protein sources on health.

High-performance liquid chromatography-tandem mass spectrometry was used to measure 20 antidepressants in human serum.

Aim: This study used high performance liquid chromatography-tandem mass spectrometry to quantify the blood concentrations of 20 antidepressants, such as bupropion and fluoxetine, in human serum samples.Methods: After direct precipitation with a 1:9 protein precipitant of methanol and acetonitrile, serum samples were examined using high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The material was separated using a Poroshell 120 EC-C18 column (3.0mm×50mm, 2.7μm) and gradient elution. The mobile phases were phase A 0.01% formic acid aqueous solution (containing 2mmol/ml ammonium acetate) and phase B methanol solution. A 0.45ml/min flow rate was used to divide the sample and inject 5μl. Electrospray ionization source positive ion mode and multiple reaction monitoring modes were used for analysis. Measurement was quantified using an internal standard technique.Results: Accuracy ranged from 90.3 to 114.3%, intra-day precision from 100.1 to 112.3%, inter-day precision from 100.4 to 112.6%, extraction recoveries from 85.5 to 114.5% and matrix effect from 85.6 to 98.7%.Conclusion: This approach is fast, accurate, sensitive and repeatable. It can identify 20 antidepressants in blood simultaneously. This can be used to monitor blood drug levels and medication metabolism.

Comparative Pharmacokinetics and Tissue Distribution of Hexahydrocurcumin Following Intraperitoneal vs Oral Administration in Mice Using LC-MS/MS.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine hexahydrocurcumin (HHC) levels in mouse plasma, brain, liver, and kidneys using a negative ion mode electrospray ionization (ESI) source. Demonstrating a lower limit of quantification (LLOQ) of 5 ng/mL, the method showed excellent linearity across a concentration range of 5-500 ng/mL in all tested matrices. Precision evaluations reported a coefficient of variation (CV%) of less than 13.19% for both intraday and interday measurements, while accuracy ranged from 95.13 to 105.07% across all quality control levels. HHC extraction recovery was consistently observed between 70.18 and 93.28%, with a CV% deviation of less than 15%. In the pharmacokinetic evaluation of HHC in mice following a single intraperitoneal (IP) or oral administration, a noncompartment analysis was utilized. After IP administration (40 mg/kg), the C max value was 47.90 times higher than that achieved via oral administration. Peak plasma concentrations were observed approximately 5 min post-IP and 15 min post-oral dosing. The observed half-lives after these administrations were approximately 1.52 and 2.17 h for IP and oral routes, respectively. Oral administration revealed a relative bioavailability of only 12.28% compared with the IP route. Furthermore, following IP administration, the half-life values in brain, liver, and kidney were not significantly different but more than the half-life value found in plasma. The liver and kidney exhibited the highest concentrations of HHC, while the brain showed the least, suggesting that the hydrophobic nature of HHC impedes its passage through the blood-brain barrier. This study is the first to provide detailed insights into the pharmacokinetics and tissue distribution characteristics of HHC following oral and IP administration in mice, setting the stage for further focus on HHC as a potential new drug candidate.

Online Supercritical Fluid Chromatography Hyphenated to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry with Quadrupole Detection for Microalgae Bio-Oil Characterization.

Microalgae are an attractive feedstock for biofuel production thanks to their renewable nature, high growth rate, and ability to use anthropogenic CO2. The conversion of microalgae by hydrothermal liquefaction (HTL) leads to a solid residue, a gaseous phase, and a biocrude. However, the bio-oil is rich in heteroatoms and requires upgrading processes to be used as biofuels. For these treatments to be effective, detailed knowledge of the sample is crucial. The bio-oil was characterized by direct introduction into a Fourier transform ion cyclotron resonance mass spectrometer (DI-FTICR MS) with an electrospray ionization source (ESI). Thousands of molecular formulas were assigned with a high level of confidence, mainly compounds with nitrogen and oxygen atoms. Additionally, the bio-oil was analyzed by coupling supercritical fluid chromatography (SFC) and FTICR to combine the separation power of SFC to the high performances of a 12 T FTICR. Quadrupole detection (2ω) was used in FTICR to have a high resolving power with a lower transient time. The coupling allowed the separation of many isomers along the chromatogram, showing the isomeric complexity of microalgae bio-oils. Moreover, classes of compounds were separated according to their heteroatom class thanks to the SFC separation. In this work, the advantages of DI-FTICR MS and SFC-FTICR MS proved complementary, and DI was useful to study the bio-oil at the molecular scale thanks to the high performances, while SFC proved useful for the characterization at the isomeric scale. This demonstrated the significant potential of this new online hyphenated technique for the characterization of complex matrices.

Metabolomics Unveils Disrupted Pathways in Parkinson's Disease: Toward Biomarker-Based Diagnosis.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by diverse symptoms, where accurate diagnosis remains challenging. Traditional clinical observation methods often result in misdiagnosis, highlighting the need for biomarker-based diagnostic approaches. This study utilizes ultraperformance liquid chromatography coupled to an electrospray ionization source and quadrupole time-of-flight untargeted metabolomics combined with biochemometrics to identify novel serum biomarkers for PD. Analyzing a Brazilian cohort of serum samples from 39 PD patients and 15 healthy controls, we identified 15 metabolites significantly associated with PD, with 11 reported as potential biomarkers for the first time. Key disrupted metabolic pathways include caffeine metabolism, arachidonic acid metabolism, and primary bile acid biosynthesis. Our machine learning model demonstrated high accuracy, with the Rotation Forest boosting model achieving 94.1% accuracy in distinguishing PD patients from controls. It is based on three new PD biomarkers (downregulated: 1-lyso-2-arachidonoyl-phosphatidate and hypoxanthine and upregulated: ferulic acid) and surpasses the general 80% diagnostic accuracy obtained from initial clinical evaluations conducted by specialists. Besides, this machine learning model based on a decision tree allowed for visual and easy interpretability of affected metabolites in PD patients. These findings could improve the detection and monitoring of PD, paving the way for more precise diagnostics and therapeutic interventions. Our research emphasizes the critical role of metabolomics and machine learning in advancing our understanding of the chemical profile of neurodegenerative diseases.