Heart-Cutting Bidimensional Liquid Chromatography for the Simultaneous Analysis of Veterinary Drugs Residues and Nucleotide Monophosphates in Sheep's Milk.

Sheep's milk is a significant source of nucleotide monophosphates (NMPs) but can also contain undesirable residues from veterinary drugs, posing a potential human health risk. This study introduces a novel application of two-dimensional liquid chromatography (2D-LC), in heart-cutting mode, for the simultaneous determination of nucleotides and veterinary drug residues in sheep's milk. 2D-LC allows for the separation of these compounds in a single chromatographic run despite their differing physicochemical properties. The proposed method separates six veterinary drug residues and five NMPs in a single injection. The compounds were separated using a C18 reversed-phase column in the first dimension and a Primesep SB analytical column in the second dimension. The method performance was evaluated in terms of linearity range, detection and quantification limits, matrix effects, precision, and accuracy. The results demonstrated good linearity and sensitivity, with quantification limits allowing for the quantification of veterinary drugs at the maximum residue level and nucleotides at typical levels found in milk samples. The method has been successfully applied to the analysis of sheep's milk samples acquired from local supermarkets, with recoveries within a range of 70-119% and 82-117% for veterinary residues and NMPs, respectively.

Simultaneous determination of favipiravir and surrogates of its metabolites by means of heart-cutting bidimensional liquid chromatography (2D-LC).

Therapeutic monitoring of drugs, particularly those with multiple metabolites, can be time-consuming and labor-intensive due to the need for different analytical methods depending on the specific metabolite or matrix of interest. In this study, we employed a heart-cutting 2D-LC separation method based on the coupling of reversed-phase and mixed-mode mechanisms to determine Favipiravir and surrogates of five main metabolites. This approach was applied to serum, plasma, urine, and human peripheral blood mononuclear cells. The method underwent validation to ensure its reliability. The findings highlight the potential of 2D-LC as a practical and efficient approach for therapeutic drug monitoring.

Real-Time Volatile Metabolomics Analysis of Dendritic Cells.

Dendritic cells (DCs) actively sample and present antigen to cells of the adaptive immune system and are thus vital for successful immune control and memory formation. Immune cell metabolism and function are tightly interlinked, and a better understanding of this interaction offers potential to develop immunomodulatory strategies. However, current approaches for assessing the immune cell metabolome are often limited by end-point measurements, may involve laborious sample preparation, and may lack unbiased, temporal resolution of the metabolome. In this study, we present a novel setup coupled to a secondary electrospray ionization-high resolution mass spectrometric (SESI-HRMS) platform allowing headspace analysis of immature and activated DCs in real-time with minimal sample preparation and intervention, with high technical reproducibility and potential for automation. Distinct metabolic signatures of DCs treated with different supernatants (SNs) of bacterial cultures were detected during real-time analyses over 6 h compared to their respective controls (SN only). Furthermore, the technique allowed for the detection of 13C-incorporation into volatile metabolites, opening the possibility for real-time tracing of metabolic pathways in DCs. Moreover, differences in the metabolic profile of naïve and activated DCs were discovered, and pathway-enrichment analysis revealed three significantly altered pathways, including the TCA cycle, α-linolenic acid metabolism, and valine, leucine, and isoleucine degradation.

UHPLC-MS/MS-Based Identity Confirmation of Amino Acids Involved in Response to and Side Effects from Antiseizure Medications.

Real-time breath analysis using secondary electrospray ionization coupled with high-resolution mass spectrometry is a fast and noninvasive method to access the metabolic state of a person. However, it lacks the ability to unequivocally assign mass spectral features to compounds due to the absence of chromatographic separation. This can be overcomed by using exhaled breath condensate and conventional liquid chromatography-mass spectrometry (LC-MS) systems. In this study, to the best of our knowledge, we confirm for the first time the presence of six amino acids (GABA, Oxo-Pro, Asp, Gln, Glu, and Tyr) previously reported to be involved in response to and side effects from antiseizure medications in exhaled breath condensate and by extension in exhaled human breath. Raw data are publicly available at MetaboLights with the accession number MTBLS6760.

Real-time pharmacokinetics via online analysis of exhaled breath.

The advantages that on-line breath analysis has shown in different fields have already made it stand as an interesting tool for pharmacokinetic studies. This review summarizes recent progress in the field, diving into the different analytical methods and the different advantages and hurdles encountered. We conclude that there is a wealth of limitations in the application of this technique, and key aspects like standardization are still outstanding. Nevertheless, this is an experimental field that has not yet been fully explored; and the advantages it offers for animal welfare, decrease in the amount of drug needed in experimental studies, and complementary insights to current pharmacological studies, warrant further exploration. Further studies are needed to overcome current limitations and incorporate this technique into the toolbox of pharmacological studies, both at an industrial and academic level.

Personalised therapeutic management of epileptic patients guided by pathway-driven breath metabolomics.

Background: Therapeutic management of epilepsy remains a challenge, since optimal systemic antiseizure medication (ASM) concentrations do not always correlate with improved clinical outcome and minimal side effects. We tested the feasibility of noninvasive real-time breath metabolomics as an extension of traditional therapeutic drug monitoring for patient stratification by simultaneously monitoring drug-related and drug-modulated metabolites. Methods: This proof-of-principle observational study involved 93 breath measurements of 54 paediatric patients monitored over a period of 2.5 years, along with an adult's cohort of 37 patients measured in two different hospitals. Exhaled breath metabolome of epileptic patients was measured in real time using secondary electrospray ionisation-high-resolution mass spectrometry (SESI-HRMS). Results: We show that systemic ASM concentrations could be predicted by the breath test. Total and free valproic acid (VPA, an ASM) is predicted with concordance correlation coefficient (CCC) of 0.63 and 0.66, respectively. We also find (i) high between- and within-subject heterogeneity in VPA metabolism; (ii) several amino acid metabolic pathways are significantly enriched (p < 0.01) in patients suffering from side effects; (iii) tyrosine metabolism is significantly enriched (p < 0.001), with downregulated pathway compounds in non-responders. Conclusions: These results show that real-time breath analysis of epileptic patients provides reliable estimations of systemic drug concentrations along with risk estimates for drug response and side effects.

Exploring polar hydrophobicity in organized media for extracting oligopeptides: application to the extraction of opiorphin in human saliva.

Supramolecular solvents (dubbed SUPRAS) are gaining momentum as extractants of compounds of interest from complex matrixes such as foodstuff and biological and environmental samples. However, their powerful extraction mechanism, based on multiligand ability for solute binding, fails when applied to very polar compounds, hindering their applicability to the extraction of highly polar metabolites. In this work, we introduce the synthesis, characterization, and application of a new kind of SUPRAS formed by heptafluorobutyric acid (HFBA). The polar hydrophobicity of this perfluorinated acid results in a SUPRAS, which coacervates at acidic pHs, that shows a great capability to extract amino acids and oligopeptides (recoveries in the range 81-105%) with nonpolar alkyl, cyclic or aromatic side chain substituents (with log D > -3.62). To further demonstrate the potential of this novel SUPRAS, an analytical methodology for the determination of opiorphin in real saliva samples was developed and fully validated. The HFBA-based SUPRAS was synthetized in situ from 950 µL of stabilized saliva, by the addition of 150 µL of HFBA and 400 µL of HCl 37% (v/v). The resulting SUPRAS was directly injected into a LC-MS/MS system for further quantification. Quantitative recoveries in the range of 87-110% were obtained with relative standard deviations below 20%. The HFBA-based SUPRAS is, therefore, capable of efficiently extracting opiorphin from saliva samples and shows a high potential for the determination of several amino acids and oligopeptides from biological samples.

LC-HRMS based on mixed-mode chromatography for the separation of teicoplanin and the unravelment of its composition.

Liquid chromatography - high resolution mass spectrometry (LC-HRMS) based on mixed-mode chromatography has proven to be a useful tool in pharmaceutical analysis for the characterization of very polar antibiotics such as teicoplanin. This compound is a semisynthetic glycopeptide antibiotic thought to be a mixture of five major compounds (named A2-1 through A2-5) and four minor ones (RS-1 to RS-4), all sharing the same glycopeptide core structure (dubbed A3-1) and differing only on the length of a hydrocarbon side chain. These nine compounds have been fully characterized in the past thirty years by means of HPLC coupled to UV and MS detectors. However, HPLC separations were all based on octadecylsilica columns (C18), which nowadays may not be the best choice for the chromatographic separation of such polar and charged compounds. In this work, several different chromatographic alternatives are tested for the separation of teicoplanin, including mixed-mode chemistries. It has been demonstrated that a C18-PFP mixed-mode column provides the best chromatographic resolution, furthermore, confirming the existence of several minor compounds in the composition of teicoplanin. Up to fourteen minor components were characterized by means of High-resolution MS/MS and confirmed by the analysis of a commercial teicoplanin product (Targocid®).

Multifunctional green supramolecular solvents for cost-effective production of highly stable astaxanthin-rich formulations from Haematococcus pluvialis.

The interest of food industry to merchandise natural astaxanthin is growing up. However, it confronts scientific and technological challenges mainly related to its poor water solubility and chemical instability. Here, we present a new quick and efficient green process to simultaneously extract, encapsulate and stabilize astaxanthin from Haematococcus pluvialis. The process is based on the hitherto unexplored combination of supramolecular solvents (SUPRAS), nanostructured liquids generated from amphiphiles through sequential self-assembly and coacervation, and nanostructured lipid carriers (NLCs). These novel nanosystems were characterized by means of dynamic light scattering, AFM and cryoSEM, revealing spherical particles of ∼100 nm. Their antioxidant activity was measured by ORAC (20.6 ±â€¯3.9 µM TE) and α-TEAC (2.92 ±â€¯0.58 µM α-TE) assays and their in vitro capacity to inhibit ROS by DHE probe. Results showed that the SUPRAS-NLCs proposed yield high extraction and encapsulation efficiencies (71 ±â€¯4%) in combination with a remarkable time stability (180 d, 4 °C).

A high thermally stable oligomer-based supramolecular solvent for universal headspace Gas Chromatography: Proof-of-principle determination of residual solvents in drugs.

Supramolecular solvent (SUPRAS) extraction is gaining attraction as a sample treatment technique because of its great performance in terms of effectiveness, versatility, sample clean-up, quickness, cost and sustainability. However, the nature of SUPRASs, being formed by amphiphile molecules, results in poor compatibility with Gas Chromatography (GC). Here, we show the hitherto unexplored development of a SUPRAS with high thermal stability (HTS) suitable for subsequent direct analysis by Headspace (HS)-GC. This novel HTS-SUPRAS, based on poly-undecylenic acid -an oligomeric surfactant-, tetraglyme -a polar aprotic solvent with excellent chemical and thermal stability- and water, was fully characterized in terms of composition and physical properties such as thermal stability. Subsequently, the HTS-SUPRAS developed was further successfully applied, as a proof-of-principle, to the extraction and determination of residual solvents in pharmaceutical drugs, including several solvents (class 2C) whose analysis by HS-GC has been shown to be highly complex. Analytical performance was demonstrated as mandated by the International Council for Harmonisation of technical requirements for pharmaceuticals for human use (ICH). Furthermore, excellent recoveries (70-120%) and high precisions (<20%, expressed as relative standard deviations) were obtained for the analysis of several different drug formulations spiked with the analyzed 37 residual solvents at their respective maximum residue levels.

Restricted Access Volatile Supramolecular Solvents for Single-Step Extraction/Cleanup of Benzimidazole Anthelmintic Drugs in Milk Prior to LC-MS/MS.

In this work, a restricted access volatile supramolecular solvent (RAM-VOL-SUPRAS) directly synthesized in milk is proposed for the first time for the simultaneous extraction and cleanup of benzimidazole anthelmintic drugs in milk meant for human consumption. The RAM-VOL-SUPRAS was formed by the self-assembly and coacervation of hexanol in tetrahydrofuran induced by the water content in milk. Benzimidazoles legislated by the European Union were quantitatively extracted (80-110%), and proteins were precipitated by the action of THF and the amphiphile; extraction of carbohydrates was avoided by a size exclusion mechanism, and lipids were removed during hexanol evaporation. The analytical methodology was fully validated according to Commission Decision 2002/657/EC. Method detection limits from 0.03 to 0.14 µg L-1 were well below the maximum residue limits legislated in milk for these drugs, with interday precisions at maximum residue levels below 13%. This novel methodology guarantees a rapid and reliable tool for daily and routine laboratory analyses in the field of food quality control.

Real-time exhaled breath analysis in patients with cystic fibrosis and controls.

We aimed at defining profiles of volatile organic compounds in exhaled breath from patients with cystic fibrosis (CF) using a novel real-time mass spectrometry technique. In this prospective matched case-control study, 30 patients with CF, and 30 healthy control subjects were matched one-to-one according to age, gender, and smoking state. We performed exhaled breath analysis by untargeted secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS). Patients with CF (mean age 26.0 ± 13.0 years) and controls (mean age 27.9 ± 14.0 years) were analyzed using SESI-HRMS. 49 exhaled breath features were found to be altered (p-value < 0.05/q-value < 0.1) in CF patients, in comparison to healthy controls. The two most discriminating features showed a prediction AUROC of 77.1% (95% CI 62.2%-87.8%) with a specificity of 80.0% and a sensitivity of 63.3%. Levels of oxidative stress metabolites such as fatty acids were found to differ significantly between patients with CF and healthy controls. Furthermore, in patients with CF, 11 features correlated with the mucus concentration of Stenotrophomonas maltophilia bacteria. Exhaled breath analysis with SESI-HRMS allows the identification of CF specific compounds in real-time and may trace bacterial strains in affected patients with CF.

SUPRAS extraction approach for matrix-independent determination of amphetamine-type stimulants by LC-MS/MS.

Monitoring of amphetamine-type stimulant (ATS) confronts clinical labs with a high number of samples involving a variety of biological matrices. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), routinely used for confirmation of ATS abuse, requires of laborious and matrix-dependent sample treatment methods, this increasing analysis time and cost. In this work, a universal and single-step sample treatment, based on supramolecular solvents (SUPRAS), was proposed for simplifying ATS confirmation in seven biological matrices. The SUPRAS was synthesized in situ in the sample (900 µL of basified oral fluid, urine, serum, sweat or breast milk or 50 mg of digested hair or fingernails) by the addition of hexanol (200 µL) and tetrahydrofuran (900 µL). The mixture was vortex-shaken and centrifuged and the SUPRAS extract was subsequently analyzed by positive ion mode electrospray LC-MS/MS. The method was fully validated for amphetamine (AMP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), N-ethyl-3,4-methylenedioxyamphetamine (MDEA) and N-methyl-3,4-methylenedioxyamphetamine (MDMA). Maximum ion suppression or enhancement was 9% and 7%, respectively, and extraction recoveries (87-111%) and within- (0.1-6.7%) and between-day (0.3-9.7%) CVs were all within required values. The lower limits of quantification (LLOQ) for biological fluids (5 ng/mL), and hair and fingernails (100 ng/g) were all well below the cut-offs established by worldwide organizations. Confirmation of MDA was carried out in five urine samples that tested positive for ATS by immunoassay. The SUPRAS-LC-MS/MS methodology succeeded in developing a hitherto unexplored and universal tool for quantifying ATS in a comprehensive pool of biological matrices of interest in forensic and clinical samples.

Comprehensive Real-Time Analysis of the Yeast Volatilome.

While yeast is one of the most studied organisms, its intricate biology remains to be fully mapped and understood. This is especially the case when it comes to capture rapid, in vivo fluctuations of metabolite levels. Secondary electrospray ionization-high resolution mass spectrometry SESI-HRMS is introduced here as a sensitive and noninvasive analytical technique for online monitoring of microbial metabolic activity. The power of this technique is exemplarily shown for baker's yeast fermentation, for which the time-resolved abundance of about 300 metabolites is demonstrated. The results suggest that a large number of metabolites produced by yeast from glucose neither are reported in the literature nor are their biochemical origins deciphered. With the technique demonstrated here, researchers interested in distant disciplines such as yeast physiology and food quality will gain new insights into the biochemical capability of this simple eukaryote.

Multicore Magnetic Nanoparticles Coated with Oligomeric Micelles: Characterization and Potential for the Extraction of Contaminants over a Wide Polarity Range.

Oligomeric micelles from sodium undecylenate (oSUD) were chemisorbed to magnetic iron oxide nanoparticles (MNPs) through a single-step synthetic route involving the simultaneous nanoparticle formation and functionalization in an aqueous medium. The resulting spherical nanoparticles (MNPs-oSUD) consisted of a concatenation of iron oxide cores, with an average size of 7.7 nm, bound by oSUD micelles (particle average diameter of ca. 200 nm). Micellar coverage was ∼50% of the MNP-oSUD (by weight) and offered multiple retention mechanisms (e.g., dispersion, hydrogen bonding, polar, and ionic) for solute solubilization while keeping it intact during analyte elution. The high density of micelles and variety of interactions provided by this sorbent rendered it highly efficient for the extraction of aromatic amines in a wide polarity range (log Kow values from -0.80 to 4.05) from textiles, urine, and wastewater. Extraction took 5 min, no cleanup or evaporation of the extracts was needed and the method, based on LC-MS/MS quantitation, proved matrix-independent. Recoveries for 17 aromatic amines in samples were in the range of 93%-123% while those with negative log Kow values were in the range of 69%-87%. Detection limits for aromatic amines in textiles (0.007-2 mg kg-1) were well below the limits legislated by the European Union (EU) (30 mg kg-1) and those in urine and wastewater (0.004-1.5 µg L-1) were at the level usually found in real-world applications. All the analyzed samples were positive in aromatic amines. The easy synthesis and excellent extraction properties of MNPs-oSUD anticipate their high potential not only for multiresidue analysis but also in other fields such as water remediation.

Real-Time Quantification of Amino Acids in the Exhalome by Secondary Electrospray Ionization-Mass Spectrometry: A Proof-of-Principle Study.

BACKGROUND: Amino acids are frequently determined in clinical chemistry. However, current analysis methods are time-consuming, invasive, and suffer from artifacts during sampling, sample handling, and sample preparation. We hypothesized in this proof-of-principle study that plasma concentrations of amino acids can be estimated by measuring their concentrations in exhaled breath. A novel breath analysis technique described here allows such measurements to be carried out in real-time and noninvasively, which should facilitate efficient diagnostics and give insights into human physiology. METHODS: The amino acid profiles in 37 individuals were determined by ion-exchange HPLC in blood plasma and simultaneously in breath by secondary electrospray ionization coupled to high-resolution mass spectrometry. Participants were split into training and test sets to validate the analytical accuracy. Longitudinal profiles in 3 individuals were additionally obtained over a 12-h period. RESULTS: Concentrations of 8 slightly volatile amino acids (A, V, I, G, P, K, F, Orn) could be determined in exhaled breath with a CV of <10%. Exhalome validation studies yielded high accuracies for each of these amino acids, on average only 3% less compared to plasma concentrations (95% CI ±13%). Higher variations were found only for amino acids with a low plasma concentration. CONCLUSIONS: This study demonstrates for the first time that amino acids can be quantified in the human breath and that their concentrations correlate with plasma concentrations. Although this noninvasive technique needs further investigation, exhalome analysis may provide significant benefits over traditional, offline analytical methods.

Determination of nucleosides and nucleotides in baby foods by hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents.

In this work we propose a rapid and efficient method for the joint determination of nucleosides and nucleotides in dairy and non-dairy baby foods based on hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of diethylammonium (DEA) as a hydrophilic ion-pairing reagent (IP-HILIC-MS/MS). Sample treatment of the baby food included dilution with water and centrifugal ultrafiltration (CUF) with an additional washing step that notably improved the global performance of the process. Later dilution of the extract with acetonitrile allowed adequate separation in the HILIC system. With the proposed treatment, we obtained extraction recoveries higher than 80% and, additionally, no matrix effects were observed. The CUF-IP-HILIC-MS/MS method was validated according to the 2002/657/EC decision and was used for the quantification of nucleotides and nucleosides in sixteen samples of commercial baby foods.

Secondary electrospray ionization coupled to high-resolution mass spectrometry reveals tryptophan pathway metabolites in exhaled human breath.

Many studies and personalized medicine in general require frequent measurements and/or rapid results of biomarker levels. Here we show that 20 low volatility metabolites of the tryptophan pathway can be detected in exhaled human breath. This real-time and non-invasive method offers an attractive alternative to blood analysis.

Capturing in Vivo Plant Metabolism by Real-Time Analysis of Low to High Molecular Weight Volatiles.

We have deployed an efficient secondary electrospray ionization source coupled to an Orbitrap mass analyzer (SESI-MS) to investigate the emissions of a Begonia semperflorens. We document how hundreds of species can be tracked with an unparalleled time resolution of 2 min during day-night cycles. To further illustrate the capabilities of this system for volatile organic compounds (VOCs) analysis, we subjected the plant to mechanical damage and monitored its response. As a result, ∼1200 VOCs were monitored displaying different kinetics. To validate the soundness of our in vivo measurements, we fully characterized some key compounds via tandem mass spectrometry (MS/MS) and confirmed their expected behavior based on prior gas chromatography/mass spectrometry (GC/MS) studies. For example, ß-caryophyllene, which is directly related to photosynthesis, was found to show a periodic day-night pattern with highest concentrations during the day. We conclude that the capability of SESI-MS to capture highly dynamic VOC emissions and wide analyte coverage makes it an attractive tool to complement GC/MS in plant studies.

Real-Time Chemical Analysis of E-Cigarette Aerosols By Means Of Secondary Electrospray Ionization Mass Spectrometry.

Chemical analysis of aerosols collected from electronic cigarettes (ECs) has shown that these devices produce vapors that contain harmful and potentially harmful compounds. Conventional analytical methods used for the analysis of electronic cigarettes do not reflect the actual composition of the aerosols generated because they usually neglect the changes in the chemical composition that occur during the aerosol generation process and after collection. The aim of this work was to develop and apply a method for the real-time analysis of electronic cigarette aerosols, based on the secondary electrospray ionization technique coupled to high-resolution mass spectrometry, by mimicking the "vaping" process. Electronic cigarette aerosols were successfully analyzed and quantitative differences were found between the liquids and aerosols. Thanks to the high sensitivity shown by this method, more than 250 chemical substances were detected in the aerosols, some of them showing a high correlation with the operating power of the electronic cigarettes. The method also allows proper quantification of several chemical components such as alkaloids and flavor compounds.