Hydrophilic Interaction Liquid Chromatography-mass Spectrometry Research Articles

Multi-class cyanobacterial toxin analysis using hydrophilic interaction liquid chromatography-mass spectrometry.

Cyanobacteria produce diverse classes of toxins including microcystins, nodularins, anatoxins, cylindrospermopsins and saxitoxins, encompassing a range of chemical properties and mechanisms of toxicity. Comprehensive analysis of these toxins in cyanobacterial, environmental and biological samples generally requires multiple methods of extraction and analysis. In this work, a method was developed for the major classes of cyanotoxins, which comprised of a three-step liquid-solid extraction method using 75 % CH3CN with 0.1 % HCOOH and a hydrophilic interaction liquid chromatography (HILIC) elution gradient that provided retention of the less polar microcystins through to the highly polar saxitoxins. Detection was performed by tandem mass spectrometry in selected reaction monitoring mode with positive and negative polarity switching. Identification criteria included matching retention times and product ion ratios with available standards. In-house validation demonstrated good performance of the method including precision ranging from 1.5 (microcystin-LA) to 5.8 (gonyautoxin-2) % RSDs, and detection limits ranging from 0.01 (cylindrospermopsin) to 0.99 (gonyautoxin-3) µg/g in freeze dried material cyanobacteria. Recovery was assessed using spiked non-toxic cyanobacterial samples (Aphanizomenon sp.) and ranged from 83 (neosaxitoxin) to 107 % ([Dha7]microcystin-LR). As a demonstration of application, toxin profiles in cyanobacterial cultures, benthic and planktonic cyanobacteria field samples, and shellfish reference materials were successfully evaluated. The procedure is also amenable for extension to other polar toxin classes including domoic acid and guanitoxin. With increasing reports of cyanobacterial blooms globally, the method represents a powerful quantitative screening tool for measuring cyanotoxins across a broad range of samples.

Metabolomics and Biochemical Benefits of Multivitamin and Multimineral Supplementation in Healthy Individuals: A Pilot Study.

Scientific evidence regarding the effectiveness of vitamin and mineral supplements in healthy individuals remains scarce. In a randomized, double-blind study, 30 healthy individuals were assigned to receive a single daily dose of multivitamin and multimineral supplementation or a double daily dose for 30 days. Before and after the intake, an untargeted metabolomics assay for serum metabolites was conducted by hydrophilic interaction liquid chromatography-mass spectrometry, and clinical assessments of peripheral blood samples were performed. A paired t-test for metabolic analysis, adjusted using the false discovery rate (FDR) and p-value correction method (rate of change > 2 and FDR < 0.05), the Shapiro-Wilk test, Student's t-test, and the Mann-Whitney U test were applied depending on the variable, with a 5% significance level. An impact on oxidative stress was observed, with a significant reduction in homocysteine levels and an increment of pyridoxic acid (vitamin B6). The effect on energy metabolism was shown by a significant increase in diverse metabolites, such as linoleoylcarnitine. Serum iron and calcium levels were also impacted. Overall, we observed a nutritional balance compatible with a good state of health. In conclusion, beneficial effects on adult health were demonstrated in relation to oxidative stress, energy metabolism, and nutritional balance.

Metabolic features of adolescent major depressive disorder: A comparative study between treatment-resistant depression and first-episode drug-naive depression

Major depressive disorder (MDD) is a psychiatric illness that can jeopardize the normal growth and development of adolescents. Approximately 40% of adolescent patients with MDD exhibit resistance to conventional antidepressants, leading to the development of Treatment-Resistant Depression (TRD). TRD is associated with severe impairments in social functioning and learning ability and an elevated risk of suicide, thereby imposing an additional societal burden. In this study, we conducted plasma metabolomic analysis on 53 adolescents diagnosed with first-episode drug-naïve MDD (FEDN-MDD), 53 adolescents with TRD, and 56 healthy controls (HCs) using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) and reversed-phase liquid chromatography-mass spectrometry (RPLC-MS). We established a diagnostic model by identifying differentially expressed metabolites and applying cluster analysis, metabolic pathway analysis, and multivariate linear support vector machine (SVM) algorithms. Our findings suggest that adolescent TRD shares similarities with FEDN-MDD in five amino acid metabolic pathways and exhibits distinct metabolic characteristics, particularly tyrosine and glycerophospholipid metabolism. Furthermore, through multivariate receiver operating characteristic (ROC) analysis, we optimized the area under the curve (AUC) and achieved the highest predictive accuracy, obtaining an AUC of 0.903 when comparing FEDN-MDD patients with HCs and an AUC of 0.968 when comparing TRD patients with HCs. This study provides new evidence for the identification of adolescent TRD and sheds light on different pathophysiologies by delineating the distinct plasma metabolic profiles of adolescent TRD and FEDN-MDD.

Assessment of Untargeted Metabolomics by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry to Define Breast Cancer Liquid Biopsy-Based Biomarkers in Plasma Samples.

An early diagnosis of cancer is fundamental not only in regard to reducing its mortality rate but also in terms of counteracting the progression of the tumor in the initial stages. Breast cancer (BC) is the most common tumor pathology in women and the second deathliest cancer worldwide, although its survival rate is increasing thanks to improvements in screening programs. However, the most common techniques to detect a breast tumor tend to be time-consuming, unspecific or invasive. Herein, the use of untargeted hydrophilic interaction liquid chromatography-mass spectrometry analysis appears as an analytical technique with potential use for the early detection of biomarkers in liquid biopsies from BC patients. In this research, plasma samples from 134 BC patients were compared with 136 from healthy controls (HC), and multivariate statistical analyses showed a clear separation between four BC phenotypes (LA, LB, HER2, and TN) and the HC group. As a result, we identified two candidate biomarkers that discriminated between the groups under study with a VIP > 1 and an AUC of 0.958. Thus, targeting the specific aberrant metabolic pathways in future studies may allow for better molecular stratification or early detection of the disease.

Diagnosing Parkinson’s disease and monitoring its progression: Biomarkers from combined GC-TOF MS and LC-MS/MS untargeted metabolomics

Parkinson's disease (PD) is a prevalent neurodegenerative disorder with a poorly understood etiology. An accurate diagnosis of idiopathic PD remains challenging as misdiagnosis is common in routine clinical practice. Moreover, current therapeutics focus on symptomatic management rather than curing or slowing down disease progression. Therefore, identification of potential PD biomarkers and providing a better understanding of the underlying disease pathophysiology are urgent. Herein, hydrophilic interaction liquid chromatography–mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-TOF MS) based metabolomics approaches were used to profile the serum metabolome of 50 patients with different stages of idiopathic PD (early, mid and advanced) and 45 age-matched controls. Levels of 57 metabolites including cysteine-S-sulfate and N-acetyl tryptophan were significantly higher in patients with PD compared to controls, with lower amounts of additional 51 metabolites including vanillic acid, and N-acetylaspartic acid. Xanthines, including caffeine and its downstream metabolites, were lowered in patients with PD relative to controls indicating a potential role caffeine and its metabolites against neuronal damage. Seven metabolites, namely cysteine-S-sulfate, 1-methylxanthine, vanillic acid, N-acetylaspartic acid, 3-N-acetyl tryptophan, 5-methoxytryptophol, and 13-HODE yielded a ROC curve with a high classification accuracy (AUC 0.977). Comparison between different PD stages showed that cysteine-S-sulfate levels were significantly increasing with the advancement of PD stages while LPI 20:4 was significantly decreasing with disease progression. Our findings provide new biomarker candidates to assist in the diagnosis of PD and monitor its progression. Unusual metabolites like cysteine-S-sulfate might point to therapeutic targets that could enhance the development of novel PD treatments, such as NMDA antagonists.

Simultaneous determination of water-soluble herbicides using hydrophilic interaction liquid chromatography-mass spectrometry.

The analysis of water-soluble herbicides, including glyphosate (Glyp), glufosinate (Gluf), paraquat (PQ), and diquat (DQ), is time-consuming and expensive because they cannot be analyzed using general toxicological screening methods. Thus, this study aimed to develop a simple and rapid method to simultaneously analyze these compounds without any derivatization nor ion-pairing reagents. The analytes were separated using hydrophilic interaction liquid chromatography and detected using tandem mass spectrometry. The developed method was applied to plant and biological samples assuming criminal damage and poisoning cases, respectively. All analytes were separated well and detected with good peak shapes. For plant samples, the herbicides were specifically detected from withered leaves using a simple extraction method. For biological samples, quantitative analysis was successfully validated, and the limit of quantification values of Glyp and Gluf were 0.2µg/mL, and those of PQ and DQ were 1ng/mL. The developed method had sufficient performance for practical forensic applications including poisoning cases and malicious uses to damage commercial crops.

Novel Adsorbents for the Determination of Amino Acids in Soil Extracts by Hydrophilic Interaction Liquid Chromatography with Mass Spectrometric Detection

Adsorbents based on various substrates—silica and a copolymer of styrene with divinylbenzene—are developed for the determination of amino acids by hydrophilic interaction liquid chromatography—mass spectrometry. The optimal version of the structure of the functional layer in two series of the obtained stationary phases was chosen, which provides the best hydrophilization for each substrate. Retention mechanisms were studied and the conditions for the mass-spectrometric detection, separation, and determination of 16 amino acids were chosen. The applicability of the obtained adsorbents and a method for determining amino acids for the analysis of soil extracts were estimated.

Analysis of Taurine in Infant Formulas and Adult Nutritionals by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry: First Action 2022.03.

Taurine is recognized as an essential growth factor and as being critical in the maintenance of functional tissue regulation. To evaluate the analytical performance of a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for compliance with AOAC Standard Method Performance Requirements (SMPR®) for taurine analysis described in SMPR 2014.013. Following protein precipitation with Carrez solutions, taurine is extracted and separated by HILIC with detection by triple quadrupole MS using multiple reaction monitoring (MRM). Stable isotope labeled (SIL) taurine internal standard is used for quantification to correct for losses in extraction and variations in ionization in the ion source. The method was shown to meet the requirements specified in the SMPR with a linear range of 0.27-2700 mg/hg RTF (ready-to-feed), a limit of detection of 0.14 mg/hg RTF, acceptable recovery of 97.2-100.1%, and acceptable repeatability of 1.6-6.4% relative standard deviation. Additionally, the method was found to have no statistically significant bias compared with reference values for National Institute of Standards and Technology (NIST) 1849a certified reference material (CRM) (P-value = 0.95) and 1869 CRM (P-value = 0.31), and with results from AOAC 997.05 (P-value = 0.10). A recent review of the method and validation data by the Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel (ERP) found that this method met all the criteria for analysis of taurine specified in SMPR 2014.013 and voted to adopt this method as First Action AOAC Official MethodSM2022.03. A method for the analysis of taurine in infant formulas and adult nutritionals by HILIC-MS/MS is described. A single-laboratory validation (SLV) study demonstrated the applicability of the method to meet requirements of SMPR 2014.013. In December 2022, the SPIFAN ERP voted to adopt this method as First Action AOAC Official Method 2022.03.

Mass spectrometry of the white adipose metabolome in a hibernating mammal reveals seasonal changes in alternate fuels and carnitine derivatives.

Mammalian hibernators undergo substantial changes in metabolic function throughout the seasonal hibernation cycle. We report here the polar metabolomic profile of white adipose tissue isolated from active and hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Polar compounds in white adipose tissue were extracted from five groups representing different timepoints throughout the seasonal activity-torpor cycle and analyzed using hydrophilic interaction liquid chromatography-mass spectrometry in both the positive and negative ion modes. A total of 224 compounds out of 660 features detected after curation were annotated. Unsupervised clustering using principal component analysis revealed discrete clusters representing the different seasonal timepoints throughout hibernation. One-way analysis of variance and feature intensity heatmaps revealed metabolites that varied in abundance between active and torpid timepoints. Pathway analysis compared against the KEGG database demonstrated enrichment of amino acid metabolism, purine metabolism, glycerophospholipid metabolism, and coenzyme A biosynthetic pathways among our identified compounds. Numerous carnitine derivatives and a ketone that serves as an alternate fuel source, beta-hydroxybutyrate (BHB), were among molecules found to be elevated during torpor. Elevated levels of the BHB-carnitine conjugate during torpor suggests the synthesis of beta-hydroxybutyrate in white adipose mitochondria, which may contribute directly to elevated levels of circulating BHB during hibernation.

Characterization of the Polar Profile of Bacon and Fuerte Avocado Fruits by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry: Distribution of Non-structural Carbohydrates, Quinic Acid, and Chlorogenic Acid between Seed, Mesocarp, and Exocarp at Different Ripening Stages.

Avocado fruit growth and development, unlike that of other fruits, is characterized by the accumulation of oil and C7 sugars (in most fruits, the carbohydrates that prevail are C6). There are five essential carbohydrates which constitute 98% of the total content of soluble sugars in this fruit; these are fructose, glucose, sucrose, d-mannoheptulose, and perseitol, which together with quinic acid and chlorogenic acid have been the analytes under study in this work. After applying an efficient extraction procedure, a novel methodology based on hydrophilic interaction liquid chromatography coupled to mass spectrometry was applied to determine the levels of these seven substances in tissues─exocarp, seed, and mesocarp─from avocado fruits of two different varieties scarcely studied, Bacon and Fuerte, at three different ripening stages. Quantitative characterization of the selected tissues was performed, and the inter-tissue distribution of metabolites was described. For both varieties, d-mannoheptulose was the major component in the mesocarp and exocarp, whereas perseitol was predominant in the seed, followed by sucrose and d-mannoheptulose. Sucrose was found to be more abundant in seed tissues, with much lower concentrations in avocado mesocarp and exocarp. Quinic acid showed a predominance in the exocarp, and chlorogenic acid was exclusively determined in exocarp samples.

Rapid Analysis of Taurine in Infant Formula and Adult Nutritionals by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Taurine is recognized as an essential growth factor and as being critical in the maintenance of functional tissue regulation. A rapid compliance method for the analysis of taurine that is applicable to infant formula and milk-based nutritional products is described. Following protein precipitation with Carrez solutions, taurine in the sample extract is separated by hydrophilic interaction liquid chromatography (HILIC) with detection by triple quadrupole mass spectrometry using multiple reaction monitoring (MRM). Stable isotope-labeled taurine internal standard is used for quantification to correct for losses in extraction and variations in ionization in the ion source. The method was shown to be accurate, with acceptable recovery of 99.6% (range = 91.1-106.5%). Results for National Institute of Standards and Technology (NIST)-certified reference materials showed no statistical bias for NIST 1849a (P = 0.96) and NIST 1869 (P = 0.88) when compared with reference values. No bias was found when results were compared with those of an international reference method, AOAC Official MethodSM997.05 (P = 0.18). Repeatability was estimated to be 3.1% RSDr (range: 2.4-4.0%, HorRat: 0.3), and intermediate precision was estimated to be 4.9% RSDiR (range: 2.2-7.7%). Successful single-laboratory validation demonstrates that this rapid method is suitable for use in high-throughput laboratories as part of routine product compliance release testing of taurine in nutritional products. A method for the analysis of taurine in infant formula and adult nutritionals by hydrophilic interaction liquid chromatography-mass spectrometry (LC-MS) is described. The method is suitable for use in high-throughput laboratories for routine product compliance testing of taurine. A single-laboratory validation study demonstrated the method to be accurate, precise, and fit for purpose.

Using Organic Contaminants to Constrain the Terrestrial Journey of the Martian Meteorite Lafayette.

A key part of the search for extraterrestrial life is the detection of organic molecules since these molecules form the basis of all living things on Earth. Instrument suites such as SHERLOC (Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals) onboard the NASA Perseverance rover and the Mars Organic Molecule Analyzer onboard the future ExoMars Rosalind Franklin rover are designed to detect organic molecules at the martian surface. However, size, mass, and power limitations mean that these instrument suites cannot yet match the instrumental capabilities available in Earth-based laboratories. Until Mars Sample Return, the only martian samples available for study on Earth are martian meteorites. This is a collection of largely basaltic igneous rocks that have been exposed to varying degrees of terrestrial contamination. The low organic molecule abundance within igneous rocks and the expectation of terrestrial contamination make the identification of martian organics within these meteorites highly challenging. The Lafayette martian meteorite exhibits little evidence of terrestrial weathering, potentially making it a good candidate for the detection of martian organics despite uncertainties surrounding its fall history. In this study, we used ultrapure solvents to extract organic matter from triplicate samples of Lafayette and analyzed these extracts via hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS). Two hundred twenty-four metabolites (organic molecules) were detected in Lafayette at concentrations more than twice those present in the procedural blanks. In addition, a large number of plant-derived metabolites were putatively identified, the presence of which supports the unconfirmed report that Lafayette fell in a semirural location in Indiana. Remarkably, the putative identification of the mycotoxin deoxynivalenol (or vomitoxin), alongside the report that the collector was possibly a student at Purdue University, can be used to identify the most likely fall year as 1919.

Metabolomics Analysis of Aqueous Humor Based on Liquid Chromatography-Mass Spectrometry.

Aqueous humor (AH) is a transparent fluid that fills the anterior segment of the eye. The composition and level of metabolites in AH are important for understanding its physiology and changes caused by the occurrence of eye disease. A simple method for the preparation and analysis of AH samples was developed using the liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) technique. The analyses were performed using two types of chromatography: reversed-phase liquid chromatography-mass spectrometry (LC-RP-MS) and hydrophilic interaction liquid chromatography-mass spectrometry (LC-HILIC-MS), in the sample prepared by one protocol.

Characterizing a novel hyposialylated erythropoietin by intact glycoprotein and glycan analysis

NeuroEPO plus is a recently developed recombinant human erythropoietin (rhEPO) without erythropoietic activity and shorter plasma half-life due to its low sialic acid content. This novel rhEPO product is under investigation as therapeutic protein in the treatment of neurodegenerative diseases owing to its neuroprotective and neurodegenerative properties. In this study, an in-depth characterization of NeuroEPO plus N-glycans was performed by a glycan isotope [12C6]/[13C6] coded aniline labeling strategy followed by capillary zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry (CapZIC-HILIC-MS). A superior amount of low sialylated glycans and less branched structures were detected in NeuroEPO plus compare to other commercial rhEPOs. At the intact glycoprotein level, NeuroEPO plus glycoforms were separated by capillary zone electrophoresis with ultraviolet detection (CE-UV), optimizing the composition and pH of the separation electrolyte. Moreover, an isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) method was also optimized for the simultaneous analysis of this basic rhEPO and conventional acidic rhEPO products. The proposed glycomic and intact glycoprotein methods provide a robust and reliable analytical platform for NeuroEPO plus characterization and for its future implementation as biopharmaceutical in neurodegenerative diseases.

Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library search using Glucose Units (GU) and accurate mass (AM) as the primary search parameters for identification. However, GU-based identifications are gradient-dependent and are not suitable for applications where separation gradients need to be optimized to analyze complex samples or achieve higher throughput. Additionally, the workflow requires calibration curves (using dextran ladder) to be generated for each analysis campaign, which in turn, are used to derive the GU values of the separated glycan species. To overcome this limitation, we employed a two-step strategy for targeted glycan analysis of a mAb expressed in Chinese Hamster Ovary (CHO) cells. The first step is to create a custom library of the glycans of interest independent of GU values (thereby eliminating the need for a calibration curve) and instead uses AM and retention time (RT) as the primary search variables. The second step is to perform targeted glycan screening using the custom-built library. The developed workflow was applied for targeted glycan analysis of a mAb expressed in CHO for 1) cell line selection 2) characterizing the day-wise glycan evolution in a model mAb during a fed-batch culture, 3) assessing the impact of different media conditions on glycosylation, and 4) evaluating the impact of two different process conditions on glycosylation changes in a model mAb grown in a bioreactor. Taken together, the data presented in this study provides insights into the sources of glycan heterogeneity in a model mAb that are seen during its commercial manufacturing.

Recent Advances in the Analysis of Protein Glycosylation by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

Recent Advances in the Analysis of Protein Glycosylation by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

HILIC-MS/MS for the Determination of Methylated Adenine Nucleosides in Human Urine.

N6-methyl-2'-deoxyadenosine (m6dA) is a newly discovered DNA epigenetic mark in mammals. N6-methyladenosine (m6A), 2'-O-methyladenosine (Am), N6,2'-O-dimethyladenosine (m6Am), and N6,N6-dimethyladenosine (m62A) are common RNA modifications. Previous studies illustrated the associations between the aberrations of these methylated adenosines in nucleic acids and cancer. Herein, we developed Fe3O4/graphene-based magnetic dispersive solid-phase extraction for the enrichment and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS/MS) for the measurements of m6dA, m6A, Am, m6Am, and m62A in human urine samples. We found that malic acid could improve the HILIC-based separation of these modified nucleosides and markedly enhance the sensitivity of their MS detection. With this method, we accurately quantified the contents of these modified adenine nucleosides in urine samples collected from gastric and colorectal cancer patients as well as healthy controls. We found that, relative to healthy controls, urinary m6dA and Am levels are significantly lower for gastric and colorectal cancer patients; while gastric cancer patients also exhibited lower levels of urinary m6A, the trend was opposite for colorectal cancer patients. Together, we developed a robust analytical method for simultaneous measurements of five methylated adenine nucleosides in human urine, and our results revealed an association between the levels of urinary methylated adenine nucleosides and the occurrence of gastric as well as colorectal cancers, suggesting the potential applications of these modified nucleosides as biomarkers for the early detection of these cancers.

Multiclass cyanotoxin analysis in reservoir waters: Tandem solid-phase extraction followed by zwitterionic hydrophilic interaction liquid chromatography-mass spectrometry

The presence of cyanobacteria and cyanotoxins in all water bodies, including ocean water and fresh water sources, represents a risk for human health as eutrophication and climate change are enhancing their level of proliferation. For risk assessment and studies on occurrence, the development of reliable and sensitive analytical approaches able to cover a wide range of cyanotoxins is essential. This work describes the development of an HILIC-MS/MS multiclass method for the simultaneous analysis of eight cyanotoxins in reservoir water samples belonging to three different classes according to their chemical structure: cyclic peptides (microcystin-LR, microcystin-RR and nodularin), alkaloids (cylindrospermopsin, anatoxin-a) and three non-protein amino acids isomers such as β-methylamino-L-alanine, 2,4-diaminobutyric acid and N-(2-aminoethyl)glycine). A SeQuant ZIC-HILIC column was employed to achieve the chromatographic separation in less than 12 min. Previously, a novel sample treatment based on a tandem solid-phase extraction (SPE) system using mixed cation exchange (MCX) and Strata-X cartridges was investigated with the aim of extracting and preconcentrating this chemically diverse group of cyanotoxins. The Strata-X cartridge, which was configured first in the line of sample flow, retained the low polar compounds and the MCX cartridge, which was at the bottom of the dual system, retained mainly the non-protein amino acids. The optimization procedure highlighted the importance of sample ion content for the recoveries of some analytes such as the isomers β-N-methylamino-L-alanine and 2-4-diaminobutyric acid. Method validation was carried out in terms of linearity, limit of detection (LOD) and quantification (LOQ), recoveries, matrix effect and precision in terms of repeatability and intermediate precision. This work represents the first analytical method for the simultaneous analysis of these multiclass cyanotoxins in reservoir water samples, achieving LOQs in the very low range of 7·10−3 – 0.1 μg L−1. Despite high recoveries obtained at the LOQ concentration levels (101.0–70.9%), relative standard deviations lower than 17.5% were achieved.

Determination of free amino acids, saccharides, and selected microbes in biogenic atmospheric aerosols – seasonal variations, particle size distribution, chemical and microbial relations

Abstract. Primary biological aerosol particles (PBAPs) play an important role in the interaction between biosphere, atmosphere, and climate, affecting cloud and precipitation formation processes. The presence of pollen, plant fragments, spores, bacteria, algae, and viruses in PBAPs is well known. In order to explore the complex interrelationships between airborne and particulate chemical tracers (amino acids, saccharides), gene copy numbers (16S and 18S for bacteria and fungi, respectively), gas phase chemistry, and the particle size distribution, 84 size-segregated aerosol samples from four particle size fractions (&lt; 1.0, 1.0–2.5, 2.5–10, and &gt; 10 µm) were collected at the SMEAR II station, Finland, in autumn 2017. The gene copy numbers and size distributions of bacteria, Pseudomonas, and fungi in biogenic aerosols were determined by DNA extraction and amplification. In addition, free amino acids (19) and saccharides (8) were analysed in aerosol samples by hydrophilic interaction liquid chromatography–mass spectrometry (HILIC-MS). Different machine learning (ML) approaches, such as cluster analysis, discriminant analysis, neural network analysis, and multiple linear regression (MLR), were used for the clarification of several aspects related to the composition of biogenic aerosols. Clear variations in composition as a function of the particle size were observed. In most cases, the highest concentration values and gene copy numbers (in the case of microbes) were observed for 2.5–10 µm particles, followed by &gt; 10, 1–2.5, and &lt; 1.0 µm particles. In addition, different variables related to the air and soil temperature, the UV radiation, and the amount of water in the soil affected the composition of biogenic aerosols. In terms of interpreting the results, MLR provided the greatest improvement over classical statistical approaches such as Pearson correlation among the ML approaches considered. In all cases, the explained variance was over 91 %. The great variability of the samples hindered the clarification of common patterns when evaluating the relation between the presence of microbes and the chemical composition of biogenic aerosols. Finally, positive correlations were observed between gas-phase VOCs (such as acetone, toluene, methanol, and 2-methyl-3-buten-2-ol) and the gene copy numbers of microbes in biogenic aerosols.

Effect of Oxime Encapsulation on Acetylcholinesterase Reactivation: Pharmacokinetic Study of the Asoxime-Cucurbit[7]uril Complex in Mice Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Oxime-based molecules are used for the treatment of patients to reactivate acetylcholinesterase (AChE) function after organophosphate intoxication. However, their efficacy is limited by low penetration through the blood-brain barrier and fast elimination. In this work, the cucurbit[7]uril (CB[7]) carrier was used for the encapsulation of the clinical agent asoxime to enhance brain bioavailability and the treatment window. We present a pharmacokinetic study of asoxime and the asoxime-CB[7] complex in an in vivo mouse model. Ultrahigh-performance liquid chromatography with electrospray ionization-mass spectrometry detection was developed to determine asoxime and CB[7] in biological fluids and tissues after thorough optimization of chromatographic conditions. The dihydroxypropane-silica stationary phase using hydrophilic interaction liquid chromatography conditions provided the best chromatographic performance. The final method was validated and applied for the pharmacokinetic study of mouse plasma, urine, bile, liver, kidney, and brain samples at different times after administration of asoxime and the asoxime-CB[7] complex. The results showed a greater than 3-fold increase in the area under the curve (AUC) in the brain for asoxime administered as a complex with CB[7] relative to that for the administration of asoxime alone. The effectiveness of the treatment strategy was evaluated using a reactivation study and a functional observatory battery. Protection of brain AChE activity is crucial for saving human lives or reducing the consequences of poisoning. The asoxime administered as a complex increased the brain activity by approximately 30% compared to that with atropine alone. CB[7] coadministration improved the AChE activity by 11%, which agrees with the higher asoxime AUC assessed in the pharmacokinetic study.