Identification and Structural Characterization of Degradation Products of Linagliptin by Mass Spectrometry Techniques.

As part of the development and production of pharmaceuticals, the purity of Active Pharmaceutical Ingredients stands as a fundamental parameter that significantly influences the quality, safety, and efficacy of the final drug product. Impurities in Active Pharmaceutical Ingredients are various unwanted substances that can appear during the whole manufacturing process, from raw materials to the final product. These impurities can stem from multiple sources, including starting materials, intermediates, reagents, solvents, and even degradation products resulting from exposure to environmental factors such as heat, light, or moisture. Their presence can potentially compromise the therapeutic effect of the drug, introduce unexpected side effects, or even pose safety risks to patients. This study aims to conduct the forced degradation of linagliptin and subsequently attempt to identify the resulting degradants. The degradation procedures were carried out in accordance with the guidelines of the International Committee for Harmonization. The degradation profile of linagliptin was investigated under various conditions, including acid hydrolysis, alkaline hydrolysis, oxidation, heat, and light exposure, utilizing ultra-performance liquid chromatography connected to a photo array detector. Identification and characterization of the degradation products were achieved using an ultra-performance liquid chromatography coupled with a single quadrupole detector mass spectrometer and also a liquid chromatography coupled with a high-resolution mass spectrometry. The identified degradation products demonstrate that linagliptin is particularly susceptible to degradation when exposed to acid and peroxide. Whereas, no significant degradation effects were observed under alkali, thermolytic, and photolytic conditions.

Coprecipitation as a One-Step Se Separation for Determination of Isotope Ratios Completed with Revised Uncertainty Evaluation.

This study introduces a simplified purification method for analyzing 82Se/78Se isotope ratios in diverse natural samples using hydride generation MC-ICP-MS. Unlike the thiol resin method, which is time-consuming and sensitive to the concentrations of reagents used at individual stages, our proposed alternative is quicker, simpler, and robust. The procedure involves coprecipitation of selenium with iron hydroxide and dissolution in hydrochloric acid. Combining hydride generation and a second cleanup stage achieves sufficient purification for Se isotope ratio measurements. The method is efficient, taking 3-4 h after sample decomposition, utilizing common reagents [HCl, Fe(NO3)3, NH4Cl] without evaporation or clean lab conditions. Results on 82Se/78Se isotope ratios in various matrices are presented, comparing them with literature data. All isotopic results have been subjected to a newly proposed state-of-the-art approach to uncertainty estimation dedicated to isotope ratio measurements. The approach is based on applying Monte Carlo simulations with consideration of different samples' results normalized by the expected value. By doing that, we obtained estimated uncertainty for any Se sample with the influence of particular measurements on the final estimation included. We employ a Monte Carlo simulation-based uncertainty estimation approach for isotope ratio measurements, providing estimated uncertainty for each selenium sample.

Dynamic adaptation of the extremophilic red microalga Cyanidioschyzon merolae to high nickel stress.

The order of Cyanidiales comprises seven acido-thermophilic red microalgal species thriving in hot springs of volcanic origin characterized by extremely low pH, moderately high temperatures and the presence of high concentrations of sulphites and heavy metals that are prohibitive for most other organisms. Little is known about the physiological processes underlying the long-term adaptation of these extremophiles to such hostile environments. Here, we investigated the long-term adaptive responses of a red microalga Cyanidioschyzon merolae, a representative of Cyanidiales, to extremely high nickel concentrations. By the comprehensive physiological, microscopic and elemental analyses we dissected the key physiological processes underlying the long-term adaptation of this model extremophile to high Ni exposure. These include: (i) prevention of significant Ni accumulation inside the cells; (ii) activation of the photoprotective response of non-photochemical quenching; (iii) significant changes of the chloroplast ultrastructure associated with the formation of prolamellar bodies and plastoglobuli together with loosening of the thylakoid membranes; (iv) activation of ROS amelioration machinery; and (v) maintaining the efficient respiratory chain functionality. The dynamically regulated processes identified in this study are discussed in the context of the mechanisms driving the remarkable adaptability of C. merolae to extremely high Ni levels exceeding by several orders of magnitude those found in the natural environment of the microalga. The processes identified in this study provide a solid basis for the future investigation of the specific molecular components and pathways involved in the adaptation of Cyanidiales to the extremely high Ni concentrations.

Breastmilk mineral composition among well-educated mothers from Central Poland - Associations with maternal dietary intake, dietary patterns and infant psychomotor development.

BACKGROUND: Maternal dietary habits could affect breastmilk mineral composition, which may influence infant development. Mineral dietary intake or supplementation slightly affects its breastmilk concentration. However, the intake of selected food groups or dietary patterns that reflect diet complexity could have a greater impact. Hence, the aim of the study was to assess breastmilk mineral composition at one, three, and six months of lactation among mothers living in urban area of Central Poland, as well as the evaluate maternal dietary determinants and associations with infant anthropometric and psychomotor development. METHODS: The study was conducted among 43 healthy and exclusively breastfeeding mothers. In the first, third, and sixth months of lactation, we collected breastmilk samples and assessed the concentration of Ca, P, Zn, Fe, Se, Ni, As, Pb, and Cd using the ICP-MS method. Maternal dietary habits were evaluated by a food frequency questionnaire in the first month of lactation, whereas in the third and sixth by the three-day food record. Based on the collected data adherence to the Polish-adapted Mediterranean (Pl-aMED; 1 month) and the DASH diet (Mellen's Index; 3 and 6 months) was assessed. In the third and sixth months of lactation infant anthropometric parameters and the sixth month of lactation psychomotor development were evaluated. RESULTS: Breastmilk Se, Ni, As, Pb, and Cd levels were under the LOQ in all the breastmilk samples at all study visits. Median breastmilk mineral concentrations of Ca, P, Zn, and Fe in the first, third, and sixth months of lactation varied from 381.9 to 332.7 mg/L, 161.6 to 139.1 mg/L, 2.2 to 0.8 mg/L, and 0.26 to 0.17 mg/L, respectively. Maternal dietary intake and supplementation did not affect breastmilk Ca, P, Zn, and Fe. Pl-aMED scores were associated with breastmilk Ca (ß = 0.489, 95% CI 0.180 - 0.799, p = 0.003) and Zn (ß = 0.499, 95% CI 0.199 - 0.798, p = 0.002) in the first month of lactation, whereas no association with the DASH diet were observed in the third and sixth month of lactation. Breastmilk Fe in the third month was associated with infant motor development (ß = 0.420, 95% CI 0.113 - 0.727, p = 0.009) in the sixth month of life, but no other associations with anthropometric or psychomotor development were observed. Moreover, we estimated that few infants meet their adequate intake (AI) requirements for P, Zn, and Fe. CONCLUSION: Our study showed that maternal adherence to Pl-aMED is a significant predictor of breastmilk Ca and Zn in the first month of lactation, which may be especially important considering that more than 75% of infants had inadequate Zn intake. Moreover, we found that breastmilk Fe positively influenced infant motor development, despite the majority of infants having inadequate intake. On the other hand, no infant had deficiency symptoms, which emphasizes the necessity to evaluate of AI norms for infants.

Investigation of the Impact of L-Phenylalanine and L-Tyrosine Pre-Treatment on the Uptake of 4-Borono-L-Phenylalanine in Cancerous and Normal Cells Using an Analytical Approach Based on SC-ICP-MS.

Boron has gained significant attention in medical research due to its B-10 isotope's high cross section for the reaction with thermal neutrons, generating ionizing particles that can eliminate cancer cells, propelling the development of boron neutron capture therapy (BNCT) for cancer treatment. The compound 4-borono-L-phenylalanine (BPA) has exhibited potential in BNCT clinical trials. Enhancing BPA uptake in cells involves proposing L-amino acid preloading. This study introduces a novel analytical strategy utilizing ICP-MS and single cell ICP-MS (SC-ICP-MS) to assess the effectiveness of L-tyrosine and L-phenylalanine preloading on human non-small cell lung carcinoma (A549) and normal Chinese hamster lung fibroblast (V79-4) models, an unexplored context. ICP-MS outcomes indicated that L-tyrosine and L-phenylalanine pre-treatment increased BPA uptake in V79-4 cells by 2.04 ± 0.74-fold (p = 0.000066) and 1.46 ± 0.06-fold (p = 0.000016), respectively. Conversely, A549 cells manifested heightened BPA uptake solely with L-tyrosine preloading, with a factor of 1.24 ± 0.47 (p = 0.028). BPA uptake remained higher in A549 compared to V79-4 regardless of preloading. SC-ICP-MS measurements showcased noteworthy boron content heterogeneity within A549 cells, signifying diverse responses to BPA exposure, including a subset with notably high BPA uptake. This study underscores SC-ICP-MS's utility in precise cellular boron quantification, validating cellular BPA uptake's heterogeneity.

Fractionation of selenium isotopes during biofortification of Saccharomyces cerevisiae and the influence of metabolic labeling with 15N.

Isotope fractionation of metals/metalloids in biological systems is an emerging research area that demands the application of state-of-the-art analytical chemistry tools and provides data of relevance to life sciences. In this work, Se uptake and Se isotope fractionation were measured during the biofortification of baker's yeast (Saccharomyces cerevisiae)-a product widely used in dietary Se supplementation and in cancer prevention. On the other hand, metabolic labeling with 15N is a valuable tool in mass spectrometry-based comparative proteomics. For Se-yeast, such labeling would facilitate the assessment of Se impact on yeast proteome; however, the question arises whether the presence of 15N in the microorganisms affects Se uptake and its isotope fractionation. To address the above-mentioned aspects, extracellularly reduced and cell-incorporated Se fractions were analyzed by hydride generation-multi-collector inductively coupled plasma-mass spectrometry (HG MC ICP-MS). It was found that extracellularly reduced Se was enriched in light isotopes; for cell-incorporated Se, the change was even more pronounced, which provides new evidence of mass fractionation during biological selenite reduction. In the presence of 15N, a weaker preference for light isotopes was observed in both, extracellular and cell-incorporated Se. Furthermore, a significant increase in Se uptake for 15N compared to 14N biomass was found, with good agreement between hydride generation microwave plasma-atomic emission spectrometry (HG MP-AES) and quadrupole ICP-MS results. Biological effects observed for heavy nitrogen suggest 15N-driven alteration at the proteome level, which facilitated Se access to cells with decreased preference for light isotopes.

Activation of trace amine-associated receptor 1 (TAAR1) transiently reduces alcohol drinking in socially housed mice.

Alcohol dependence is characterized by the abnormal release of dopamine in the brain reward-related areas. Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor that negatively regulates dopamine neurotransmission and thus is a promising target in the treatment of drug addiction. However, the role of TAAR1 in the regulation of alcohol abuse remains understudied. Here, we assessed the effect of TAAR1 activation on alcohol drinking behaviours of C57Bl/6J female mice housed in IntelliCages. The animals were administered with either vehicle or TAAR1 full selective agonist, RO5256390, and tested for alcohol consumption, alcohol preference and motivation for alcohol seeking. We found that mice with the highest preference for alcohol (high drinkers) in the RO5256390 group consumed less alcohol and had lower alcohol preference in comparison with high drinkers in the vehicle group, during 20 h of free alcohol access (FAA). We also found decreased alcohol consumption and alcohol preference comparing all animals in the RO5256390 to all animals in the vehicle group, during 20 h of FAA performed after the abstinence. These effects of RO5256390 lasted for the first 24 h after administration that roughly corresponded to the compound level in the brain, measured by mass spectrometry. Finally, we found that administration of RO5256390 may attenuate motivation for alcohol seeking. Taken together, our findings reveal that activation of TAAR1 may transiently reduce alcohol drinking; thus, TAAR1 is a promising target for the treatment of alcohol abuse and relapse.

Elemental imaging approach to comparative analysis of historical inks. Proof of concept based on non-invasive examinations of the Chopin manuscript as a case study.

This work presents an indirect method of investigating the elemental diversity of historical inks. The manuscript of Fryderyk Chopin's Impromptu in A flat major, Op. 29 was examined as an example of the proposed approach for testing documents containing different inks. Preliminary in situ X-ray fluorescence (XRF) measurements carried out it the museum storage room provided qualitative reference data for the object. Then, selected areas on the item were examined with indicator papers soaked in 4,7-diphenyl-1,10-phenanthroline (Bphen). The reaction with the ligand enabled the immediate colorimetric detection of Fe(II) in the form of magenta Fe(Bphen)3 complex. The overall condition of the manuscript with regard to the risk of ink corrosion was assessed in this way. More chemical information were obtained by the proposed elemental imaging-based approach to evaluate chemical heterogeneity of the used indicator paper samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The recorded data were visualised in the form of elemental distribution maps. The Fe enriched areas indicated Regions of Interest (ROIs) to approximate composition of the inks from the manuscript. All calculations were done solely for the data mathematically singled out from these areas. The varying proportions of AI, Mn, Co and Cu relative to Fe correlated with the ROIs of the composer's handwriting, editor's notes and stave lines, demonstrating the applicability of the proposed approach for comparative studies.

A Standardized Protocol for Assuring the Validity of Proteomics Results from Liquid Chromatography-High-Resolution Mass Spectrometry.

Significant advances in the technological development of mass spectrometry in the field of proteomics and the generation of extremely large amounts of data require a very critical approach to assure the validity of results. Commonly used procedures involved liquid chromatography followed by high-resolution mass spectrometry measurements. Proteomics analysis is used in many fields including the investigation of the metabolism of biologically active substances in organisms. Thus, there is a need to care about the validity of the obtained results. In this work, we proposed a standardized protocol for proteomic analysis using liquid chromatography-high-resolution mass spectrometry, which covers all of these analytical steps to ensure the validity of the results. For this purpose, we explored the requirements of the ISO/IEC 17025:2017 standard as a reference document for quality control in biochemistry research-based mass spectrometry.

Maternal diet during breastfeeding in correlation to calcium and phosphorus concentrations in human milk.

BACKGROUND: The impact of maternal diet on mineral concentration in human milk (HM) remains unclear. The main aim of this study was to investigate the relationship between maternal dietary intake and calcium and phosphorus concentrations in HM. Furthermore, we aimed to evaluate the intake of both minerals by exclusively breastfed infants. METHODS: HM samples were obtained from 30 mothers at 6-8 weeks postpartum. Each mother was asked to express pre- and postfeeding milk four times during a 24-h period (6.00-12.00, 12.00-18.00, 18.00-24.00, 24.00-6.00). Maternal dietary assessment was based on a food frequency questionnaire and 3-day dietary records. Analysed minerals were determined using an inductively coupled plasma mass spectrometer (NexION 300D ICP mass spectrometer, Perkin Elmer SCIEX). RESULTS: The mean concentrations of calcium and phosphorus in HM samples were 278.7 ± 61.0 and 137.1 ± 21.9 mg/L, respectively, maintaining 2:1 ratio by weight. The concentration of both minerals was correlated with each other (r = 0.632, p = <0.001). The infants' mean calcium intake was 149.53 ± 36.41 mg/L, and their mean phosphorus intake was 74.62 ± 19.41 mg/L. The risk of insufficient intake of calcium was reported in 60% of infants (n = 18). Spearman's/Pearson's correlation coefficients did not reveal any correlations between HM calcium concentration and maternal diet, contrary to HM phosphorus concentration, which was positively correlated with energy (r = 0.369, p = 0.045), total protein (r = 0.464, p = 0.01), calcium (r = 385, p = 0.036), phosphorus (r = 501, p = 0.005), niacin (p < 0.001) and pyridoxine (r = 382, 0.037) intake. However, in multivariable analysis we observed that maternal dietary intake of both minerals had a positive influence on their concentration in HM. CONCLUSIONS: Maternal calcium and phosphorus intake influenced the concentration of both minerals in HM; however, the relationship was rather weak. In addition, we observed that calcium intake by most of the exclusively breastfed infants was insufficient to meet the recommended daily intake.

Cfap91-Dependent Stability of the RS2 and RS3 Base Proteins and Adjacent Inner Dynein Arms in Tetrahymena Cilia.

Motile cilia and eukaryotic flagella are specific cell protrusions that are conserved from protists to humans. They are supported by a skeleton composed of uniquely organized microtubules-nine peripheral doublets and two central singlets (9 × 2 + 2). Microtubules also serve as docking sites for periodically distributed multiprotein ciliary complexes. Radial spokes, the T-shaped ciliary complexes, repeat along the outer doublets as triplets and transduce the regulatory signals from the cilium center to the outer doublet-docked dynein arms. Using the genetic, proteomic, and microscopic approaches, we have shown that lack of Tetrahymena Cfap91 protein affects stable docking/positioning of the radial spoke RS3 and the base of RS2, and adjacent inner dynein arms, possibly due to the ability of Cfap91 to interact with a molecular ruler protein, Ccdc39. The localization studies confirmed that the level of RS3-specific proteins, Cfap61 and Cfap251, as well as RS2-associated Cfap206, are significantly diminished in Tetrahymena CFAP91-KO cells. Cilia of Tetrahymena cells with knocked-out CFAP91 beat in an uncoordinated manner and their beating frequency is dramatically reduced. Consequently, CFAP91-KO cells swam about a hundred times slower than wild-type cells. We concluded that Tetrahymena Cfap91 localizes at the base of radial spokes RS2 and RS3 and likely plays a role in the radial spoke(s) positioning and stability.

Study on Tissue Homogenization Buffer Composition for Brain Mass Spectrometry-Based Proteomics.

Mass spectrometry-based proteomics aims to study the proteome both qualitatively and quantitatively. A key step in proteomic analysis is sample preparation, which is crucial for reliable results. We investigated the effect of the composition of the homogenization buffer used to extract proteins from brain tissue on the yield of protein extraction and the number and type of extracted proteins. Three different types of buffers were compared-detergent-based buffer (DB), chaotropic agent-based buffer (CAB) and buffer without detergent and chaotropic agent (DFB). Based on label-free quantitative protein analysis, detergent buffer was identified as the most suitable for global proteomic profiling of brain tissue. It allows the most efficient extraction of membrane proteins, synaptic and synaptic membrane proteins along with ribosomal, mitochondrial and myelin sheath proteins, which are of particular interest in the field of neurodegenerative disorders research.

Label-Free Mass Spectrometry-Based Quantitative Proteomics to Evaluate the Effects of the Calcium-Sensing Receptor Agonist Cinacalcet on Protein Expression in Rat Brains and Livers.

BACKGROUND Cinacalcet is a calcium-sensing receptor agonist that is clinically approved for the treatment of secondary hyperparathyroidism in chronic kidney disease and hypercalcemia in patients with parathyroid carcinoma. This study aimed to use quantitative mass spectrometry-based label-free proteomics to evaluate the effects of cinacalcet on protein expression in rat brains and livers. MATERIAL AND METHODS We randomly assigned 18 Wistar rats to 2 groups: an untreated control group (n=6) and a group treated with cinacalcet at a dose corresponding to the maximum dose used in humans (2 mg/kg/body weight, 5 days/week) divided into 7-day (n=6) and 21-day (n=6) treatment subgroups. A mass-spectrometry-based label-free quantitative proteomics approach using peptides peak area calculation was used to evaluate the changes in protein expression in examined tissues. Bioinformatics analysis of quantitative proteomics data was done using MaxQuant and Perseus environment. RESULTS No changes in protein expression were revealed in the 7-day treatment subgroup. We detected 10 upregulated and 3 downregulated proteins in the liver and 1 upregulated protein in the brain in the 21-day treatment subgroup compared to the control group. Based on Gene Ontology classification, all identified differentially expressed proteins were indicated as molecular functions involved in the enzyme regulator activity (36%), binding (31%), and catalytic activity (19%). CONCLUSIONS These findings indicate that long-term cinacalcet therapy can impair phase II of enzymatic detoxication and can cause disturbances in blood hemostasis, lipid metabolism, and inflammatory mediators or contribute to the acceleration of cognitive dysfunction; therefore, appropriate patient monitoring should be considered.

Label-Free Mass Spectrometry-Based Proteomic Analysis in Lamb Tissues after Fish Oil, Carnosic Acid, and Inorganic Selenium Supplementation.

Selenium is an essential nutrient, building twenty five identified selenoproteins in humans known to perform several important biological functions. The small amount of selenium in the earth's crust in certain regions along with the risk of deficiency in organisms have resulted in increasingly popular dietary supplementation in animals, implemented via, e.g., inorganic selenium compounds. Even though selenium is included in selenoproteins in the form of selenocysteine, the dietary effect of selenium may result in the expression of other proteins or genes. Very little is known about the expression effects modulated by selenium. The present study aimed to examine the significance of protein expression in lamb tissues obtained after dietary supplementation with selenium (sodium selenate) and two other feed additives, fish oil and carnosic acid. Label-free mass spectrometry-based proteomic analysis was successfully applied to examine the animal tissues. Protein-protein interaction network analysis of forty differently-expressed proteins following inorganic selenium supplementation indicated two significant clusters which are involved in cell adhesion, heart development, actin filament-based movement, plasma membrane repair, and establishment of organelle localization.

Fluorine-Containing Drug Administration in Rats Results in Fluorination of Selected Proteins in Liver and Brain Tissue.

In many pharmaceuticals, a hydrogen atom or hydroxyl group is replaced by a fluorine to increase bioavailability and biostability. The fate of fluorine released from fluorine-containing drugs is not well investigated. The aim of this study was to examine possible fluorination of proteins in rat liver and brain after administration of the fluorinated drug cinacalcet. We assigned 18 Wistar rats to a control group (n = 6) and a group treated with cinacalcet (2 mg kg-1/body weight, 5 days/week), divided into 7 day (n = 6) and 21 day (n = 6) treatment subgroups. Fluorinated proteins were identified using a free proteomics approach; chromatographic separation and analysis by high-resolution mass spectrometry; peptide/protein identification using the Mascot search algorithm; manual verification of an experimentally generated MS/MS spectrum with the theoretical MS/MS spectrum of identified fluorinated peptides. Three fluorinated proteins (spectrin beta chain; carbamoyl-phosphate synthase [ammonia], mitochondrial; 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 1) were identified in the liver and four (spectrin beta chain, dihydropyrimidinase-related protein 4, prominin-2, dihydropyrimidinase-related protein 4) in the brain tissue after 21 days of cinacalcet treatment, but not in the control group. Introduction of fluorine into an organism by administration of fluorinated drugs results in tissue-specific fluorination of proteins.

Cancer Influences the Elemental Composition of the Myocardium More Strongly than Conjugated Linoleic Acids-Chemometric Approach to Cardio-Oncological Studies.

The aim of the study was to verify in a cardio-oncological model experiment if conjugated linoleic acids (CLA) fed to rats with mammary tumors affect the content of selected macro- and microelements in their myocardium. The diet of Sprague-Dawley females was supplemented either with CLA isomers or with safflower oil. In hearts of rats suffering from breast cancer, selected elements were analyzed with a quadrupole mass spectrometer with inductively coupled plasma ionization (ICP-MS). In order to better understand the data trends, cluster analysis, principal component analysis and linear discriminant analysis were applied. Mammary tumors influenced macro- and microelements content in the myocardium to a greater extent than applied diet supplementation. Significant influences of diet (p = 0.0192), mammary tumors (p = 0.0200) and interactions of both factors (p = 0.0151) were documented in terms of Fe content. CLA significantly decreased the contents of Cu and Mn (p = 0.0158 and p = 0.0265, respectively). The level of Ni was significantly higher (p = 0.0073), which was more pronounced in groups supplemented with CLA. The obtained results confirmed antioxidant properties of CLA and the relationship with Se deposition. Chemometric techniques distinctly showed that the coexisting pathological process induced differences to the greater extent than diet supplementation in the elemental content in the myocardium, which may impinge on cardiac tissue's susceptibility to injuries.

Replicated, urban-driven exposure to metallic trace elements in two passerines.

While there are increasing examples of phenotypic and genotypic differences between urban and non-urban populations of plants and animals, few studies identified the mechanisms explaining those dissimilarities. The characterization of the urban landscape, which can only be achieved by measuring variability in relevant environmental factors within and between cities, is a keystone prerequisite to understand the effects of urbanization on wildlife. Here, we measured variation in bird exposure to metal pollution within 8 replicated urbanization gradients and within 2 flagship bird species in urban evolutionary ecology: the blue tit (Cyanistes caeruleus) and the great tit (Parus major). We report on a highly significant, positive linear relationship between the magnitude of urbanization-inferred as either tree cover, impervious surface cover, or an urbanization score computed from several environmental variables, and copper, zinc and lead concentrations in bird feathers. The reverse relationship was measured in the case of mercury, while cadmium and arsenic did not vary in response to the urbanization level. This result, replicated across multiple cities and two passerine species, strongly suggests that copper, zinc, lead and mercury pollution is likely to trigger the emergence of parallel responses at the phenotypic and/or genotypic level between urban environments worldwide.

A Novel Approach for the Determination of the Ge Isotope Ratio Using Liquid-Liquid Extraction and Hydride Generation by Multicollector Inductively Coupled Plasma Mass Spectrometry.

In this work, a method for the accurate and precise determination of the Ge isotope ratio in synthetic water and natural samples of geological origin using multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) with hydride generation was developed. The method was based on the liquid-liquid extraction of Ge to eliminate all elements affecting the generation of germanium hydrides. The standard-sample bracketing method was used to correct instrumental bias. Registration of analytical signal in time-resolved mode gave way to choose signals with best parameters and improved the precision of the results. Controlling the pH by using acetic buffer boosted the sensitivity by nearly five times in comparison to hydride generation methods suggested by other authors. The newly developed method is much simpler and quicker, does not need laborious Ge separation with ion-exchange resins, and thanks to its superior sensitivity, allows measurements of the Ge isotopic ratio in materials with relatively low Ge content. Delta values of the 74Ge/70Ge isotope ratio were measured in standard reference materials for which reference values were available in the GeoREM database. We demonstrated that the accuracy and precession of this method are equally good or better than methods proposed by other authors.