Mass Spectrometric Method Research Articles

New sensitive ambient mass spectrometric method combined with chemometric modelling for the analysis of Equisetum palustre L. contaminations in the traditional herb Equiseti herba

The aim of this study was to develop a new and sensitive ambient mass spectrometric method in combination with multivariate data analysis to classify and quantify contaminations of the traditional herb Equiseti herba through the similar looking E. palustre. Due to its morphological similarity and the often-overlapping habitats, mix-ups of the two plants are common. This is concerning since E. palustre contains substances that are potentially toxic to humans, such as the piperidine alkaloids palustrine and palustridiene. Next to macroscopic and microscopic examination, the identification of Equiseti herba in the European Pharmacopoeia is carried out by means of thin layer chromatography, which must detect up to 5% of foreign compounds. The results of the chemometric models were built from the mass spectrometric data. Furthermore, they were compared with a near infrared spectroscopy method combined with multivariate data analysis, since this type of spectroscopy has already been established in quality control. As a result, the linear discriminant model obtained from the mass spectrometric data could classify an independent set of artificial contaminations of E. arvense through E. palustre with high correctness as low as 1% dry weight contamination grade. In addition, quantification through a partial least square model showed adequate results for a location of the grade of contamination. In conclusion, the new ambient mass spectrometric method presents itself superior to the respective near infrared method and the chemometric models built from its data as well as the official thin layer chromatography of the European Pharmacopoeia.

A Sensitive LC-MS/MS Method for the Simultaneous Determination of Two Thia-Analogous Indirubin N-Glycosides and Indirubin-3'-Monoxime in Plasma and Cell Culture Medium.

Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3′-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3′-oxo-benzo[b]thiophen-2′-(Z)-ylidene]-1-(β-d-glucopyranosyl)-oxindole) and KD85 (3-[3′-oxo-benzo[b]thiophen-2′-(Z)-ylidene]-1-(β-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.

Rare earth elements in surface waters and sediments of the Mgoua watershed, south western Cameroon

The concentrations and distribution spectra of 14 stable rare earth elements (REE) were studied for surface waters (n = 30) and sediments (n = 30) of the tropical zone Mgoua watershed in southwest of Cameroon. Samples were analyzed using the inductively coupled plasma mass spectrometer (ICP-MS) method. The ∑REE concentrations of the surface waters vary from 0.11 to 6.60 µg/L with an average value of 1.18 µg/L. This average is 150 times lower than the average values of the Post-Archean Australian Average Shale (PAAS). In the sediments, the concentrations range from 282.12 to 727.67 ppm with an average value of 550.05 ppm, making it 2.5 times higher than the PAAS. Rare earth spectra normalized to PAAS are spread out and show heavy rare earth enrichment; positive Eu anomalies; negative, low, or no Ce anomalies; and a gradual decrease in heavy earths in river and well waters and sediments, but are sawtooth with strong negative Ce anomalies in effluent waters and sediments. A low rare earth fractionation is noted in river and well waters [(La/Yb)N ~ 0.35–1.19], while it is high in effluent waters [(La/Yb)N ~ 0.42–56.99]; a slight depletion of rare earths in all environments [(La/Sm)N ~ 0.69–2.35], as well as a depletion of heavy rare earths [(Gd/Yb)N ~ 0.58–1.94]. In the sediments, the same characteristics are found with elemental ratio values differing slightly [(La/Yb)N ~ 1.15–1.99]; [(La/Sm)N ~ 0.75–1.36]; and [(Gd/Yb)N ~ 1.15–2.01]. This study shows that the Mgoua watershed does not have a high concentration of REE, although in the sediments they are well above the average PASS value while they are insignificant in the waters.

W247 A simple and robust mass spectrometric method for simultaneous determination of antidiabetic, antihypertensive, antidepressive, analgesic and antihyperlipidemic drug levels

W247 A simple and robust mass spectrometric method for simultaneous determination of antidiabetic, antihypertensive, antidepressive, analgesic and antihyperlipidemic drug levels

Search and investigation of potential peptide agents of interaction between human organism and its microbiome

Symbiotic interaction between the human body and its microbiota is an important issue of modern biomedicine and personalized medicine. However, little is known on molecular mechanisms of that relationship. Bearing in mind the ubiquitous participation of peptides in biomolecular interactions and regulatory processes we attempted direct search of blood peptides originated from microbial proteins. LC‐MS/MS analysis was carried out of blood serum and plasma samples taken from 20 healthy donors on Q Exactive HF‐X Hybrid Quadrupole‐Orbitrap mass‐spectrometer. Sample preparation was carried out based on our previously developed method of peptide desorption from the surface of major blood plasma proteins followed by standard chromatographic steps. Human microbiota protein sequences were taken from NIH Human Microbiome Project.As a result, out of 13,625 identified peptides 912 were unique fragments of microbial precursors, which is about 6.69% of the total amount of detected bloodstream peptides. In 30 cases peptide identification was confirmed by mass‐spectral study of individual synthetic samples. Absolute quantification by the mass‐spectrometric method of multiple reaction monitoring (MRM) confirmed the presence of bacterial peptides in plasma and serum in the range of approximately 0.1 nM to 1 uM, which is comparable to physiologically significant hormone concentrations in human blood in normal conditions. The abundance of microbiota peptides reaches its maximum 5h after a meal. Most of the peptides correlate with the bacterial composition of the small intestine and are likely obtained by hydrolysis of membrane proteins with trypsin, chymotrypsin and pepsin — the main proteases of the gastrointestinal tract.The isolated fraction of peripheral blood mononuclear cells showed increase secretion of proinflammatory cytokines, colony stimulating factors and chemoattractants as the response to the addition of some of the identified microbiotic peptides. Such peptides — identified both in the plasma and in the serum — have properties of resistance to fibrinolysis, since the pool of peptides is preserved after passing through the digestive tract. The physicochemical properties of the identified bacterial peptides are consistent with those required for the selective permeability of mucosal barriers. Our approach to the identification of microbiota peptides in the blood serum and plasma may be useful for determining the microbiota composition of hard‐to‐reach intestinal areas, such as the small intestine, and for monitoring the permeability of the intestinal mucosal barrier.

Mass spectrometric study and modeling of the thermodynamic properties in the Gd2 O3 -ZrO2 -HfO2 system at high temperatures.

Materials based on the Gd2 O3 -ZrO2 -HfO2 system are promising for a wide range of high-temperature technological applications, such as for obtaining thermal barrier coatings in the aviation and space industry, as well as advanced materials in nuclear power applications. Experimental studies of the ceramics based on this system by the Knudsen effusion mass spectrometric method provides such valuable information as the vapor composition over the samples and enables derivation of the thermodynamic functions. Samples of ceramics in the Gd2 O3 -ZrO2 -HfO2 system were synthesized and analyzed by X-ray fluorescence and diffraction techniques. The vaporization processes and partial pressures of the vapor species over the samples were obtained by the high-temperature mass spectrometric method using the ion current comparison method. The derived thermodynamic functions were optimized within the Generalized Lattice Theory of Associated Solutions (GLTAS) approach. At the temperature of 2600 K, the GdO, ZrO, ZrO2 , HfO, and O vapor species were found over the samples, but only for the GdO and ZrO species were accurate experimental data on the partial pressures obtained. In all the ceramic samples, the Gd2 O3 activity was determined. On this experimental basis, modeling within the GLTAS approach was performed. It allowed the evaluation of the Gd2 O3 , ZrO2 , and HfO2 activities in the system under study and a rough approximation of the excess Gibbs energy as a function of composition to be obtained. At 2600 K the Gd2 O3 -ZrO2 -HfO2 system is characterized by negative deviations of its thermodynamic properties from the ideal behavior. Consistency of the obtained modeling results indicate reasonable uniformity of the energy parameters of the lattice model derived in calculations of the hafnia-containing oxide systems, which may be used in further modeling of multicomponent systems.

Development of a liquid chromatography mass spectrometry method for the determination of vitamin K1, menaquinone-4, menaquinone-7 and vitamin K1-2,3 epoxide in serum of individuals without vitamin K supplements.

Vitamin K and metabolites have a beneficial role in blood coagulation, bone metabolism and growth. However, the determination of vitamin K concentrations inthe blood in patients consuming a diet with naturally occurring vitamin K is currently challenging. We aim to develop a cost-effective and rapid method to measure vitamin K metabolites with potential application for clinics and research. We developed a simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of vitamin K1, menaquinone-4 (MK-4), menaquinone-7 (MK-7) and vitamin K1-2,3 epoxide in human serum and validated the method in a study cohort of 162 patients tested for carbohydrate malabsorption and in 20 patients with oral phenprocoumon intake. The overall precision (CVs) ranged between 4.8 and 17.7% in the specified working range (0.06-9.0nmol/L for all analytes except for MK-7 with 0.04-6.16nmol/L). In the malabsorption cohort samples, measured values were obtained for all different vitamin K metabolites except for vitamin K1-2,3 epoxide. This metabolite could be detected only in patients with phenprocoumon intake. The good performance of the method is especially achieved by the interaction of three factors: the use of lipase in the sample preparation, the use of an atypical fluorinated reversed phase column, and a logarithmic methanol gradient. The described method is able to determine the concentration of four vitamin K metabolites in a time-efficient, simple and cost-effective manner. It can be suitable for both routine clinics and research.

Simultaneous determination of novel β-lactamase inhibitor WCK 4234 and Meropenem in dog plasma by LC-MS/MS and its application to preclinical pharmacokinetic study.

A precise and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) bioanalytical method has been developed and validated for the simultaneous quantification of WCK 4234 and meropenem (MEM) in dog plasma. Protein precipitation using acetonitrile was employed as a sample preparation approach. Cefepime was used as an internal standard. The developed method was selective, sensitive (limit of quantification, 0.075 μg/ml for both drugs), accurate (recovery > 90%), precise (CV <10%) and linear (r2 ≥0.99, concentration range 0.075-120 μg/ml for both analytes). The developed method was successfully applied for the determination of both drugs in plasma to assess the pharmacokinetics in beagle dogs. WCK 4234 + MEM in a 1:1 ratio at 15 + 15 and 30 + 30 mg/kg doses were administered by the intravenous route. The mean plasma concentration and area under the concentration-time curve of WCK 4234 ranged from 38.3 to 77.4μg/ml and from 47.8 to 77.1μg h/ml, respectively, and the values for MEM ranged from 52.2 to 115.3μg/ml and 70.5 to 133.6μg h/ml respectively. The elimination half-life of WCK 4234 and MEM was around 0.8h.

Inter-laboratory Validation of an HPLC–MS/MS Method for the Detection of Microbial Transglutaminase in Meat and Meat Products

Microbial transglutaminase (TG) is an enzyme isolated on an industrial scale from Streptomyces mobaraensis. Technical TG, a formulated powder, is primarily used to restructure meat in the meat-processing industry, typically at a 1% concentration and is often referred to as “meat glue.” In the European Union, meat restructured with TG requires the indication “formed meat” on the label according to Regulation (EU) No 1169/2011. In order to detect food fraud like the undeclared TG usage in meat and meat products, a qualitative mass spectrometric method using specific tryptic marker peptides has been published in 2017. Here the successful inter-laboratory validation and first-time standardization of a proteomics method for food control is described, which was subsequently included into the Official Collection of Analysis Methods according to the German Food and Feed Code (§ 64 LFGB). Thirteen laboratories from governmental, academic, and private institutions participated in the study, whereas four laboratories did not meet the minimal quality criteria and therefore their results had to be excluded. Three different test materials containing between 0.2 and 2% technical TG as well as blank samples were produced and tested. The laboratories used triple-quadrupole mass spectrometers from several vendors as well as quadrupole time-of-flight instruments. The detection of TG was considered to be positive, if three mass transitions for the marker peptides VTPPAEPLDR (TG-1) and SPFYSALR (TG-2), each, showed a signal-to-noise ratio of at least 3. The level of detection LOD95% for the median laboratory with intermediate performance was 0.31%, the false-positive rate was 0% and the false-negative rate was 2.1%.

Proteomic characterisation and phylogenetic derivation of ovine αS1-CN B and αS1-CN G genetic variants

A combination of chromatographic, immunoelectrophoretic and mass spectrometric methods was applied to healthy individual milk of ovine massese breed. Caseome of αS1-casein (αS1-CN) fraction was characterised in homozygous and heterozygous phenotypes for the novel B and G genetic variants. Using ovine αS1-CN C counterpart as reference, amino acid substitutions and phosphorylated components were determined. The replacement of p.Arg90>His and p.Ile194>Thr were found in αS1-CN B variant, while the single p.Ile194>Thr substitution occurred in αS1-CN G variant. The quali-quantitative composition of phosphorylated αS1-CN components in milk of animals bearing each variant was defined, highlighting possible genetic markers for assessment of dairy product quality. The heterogeneity of the electrophoretic profiles of the corresponding caseomes was due to the number of phosphorylated components and their net charge and pI values, and updated the ovine αS1-CN phylogenetic tree, suggesting αS1-CN G as the ancestral variant of αS1-CN B and other variants.

Unexpected sensitivity enhancement in analysing alfatoxin M1 using LC-IDMS

Aflatoxin M1 (AFM1) and patulin (PAT) are regulated mycotoxins, which are generally analyzed in food samples alone (“one analyte at a time”). This paper presents the development of a liquid chromatography isotope dilution tandem mass spectrometric (LC-IDMS) method for the determination of AFM1 in milk-based products, which method was then successfully applied to PAT in apple-based food samples. The method involved a selective solid-phase extraction clean-up at acidic pH employing mixed-mode cation exchange cartridges. The purification allowed the adequate adsorption of neutral target compounds and basic matrix constituents of the sample to the reversed-phase (RP) and to the cation exchange phase of the cartridge, respectively. The sample elution with pure methanol yielded an extract free from basic matrices. The remaining acidic and neutral matrix constituents of the samples were separated from the analytes using mixed-mode anion exchange HPLC column. Mobile phases without any modifiers were used for the highest sensitivity and better selectivity. The intensity enhancement obtained by neutral eluent composition was inter-laboratory tested and confirmed on same type of instruments. The sample preparation requires low amount of organic solvent, thus adhered to green analytical chemistry. The developed method for analyzing AFM1 resulted in low matrix effect of LC-IDMS separation −4.9-(−1.5%) and high precision (2.7–5.9%), calculated under method validations at spiking levels between 0.005 and 0.050 µg/kg. Method performance characteristics for analyzing PAT were also evaluated at 10 and 25 µg/kg levels. Finally, the advantageous features of the newly developed method were briefly compared and discussed with the existing approaches.

IDF21-0121 NDMA Analytics in Metformin Products: Comparison of Methods and Pitfalls

Background: Since late 2019 concerns regarding trace levels of the N-nitrosamine NDMA in metformin-containing pharmaceuticals have been an issue, as they exceeded the maximum allowable intake of 96 ng/day for a medicine with long-term intake. This has resulted in thorough assessment of the safety of metformin products in this regard by manufactures worldwide. From the early focus on trace NDMA levels in drug substance, the scope of concern quickly expanded to drug products, associated excipients, and the potential for nitrosamine formation in packaging processes. As the scope expanded, the trace analysis challenge has grown. Metformin is made from the starting materials 2-cyanoguanidine and DMA, hence low residual amounts of DMA in the API are unavoidable. Inorganic nitrite is present in trace amounts in many excipients and can react with DMA to form the mutagenic N-nitrosamine NDMA, which needs to be measured in complex drug product formulations. The methods developed in the pharmaceutical industry are validated according to the applicable guidelines. Nevertheless, incorrect measurements can never be fully excluded. In food analysis, environmental analysis and in medical diagnostics, proficiency tests are a common tool to identify and eliminate systematic errors by comparing measurement results from individual laboratories. Aim: Here, we describe the outcome of an across-industry method comparison study for determination of NDMA in metformin-containing drug products between four labs, employing orthogonal mass spectroscopic methods, and discuss how it has helped to identify and mitigate artefactual results due to NDMA in-situ formation in the analytical process. Method: Proficiency testing employing orthogonal mass spectrometric methods was performed between four laboratories in three countries through analysis of different branded metformin-containing products. To exclude bias from sample inhomogeneity, fine powders of test tablets were prepared by one of the participating laboratories. To avoid bias from sample aging, experiments were performed contemporarily. Results: A risk of overestimating NDMA levels has been identified. Notably, special attention must be given to the sample preparation to prevent artefactual formation of NDMA in the extracted sample. Metformin medicines contain DMA in the ppm range, and nitrite in the ppb range, which react in the standard organic solvent DCM to form elevated levels of NDMA. This study has demonstrated that both removal of DMA and nitrite via solvent extraction, and “quenching” the reaction by addition of a nitrosation scavenger are effective remedies. Discussion: Without proper mitigation, NDMA in-situ formation causes overestimation of the NDMA content, which may in turn lead to an overestimation of affected batches and, ultimately, the risk to patients.

Determination of arsenic, cadmium, mercury, lead and nickel as elemental impurities in atorvastatin calcium by inductively coupled mass spectrometer.

Atorvastatin calcium is employed as front-line treatment for cardiovascular diseases. According to the international conference on harmonization (ICH) guideline ICH Q3D, elemental impurities can come into drug products from various sources. These elemental impurities do not have any therapeutic benefit to the end-user. On the contrary, it harms the normal physiological system. Class 1 elements like arsenic, cadmium, mercury, and lead are inorganic impurities that can cause toxic effects on the human body. Nickel was used as a catalyst during the synthesis process of atorvastatin calcium. It comes under the Class-2A, can cause toxicity to humans, and must be quantified. A simple, fast, reliable inductively coupled plasma mass spectrometric method for the estimation of elemental impurities like arsenic, cadmium, mercury, lead, and nickel in atorvastatin calcium by open sample digestion technique was developed and validated in accordance with ICH Q3D and USP < 232 > and USP <233 > general chapter. Internal standards like indium, terbium, thallium, bismuth and yttrium were used to correct the non-spectral interferences that were generated during analysis. Gold was added to all solutions as it preserves mercury by amalgamation. The system performance was evaluated every time my performing system suitability parameters. The limits for all the elements were fixed in accordance with ICH Q3D. The limit of detection and the limit of quantification for all the five elements were estimated. Method specificity was proven by checking for interferences due to the sample matrix and other elements. Linearity of each element in standards was established from 25% to 200% of sample concentration, and correlation coefficients were found to be not less than 0.999. The accuracy of the method was demonstrated at three spiking levels at 50%, 100%, and 150% of the J-value for all the elements. The recoveries for all elements at each level were within the range of 90-120%. Method precision was proved at 100% J-value. The relative standard deviation of all elements was less than 5%. It concludes that this newly developed and validated reliable inductively coupled plasma mass spectrometric method for estimating of elemental impurities like arsenic, cadmium, mercury, lead, and nickel in atorvastatin calcium was within the permitted limit and suitable for routine use.

Determination of Aminophosphonate Herbicides in Glutamate Loaded Spice Mix by LC-IDMS and Method Extension to Other Food Matrices

The accumulation of organophosphorus type herbicides has been observed worldwide in the environment (i.e. soil, water), together with their appearance in foods of plant origin. This paper reports a new liquid chromatography–isotope dilution–tandem mass spectrometric method (LC-IDMS) for the analysis of glufosinate (GLUF), glyphosate (GLY) and its main metabolite, aminomethylphosphonic acid (AMPA), in challenging food samples. Sample preparation is based on aqueous extraction with ethylenediaminetetraacetic acid solution, followed by solid-phase extraction (SPE) on mixed-mode cation exchange cartridges to remove matrix constituents before derivatization with 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl). Derivatized samples were cleaned up on hydrophilic modified polymeric SPE cartridge. This two-step SPE supported sample preparation approach, and the LC-IDMS separation carried out in negative ionization mode resulted in fit-for-purpose recovery (81–118%) and precision (4–18%) in the validation of glutamate loaded spice mix, mushroom, maize and cherry samples. Amino acid content influencing FMOC derivatization efficiency was estimated with a HILIC-MS/MS setup. Multiple reaction monitoring (MRM) was assisted with high-resolution (QTOF) accurate mass data on the FMOC-derivatized GLUF, GLY and AMPA standards. The limit of quantification (LOQ) was 0.005 mg/kg for all the three analytes. The method was successfully applied on quality control samples (oat and arugula) with fit-for-purpose accuracy (99–120%) and on other nineteen real samples, where GLY and AMPA were detected in the range between 0.005 and 0.069 mg/kg.

Dihydrogen phosphate anion boosts the detection of sugars in electrospray ionization mass spectrometry: A combined experimental and computational investigation.

Sugars are key molecules of life but challenging to detect via electrospray ionization mass spectrometry (ESI-MS). Unfortunately, sugars are challenging analytes for mass spectrometric methods due to their high gas-phase deprotonation energies and low gas-phase proton affinities which make them difficult to ionize in high abundance for MS detection. Hydrogen-bond interactions in H2 PO4 - -saccharide anionic systems were studied both experimentally (via electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, ESI-FT-ICR-MS) and computationally by several sophisticated density-functional theoretical methods (DFT and DFT-D3). The H2 PO4 - dopant boosts the detection of sugars up to 51-times in the case of sucrose and up to 263-times for glucose (at 0.1 ppm concentration level). H2 PO4 - binds toward sugar molecules with noticeably more hydrogen bonds than the established dopant chloride Cl- does, with increasing binding energies in the order: Monosaccharides < Trisaccharides < Disaccharides. Analysis of a complex oak plant sample revealed that NH4 H2 PO4 specifically labeled a diverse set of sugar-type plant metabolites in the form of [M + H2 PO4 ]- complexes. We reveal the mechanism of interaction of H2 PO4 - with different sugars and glycosylated organic compounds, which significantly enhances their ionization in mass spectrometry. A computational and experimental investigation is presented. A strong correlation between the MS signal intensities of detected [M + H2 PO4 ]- anions of different saccharides and their calculated dissociation enthalpies was revealed. Thus, the variation in MS signal intensities can be very well described to a large extent by the variation in calculated saccharide affinities toward the H2 PO4 - dopant anion, showing that DFT-D3 can very well describe experimental FT-ICR-MS observations.

Acetylation, Phosphorylation, Ubiquitination (Oh My!): Following Post-Translational Modifications on the Ubiquitin Road.

Ubiquitination is controlled by a series of E1, E2, and E3 enzymes that can ligate ubiquitin to cellular proteins and dictate the turnover of a substrate and the outcome of signalling events such as DNA damage repair and cell cycle. This process is complex due to the combinatorial power of ~35 E2 and ~1000 E3 enzymes involved and the multiple lysine residues on ubiquitin that can be used to assemble polyubiquitin chains. Recently, mass spectrometric methods have identified that most enzymes in the ubiquitination cascade can be further modified through acetylation or phosphorylation under particular cellular conditions and altered modifications have been noted in different cancers and neurodegenerative diseases. This review provides a cohesive summary of ubiquitination, acetylation, and phosphorylation sites in ubiquitin, the human E1 enzyme UBA1, all E2 enzymes, and some representative E3 enzymes. The potential impacts these post-translational modifications might have on each protein function are highlighted, as well as the observations from human disease.

An Alternative Strategy for Screening and Confirmation of 330 Pesticides in Ground- and Surface Water Using Liquid Chromatography Tandem Mass Spectrometry.

The presence of pesticide residues in water is a huge worldwide concern. In this paper we described the development and validation of a new liquid chromatography tandem mass spectrometric (LC-MS/MS) method for both screening and quantification of pesticides in water samples. In the sample preparation stage, the samples were buffered to pH 7.0 and pre-concentrated on polymeric-based cartridges via solid-phase extraction (SPE). Highly sensitive detection was carried out with mobile phases containing only 5 mM ammonium formate (pH of 6.8) as an eluent additive and using only positive ionization mode in MS/MS instrument. Hence, only 200-fold sample enrichment was required to set a screening detection limit (SDL) and reporting limit (RL) of 10 ng/L. The confirmatory method was validated at 10 and 100 ng/L spiking levels. The apparent recoveries obtained from the matrix-matched calibration (5–500 ng/L) were within the acceptable range (60–120%), also the precision (relative standard deviation, RSD) was not higher than 20%. During the development, 480 pesticides were tested and 330 compounds fulfilled the requirements of validation. The method was successfully applied to proficiency test samples to evaluate its accuracy. Moreover, the method robustness test was carried out using higher sample volume (500 mL) followed by automated SPE enrichment. Finally, the method was used to analyze 20 real samples, in which some compounds were detected around 10 ng/L, but never exceeded the assay maximum level.

Characterisation of riverine dissolved organic matter using a complementary suite of chromatographic and mass spectrometric methods

Dissolved organic matter (DOM) plays a fundamental role in nutrient cycling dynamics in riverine systems. Recent research has confirmed that the concentration of riverine DOM is not the only factor regulating its functional significance; the need to define the chemical composition of DOM is a priority. Past studies of riverine DOM rested on bulk quantification, however technological advancements have meant there has been a shift towards analytical methods which allow the characterisation of DOM either at compound class or more recently molecular level. However, it is important to consider that all analytical methods only consider a defined analytical window. Thus, herein, we explore the use of a hierarchy of methods which can be used in combination for the investigation of a wide range of DOM chemistries. By using these methods to investigate the DOM composition of a range of streams draining catchments of contrasting environmental character, a wide range of compounds were identified across a range of polarities and molecular weight, thereby extending the analytical window. Through the elucidation of the DOM character in stream samples, information can be collected about likely the sources of DOM. The identification of individual key compounds within the DOM pool is a key step in the design of robust and informative bioassay experiments, used to understand in-stream ecosystem responses. This is critical if we are to assess the role of DOM as a bioavailable nutrient resource and/or ecotoxicological factor in freshwater.

Cancer Conformational Landscape Shapes Tumorigenesis.

During tumorigenesis, DNA mutations in protein coding sequences can alter amino acid sequences which can change the structures of proteins. While the 3D structure of mutated proteins has been studied with atomic resolution, the precise impact of somatic mutations on the 3D proteome during malignant transformation remains unknown because methods to reveal in vivo protein structures in high throughput are limited. Here, we measured the accessibility of the lysine ε-amine for chemical modification across proteomes using covalent protein painting (CPP) to indirectly determine alterations in the 3D proteome. CPP is a novel, high-throughput quantitative mass spectrometric method that surveyed a total of 8052 lysine sites across the 60 cell lines of the well-studied anticancer cell line panel (NCI60). Overall, 5.2 structural alterations differentiated any cancer cell line from the other 59. Structural aberrations in 98 effector proteins correlated with the selected presence of 90 commonly mutated proteins in the NCI60 cell line panel, suggesting that different tumor genotypes reshape a limited set of effector proteins. We searched our dataset for druggable conformational aberrations and identified 49 changes in the cancer conformational landscape that correlated with the growth inhibition profiles of 300 drug candidates out of 50,000 small molecules. We found that alterations in heat shock proteins are key predictors of anticancer drug efficacy, which implies that the proteostasis network may have a general but hitherto unrecognized role in maintaining malignancy. Individual lysine sites may serve as biomarkers to guide drug selection or may be directly targeted for anticancer drug development.

SARS-CoV-2 spike antigen quantification by targeted mass spectrometry of a virus-based vaccine

The spike glycoprotein mediates virus binding to the host cells and is a key target for vaccines development. One SARS-CoV-2 vaccine is based on vesicular stomatitis virus (VSV), in which the native surface glycoprotein has been replaced by the SARS-CoV-2 spike protein (VSV-ΔG-spike). The titer of the virus is quantified by the plaque forming unit (PFU) assay, but there is no method for spike protein quantitation as an antigen in a VSV-based vaccine. Here, we describe a mass spectrometric (MS) spike protein quantification method, applied to VSV-ΔG-spike based vaccine. Proof of concept of this method, combining two different sample preparations, is shown for complex matrix samples, produced during the vaccine manufacturing processes. Total spike levels were correlated with results from activity assays, and ranged between 0.3−0.5 μg of spike protein per 107 PFU virus-based vaccine. This method is simple, linear over a wide range, allows quantification of antigen within a sample and can be easily implemented for any vaccine or therapeutic sample.