Single Sample Preparation Research Articles

Disposition and fate of polyoxyethylene glycerol ricinoleate as determined by LC-Q-TOF MS coupled with MSALL, SWATH and HR MS/MS techniques

Polyoxyethylene glycerol ricinoleate (PGR) serves as a solubilizer/emulsifier that is commonly used in pharmaceutical formulations despite being associated with severe anaphylactoid hypersensitivity reactions. Cremophor EL® (CrEL) is the most representative PGR produced from reacting ethylene oxide with castor oil. To help clarify the cause of side effects and potentially improve the safety of PGR-based drug delivery vehicle, we have developed separate but related analytical methods for the quantitation of CrEL and its main metabolites, glycerol ethoxylate (GE) and ricinoleic acid (RA). Since CrEL and GE are highly disperse mixtures of polymers that are not amenable to analysis by conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS), we used liquid chromatography-triple-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF MS) combined with product ion data acquisition by MSALL and sequential window acquisition of all theoretical fragments mass spectrometry (SWATH MS), respectively to perform the analysis. In contrast, RA is a single molecular entity that could be readily analyzed using conventional LC-HR MS/MS. Selection of specific fragment ions for CrEL, GE, RA and their internal standards enabled a precise quantitation of such a complex analytes system in rat plasma after a single and simple sample preparation method. Assay validation indicated linearity for CrEL, GE and RA over the concentration ranges 0.2~20.0 μg/mL, 0.1~10.0 μg/mL and 0.1~20.0 μg/mL, respectively with satisfactory results for other validation parameters. A subsequent pharmacokinetic study involving single intravenous 200 mg/kg injections of CrEL to rats showed the methods enable comprehensive and high throughput quantitation of CrEL and its metabolites in a biological matrix. Our combination of assays provides effective application in investigating the cause of the hypersensitivity reaction of PGR and potentially to improve its safety for using as a vehicle in drug formulations.

Development of a Novel, Fast, Simple HPLC Method for Determination of Atorvastatin and its Impurities in Tablets

Our main target and concept was to develop a method for the determination of the most prescribed antilipemic drug, atorvastatin, together with its related substances, with a single sample preparation and during a single chromatographic run, in the shortest possible period of time, with the lowest possible mobile phase consumption. A new rapid, simple chromatographic method for the determination of atorvastatin and its main specified impurities was developed, using different chromatographic columns. With this new concept of a mobile phase and a powerful core–shell, or a superficially porous silica-based column, satisfactory results for targeted parameters, such as critical peak resolution, run time length, and column backpressure, were achieved. The analysis is performed within a run duration of less than 15 min, which is about six times shorter than the official European Pharmacopoeia method. The chromatogram performances suggests that the method limit of quantification (LOQ) can be about 7 times lower, and the limit of detection (LOD) about 20 times lower, using an injection volume of only 2 µl. This was confirmed by the performed method validation in accordance with the International Conference on Harmonization (ICH) guideline for the validation of analytical procedures Q2(R1), where the selectivity, linearity, accuracy, precision, limit of quantification, and limit of detection were tested and confirmed.

First person – Marte Molenaars and Bauke Schomakers

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Marte Molenaars and Bauke Schomakers are co-first authors on ‘Metabolomics and lipidomics in Caenorhabditis elegans using a single-sample preparation’, published in DMM. Marte conducted the research described in the article while a PhD student in the lab of Riekelt Houtkooper at Amsterdam UMC, Amsterdam, The Netherlands, and is now a postdoc in the lab of Richard Possemato at New York University School of Medicine, New York, NY, USA. During her PhD, she was investigating cellular metabolic pathways and how to use them to target ageing and age-related diseases, and currently she focuses on metabolic pathways in cancer cells as a postdoc. Bauke is a Research Analyst in the lab of Riekelt Houtkooper at Amsterdam UMC, Amsterdam, The Netherlands, investigating liquid chromatography–mass spectrometry-based omics methods for the comprehensive analysis of biological samples.

Metabolomics and lipidomics in Caenorhabditis elegans using a single-sample preparation.

ABSTRACTComprehensive metabolomic and lipidomic mass spectrometry methods are in increasing demand; for instance, in research related to nutrition and aging. The nematode Caenorhabditis elegans is a key model organism in these fields, owing to the large repository of available C. elegans mutants and their convenient natural lifespan. Here, we describe a robust and sensitive analytical method for the semi-quantitative analysis of >100 polar (metabolomics) and >1000 apolar (lipidomics) metabolites in C. elegans, using a single-sample preparation. Our method is capable of reliably detecting a wide variety of biologically relevant metabolic aberrations in, for example, glycolysis and the tricarboxylic acid cycle, pyrimidine metabolism and complex lipid biosynthesis. In conclusion, we provide a powerful analytical tool that maximizes metabolic data yield from a single sample.This article has an associated First Person interview with the joint first authors of the paper.

Single‐run DSC method for determining surface crystal growth rate in glasses generalized for samples of different shapes

AbstractWe propose a new generalized method for the determination of surface crystallization growth rates in a wide temperature range using a single Differential Scanning Calorimetry (DSC) experiment. It can be applied to different sample geometries—we have tested the method for powdered and millimetric cubic samples. The crystal growth rate is calculated from the DSC crystallization peak profile and the average particle size. The method was successfully tested using diopside (MgO·CaO·SiO2) glass powders and a sodium disilicate glass (Na2O·2SiO2) millimetric cube, which undergoes exclusive stoichiometric surface crystallization. The resulting crystal growth rates spanned ranges of approximately 60 K for diopside glass and 170 K for sodium disilicate. The method is very convenient, requiring a single DSC run and simple sample preparation. The crystal growth rates, , proved to be accurate when compared to literature optical microscopy measurements, with a root mean square deviation of 0.22 in for diopside glass.

Determination of amlodipine, indapamide and perindopril in human plasma by a novel LC–MS/MS method: Application to a bioequivalence study

A robust and rapid UPLC-MS/MS method has been developed, optimized and validated for determination of amlodipine (AML), indapamide (IND) and perindopril (PRN) in human plasma. A positive electrospray ionization mode was used in a Xevo TQD LC-MS/MS instrument. A single sample preparation step using extraction technique was applied to extract the three analytes from plasma samples. There was no need to extract indapamide from blood samples in a further step. Extraction of the three drugs and internal standards was done using a solvent mixture composed of methyl tertiary butyl ether, dichloromethane and ethyl acetate. The prepared samples were analyzed using an Acquity UPLC HSS C18 (100 × 2.1 mm, 1.7 μm) column. Ammonium acetate and methanol, pumped at a flow rate of 0.3 ml/min, were used as a mobile phase. Method validation was done as per the US Food and Drug Administration guidelines. Linearity was achieved in the range of 0.2-15 ng/ml for AML, 0.5-50 ng/ml for IND and 0.5-120 ng/ml for PRN. Accuracy and precision were estimated and found to be within the acceptable ranges. The rapid chromatography permits analysis of many samples per batch, making the method suitable for clinical and pharmacokinetic investigations. The developed and validated method was applied to estimate AML, IND, and PRN in a fasting bioequivalence study in healthy human volunteers.

Optimization of Sample Preparation for the Observation of Macromolecular Complexes by Electron (cryo-)Microscopy.

Electron microscopy is a powerful tool for studying the homogeneity and structure of biomolecular complexes. The small wavelength of electron and the availability of electron optics enable the direct visualization of macromolecular assemblies in a large range of sizes between 5 and 100nm. This informs us about the degree of multimerization or aggregation and provides precise information about their general shape and dimensions. When combined with sophisticated image analysis protocols, three-dimensional (3D) information can be gained from 2D projections of the sample, leading to a structural description. When intermediate steps of a reaction can be imaged, insights into the mode of action of macromolecules can be gained, and structure-function relations can be established. However, the way the sample is prepared for its observation within the vacuum of an electron microscope determines the information that can be retrieved from the experiment. We will review two commonly used specimen preparation protocols for subsequent single-particle electron microscopy observation, namely negative staining and vitrification.

BFRs (PBDEs and HBCDs) in freshwater species from Lake Trasimeno (Italy): The singular case of HBCDs in red swamp crayfish

Eighty-six samples belonging to five different species (crucian carp, Carassius carassius; European perch, Perca fluviatilis; tench, Tinca tinca; eel, Anguilla anguilla; red swamp crayfish, Procambarus clarkii) collected from Lake Trasimeno (Italy) were analyzed to assess polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs) contamination. The Trasimeno is the largest Italian peninsular lake located in Umbria (Central Italy), in a rural area with low anthropogenic impact. All the samples were analyzed by an in-house developed analytical procedure involving a single sample preparation with dual detection: Gas- and Liquid-Chromatography coupled to tandem Mass Spectrometry (GC–MS/MS for PBDEs and LC-MS/MS for HBCDs). BFRs levels in crucian carp, tench and European perch were negligible and mostly below quantification limits (LOQs). In eel, the species with the higher fat content, PBDE sum (15 congeners) ranged from 0.269 to 0.916 ng/g w.w. BDE-47, -100 and -154 accounted for roughly 57%, 16% and 11% of the PBDE sum, respectively, while BDE-99 (usually one of the most abundant congeners in biota), only for 3%. HBCDs (sum of α-, β-, γ-isomers) were found between 0.157 and 1.14 ng/g w.w. with α- as predominant isomer (92% of the sum), followed by γ- (5%) and β- (2%). Peculiar was the contamination in red swamp crayfish characterized by negligible PBDEs and very high HBCDs levels with a singular contamination pattern. In female pools (n = 9) the mean HBCDs sum was 0.150 ng/g w.w., while in males higher concentrations were measured (mean = 2.77 ng/g w.w.). A significant correlation seems to exist between the contamination level and the seasonal cycle only in male crayfish. Interestingly, among the HBCDs, the γ-isomer was the highest (67% of the total) while α- contributes only for 20%.

Method optimization and validation for multi-class residue analysis in turmeric

Turmeric (Curcuma longa L.) is an economically important food and medicinal plant that grows in tropical and subtropical regions consumed worldwide. Turmeric is a difficult matrix for residue/contaminant analysis due to the high content of potentially interfering polyphenols (curcuminoids) and essential oils with physicochemical properties similar to target analytes. To solve this problem, a simple and straightforward sample preparation protocol based on ethyl acetate extraction and dispersive solid phase clean-up, followed by gas and liquid chromatography coupled to tandem mass spectrometry analysis, was optimized and validated, after selecting a representative blank material using gas chromatography coupled to ion mobility spectrometry and multivariate data analysis. Method validation was performed according to Codex guidelines for 67 pesticide residues, including 12 persistent organic pollutants, 4 aflatoxins and 3 dyes at 0.01, 0.02 and 0.05 mg kg−1level. Recoveries ranged between 60 and 120%, with relative standard deviations for repeatability and reproducibility below 20% and 32% respectively. Method performance was tested collaboratively between laboratories in Austria and Uruguay which helped proving the robustness of the methodology. The method was applied to the analysis of commercial samples and chlorpyrifos was detected in most of them.The novelty of the work relies in the optimization and validation of a multiclass method that allows analysis of four types of organic residues/contaminants, of paramount importance for food safety, using a single sample preparation step followed by gas and liquid chromatography coupled to tandem mass spectrometry determination.

Abstract 6197: Improving sample preparation workflows for single cell multiomic analysis of infiltrating leukocytes from solid tumors

Abstract Single cell multiomic (scM) methods are powerful tools to characterize patient-specific tumor-immune microenvironment, but current methods of sample handling and preparation can introduce bias. Obtaining non-biased results is particularly important in studies to identify new biomarkers for patient stratification of responders versus non-responders for immunotherapies in oncology. This problem is becoming acute as the number of immune-therapy modalities and combinations in clinical trials and in medical practice continue to expand. Current methodologies used to analyze patient specimens in routine pathology are not adequate to address heterogeneity and complexity of immune cell ecosystem of individuals. SCM analysis of immune cells from tumor microenvironment using technologies that allow high parameter analysis of genes and proteins will allow a comprehensive atlas of immune cells in the tumor tissue, however the quality of single cell sample preparation is critical. Here, we report evaluation of several available solid tumor dissociation methods, including mechanical dissociation by scalpel, BD™ Medimachine System, Miltenyi gentleMACS™ dissociator. Mechanical dissociation was followed by enzymatic dissociation using BD Horizon™ Dri Tumor & Tissue Dissociation Reagent or, in the case of gentleMACS™, the Miltenyi Tumor Dissociation Kit. Metrics evaluated were cell recovery, viability, and proportion of various cell lineage populations using flow cytometry; and expression of 399 mRNA transcripts and 31 proteins using single cell multiomics (BD Rhapsody™, BD Rhapsody™ Immune Response Targeted Panel, and BD™ AbSeq). Tumor tissues were from a mouse a pancreatic (PDAC) tumor model and humanized Patient-Derived Xenograft (PDX; from Onco-hu® mice) models of colon and lung cancer. Method dependent differences in viable cell yield and proportions of immune vs. tumor cells were observed. SCM analysis of FACS sorted CD45+ tumor-infiltrating lymphocytes (TILs) showed differences in gene and protein expression profiles, particularly for expression of rare vs. abundant genes depending on the dissociation method. Our data identify protocols to interrogate tumor immune microenvironment starting from tumor tissue specimen through scM all the way to informatics analysis with consistent results. Similar studies and further optimization are recommended for other tumor types. Consistent adoption of standardized protocols will enable robust findings in research and clinical trials, with the ultimate goal of improved therapies, biomarkers and outcomes. For Research Use Only. Not for use in diagnostic or therapeutic procedures. GentleMACS™ is trademark of Miltenyi Biotec. Onco-hu® is a registered trademark of The Jackson Laboratory. BD, the BD Logo, Horizon, Medimachine, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Aaron J. Middlebrook, Wei Huang, Margaret Nakamoto, Xiaoshan Shi, Evelyn Lo, Patrick Neuhoefer, Scott Bornheimer, Smita Ghanekar. Improving sample preparation workflows for single cell multiomic analysis of infiltrating leukocytes from solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6197.

A comprehensive approach for the simultaneous analysis of all main water-soluble vitamins in multivitamin preparations by a stability-indicating HPLC-DAD method

A stability-indicating HPLC-DAD method for simultaneous determination of all nine main water-soluble vitamins, in addition to two commonly used vitamers, was developed and fully validated in analytical ranges, adjusted to their recommended dietary allowance values. An XSelect CSH C18 column with gradient elution using phosphate buffer and methanol was used for their optimal separation. The results from forced degradation studies along with peak purity tests and response ratios at dual wavelengths for the individual vitamins in all tested samples confirmed the method’s stability-indicative nature. The complete developed methodology, including a single sample preparation for the vitamins simultaneous analysis, was applied to their assay in 13 commercial multivitamin preparations, revealing mostly higher amounts than the label claims. The developed method is applicable for stability testing, multivitamin products shelf-life determination as well as routine assay analysis of all water-soluble vitamins in their most common forms, including the analytically most demanding flavin mononucleotide.

Single cell dissociation from mouse tumor samples and analysis on BD FACSLyric Flow Cytometer.

e15245 Background: Single cell multiomics analysis of tumor cells and tumor infiltrating immune cells using high parameter flow cytometry and single cell genomics technologies is a powerful approach in the investigation of tumor biology and in the discovery of new drug targets and biomarkers. The preparation of single cells from solid tumor samples often involves a physical dissociation step in combination with an enzymatic digestion step. Methods: The effect of the tumor cell dissociation process on the phenotypic properties of single cells isolated from solid tumor samples were analyzed using a 12-color reagent panel on the BD FACSLyric flow cytometer. Specifically, spontaneous pancreatic tumor samples from genetically modified mice were processed using a manual dissociation process with a scalpel blade as well as an integrated tumor sample dissociation process with the prototype Singulator instrument with various protocol settings. The BD Horizon Dri Tumor & Tissue Dissociation Reagent (TTDR) reagent was used for the enzymatic step in all sample preparations. The yield, viability, and frequency of tumor cells and different subsets of leukocytes were measured for each tumor-derived single cell sample preparation. On the Singulator system, cell yield reached a plateau with highest viability at the 30-min process time and a higher percentage of tumor cells was obtained compared to the manual sample processing method. Results: The relative frequencies of T cells, B cells, myeloid cells and macrophages also changed in different sample preparation methods. However, the spatial heterogeneity in the tumor section and cell composition may contribute to these differences and would require more in-depth analysis. In summary, these results suggest that there are many variables in the tumor sample single cell dissociation process that could impact the downstream study results, particularly for multiomics studies. Conclusions: This study highlights the need for optimization and standardization of the sample processing methodologies for downstream analysis. Disclaimers: For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD, the BD Logo, FACSLyric, and Horizon are trademarks of Becton, Dickinson and Company. © 2020 BD and its subsidiaries. All rights reserved.

Evaluation of the quantity and the carbon isotopic composition of amino acids by using diverse sample residues after lipid extraction.

Compound-specific stable isotope analysis (CSIA) becomes a critical tool in the fields of ecology and biogeochemistry to trace basal carbon sources such as amino acids (AAs) and fatty acids (FAs) in natural environments. However, in many studies, either AAs or FAs have been analyzed due to restricted sample amounts. The aim of this study is to report a single-sample preparation method by identifying AAs using lipid-free residues derived from a typical FA analytic procedure. The capability of the lipid-free residues was evaluated by determining AA quantities and carbon isotopic compositions (δ13C) across diverse sample matrices, including soil, suspended particulate organic matter (SPM), fish tissue, and cultured cyanobacteria (N = 3). Our result showed that the use of lipid-free residues after a typical lipid extraction procedure in most samples did not significantly reduce the AA quantities, relative to the conventional AA method (done by using whole samples), except in case of samples of soil. Moreover, δ13C compositions of the most AAs (from essential to nonessential AAs) in all samples were not significantly different, with an average isotope difference of < 2.1‰ between the two methods. Overall results indicate that the residual parts after extracting lipid fraction are useful for reliable AA quantification and individual isotopic information. Ultimately, one sample preparation for determining FA and AA data enables us to characterize one (or multiple) basal carbon source(s) within rare samples despite their limited abundance. Graphical abstract.

Single sample preparation for brominated flame retardants in fish and shellfish with dual detection: GC-MS/MS (PBDEs) and LC-MS/MS (HBCDs).

An analytical method for the simultaneous determination of polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs) in fish, shellfish and muscle of terrestrial animals was developed as an extension of a previously validated method for PBDE analysis. A single sample preparation based on QuEChERS-like extraction and a two-step clean-up, followed by a dual instrumental detection, was implemented. GC-MS/MS was used for PBDEs and LC-MS/MS for HBCDs. The method allows the quantification of fifteen PBDEs (28, 47, 49, 66, 77, 85, 99, 100, 138, 153, 154, 183, 197, 206, 209) and three HBCD isomers (α, β and γ), reaching 10 pg/g LOQs for all the analytes except BDE 206 and 209 (100 pg/g LOQ). The validated method was applied to the analysis of 12 fish and shellfish species (sole, spiny dogfish, smooth-hound, mackerel, swordfish, grey mullet, cod, anchovy, red mullet, Atlantic horse mackerel, tuna fish and mussel) collected in central Italian markets accounting for a total of 44 samples. Generally, ΣPBDEs showed higher concentration than ΣHBCDs except in the case of mussels in which ΣHBCDs > ΣPBDEs. Cod and smooth-hound are the least contaminated species among those analysed in the present study, whereas the highest brominated flame retardant (BFR) levels were found in spiny dogfish samples. The measured contamination levels were generally comparable to or lower than those already published in European studies. Literature data for HBCDs in European fish and shellfish were hard to find; therefore, PBDE and HBCD levels were also compared with European Environmental Quality Standard (EQS) in biota (Directive 2013/39/EU). EQSs for HBCDs are six orders of magnitude higher than those for PBDEs; therefore, while no exceedance was observed for the first, almost all the samples analysed for PBDEs were above EQSs. The presented preliminary data on PBDEs and HBCDs are among the first published in marine fish and shellfish commercialised in Central Italy. Graphical abstrac.

36Cl preparation method for Chinese Karst samples (Tiankeng)

Tiankeng, a typical Karst relief of the Late Quaternary Period, in the south of China, plays an important role when researching the surface shape and evolution of the Karst relief. The accumulation of Chlorine-36 (T1/2 = 301 ka) through the interaction of cosmic ray particles with Ca, K and Cl in rocks allow it to be used as the ideal nuclide for determination of the exposure age and erosion rate of limestone. In this work, an improved 36Cl sample preparation method for Tiankeng limestone was presented. The Isotope Dilution-AMS technique allows for the simultaneous measurement of 36Cl and Cl with a single AMS sample preparation. The barium introduced during the sample preparation was removed to avoid ionizer poisoning and attain a higher and more stable beam current. The method is particularly suitable for limestone samples, as well as for soil, sediment and water samples.

Triazine-Based Covalent Organic Framework: A Promising Sorbent for Efficient Elimination of the Hydrocarbon Backgrounds of Organic Sample for GC-MS and 1H NMR Analysis of Chemical Weapons Convention Related Compounds.

The strict monitoring and precise measurements of chemical warfare agents (CWAs) in environmental and other complex samples with high accuracy have great practical significance from the forensic and Chemical Weapons Convention (CWC) verification point of view. Therefore, this study was aimed to develop an efficient extraction and enrichment method for identification and quantification of toxic agents, especially with high sensitivity and multidetection ability in complex samples. It is the first study on solid-phase extraction (SPE) of CWAs and their related compounds from hydrocarbon backgrounds using covalent triazine-based frameworks (CTFs). This nitrogen-rich CTF sorbent has shown an excellent SPE performance toward sample cleanup by selective elimination of hydrocarbon backgrounds and enrich the CWC related analytes in comparison with the conventional and other reported methods. The best enrichment of the analytes was found with the washing solvent (1 mL of n-hexane) and the extraction solvent (1 mL of dichloromethane). Under the optimized conditions, the SPE method had good linearity in the concentration range of 0.050-10.0 μg mL-1 for organophosphorus esters, 0.040-20.0 μg mL-1 for nerve agents, and 0.200-20.0 μg mL-1 for mustards with correlation coefficients ( r2) between 0.9867 and 0.9998 for all analytes. Limits of detection ( S/ N = 3:1) in the SIM mode werefound to be in the range of 0.015-0.050 μg mL-1 for organophosphorus esters, 0.010-0.030 μg mL-1 for nerve agents, and 0.050-0.100 μg mL-1 for blister agents. Limits of quantification ( S/ N = 10:1) were found in the range of 0.050-0.200 μg mL-1 for organophosphorus esters, 0.040-0.100 μg mL-1 for nerve agents, and 0.180-0.350 μg mL-1 for blister agents in the SIM mode. The recoveries of all analytes ranged from 87 to 100% with the relative standard deviations ranging from 1 to 8%. This method was also successfully applied for the sample preparation of 1H NMR analysis of sulfur and nitrogen mustards in the presence of hydrocarbon backgrounds. Therefore, this SPE method provides the single sample preparation for both NMR and GC-MS analyses.

Determination of cardiovascular drugs in sewage sludge by matrix solid-phase dispersion and ultra-performance liquid chromatography tandem mass spectrometry.

The current study presents a single step sample preparation procedure for the simultaneous determination of five antihypertensive (propranolol, losartan, irbesartan, telmisartan, and valsartan), three antiarrhythmic drugs (flecainide, dronedarone, and amiodarone), and one of their metabolites (N-desethylamiodarone) in sludge from municipal sewage treatment plants (STPs). Matrix solid-phase dispersion (MSPD) and ultra-performance liquid chromatography (UPLC) with tandem mass spectrometry (MS/MS) detection were selected as sample preparation and determination techniques, respectively. Under optimal conditions, MSPD extractions were carried out with freeze-dried samples (0.5g) dispersed on 2g of C18. Exhaustive extraction of target compounds was achieved with 10mL of a methanol/acetonitrile/formic acid (30:69:1) solution. The obtained extract was ready for UPLC-MS/MS analysis without any further treatment, except filtration. The overall recoveries of the method (calculated against solvent-based standards) varied from 82 to 124%, with standard deviations in the range from 2 to 16%. Thus, the method was free of matrix effects during electrospray ionization. The achieved limits of quantification stayed between 2 and 10ngg-1, and the linear response range extended to 5000ngg-1. The occurrence of target compounds was investigated in sludge from 14 different STPs. High detection frequencies were observed for all compounds, with average concentrations above 100ngg-1 for six cardiovascular drugs.

Anion-exchange polymer filament coating for ultra-trace isotopic analysis of plutonium by thermal ionization mass spectrometry

A new sample loading procedure was developed for isotope measurements of ultra-trace amounts of Pu with thermal ionization mass spectrometry (TIMS) that is based on a polymer thin film architecture. The goals were to simplify single filament TIMS sample preparation for Pu, while preserving the sensitivity and accuracy of the resin bead loading method, and to eliminate sample losses experienced with the bead loading method. Rhenium filaments were degassed, dip-coated with a thin (~ 120 nm) hydrophobic base layer of poly(vinylbenzyl chloride) (PVBC), and spotted with an aqueous solution comprising triethylamine-quaternized PVBC and diazabicyclo[2.2.2]octane crosslinker. This procedure formed a toroidal, hydrophilic anion-exchange polymer spot surrounded by the hydrophobic base polymer. The thin film-coated filaments were direct loaded with 10 pg of New Brunswick Laboratory certified reference material (NBL CRM) 128 from a 9 M HCl matrix. Aqueous sample droplets adhered to the anion-exchange polymer spot, facilitating sample loading. Toroidal spots with a thickness of 20–30 µm generated the highest sample utilization, surpassing the sample utilization of the standard bead loading method by 175%. Measured isotopic ratios were in good agreement with the certified value of the 239Pu/242Pu ratio for NBL CRM 128. The use of dimpled filaments further aided sample loading by providing a well-shaped substrate to deposit the sample droplet. No sample losses were experienced with the thin film loading method over 65 sample analyses. Finally, polymer coatings suppressed filament aging under atmospheric conditions, enabling the bulk production of filaments with adequate shelf life for future analyses.

Comprehensive analysis by liquid chromatography Q-Orbitrap mass spectrometry: Fast screening of peptides and organic molecules.

The number of substances nominally listed in the prohibited list of the World Anti-Doping Agency increases each year. Moreover, many of these substances do not have a single analytical target and must be monitored through different metabolites, artifacts, degradation products, or biomarkers. A new analytical method was developed and validated for the simultaneous analysis of peptides and organic molecules using a single sample preparation and LC-Q-HRMS detection. The simultaneous analysis of 450 target molecules was performed after cleanup on a mixed-mode solid-phase extraction cartridge, combined with untreated urine. The cleanup solvent and reconstitution solvent were the most important parameters for achieving a comprehensive sample preparation approach. A fast chromatographic run based on a multistep gradient was optimized under different flows; the detection of all substances without isomeric coelution was achieved in 11minutes, and the chromatographic resolution was considered a critical parameter, even in high-resolution mass spectrometry detection. The mass spectrometer was set to operate by switching between positive and negative ionization mode for FULL-MS, all-ion fragmentation, and FULL-MS/MS2 . The suitable parameters for the curved linear trap (c-trap) conditions were determined and found to be the most important factors for the development of the method. Only FULL-MS/MS2 enables the detection of steroids and peptides at concentrations lower than the minimum required performance levels set by World Anti-Doping Agency (1ngmL-1 ). The combination of the maximum injection time of the ions into the c-trap, multiplexing experiments, and loop count under optimized conditions enabled the method to be applied to over 10000 samples in only 2months during the 2016 Rio Summer Olympic and Paralympic Games. The procedure details all aspects, from sample preparation to mass spectrometry detection. FULL-MS data acquisition is performed in positive and negative ion mode simultaneously and can be applied to untargeted approaches.

Capillary electrophoresis analysis of affinity to assess carboxylation of multi-walled carbon nanotubes

Surface oxidation improves the dispersion of carbon nanotubes in aqueous solutions and plays a key role in the development of biosensors, electrochemical detectors and polymer composites. Accurate characterization of the carbon nanotube surface is important because the development of these nano-based applications depends on the degree of functionalization, in particular the amount of carboxylation. Affinity capillary electrophoresis is used to characterize the oxidation of multi-walled carbon nanotubes. A polytryptophan peptide that contains a single arginine residue (WRWWWW) serves as a receptor in affinity capillary electrophoresis to assess the degree of carboxylation. The formation of peptide-nanotube receptor-ligand complex was detected with a UV absorbance detector. Apparent dissociation constants (KD) are obtained by observing the migration shift of the WRWWWW peptide through background electrolyte at increasing concentrations of multi-walled carbon nanotubes. A 20% relative standard deviation in method reproducibility and repeatability is determined with triplicate analysis within a single sample preparation and across multiple sample preparations for a commercially available carbon nanotube. Affinity capillary electrophoresis is applied to assess differences in degree of carboxylation across two manufacturers and to analyze acid treated carbon nanotubes. The results of these studies are compared to X-ray photoelectron spectroscopy and zeta potential. Affinity capillary electrophoresis comparisons of carbon nanotube samples prepared by varying acid treatment time from 30 min to 3 h yielded significant differences in degree of carboxylation. X-ray photoelectron spectroscopy analysis was inconclusive due to potential acid contamination, while zeta potential showed no change based on surface charge. This work is significant to research involving carbon nanotube-based applications because it provides a new metric to rapidly characterize carbon nanotubes obtained from different vendors, or synthesized in laboratories using different procedures.