Stepping-up accurate quantification of chlorinated paraffins: Successful certification of the first matrix reference material.

BACKGROUND: Chlorinated paraffins (CPs) are industrial chemicals categorised as persistent organic pollutants because of their toxicity, persistency and tendency to long-range transport, bioaccumulation and biomagnification. Despite having been the subject of environmental attention for decades, analytical methods for CPs still struggle reaching a sufficient degree of accuracy. Among the issues negatively impacting the quantification of CPs, the unavailability of well-characterised standards, both as pure substances and as matrix (certified) reference materials (CRMs), has played a major role. The focus of this study was to provide a matrix CRM as quality control tool to improve the comparability of CPs measurement results. RESULTS: We present the process of certification of ERM®-CE100, the first fish reference material assigned with certified values for the mass fraction of short-chain and medium-chain chlorinated paraffins (SCCPs and MCCPs, respectively). The certification was performed in accordance with ISO 17034:2016 and ISO Guide 35:2017, with the value assignment step carried out via an intercomparison of laboratories of demonstrated competence in CPs analysis and applying procedures based on different analytical principles. After confirmation of the homogeneity and stability of the CRM, two certified values were assigned for SCCPs, depending on the calibrants used: 31 ± 9 µg kg-1 and 23 ± 7 µg kg-1. The MCCPs certified value was established as 44 ± 17 µg kg-1. All assigned values are relative to wet weight in the CRM that was produced as a fish paste to enhance similarity to routine biota samples. SIGNIFICANCE AND NOVELTY: The fish tissue ERM-CE100 is the first matrix CRM commercially available for the analysis of CPs, enabling analytical laboratories to improve the accuracy and the metrological traceability of their measurements. The certified CPs values are based on results obtained by both gas and liquid chromatography coupled with various mass spectrometric techniques, offering thus a broad validity to laboratories employing different analytical methods and equipment.

Synthesis, identification, chiral separation and crystal structure of (3R,4R,7S,8S)-3,4,7,8-tetrachlorodecane and its stereoisomers.

Chlorinated paraffins (CPs) are a notoriously known class of compounds that stand amongst the most wide-spread persistent organic pollutants. Therefore, their reliable, repeatable, and reproducible quantitative analysis using well-defined reference standards is of utmost importance. In view of the increasing demand for constitutionally and stereochemically defined CP standards, we have synthesized a stereoisomeric mixture of 3,4,7,8-tetrachlorodecane. One stereoisomer - (3R,4R,7S,8S)-3,4,7,8-tetrachlorodecane was separated from the mixture, and enriched fractions of residual stereoisomers were achieved through crystallisation of the residual mother liquors. The molecular structure of the single isolated stereoisomer was confirmed through single-crystal X-ray crystallographic data. One fraction of 3,4,7,8-tetrachlorodecane stereoisomers was successfully separated on a chiral stationary phase using supercritical fluid chromatography hyphenated to mass spectrometry (column: Chiral ART Amylose-C; mobile phase: CO2/MeOH (96/4 v/v) with 0.1% diethylamine). The reported separation of stereoisomers is unprecedented in CP analysis so far.

Innovative reference materials for method validation in microplastic analysis including interlaboratory comparison exercises.

Reference materials (RMs) are vital tools in the validation of methods used to detect environmental pollutants. Microplastics, a relatively new environmental pollutant, require a variety of complex approaches to address their presence in environmental samples. Both interlaboratory comparison (ILC) studies and RMs are essential to support the validation of methods used in microplastic analysis. Presented here are results of quality assurance and quality control (QA/QC) performed on two types of candidate microplastic RMs: dissolvable gelatin capsules and soda tablets. These RMs have been used to support numerous international ILC studies in recent years (2019-2022). Dissolvable capsules containing polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), and polystyrene (PS), in different size fractions from 50 to 1000 µm, were produced for one ILC study, obtaining relative standard deviation (RSD) from 0 to 24%. The larger size fraction allowed for manual addition of particles to the capsules, yielding 0% error and 100% recovery during QA/QC. Dissolvable capsules were replaced by soda tablets in subsequent ILC studies and recovery test exercises because they were found to be a more reliable carrier for microplastic RMs. Batches of soda tablets were produced containing different single and multiple polymer mixtures, i.e., PE, PET, PS, PVC, polypropylene (PP), and polycarbonate (PC), with RSD ranging from 8 to 21%. Lastly, soda tablets consisting of a mixture of PE, PVC, and PS (125-355 µm) were produced and used for recovery testing during pretreatment of environmental samples. These had an RSD of 9%. Results showed that soda tablets and capsules containing microplastics >50 µm could be produced with sufficient precision for internal recovery tests and external ILC studies. Further work is required to optimize this method for smaller microplastics (< 50 µm) because variation was found to be too large during QA/QC. Nevertheless, this approach represents a valuable solution addressing many of the challenges associated with validating microplastic analytical methods.

Nuclear magnetic resonance as a tool to determine chlorine percentage of chlorinated paraffin mixtures.

A new simple method for chlorine percentage calculations (method C), from proton nuclear magnetic resonance (1H NMR) spectroscopy, has been established and applied to an industrial chlorinated paraffin (CP) mixture and 13 single-chain CPs of known carbon chain lengths. Two modified methods (method A and B), originating from the work of Sprengel et al., have been utilized on the same single-chain mixtures. All samples were analysed by 1H NMR and two-dimensional heteronuclear quantum coherence (HSQC) for this purpose. All three methods worked well for medium chlorinated (45-55% Cl) single-chain mixtures of known carbon chain lengths. Method A yielded the best result for mixtures of lower chlorine content (<45% Cl), method C gave better estimations for higher chlorine contents (>55% Cl). Compared to Mohr's titration, method A showed a deviation of 0.7-7.8% (3.6% average), method B 4.1-11.3% (7.0% average) and method C 0.6-11.6% (5.2% average), for all 13 single-chain mixtures. The new method C is the only method that could be applied for determining the chlorine percentage of industrial mixtures of multiple, unknown chain lengths.

Preparation of a phosphotyrosine-protein standard for use in semiquantitative western blotting with enhanced chemiluminescence.

Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is identification of those tumors that express non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a reference standard and facilitate comparisons between samples.

Correction to: 2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications.

This chapter was previously published as non-open access. This is updated to an open access chapter now.

2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications.

Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS PAGE) is a method that separates proteins according to their isoelectric points in the first dimension and molecular masses in the second dimension. Evidence is provided that 2D SDS PAGE is reproducible, robust and compatible with SDS in both dimensions including isoelectric focusing in tube gels, the first dimension. The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer and is better yet when dissolved in a mixture of both buffers. Quantification of 60 proteins in rat liver cytosol over a wide range of pI and MW gave linear plots of spot density versus total protein for loads of 200, 400 and 600 µg protein dissolved in SDS buffer and run in triplicate on 2D gels (Average R2 = 0.987). Examples of biomedical applications are provided in which 2D proteins of interest found by comparing stained or western blotted 2D gel patterns were identified by mass spectrometry (MS).

An ethnographic study of the effects of cognitive symptoms in patients with major depressive disorder: the IMPACT study.

BACKGROUND: The manifestation of major depressive disorder (MDD) may include cognitive symptoms that can precede the onset of MDD and persist beyond the resolution of acute depressive episodes. However, little is known about how cognitive symptoms are experienced by MDD patients and the people around them. METHODS: In this international (Brazil, Canada, China, France, and Germany) ethnographic study, we conducted semi-structured interviews and observations of remitted as well as symptomatic MDD patients (all patients self-reported being diagnosed by an HCP and self-reported being on an antidepressant) aged 18-60 years with self-reported cognitive symptoms (N = 34). In addition, participating depressed patients' close family or friends (N = 31) were interviewed. Separately recruited from depressed participants, work colleagues (N = 21) and healthcare providers (HCPs; N = 13) of depressed individuals were interviewed. RESULTS: Key insights were that: (1) patients were generally unaware that their cognitive symptoms were linked to their depression and, instead, attributed these symptoms to negative aspects of their person (e.g., age, separate disease, laziness, exhaustion); (2) cognitive symptoms in MDD appeared to negatively impact patients' social relationships and patients' ability to handle daily tasks at work and at home; (3) patients' cognitive symptoms also impacted relationships with family members and coworkers; (4) patients' cognitive symptoms increased stress and feelings of failure, which in turn seemed to worsen the cognitive symptoms, thereby creating a destructive cycle; and (5) although HCPs recommended that patients re-engage in everyday activities to help overcome their depression, cognitive symptoms seemed to impede such functional recovery. CONCLUSIONS: Taken together, these findings highlight a negative impact of patients' cognitive symptoms on their social functioning, work performance, and quality of life on the people close to them, and consequently on the degree of functional recovery after depression.

Economical synthesis of 13C-labeled opiates, cocaine derivatives and selected urinary metabolites by derivatization of the natural products.

The illegal use of opiates and cocaine is a challenge world-wide, but some derivatives are also valuable pharmaceuticals. Reference samples of the active ingredients and their metabolites are needed both for controlling administration in the clinic and to detect drugs of abuse. Especially, (13)C-labeled compounds are useful for identification and quantification purposes by mass spectroscopic techniques, potentially increasing accuracy by minimizing ion alteration/suppression effects. Thus, the synthesis of [acetyl-(13)C4]heroin, [acetyl-(13)C4-methyl-(13)C]heroin, [acetyl-(13)C2-methyl-(13)C]6-acetylmorphine, [N-methyl-(13)C-O-metyl-(13)C]codeine and phenyl-(13)C6-labeled derivatives of cocaine, benzoylecgonine, norcocaine and cocaethylene was undertaken to provide such reference materials. The synthetic work has focused on identifying (13)C atom-efficient routes towards these derivatives. Therefore, the (13)C-labeled opiates and cocaine derivatives were made from the corresponding natural products.

Synthesis of [13C4]-labeled ∆9-tetrahydrocannabinol and 11-nor-9-carboxy-∆9-tetrahydrocannabinol as internal standards for reducing ion suppressing/alteration effects in LC/MS-MS quantification.

(-)-∆9-Tetrahydrocannabinol is the principal psychoactive component of the cannabis plant and also the active ingredient in some prescribed drugs. To detect and control misuse and monitor administration in clinical settings, reference samples of the native drugs and their metabolites are needed. The accuracy of liquid chromatography/mass spectrometric quantification of drugs in biological samples depends among others on ion suppressing/alteration effects. Especially, 13C-labeled drug analogues are useful for minimzing such interferences. Thus, to provide internal standards for more accurate quantification and for identification purpose, synthesis of [13C4]-∆9-tetrahydro-cannabinol and [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was developed via [13C4]-olivetol. Starting from [13C4]-olivetol the synthesis of [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was shortened from three to two steps by employing nitromethane as a co-solvent in condensation with (+)-apoverbenone.

Evaluation of ¹³C- and ²H-labeled internal standards for the determination of amphetamines in biological samples, by reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry.

Stable isotope-labeled internal standards (SIL-ISs) are often used when applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze for legal and illegal drugs. ISs labeled with (13)C, (15)N, and (18)O are expected to behave more closely to their corresponding unlabeled analytes, compared with that of the more classically used (2)H-labeled ISs. This study has investigated the behavior of amphetamine, (2)H3-, (2)H5, (2)H6-, (2)H8-, (2)H11-, and (13)C6-labeled amphetamine, during sample preparation by liquid-liquid extraction and LC-MS/MS analyses. None or only minor differences in liquid-liquid extraction recoveries of amphetamine and the SIL-ISs were observed. The chromatographic resolution between amphetamine and the (2)H-labeled amphetamines increased with the number of (2)H-substitutes. For chromatographic studies we also included seven additional (13)C6-amphetamines and their analytes. All the (13)C6-labeled ISs were co-eluting with their analytes, both when a basic and when an acidic mobile phase were used. MS/MS analyses of amphetamine and its SIL-ISs showed that the ISs with the highest number of (2)H-substitutes required more energy for fragmentation in the collision cell compared with that of the ISs with a lower number. The findings, in this study, support those of previous studies, showing that (13)C-labeled ISs are superior to (2)H-labeled ISs, for analytical purposes.

Synthesis of [13C6]-labelled phenethylamine derivatives for drug quantification in biological samples.

The availability of high-quality (13)C-labelled internal standards will improve accurate quantification of narcotics and drugs in biological samples. Thus, the synthesis of 10 [(13)C6]-labelled phenethylamine derivatives, namely amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 4-methoxyamphetamine, 4-methoxymethamphetamine, 3,5-dimethoxyphenethylamine 4-bromo-2,5-dimethoxyphenethylamine and 2,5-dimethoxy-4-iodophenethylamine, have been undertaken. [(13)C6]-Phenol proved to be an excellent starting material for making (13)C-labelled narcotic substances in the phenethylamine class, and a developed Stille-type coupling enabled an efficient synthesis of the 3,4-methylenedioxy and 4-methoxy derivatives. The pros and cons of alternative routes and transformations are also discussed. The [(13)C6]-labelled compounds are intended for use as internal standards in forensic analysis, health sciences and metabolomics studies by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry.

NMR spectral assignment of 2α- and 3ß-methylhopanes and evidence for boat conformation in D ring of 17α(H),21α(H)-hopanes.

The full (1)H and (13)C NMR chemical shift assignment of 2α-methyl-17α(H),21ß(H)-hopane is presented. This compound is formed in mature sediments from biogenic sources of 2ß-methyl-17ß(H),21ß(H)-hopanoids, which include several cyanobacteria. In addition, full (1)H and (13)C NMR chemical shift data of all four 17,21 isomers of 3ß-methylhopane have been assigned. The thermodynamically most stable 3ß-configuration corresponds to that found in bacterial sources. The data presented here suggest minor corrections to the (13)C chemical assignments reported earlier for 17α(H)-hopanes. Moreover, spectral evidence indicates an unexpected ring-D boat conformation of 17α(H),21α(H)-hopanes, which may serve to explain the steric strain reported for this isomer.

Monofluorinated analogues of polybrominated diphenyl ethers as analytical standards: synthesis, NMR, and GC-MS characterization and molecular orbital studies.

Polybrominated diphenyl ethers (PBDEs), a group of 209 individual congeners distinguishable by the number and position of bromines, are produced for use as flame retardants in consumer goods. PBDEs have become ubiquitous environmental contaminants, present in increasing levels in the environment and humans. In the present study, 10 individual monofluorinated analogues of PBDEs (F-PBDEs) and one difluorinated PBDE (FF-PBDE) were synthesized and characterized, and their gas chromatographic (GC) and mass spectrometric (MS) characteristics determined. The synthesis method utilized a nucleophilic reaction of bromophenols with diphenyliodonium salts and the perbromination of fluorosubstituted diphenyl ethers. Reaction yields were between 10% and 59% with > or = 98% purity. Apart from the aromatic ring carrying the fluorine atom, only minor chemical nuclear magnetic resonance (NMR) shift changes were observed in comparison to the corresponding parent PBDEs, with the exception that the J(F,H) coupling was stronger. Our preliminary data show that F-PBDEs and PBDEs have comparable retention times in gas chromatography with F-PBDEs demonstrating in general shorter or identical retention times, depending on the pattern of fluorine substitution. We also calculated the torsion angles and the dipole moments for both and report that there is a good correlation between GC retention times and the torsion angles but not with dipole moments. In MS, the difference of the ion peaks of the F-PBDE/ PBDE pairs is m/z 19 (F), which allows a simultaneous MS detection without separation. On the basis of GC separation, simultaneous MS detection, and the stability of fluorine due to its generally resistance to nucleophilic displacement, we propose that F-PBDEs may function as valuable potential standards, markers, and tracers in environmental analysis.

Synthesis of fluorinated polybrominated diphenyl ethers (F-PBDEs) as internal standards for environmental analysis.

Polybrominated diphenyl ethers (PBDEs) have become widely distributed as environmental contaminants due to their wide-spread use as flame retardants. Their structural similarity to other halogenated organic pollutants, for example polychlorinated biphenyls (PCBs), has led to speculation that they may have similar toxicological properties and effects. Recent focus on PBDEs as possible priority pollutants has also led to an increasing need for reference standards of PBDEs for toxicological studies and for environmental analysis. In this work we synthesized a series of fluorinated PBDEs (F-PBDEs) which can be used as possible internal standards, as an alternative to high-cost alternatives, such as the (13)C-labelled analogues. F-PBDEs have been synthesized by using different coupling reactions and by bromination of fluorinated starting materials.

Characterization of monofluorinated polycyclic aromatic compounds by 1H, 13C and 19F NMR spectroscopy.

Monofluorinated polycyclic aromatic hydrocarbons (F-PAHs) have attracted much attention in analytical, environmental, toxicological and mechanistic studies because of their physico-chemical properties, which are closely similar to those of the parent PAHs. Because of this, full NMR characterization has become of interest. Complete 1H, 13C and 19F NMR chemical shifts, and also 1J(H,C), (n)J(C,F), (n)J(H,F) and (n)J(H,H) coupling constants, have been assigned for the F-PAHs 1-fluoronaphthalene, 2-fluorofluorene, 5-fluoroacenaphthylene, 2-fluorophenanthrene, 3-fluorophenanthrene, 3-fluorofluoranthene, 1-fluoropyrene, 1-fluorochrysene, 2-fluorochrysene, 3-fluorochrysene and 9-fluorobenzo[k]fluoranthene. To allow comparison with the corresponding parent PAHs, the 1H and 13C chemical shifts of acenaphthylene, phenanthrene, fluoranthene, pyrene and benzo[k]fluoranthene were determined. Chemical shift increments and the effects on the coupling constants from the fluorine substitution are discussed.