Identifying antimicrobials and their metabolites in wastewater and surface water with effect-directed analysis.

This study aimed to identify antimicrobial contaminants in the aquatic environment with effect-directed analysis. Wastewater influent, effluent, and surface water (up- and downstream of the discharge location) were sampled at two study sites. The samples were enriched, subjected to high-resolution fractionation, and the resulting 80 fractions were tested in an antibiotics bioassay. The resulting bioactive fractions guided the suspect and nontargeted identification strategy in the high-resolution mass spectrometry data that was recorded in parallel. Chemical features were annotated with reference databases, assessed on annotation quality, and assigned identification confidence levels. To identify antibiotic metabolites, Phase I metabolites were predicted in silico for over 500 antibiotics and included as a suspect list. Predicted retention times and fragmentation patterns reduced the number of annotations to consider for confirmation testing. Overall, the bioactivity of three fractions could be explained by the identified antibiotics (clarithromycin and azithromycin) and an antibiotic metabolite (14-OH(R) clarithromycin), explaining 78% of the bioactivity measured at one study site. The applied identification strategy successfully identified antibiotic metabolites in the aquatic environment, emphasizing the need to include the toxic effects of bioactive metabolites in environmental risk assessments.

Dataset of near-infrared spectral data of illicit-drugs and forensic casework samples analyzed by five portable spectrometers operating in different wavelength ranges.

The increasing amount of globally seized controlled substances in combination with the more diverse drugs-of-abuse market encompassing many new psychoactive substances (NPS) provides challenges for rapid and reliable on-site presumptive drug testing. Long-established colorimetric spot tests tend to fail due to the unavailability of reliable tests for novel drugs and to false-positive reactions on commonly encountered substances. In addition, handling of samples and chemicals is required. Spectroscopic techniques do not have these disadvantages as spectra are compound-specific and non-invasive tests are possible. Near-infrared (NIR) spectroscopy is a promising technique for on-scene forensic drug detection. Numerous portable devices were introduced in the market in recent years. However, most handheld spectrometers operate in different and relatively confined wavelength ranges compared to the full 780 - 2500 nm NIR wavelength range. In addition, their spectral resolution is limited compared to benchtop instruments. This dataset presents the NIR spectra of 430 forensic samples, including regularly encountered illicit-drugs, NPS, commonly used adulterants, bulking-agents and excipients, and seized casework materials (powders and tablets). Data is available from 5 different NIR spectrometers; including a benchmark high-resolution, full range 350-2500 nm laboratory grade instrument and 4 portable spectrometers operating in the ranges of 1300-2600 nm, 1550-1950 nm, 950-1650 nm and 740-1070 nm. Via this dataset, spectra of illicit-drugs become available to institutes that typically do not have access to controlled substances. This data can be used to develop chemometric detection and classification models for illicit-drugs and provide insight in diagnostic spectral features that need to be recorded for reliable detection models. Additionally, the high-resolution, full range VIS-NIR spectra of the benchmark ASD instrument can be used for in-silica predictions of spectra in a certain wavelength range to provide insight in the optimal resolution and wavelength range of a prospective portable device.

Evaluation of Adverse Effects of Resorbable Hyaluronic Acid Fillers: Determination of Macrophage Responses.

Resorbable tissue fillers for aesthetic purposes can induce severe complications including product migration, late swelling, and inflammatory reactions. The relation between product characteristics and adverse effects is not well understood. We hypothesized that the degree of cross-linking hyaluronic acid (HA) fillers was associated with the occurrence of adverse effects. Five experimental HA preparations similar to HA fillers were synthesized with an increasing degree of cross-linking. Furthermore, a series of commercial fillers (Perfectha®) was obtained that differ in degradation time based on the size of their particulate HA components. Cytotoxic responses and cytokine production by human THP-1-derived macrophages exposed to extracts of the evaluated resorbable HA fillers were absent to minimal. Gene expression analysis of the HA-exposed macrophages revealed the responses related to cell cycle control and immune reactivity. Our results could not confirm the hypothesis that the level of cross-linking in our experimental HA fillers or the particulate size of commercial HA fillers is related to the induced biological responses. However, the evaluation of cytokine induction and gene expression in macrophages after biomaterial exposure presents promising opportunities for the development of methods to identify cellular processes that may be predictive for biomaterial-induced responses in patients.

Performance evaluation of handheld Raman spectroscopy for cocaine detection in forensic case samples.

Handheld Raman spectroscopy is an emerging technique for rapid on-site detection of drugs of abuse. Most devices are developed for on-scene operation with a user interface that only shows whether cocaine has been detected. Extensive validation studies are unavailable, and so are typically the insight in raw spectral data and the identification criteria. This work evaluates the performance of a commercial handheld Raman spectrometer for cocaine detection based on (i) its performance on 0-100 wt% binary cocaine mixtures, (ii) retrospective comparison of 3,168 case samples from 2015 to 2020 analyzed by both gas chromatography-mass spectrometry (GC-MS) and Raman, (iii) assessment of spectral selectivity, and (iv) comparison of the instrument's on-screen results with combined partial least square regression (PLS-R) and discriminant analysis (PLS-DA) models. The limit of detection was dependent on sample composition and varied between 10 wt% and 40 wt% cocaine. Because the average cocaine content in street samples is well above this limit, a 97.5% true positive rate was observed in case samples. No cocaine false positives were reported, although 12.5% of the negative samples were initially reported as inconclusive by the built-in software. The spectral assessment showed high selectivity for Raman peaks at 1,712 (cocaine base) and 1,716 cm-1 (cocaine HCl). Combined PLS-R and PLS-DA models using these features confirmed and further improved instrument performance. This study scientifically assessed the performance of a commercial Raman spectrometer, providing useful insight on its applicability for both presumptive detection and legally valid evidence of cocaine presence for law enforcement.

Benchtop NMR spectroscopy in the analysis of substandard and falsified medicines as well as illegal drugs.

Substandard and falsified medical products may cause harm to patients and fail to treat the diseases or conditions for which they were intended. It is therefore required to have analytical methods available to assess medical product quality. Benchtop NMR spectroscopy provides a generic, inherently quantitative, analytical method capable of separating specific signals from those of a matrix. We have developed an analytical method for the analysis of active ingredients in pharmaceutical products and illegal drugs, based on benchtop NMR spectroscopy. Within its resolution limits, benchtop NMR spectroscopy is useful in determining the identity of the active ingredients in products containing acetaminophen, aspirin, caffeine, diclofenac, ibuprofen, naproxen, sildenafil, tadalafil and sibutramine, cocaine, and gamma hydroxybutyric acid, with a limit of detection of about 1 mg/mL. Furthermore, the content of the active ingredient can be determined with an error of 10%. Additionally, a chemometrics approach is shown to be useful to classify spectra in order to identify the active substances present in the sample, reducing the need for expert interpretation of the spectra acquired.

Fora fuelling the discovery of fortified dietary supplements - An exploratory study directed at monitoring the internet for contaminated food supplements based on the reported effects of their users.

Dietary supplements are products that are widely used for instance as energisers or to lose weight. There have been cases reported where undeclared ingredients present in such supplements have caused adverse effects on the health of the user. As there are many different products to choose from, it seems impossible to predict which might contain harmful components and to ban them from the market. Nonetheless, the use of dietary supplements and the experiences of users are shared in online discussions. We describe the development of a search engine to retrieve products associated with certain effects. Upon application we were able to retrieve a list of dietary supplements that are repeatedly associated with excessive effects by users on public fora. The top of the list contains supplements that have previously been banned because they contained undeclared harmful components. The use of the search engine as described here is a powerful method for making a risk-based selection of dietary supplements which can then be analysed for the presence of illegal or other unwanted components.

The stimulant higenamine in weight loss and sports supplements.

BACKGROUND: Higenamine is a stimulant with cardiovascular properties recently prohibited in sport by the World Anti-Doping Agency (WADA). Higenamine is also a natural constituent of several traditional botanical remedies and is listed as an ingredient in weight loss and sports supplements sold over-the-counter in the United States. OBJECTIVES: We analyzed dietary supplements available for sale in the United States prior to WADA's prohibition of higenamine in sport for the presence and quantity of higenamine. METHODS: All supplements labeled as containing higenamine or a synonym (i.e., norcoclaurine or demethylcoclaurine) available for sale in the United States were identified. For each brand, one sample was analyzed by NSF International (Ann Arbor, MI) and one sample by the Netherland's National Institute for Public Health and the Environment (RIVM). NSF International carried out qualitative and quantitative analyses using ultra high performance liquid chromatography (UHPLC) with tandem mass spectrometry. RIVM carried out qualitative analysis using UHPLC quadrupole time of flight mass spectrometry for an independent confirmation of identity. RESULTS: Twenty-four products were analyzed. The majority of supplements were marketed as either weight loss (11/24; 46%) or sports/energy supplements (11/24; 46%); two brands did not list a labeled indication. The quantity of higenamine (±95% CI) ranged from trace amounts to 62 ± 6.0 mg per serving. Consumers could be exposed to up to 110 ± 11 mg of higenamine per day when following recommended serving sizes provided on the label. Five products (5/24; 21%) listed an amount of higenamine, but none were accurately labeled; the quantity in these supplements ranged from <0.01% to 200% of the quantity listed on the label. CONCLUSION: Dosages of up to 62 ± 6.0 mg per serving of the stimulant higenamine were found in dietary supplements sold in the United States.

A high crosslinking grade of hyaluronic acid found in a dermal filler causing adverse effects.

Facial treatments with dermal fillers for medical or esthetic purposes occasionally give rise to adverse effects, ranging from temporary effects such as reddening of the skin, to long term effects such as hardening of tissue. There appears to be a relationship between the lifetime of the filler product and the risk for adverse effects. The lifetime of hyaluronic acid-based fillers is dependent on the presence and amount of crosslinking agents such as 1,4-butanediol diglycidyl ether (BDDE). It would therefore make sense to establish methodology to analyze the crosslinking grade of HA-based filler products on a routine basis. To this end, an analytical method was developed and validated to identify HA-BDDE-based fillers and to quantify their modification and crosslinking grade. The method was subsequently applied to products from the legal supply chain and the illegal market. It was found that the product Hyacorp H 1000, previously taken from the market, indeed contains a high modification grade and crosslinking grade, as was the assumed reason for the increased risk for adverse effects of this product. However, it was also shown that the Hyacorp products are highly unreliable in relation to their product composition in general. In this study, authentic products could not be distinguished from the illegal market products based on their modification and crosslinking grade.

Four experimental stimulants found in sports and weight loss supplements: 2-amino-6-methylheptane (octodrine), 1,4-dimethylamylamine (1,4-DMAA), 1,3-dimethylamylamine (1,3-DMAA) and 1,3-dimethylbutylamine (1,3-DMBA).

BACKGROUND: The United States Food and Drug Administration banned the stimulant 1,3-dimethylamylamine (1,3-DMAA) from dietary supplements and warned consumers that the stimulant can pose cardiovascular risks ranging from high blood pressure to heart attacks. OBJECTIVES: We designed our study to determine if a new stimulant similar in structure to 1,3-DMAA has been introduced as an ingredient in supplements sold in the United States (US). METHODS: We analyzed six brands of supplements that listed an ingredient on the label (e.g., Aconitum kusnezoffii, DMHA or 2-amino-isoheptane) that might refer to an analog of 1,3-DMAA. Supplements were analyzed by two separate laboratories using ultra-high-performance liquid chromatography mass spectrometry and reference standards. RESULTS: Two previously unidentified 1,3-DMAA analogs (2-amino-6-methylheptane [octodrine] and 1,4-dimethylamylamine [1,4-DMAA]) and two banned stimulants (1,3-DMAA and 1,3-dimethylbutylamine [1,3-DMBA]) were identified. Octodrine was found at a dose (±95% CI) of 72 ± 7.5 mg per serving. In Europe, octodrine was previously sold as a pharmaceutical in multi-ingredient medications at dosages from 8 to 33 mg. The quantity of octodrine found in our study was more than twice the largest pharmaceutical dose. The other new stimulant, 1,4-DMAA, has not previously been approved for human consumption, and its safety in humans is unknown. 1,4-DMAA was found at dosages between 21 ± 11 mg to 94 ± 48 mg per serving. In addition, two banned stimulants - 1,3-DMAA and 1,3-DMBA - were also identified: 24 ± 7.6 mg to 35 ± 11 mg of 1,3-DMAA and 51 ± 16 mg of 1,3-DMBA. In one product, 24 ± 7.6 mg of 1,3-DMAA was combined with 21 ± 11 mg of 1,4-DMAA. 1,3-DMAA has been investigated as potentially contributing to hemorrhagic strokes and sudden death, whereas the safety of 1,3-DMBA in humans is unknown. CONCLUSION: Two banned stimulants (1,3-DMAA and 1,3-DMBA) and two previously unidentified stimulants (1,4-DMAA and octodrine) were identified in supplements sold in the United States.

The quality of sildenafil active substance of illegal source.

There must be a large market for active pharmaceutical ingredients of illegal source to support the huge and lucrative business of trade in illegal medicines. The active substances found in illegal pharmaceuticals may differ from their legal counterparts concerning purity and associated risks for the health of the user. In this study we show two examples in which the active substance sildenafil, used in erectile dysfunction products, was not of European Pharmacopeia quality. In one case milligram-scale amounts of a 2-mercaptobenzothiazole contamination were found, in another case the mesylate salt rather than the monograph based citrate was used. For the user of products containing these active substances, the risks of side effects increase through the inherent properties of the impurity and the chance of overdosing. The fact that the users are most likely not aware of the poor quality of the products adds up to the health risk of using prescription medication without consulting medical professionals.

History full circle: 'Novel' sympathomimetics in supplements.

Since the banning of ephedrine in over-the-counter nutritional supplements a decade ago, a plethora of untested and/or unsafe sympathomimetic stimulants have taken its place. This paper argues that these 'novel' stimulants in supplements recapitulate the work of synthetic chemists at commercial pharmaceutical firms during the 1930s and 1940s, all seeking substitutes for recently successful products based on ephedrine and amphetamine. Copyright © 2015 John Wiley & Sons, Ltd.

Operation resistance: A snapshot of falsified antibiotics and biopharmaceutical injectables in Europe.

Operation Pangea is an annual international week of action combating pharmaceutical crime. In this study, called Operation Resistance, we asked the national agencies in Europe to search for falsified antibiotics and biopharmaceutical injectables (peptides and proteins) amongst the medicines seized in Pangea 7 (2014). Reports were received from Belgium, Cyprus, Czech Republic, Denmark, France, the Netherlands, Portugal, Sweden, Spain, the United Kingdom, Norway, and Switzerland. The countries reported seizing about 21,000 dose units (e.g. tablets, capsules) of falsified antibiotics in total. Most of the antibiotics were unlicensed medicines with common antibiotic drugs. In this study week, very few falsified biopharmaceutical injectables were reported. Laboratories reported human growth hormone, sermorelin, melanotan II, and no active ingredients. The average shipment size seemed too large for personal use indicating that a substantial part was intended for resale. This study provides a snapshot of the falsified antibiotics and biopharmaceuticals that enter European countries. How much is actually reaching users during Pangea week - in on other weeks - remains unknown. The shipment sizes indicate falsified antibiotics and biopharmaceuticals are imported for both personal use and resale. The use of antibiotics from unreliable sources is a health risk, contributes to antimicrobial resistance, and may obscure a source of infection from health agencies. The falsified biopharmaceuticals are a health risk because they lack all labelling and may contain unlicensed drugs for injection. It seems important to raise awareness among health-care professionals that falsified medicines in Europe are not restricted to erectile dysfunction drugs. Copyright © 2015 John Wiley & Sons, Ltd.

A Focal Adhesion Kinase-Derived Peptide Binds the Src SH3 Domain in Two Orientations, As Demonstrated Using Paramagnetic Nuclear Magnetic Resonance.

SH3 binding peptides contain polyproline helices and are classified according to their binding orientations as N-to-C-terminal or C-to-N-terminal. We have tested the hypothesis that such a peptide binds in both orientations but with different populations. A focal adhesion kinase (FAK)-derived peptide was tested for its binding orientation on the Src SH3 domain. Paramagnetic tags were introduced at several positions on the SH3 domain, and on the basis of the paramagnetic relaxation enhancements (PREs) of the amide protons, the positions of the paramagnetic centers were determined. Two peptides were synthesized with (13)C-enriched Ala or Pro, at the N-terminal or C-terminal side of the peptide, and the intermolecular PREs were measured. The results provide compelling evidence that the FAK-derived peptide binds the SH3 domain in two orientations. In the major state, the SH3 domain binds the peptide in the N-C orientation, whereas 20% of the time, the peptide binds in the C-N orientation. We conclude that the distinction between N-C and C-N orientations, which is based on crystal structures, might be artificial. The pseudosymmetric nature of the polyproline helix might allow for binding in both orientations in the solution state.

Chemical fingerprinting of silicone-based breast implants.

With millions of women worldwide carrying them, silicone-based breast implants represent a large market. Even though silicone breast implants already have a history of use of more than 50 years, the discussion on their safety has not yet come to an end. To improve safety assessment, regulatory authorities should have the availability of a set of tests to be able to determine parameters of implant identity and quality. Therefore, the gels and envelopes of various brands and types of silicone-based breast implants have been subjected to infrared, Raman and NMR spectroscopy. We show that by using a combination of complementary spectroscopic techniques breast implants of various origins can be distinguished on typical chemical hallmarks. It was found that typical silicone-based implants display a surplus of vinyl signals in the gel, cyclosiloxane impurities are tolerable at low levels only and a barrier layer is present in the implant envelope. The techniques presented here and the results obtained offer a good starting point for market surveillance studies.

PARAssign--paramagnetic NMR assignments of protein nuclei on the basis of pseudocontact shifts.

The use of paramagnetic NMR data for the refinement of structures of proteins and protein complexes is widespread. However, the power of paramagnetism for protein assignment has not yet been fully exploited. PARAssign is software that uses pseudocontact shift data derived from several paramagnetic centers attached to the protein to obtain amide and methyl assignments. The ability of PARAssign to perform assignment when the positions of the paramagnetic centers are known and unknown is demonstrated. PARAssign has been tested using synthetic data for methyl assignment of a 47 kDa protein, and using both synthetic and experimental data for amide assignment of a 14 kDa protein. The complex fitting space involved in such an assignment procedure necessitates that good starting conditions are found, both regarding placement and strength of paramagnetic centers. These starting conditions are obtained through automated tensor placement and user-defined tensor parameters. The results presented herein demonstrate that PARAssign is able to successfully perform resonance assignment in large systems with a high degree of reliability. This software provides a method for obtaining the assignments of large systems, which may previously have been unassignable, by using 2D NMR spectral data and a known protein structure.

Small-molecule binding sites on proteins established by paramagnetic NMR spectroscopy.

Determining the three-dimensional structure of a small molecule-protein complex with weak affinity can be a significant challenge. We present a paramagnetic NMR method to determine intermolecular structure restraints based on pseudocontact shifts (PCSs). Since the ligand must be in fast exchange between free and bound states and the fraction bound can be as low as a few percent, the method is ideal for ligands with high micromolar to millimolar dissociation constants. Paramagnetic tags are attached, one at a time, in a well-defined way via two arms at several sites on the protein surface. The ligand PCSs were measured from simple 1D (1)H spectra and used as docking restraints. An independent confirmation of the complex structure was carried out using intermolecular NOEs. The results show that structures derived from these two approaches are similar. The best results are obtained if the magnetic susceptibility tensors of the tags are known, but it is demonstrated that with two-armed probes, the magnetic susceptibility tensor can be predicted with sufficient accuracy to provide a low-resolution model of the ligand orientation and the location of the binding site in the absence of isotope-labeled protein. This approach can facilitate fragment-based drug discovery in obtaining structural information on the initial fragment hits.

A pH-sensitive, colorful, lanthanide-chelating paramagnetic NMR probe.

Paramagnetic lanthanides ions are broadly used in NMR spectroscopy. The effects of unpaired electrons on NMR spectral parameters provide a powerful tool for the characterization of macromolecular structures and dynamics. Here, a new lanthanide-chelating NMR probe, Caged Lanthanide NMR Probe-7 (CLaNP-7), is presented. It can be attached to protein surfaces via two disulfide bridges, yielding a probe that is rigid relative to the protein backbone. CLaNP-7 extends the application range of available probes. It has a yellow color, which is helpful for sample preparation. Its effects are comparable to those of CLaNP-5, but its charge is two units lower (+1) than that of CLaNP-5 (+3), reducing the change in surface potential after probe attachment. It also has a different magnetic susceptibility tensor, so by using both tags, two sets of structural restraints can be obtained per engineered cysteine pair. Moreover, it was found that the orientation of the magnetic susceptibility tensor is pH dependent (pK(a) ≈ 7) when a histidine residue is located in the neighborhood of the probe attachment site. The results show that the His imidazole group interacts with the CLaNP-7 tag. It is proposed that the histidine residue forms a hydrogen bond to a water/hydroxyl molecule that occupies the ninth coordination position on the lanthanide, thus breaking the two-fold symmetry of the CLaNP tag in a pH-dependent way.