Production and certification of BOTS-1: bovine muscle-certified reference material for incurred veterinary drug residues.

A freeze-dried bovine muscle-certified reference material (CRM), known as BOTS-1 (DOI: https://doi.org/10.4224/crm.2018.bots-1 ), containing incurred residues of commonly used veterinary drugs was produced and certified for the mass fraction of eight veterinary drug residues. Value assignment was carried out using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods in conjunction with isotope dilution and standard addition approaches involving stable isotope internal standards. Data from the National Research Council of Canada (NRC), Canadian Food Inspection Agency (CFIA), United States Department of Agriculture (USDA), and the Federal Office of Consumer Protection and Food Safety in Germany (BVL) were used for value assignment. Results for two drug residues were also obtained through an international inter-laboratory comparison CCQM-K141/P178 organized under the auspices of the International Bureau of Weights and Measures (BIPM). Quantitative NMR (1H-qNMR) was used to characterize primary standards of all veterinary drugs certified. The certified mass fractions of the veterinary drug residues were 490 ± 100 µg/kg for chlorpromazine, 44 ± 4.4 µg/kg for ciprofloxacin, 3.3 ± 1.4 µg/kg for clenbuterol, 9.5 ± 0.8 µg/kg for dexamethasone, 57 ± 4.8 µg/kg for enrofloxacin, 3.0 ± 0.4 µg/kg for meloxicam, 12.4 ± 1.2 µg/kg for ractopamine, and 2290 ± 120 µg/kg for sulfadiazine with expanded uncertainties quoted (95% confidence) which include the effects due to between-bottle inhomogeneity, instability during long-term storage and transportation, and characterization.

Quantification and Stability Determination of Surface Amine Groups on Silica Nanoparticles Using Solution NMR.

Surface chemistry is a critical factor for determining the behavior of a nanomaterial after incorporation in composites, devices, and biomedical products, and is also important for nanotoxicology studies. We have developed an optimized protocol for dissolution of aminated silicas and determination of functional-group contents by quantitative 1H NMR (qNMR) analysis of the released amines. A number of variables were optimized for the dissolution protocol, including the base concentration, mass of silica, time, temperature, and method of sample agitation, in order to achieve adequate NMR signals for quantification. The protocol was tested using nanoparticles from a single commercial supplier with sizes ranging from 20 to 120 nm that were functionalized with 3-aminopropyl groups. Interestingly the batch-to-batch variability for some sizes of these aminated silicas was as high as 50%. Amine contents measured by a ninhydrin colorimetric assay were typically ∼20% lower than those measured by qNMR, consistent with measurement of only ninhydrin-reagent accessible amines. The dissolution-qNMR protocol was compatible with aminated silicas from other commercial suppliers, and in these cases, an even larger variability in surface coverage was observed. Silica nanoparticles with longer-chain amines and variable amine loadings were synthesized to demonstrate the ability to quantify amines with more complex structures and to assess the limit of quantification for the dissolution-qNMR method. Finally, the stability of the aminated nanoparticles was examined. Loss of 3-aminopropyl groups occurred in water at room temperature and was significantly more rapid at higher temperatures. Amine loss increased with increasing surface coverage and was slower for long-chain amines, consistent with studies of amine stability on planar silica. Overall, this work highlights the importance of developing methods for quantifying surface functionalization, particularly given the variability in surface coverage for commercial samples, and for ensuring that the amine group is stable under its usage conditions.

Purity assignment for peptide certified reference materials by combining qNMR and LC-MS/MS amino acid analysis results: application to angiotensin II.

The purity value assignment of metrologically traceable peptide reference standards requires specialized primary methods. Conventionally, amino acid analysis by isotope dilution tandem mass spectrometry (LC-MS/MS) following peptide hydrolysis is employed as a reference method. By contrast, quantitative nuclear magnetic resonance (qNMR) spectroscopy allows for quantitation of intact peptides, thus eliminating potential bias due to hydrolysis. Both methods are susceptible to interference from related peptide impurities, which need to be accurately measured and accounted for. The mass balance approach has also been employed for peptide purity measurements, whereby the purity is defined by the sum of the mass fraction of all impurities identified. Ideally, results from these three orthogonal methods can be combined for final purity assignment of peptide reference standards. Here we report a novel strategy for correcting both LC-MS/MS and 1H-qNMR results for related peptide impurities and combining results from both methods using a Bayesian statistical approach using mass balance results as prior knowledge. The mass balance method relied on a validated 19F-qNMR method to measure the trifluoroacetic acid (TFA) counter-ion, considered an impurity in this case at nearly 25% by mass. Using a candidate certified reference material (CRM) for angiotensin II, excellent agreement was achieved with the three methods. The final purity value assignment of the candidate CRM was 691 ± 9 mg/g (k = 2).

Determination of chemical purity and isotopic composition of natural and carbon-13-labeled arsenobetaine bromide standards by quantitative(1)H-NMR.

In this study, we report the characterization of three arsenobetaine-certified reference materials by quantitative NMR. We have synthesized an arsenobetaine bromide high-purity standard of natural isotopic composition (ABET-1) and two carbon-13-labeled isotopic standards (BBET-1 and CBET-1). Assignments of the chemical purity and isotopic composition are not trivial in the case of arsenobetaine, and in this study we utilized quantitative(1)H-NMR techniques for the determination of the mass fractions (chemical purity). The isotopic purity of all three standards was also assessed by NMR from the carbon-13 satellite signals. The standards are non-hygroscopic, high-purity (ca. 0.99 g/g), and the carbon-13 enrichment for both isotopic standards is x((13)C)≈0.99. These standards are designed for use as primary calibrators for mass spectrometric determination of arsenobetaine in environmental samples.

Methanol incorporation in clathrate hydrates and the implications for oil and gas pipeline flow assurance and icy planetary bodies.

One of the best-known uses of methanol is as antifreeze. Methanol is used in large quantities in industrial applications to prevent methane clathrate hydrate blockages from forming in oil and gas pipelines. Methanol is also assigned a major role as antifreeze in giving icy planetary bodies (e.g., Titan) a liquid subsurface ocean and/or an atmosphere containing significant quantities of methane. In this work, we reveal a previously unverified role for methanol as a guest in clathrate hydrate cages. X-ray diffraction (XRD) and NMR experiments showed that at temperatures near 273 K, methanol is incorporated in the hydrate lattice along with other guest molecules. The amount of included methanol depends on the preparative method used. For instance, single-crystal XRD shows that at low temperatures, the methanol molecules are hydrogen-bonded in 4.4% of the small cages of tetrahydrofuran cubic structure II hydrate. At higher temperatures, NMR spectroscopy reveals a number of methanol species incorporated in hydrocarbon hydrate lattices. At temperatures characteristic of icy planetary bodies, vapor deposits of methanol, water, and methane or xenon show that the presence of methanol accelerates hydrate formation on annealing and that there is unusually complex phase behavior as revealed by powder XRD and NMR spectroscopy. The presence of cubic structure I hydrate was confirmed and a unique hydrate phase was postulated to account for the data. Molecular dynamics calculations confirmed the possibility of methanol incorporation into the hydrate lattice and show that methanol can favorably replace a number of methane guests.

In-situ observation of nucleation and growth of PbSe magic-sized nanoclusters and regular nanocrystals.

In-situ observation of the temporal evolution of the absorption of PbSe nanocrystals (NCs) via a low-temperature noninjection approach is presented. Based on a model reaction of lead oleate (Pb(OA)(2) ) and n-trioctylphosphine selenide (TOPSe) in 1-octadecene at 35-80 °C, the use of commercially available TOP (90 or 97%) in affecting the formation of the NCs is explored. TOPSe solutions made from TOP 90% exhibited higher reactivity than those made from TOP 97%. (31)P NMR spectroscopy detected no dioctylphosphine selenide (DOPSe) but some DOP in ≈1.0 M TOPSe/TOP solution (made from TOP 90%), as well as no diphenylphosphine selenide (DPPSe) when DPP was added to the ≈1.0 M solution. Hence, it is proposed that, for the formation of PbSe monomers, an indirect pathway dominates with the formation of a Pb-P complex/intermediate, which results from the activation of Pb(OA)(2) by a phosphine compound (such as DPP, DOP, or TOP) and in turn reacts with TOPSe. With the use of TOP 90% and the addition of secondary phosphine DPP, the formation of PbSe magic-sized nanoclusters (MSNCs) and regular NCs (RNCs) is investigated. With proper tuning of the synthesis conditions, the formation of various PbSe MSNCs versus RNCs is monitored in situ with versus without the addition of DPP, or at different reaction temperatures but otherwise identical synthetic formulation and reaction parameters. Accordingly, the degree of supersaturation (DS) of the PbSe monomer affecting the development of these PbSe MSNCs versus RNCs is proposed; the higher the DS, the more the MSNCs are favored. Also, surface-determined cluster-cluster aggregation is proposed to be the growth mechanism for both the RNCs and MSNCs. For the former, quantized growth is followed by continuous growth. For the latter, the sizes of the magic-sized families are calculated.

The effect of aminoglycoside antibiotics on the thermodynamic properties of liposomal vesicles.

Liposomes are ideal drug-delivery systems because they can alter the pharmacokinetic characteristics and biodistribution profile of the incorporated bioactive molecule. The effect of the aminoglycoside antibiotics, gentamicin (GN), tobramycin (TOB), and amikacin (AMI), on the thermodynamic properties of multilamellar vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied by using differential scanning calorimetry (DSC), electron paramagnetic resonance (EPR), and (31)P nuclear magnetic resonance (NMR) spectroscopy. The relationship between the structure of aminoglycoside antibiotics and their effect on the physical properties of the liposomal bilayers was investigated. The incorporation of the drugs was achieved and an osmotic gradient created by controlling the mole ratio of the drug inside to that outside of the DPPC vesicles so that [drug(inside DPPC)]/[drug(outside DPPC)] was 1:0, 1:0.2, 1:1, or 1:2.5. Incorporation of the drugs into liposomes caused the T(m) to shift to a higher temperature and the delta H(m) and delta T(1/2) values to decrease. The 2A(max) and the order parameter (S), obtained from the EPR spectra, indicated that the fluidity of the liposomal membrane was affected by the type of drug and by the concentration used; GN and TOB decreased the fluidity and disturbed chain packing at mole ratios of [drug(inside DPPC)]/[drug(outside DPPC)] ranging from 1:0 to 1:0.2, while AMI increased the fluidity and disrupted chain packing at an osmotic gradient of 1:2.5. In conclusion, the molecular organization and thermotropic properties of the multilamellar DPPC vesicles were dependent on the osmotic gradient and structure of the aminoglycoside.

Inclusion complexes of coumarin in cucurbiturils.

Coumarin was found to form stable inclusion complexes with cucurbiturils. In the presence of cucurbit[7]uril (CB[7]), 1 : 1 inclusion complexes were observed in aqueous solution, as monitored by (1)H NMR and UV-visible absorption spectroscopies, and further supported by ab initio calculations, whereas with cucurbit[8]uril (CB[8]) a solid phase 1 : 2 host : guest complex was found in a single crystal X-ray diffraction structure determination.

Discovery of indoline-based, natural-product-like compounds as probes of focal adhesion kinase signaling pathways.

With the goal of identifying small molecule modulators of protein-protein interactions, we developed a solid-phase synthesis method, which was then successfully utilized in a library generation of 164 aminoindoline-derived, natural-product-like compounds. This library and several other related intermediates synthesized during this project were then subjected to different biological assays in search of small molecule modulators of focal adhesion kinase (FAK)-mediated signaling pathways. This study included (i) an in vitro, full length FAK inhibition assay, (ii) a cell proliferation assay, and (iii) a wound healing assay. In FAK inhibition assay, eight library members (5-12) and three aminoindoline derivatives (13, 14, and 2) were identified as promising candidates. Compounds 13 and 2 inhibited the FAK activity by 25-45% at 10 microM. These two lead compounds also showed activity in a wound healing assay. To our knowledge, these aminoindoline-derived small molecules belong to a new family of FAK inhibitors.

Benzofuran-derived cyclic beta-amino acid scaffold for building a diverse set of flavonoid-like probes and the discovery of a cell motility inhibitor.

We report here a practical, enantioselective synthesis of benzofuran-derived, cyclic trans-beta-amino acid scaffold. In two cases, tricyclic derivatives having six- and eight-membered unsaturated lactams were obtained from this versatile scaffold. To explore the biological applications, these compounds were subjected to cell-based assays, using NIH3T3 mouse cells to examine their potency as cell motility inhibitors and identified 18 as a potent cell motility inhibitor (IC50 approximately 40 microM in chamber cell migration assay).

Single-crystal to single-crystal phase transition of cucurbit[5]uril hydrochloride hydrates: large water-filled channels transforming to layers of unusual stability.

Cucurbit[5]uril hydrochloride hydrate crystals with large water-filled channels transform to a highly stable layer structure via a single-crystal to single-crystal mechanism; (129)Xe NMR showed that porosity in CB[5] samples depends critically on the method of preparation.

Building skeletally diverse architectures on the Indoline Scaffold: the discovery of a chemical probe of focal adhesion kinase signaling networks.

Inspired by bioactive indoline alkaloid natural products, here, we report a divergent synthesis approach that led to skeletally diverse indoline alkaloid-inspired compounds. The natural product-inspired compounds obtained were then subjected to a series of in vitro and cellular assays to examine their properties as modulators of focal adhesion kinase (FAK) activity. This study resulted in the identification of a promising lead inhibitor of FAK (42), which also showed activity in a wound healing and cell invasion assay. The in silico study of the lead compound (42) was also undertaken.

Reagent-based, modular, tandem Michael approach for obtaining different indoline alkaloid-inspired polycyclic architectures.

A modular, reagent-based approach to obtain different indoline alkaloid-inspired, tetracyclic architectures is developed. With the use of TBSOTf as a Lewis acid, we report here a tandem Michael-based approach that led to the synthesis of a diastereomeric mixture of tetracyclic derivatives with two additional six-membered rings. By simply changing the Lewis acid to TMSOTf, we were able to obtain a different tetracyclic compound having additional functionalized 5- and 7-membered rings with complete stereocontrol.

Determination of cyanocobalamin by isotope dilution LC-MS/MS.

Cyanocobalamin (CNCbl) is an active form of vitamin B12, commonly employed for the preparation of multivitamin supplements and fortified food. In this study, we present a novel analytical method for its determination based on stable isotope dilution liquid chromatography electrospray tandem mass spectrometry (ID LC-MS/MS). Isotopically enriched 13C15NCbl was synthesized in-house and used as internal standard. The method was validated using NIST SRM 3280 multivitamin reference material and by comparison with an independent methodology based on LC-ICPMS. The proposed method provided a detection limit of 57 pg/g and could be applied for the determination of trace level of CNCbl in multivitamin supplements with a relative standard uncertainty better than 3%. The novel ID LC-MS/MS is a primary ratio method that could become a reference for CNCbl determination in multivitamins and food supplements. The method was applied for the characterization of two NRC multivitamin tablet Certified Reference Material (CRM) candidates, VITA-1 and VITB-1 whose CNCbl levels were quantified as 2.64 ± 0.09 and 1.75 ± 0.12 µg/g, respectively.

Supramolecular stabilization of hydroxylamine TEMPOH by complexation with an amphiphilic calixarene.

In an ethanol/water mixture, the nitroxyl radical TEMPO abstracts a hydrogen atom from a phenolic OH group of the amphiphilic para-hexanoylcalix[4]arene, and the hydroxylamine TEMPOH formed yields a stable inclusion complex with another molecule of the calixarene.

Low-temperature approach to highly emissive copper indium sulfide colloidal nanocrystals and their bioimaging applications.

We report our newly developed low-temperature synthesis of colloidal photoluminescent (PL) CuInS2 nanocrystals (NCs) and their in vitro and in vivo imaging applications. With diphenylphosphine sulphide (SDPP) as a S precursor made from elemental S and diphenylphosphine, this is a noninjection based approach in 1-dodecanethiol (DDT) with excellent synthetic reproducibility and large-scale capability. For a typical synthesis with copper iodide (CuI) as a Cu source and indium acetate (In(OAc)3) as an In source, the growth temperature was as low as 160 °C and the feed molar ratios were 1Cu-to-1In-to-4S. Amazingly, the resulting CuInS2 NCs in toluene exhibit quantum yield (QY) of ~23% with photoemission peaking at ~760 nm and full width at half maximum (FWHM) of ~140 nm. With a mean size of ~3.4 nm (measured from the vertices to the bases of the pyramids), they are pyramidal in shape with a crystal structure of tetragonal chalcopyrite. In situ (31)P NMR (monitored from 30 °C to 100 °C) and in situ absorption at 80 °C suggested that the Cu precursor should be less reactive toward SDPP than the In precursor. For our in vitro and in vivo imaging applications, CuInS2/ZnS core-shell QDs were synthesized; afterwards, dihydrolipoic acid (DHLA) or 11-mercaptoundecanoic acid (MUA) were used for ligand exchange and then bio-conjugation was performed. Two single-domain antibodies (sdAbs) were used. One was 2A3 for in vitro imaging of BxPC3 pancreatic cancer cells. The other was EG2 for in vivo imaging of a Glioblastoma U87MG brain tumour model. The bioimaging data illustrate that the CuInS2 NCs from our SDPP-based low-temperature noninjection approach are good quality.