The qNMR Summit 5.0: Proceedings and Status of qNMR Technology.

The goal of the qNMR Summit is to take stock of the status quo and the recent developments in qNMR research and applications in a timely and accurate manner. It provides a platform for both advanced and novice qNMR practitioners to receive a well-rounded update and discuss potential qNMR-related applications and collaborations. For over a decade, scientists from academia, industry, nonprofit institutions, and governmental bodies have focused on the standardization of qNMR methodology, as well as its metrological and pharmacopeial utility. This paper reviews key content of qNMR Summits 1.0 to 4.0 and puts into perspective the outcomes and available transcripts of the October 2019 Summit 5.0, with attendees from the United States, Canada, Japan, Korea, and several European countries. Summit presentations focused on qNMR methodology in the pharmaceutical industry, advanced quantitation algorithms, and promising developments.

A nontargeted approach to determine the authenticity of Ginkgo biloba L. plant materials and dried leaf extracts by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and chemometrics.

The lack of stringent regulations regarding raw materials for herbal supplements used for medicinal purposes has been a constant challenge in the industry. Ginkgo biloba L. leaf extracts attract consumers because of the supposed positive effect on mental performance and memory. Supplements are produced using dried leaf materials and standardized leaf extracts such as EGb 761. Adulteration of Ginkgo biloba L. plants and extracts are becoming more and more common practice due to economically driven motivation from increasing demand in the market and the high cost of raw materials and production. Reinforcement in quality control (QC) to avoid adulterations is necessary to ensure the efficacy of the supplements. In this study, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was used with principal component analysis (PCA) as an unsupervised exploratory method to analyze, identify, and evaluate the adulterated Ginkgo biloba L. plant materials and dried leaf extracts using the PCA scores and loadings obtained and compound identification.

NIST Spectroscopic Measurement Standards.

Ultraviolet (UV) absorbance measurements provide a rapid and reliable method to determine protein concentrations. the National Institute of standards and technology (NIST) has developed Standard Reference Material (SRM) 2082 as a pathlength standard for UV absorbance measurements for use with the new generation of microvolume spectrophotometers and short-pathlength cuvettes. short pathlengths are used with high-concentration targets to ensure that absorbance values are within the optimal range. the short-pathlength instruments and cuvettes also reduce the required volumes to conserve valuable samples. the authors compared the results obtained with high-quality dual-beam spectrophotometers and short-pathlength cuvettes to the results obtained from a microvolume spectrophotometer and a microvolume plate reader. SRM 2082 can be used to accurately calculate pathlength values, thereby increasing the accuracy in subsequent measurements using the short-pathlength cuvettes and microvolume absorbance instruments. RM 8671 (reference material, the NISTmAb) can then be used to ensure the accuracy and reproducibility of protein concentration measurements by providing an industrially relevant reference material, a well-characterized monoclonal antibody.

Development of NIST Standard Reference Material 2082, a Pathlength Standard for Measurements in the Ultraviolet Spectrum.

New spectrophotometers and cuvettes have been designed to allow the measurement of absorbance values from samples using microliter volume sizes. These measurements are done using short pathlengths to decrease the sample volumes required. The major applications for these spectrophotometers and cuvettes are samples that are difficult to obtain in large amounts, such as proteins and nucleic acids that absorb light in the ultraviolet range. Existing ultraviolet absorbance standards have been designed for longer pathlength measurements. Standard Reference Material (SRM) 2082 was developed to validate the pathlengths of short-pathlength cuvettes and instruments using materials with absorbance spectra that are similar to the most commonly used samples. SRM 2082 consists of three individual components: a blank buffer solution, a solution of the amino acid tryptophan in the buffer, and a solution of the nucleobase uracil in the buffer. The tryptophan solution has an absorbance spectrum (peak at 280 nm) similar to proteins, and the uracil has an absorbance spectrum (peak at 260 nm) similar to nucleic acids. The absorbance values of these solutions were determined using a series of cuvettes with pathlengths from 0.1 mm to 2 mm. The pathlengths of the cuvettes used for the absorbance measurements were determined at the National Institute of Standards and Technology by physical and optical measurements. The effects of temperature and spectral bandwidth variations on the absorbance values of SRM 2082 were also investigated.

Particle Fabrication Using Inkjet Printing onto Hydrophobic Surfaces for Optimization and Calibration of Trace Contraband Detection Sensors.

A method has been developed to fabricate patterned arrays of micrometer-sized monodisperse solid particles of ammonium nitrate on hydrophobic silicon surfaces using inkjet printing. The method relies on dispensing one or more microdrops of a concentrated aqueous ammonium nitrate solution from a drop-on-demand (DOD) inkjet printer at specific locations on a silicon substrate rendered hydrophobic by a perfluorodecytrichlorosilane monolayer coating. The deposited liquid droplets form into the shape of a spherical shaped cap; during the evaporation process, a deposited liquid droplet maintains this geometry until it forms a solid micrometer sized particle. Arrays of solid particles are obtained by sequential translation of the printer stage. The use of DOD inkjet printing for fabrication of discrete particle arrays allows for precise control of particle characteristics (mass, diameter and height), as well as the particle number and spatial distribution on the substrate. The final mass of an individual particle is precisely determined by using gravimetric measurement of the average mass of solution ejected per microdrop. The primary application of this method is fabrication of test materials for the evaluation of spatially-resolved optical and mass spectrometry based sensors used for detecting particle residues of contraband materials, such as explosives or narcotics.

Probing the intracellular glutathione redox potential by in-cell NMR spectroscopy.

Non-invasive and real-time analysis of cellular redox processes has been greatly hampered by lack of suitable measurement techniques. Here we describe an in-cell nuclear magnetic resonance (NMR) based method for measuring the intracellular glutathione redox potential by direct and quantitative measurement of isotopically labeled glutathione introduced exogenously into living yeast. By using this approach, perturbations in the cellular glutathione redox homeostasis were also monitored as yeast cells were subjected to oxidative stress.

Quantitative measurements of glutathione in yeast cell lysate using 1H NMR.

Methods for quantifying the level of glutathione (GSH) in yeast cell lysate are described using (1)H NMR analysis. For quantification purposes, the (1)H resonances corresponding to the Cys ßCH2 of GSH were identified as having the fewest overlapping spectral interferences from lysate matrix components using GSH spiked yeast lysate samples. Two methods, standard addition based on peak integration and a spectral subtraction approach, were evaluated for quantifying GSH in lysate samples. The peak integration procedure required baseline estimation and a peak fitting step to correct for background interferences while the spectral subtraction procedure was comparatively straightforward. The level of GSH measured by (1)H NMR was in good agreement with the concentration measured by the DTNB-GSSG reductase recycling assay. The proposed NMR method can lead to a reliable quantitation of GSH and could be applicable to a variety of other analytes of interest in complex biological matrices.

A Collaborative Platform for Novel Compound Identification - Characterization of Designer Phencyclidines (PCPs) POXP, PTHP, and P2AP.

With the sustained prevalence and introduction of new emerging drugs throughout the world there is a need for continued development and maintenance of platforms that enable rapid identification and characterization of unknown compounds. To complement existing efforts, a collaborative platform between the National Institute of Standards and Technology (NIST) and practicing forensic agencies is being deployed which enables laboratories to leverage techniques and expertise that may not exist at their facilities. Using this approach, unknown compounds are identified and characterized using a suite of analytical tools to obtain (1) a rapid preliminary identification followed by (2) a more complete characterization and confirmation of the preliminary identification. To demonstrate this platform, the characterization of three previously unreported analogs of phencyclidine (PCP) - POXP, PTHP, and P2AP - are described. A preliminary identification of the three substances was obtained using direct analysis in real time mass spectrometry (DART-MS) with confirmation by nuclear magnetic resonance (NMR) spectroscopy, gas chromatography mass spectrometry (GC-MS) and gas chromatography flame ionization detection (GC-FID).

Expanding NIST Calibration of Fluorescent Microspheres for Flow Cytometry to More Fluorescence Channels and Smaller Particles.

The National Institute of Standards and Technology (NIST), the National Institutes of Health (NIH) and other industry stakeholders have been working together to enable fluorescence intensities of flow cytometer calibration beads to be assigned quantitative equivalent reference fluorophore (ERF) values with high accuracy and precision. The ultimate goal of this effort is to accurately quantify the number of antibodies bound to individual living cells. The expansion of this effort to assign ERF values to more than 50 fluorescence channels and particles with diameters ranging from 10 µm down to 80 nm is reported here.

Anti-HER2 IgY antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells.

BACKGROUND: Nanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for these applications, strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and demonstrated its dual functionality for both detection and selective destruction of cancer cells in an in vitro model consisting of HER2-expressing SK-BR-3 cells and HER2-negative MCF-7 cells. METHODS: The complex was constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken IgY antibody, which is more specific and sensitive than mammalian IgGs. Raman signals were recorded on Raman spectrometers with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 5 W/cm2 for 2 min. Cell viability was examined by the calcein AM/ethidium homodimer-1 (EthD-1) staining. RESULTS: Using a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated SK-BR-3 cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast cancer cells without harming receptor-free cells. The cell death was effectuated without the need of internalization of SWNTs by the cancer cells, a finding that has not been reported previously. CONCLUSION: We have demonstrated that the HER2 IgY-SWNT complex specifically targeted HER2-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells. The complex can be potentially used for both detection and selective photothermal ablation of receptor-positive breast cancer cells without the need of internalization by the cells. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of IgY antibodies can lead to new strategies for cancer detection and therapy.

Differentiation of fentanyl analogues by low-field NMR spectroscopy.

Forensic laboratories commonly receive new psychoactive substances such as fentanyl analogues and other synthetic opioids that are difficult to identify. Slight changes to chemical structures, e.g. shifting the position of functional groups such as methyl groups or halogens on the aromatic ring, may not be distinguished using traditional methods. NMR is a powerful tool used to elucidate distinctive structural information needed to differentiate regioisomers. However, the cost, size, and cryogen maintenance of superconducting NMR spectrometers can be impractical for some forensic laboratories. Recent studies have shown potential applications of low-field NMR as an alternative in forensic drug analysis. These benchtop, semi-portable instruments are less costly, have a smaller footprint, do not use cryogens, and require little maintenance. In this study, we show that 65 fentanyl and related substances, including various types of positional isomers, were readily differentiated using low-field (62 MHz) 1H NMR spectroscopy. In addition, the use of quantum mechanical spin system analysis was investigated for the purposes of translating experimentally observed high-field 1H spectra to lower field strengths. Spin system analysis of 600 MHz NMR spectra was conducted on a subset (15) of the reference materials analyzed. The results were used to calculate 62 MHz spectra for comparison purposes with the experimental spectra. This was successfully demonstrated, showing that field-strength independent 1H NMR spectral libraries are feasible and can facilitate reference material data dissemination across forensic drug laboratories.

Efficient electrochemical degradation of multiwall carbon nanotubes.

As the production mass of multiwall carbon nanotubes (MWCNT) increases, the potential for human and environmental exposure to MWCNTs may also increase. We have shown that exposing an aqueous suspension of pristine MWCNTs to an intense oxidative treatment in an electrochemical reactor, equipped with an efficient hydroxyl radical generating Boron Doped Diamond (BDD) anode, leads to their almost complete mineralization. Thermal optical transmittance analysis showed a total carbon mass loss of over two orders of magnitude due to the electrochemical treatment, a result consistent with measurements of the degraded MWCNT suspensions using UV-vis absorbance. Liquid chromatography data excludes substantial accumulation of the low molecular weight reaction products. Therefore, up to 99% of the initially suspended MWCNT mass is completely mineralized into gaseous products such as CO2 and volatile organic carbon. Scanning electron microscopy (SEM) images show sporadic opaque carbon clusters suggesting the remaining nanotubes are transformed into structure-less carbon during their electrochemical mineralization. Environmental toxicity of pristine and degraded MWCNTs was assessed using Caenorhabditis elegans nematodes and revealed a major reduction in the MWCNT toxicity after treatment in the electrochemical flow-by reactor.

Magnetoelastic transducers for monitoring coagulation, clot inhibition, and fibrinolysis.

Magnetoelastic transduction has been used to detect and monitor the viscosity changes that occur during the biological reactions of coagulation and fibrinolysis. Magnetoelastic sensors can be used, because the characteristic resonance frequency of the magnetoelastic strip shifts in response to the changes in fluid viscosity. At a set frequency, the output signal can be obtained over time to develop a coagulation and/or dissolution profile, which display the change in viscosity of a plasma sample that has undergone either coagulation or fibrinolysis. For coagulation screening, an exogenous tissue factor is added to an anticoagulated plasma sample to initiate coagulation. Further studies were performed to investigate fibrinolysis through the addition of plasmin. Plasmin is used in two different ways-as a competitive inhibitor before the initiation of clotting and also as a protease to dissolve the previously formed clot. This method is a viable option for the monitoring of processes that are paramount to maintaining hemostasis.

Automated spectral smoothing with spatially adaptive penalized least squares.

A variety of data smoothing techniques exist to address the issue of noise in spectroscopic data. The vast majority, however, require parameter specification by a knowledgeable user, which is typically accomplished by trial and error. In most situations, optimized parameters represent a compromise between noise reduction and signal preservation. In this work, we demonstrate a nonparametric regression approach to spectral smoothing using a spatially adaptive penalized least squares (SAPLS) approach. An iterative optimization procedure is employed that permits gradual flexibility in the smooth fit when statistically significant trends based on multiscale statistics assuming white Gaussian noise are detected. With an estimate of the noise level in the spectrum the procedure is fully automatic with a specified confidence level for the statistics. Potential application to the heteroscedastic noise case is also demonstrated. Performance was assessed in simulations conducted on several synthetic spectra using traditional error measures as well as comparisons of local extrema in the resulting smoothed signals to those in the true spectra. For the simulated spectra, a best case comparison with the Savitzky-Golay smoothing via an exhaustive parameter search was performed while the SAPLS method was assessed for automated application. The application to several dissimilar experimentally obtained Raman spectra is also presented.

Molecular factor computing for predictive spectroscopy.

PURPOSE: The concept of molecular factor computing (MFC)-based predictive spectroscopy was demonstrated here with quantitative analysis of ethanol-in-water mixtures in a MFC-based prototype instrument. METHODS: Molecular computing of vectors for transformation matrices enabled spectra to be represented in a desired coordinate system. New coordinate systems were selected to reduce the dimensionality of the spectral hyperspace and simplify the mechanical/electrical/computational construction of a new MFC spectrometer employing transmission MFC filters. A library search algorithm was developed to calculate the chemical constituents of the MFC filters. The prototype instrument was used to collect data from 39 ethanol-in-water mixtures (range 0-14%). For each sample, four different voltage outputs from the detector (forming two factor scores) were measured by using four different MFC filters. Twenty samples were used to calibrate the instrument and build a multivariate linear regression prediction model, and the remaining samples were used to validate the predictive ability of the model. RESULTS: In engineering simulations, four MFC filters gave an adequate calibration model (r2 = 0.995, RMSEC = 0.229%, RMSECV = 0.339%, p = 0.05 by f test). This result is slightly better than a corresponding PCR calibration model based on corrected transmission spectra (r2 = 0.993, RMSEC = 0.359%, RMSECV = 0.551%, p = 0.05 by f test). The first actual MFC prototype gave an RMSECV = 0.735%. CONCLUSION: MFC was a viable alternative to conventional spectrometry with the potential to be more simply implemented and more rapid and accurate.

Centrifugal microfluidics with integrated sensing microdome optodes for multiion detection.

An array of four sensing microdome optodes (potassium, sodium, calcium, and chloride) was incorporated into a centrifugal microfluidics platform to obtain a multiion analysis system. The behavior of each sensing microdome was in good agreement with a theoretical model describing the response. The selectivity of each optode over common interfering ions was established and was used to identify calibrant solutions that can be employed for the simultaneous calibration of all four optodes without significant cross-interference. The microfluidic platform was designed to facilitate both three-point calibration of the optodes and triplicate analysis of a sample within a single run, which increases the accuracy of the determination. The optimized microfluidic system was used to determine simultaneously the concentration of potassium, sodium, calcium, and chloride in aquarium water (with the composition of Lake Tanganyika water) with less than 6% error. The simple process of fabrication of these microdomes and their incorporation into a centrifugal microfluidic platform should facilitate the development of portable ion-sensing analysis systems.

Decyl methacrylate-based microspot optodes.

Optode sensing membranes employing decyl methacrylate cross-linked with 1,6-hexanediol dimethacrylate as the polymer support were fabricated by a direct microspotting method on several surfaces. Photopolymerization was used to attach the microspots to the substrate. Using this method, diameters in the micrometer domain were obtained. Silanized glass, poly(methyl methacrylate) (PMMA), polycarbonate, and poly(dimethylsiloxane) were tested as possible substrates. Both polypropylene tips and the steel tips of drafting pens were used for spotting. It was determined that both silanized glass and PMMA gave working optodes, but the ones on PMMA did not fit the theoretical model. Diameters of 994 +/- 80 and 1279 +/- 85 microm were obtained on silanized glass and PMMA, respectively, using the polypropylene tips for spotting. Different size optodes were fabricated using 0.35- and 0.50-mm steel drafting pen tips. The 0.35-mm tips produced diameters of 895 +/- 26 and 688 +/- 54 microm on silanized glass and PMMA, respectively, and the 0.50-mm tips produced diameters of 1274 +/- 94 microm on silanized glass and 839 +/- 28 microm on PMMA. Thus, the microspot size can be controlled based on the hydrophobicity of the surface and the size of the tip used for spotting. Calibration plots of potassium optode microspots indicated that miniaturization does not alter response characteristics, such as selectivity, response time, and dynamic range, of the optodes.

Near-infrared spectrometry of abdominal aortic aneurysm in the ApoE-/- mouse.

Abdominal aortic aneurysms (AAAs) occur in 5-7% of people over age 60 in the United States. Early intervention in the disease process could have a significant impact on the incidence of complications and on patient survival, but identifying incipient aneurysms can be difficult. ApoE knockout mice develop AAAs following infusion of angiotensin II (AngII) by osmotic minipump into the subcutaneous space of mice at doses ranging from 500 to 1000 ng kg(-1) min(-1) for 7-28 days. These mice are used as models of AAA development. This study tested the hypothesis that near-IR spectrometry and PCR can determine AngII dose (SEE = 26 ng kg(-1) min(-1), SEP = 37 ng kg(-1) min(-1), r2 = 0.99) and collagen/elastin (C/E) ratio (SEE = 0.38, SEP = 0.39, r2 = 0.85) in mouse aortas.