Sequential Multimaterial Additive Manufacturing of Functionally Graded Biopolymer Composites.

Cellulose, chitin, and pectin are three of the most abundant natural materials on Earth. Despite this, large-scale additive manufacturing with these biopolymers is used only in limited applications and frequently relies on extensive refinement processes or plastic additives. We present novel developments in a digital fabrication and design approach for multimaterial three-dimensional printing of biopolymers. Specifically, our computational and digital fabrication workflow-sequential multimaterial additive manufacturing-enables the construction of biopolymer composites with continuously graded transitional zones using only a single extruder. We apply this method to fabricate structures on length scales ranging from millimeters to meters. Transitional regions between materials created using these methods demonstrated comparable mechanical properties with homogenous mixtures of the same composition. We present a computational workflow and physical system support a novel and flexible form of multimaterial additive manufacturing with a diverse array of potential applications.

Design and results of the ice sheet model initialisation experiments initMIP-Greenland: an ISMIP6 intercomparison.

Earlier large-scale Greenland ice sheet sea-level projections (e.g., those run during the ice2sea and SeaRISE initiatives) have shown that ice sheet initial conditions have a large effect on the projections and give rise to important uncertainties. The goal of the initMIP-Greenland intercomparison exercise is to compare, evaluate and improve the initialisation techniques used in the ice sheet modelling community and to estimate the associated uncertainties in modelled mass changes. initMIP-Greenland is the first in a series of ice sheet model intercomparison activities within ISMIP6 (the Ice Sheet Model Intercomparison Project for CMIP6), which is the primary activity within the Coupled Model Intercomparison Project - phase 6 (CMIP6) focusing on the ice sheets. Two experiments for the large-scale Greenland ice sheet have been designed to allow intercomparison between participating models of 1) the initial present-day state of the ice sheet and 2) the response in two idealised forward experiments. The forward experiments serve to evaluate the initialisation in terms of model drift (forward run without additional forcing) and in response to a large perturbation (prescribed surface mass balance anomaly), and should not be interpreted as sea-level projections. We present and discuss results that highlight the diversity of data sets, boundary conditions and initialisation techniques used in the community to generate initial states of the Greenland ice sheet. We find good agreement across the ensemble for the dynamic response to surface mass balance changes in areas where the simulated ice sheets overlap, but differences arising from the initial size of the ice sheet. The model drift in the control experiment is reduced for models that participated in earlier intercomparison exercises.

Rapid analysis of fentanyls and other novel psychoactive substances in substance use disorder patient urine using paper spray mass spectrometry.

RATIONALE: Drug overdose deaths due to fentanyls and other novel psychoactive substances (NPS) are on the rise. The higher potencies of fentanyl analogs compared with morphine require new technologies to identify and quantitate NPS. METHODS: Paper spray tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry were used to identify and measure fentanyl analogs as well as common drugs of abuse in urine samples from substance use disorder clinics. Ten-microliter urine samples were deposited directly on paper spray cartridges previously loaded with internal standards, dried, and analyzed with no other sample treatment. Quantitative results were obtained using MS/MS. Individual drugs were identified using high-resolution accurate mass spectrometry, and confirmed by data-dependent MS/MS. RESULTS: Calibration curves in urine were linear over a range of 0.5-50 ng/mL with R2 of 0.99 or better for eight representative fentanyl analogs. Cartridges preloaded with internal standards demonstrated satisfactory quantitative results compared with LC/MS. Direct identification and confirmation of fentanyl analogs and other common drugs of abuse in urine using high-resolution accurate mass and MS/MS fragmentation were demonstrated at low picogram levels. CONCLUSIONS: Paper spray mass spectrometry can reliably identify and quantitate fentanyl analogs and other drugs of abuse in urine. Using paper spray cartridges as collection devices reduces exposure and transportation risks associated with biological fluids. Cartridges preloaded with labeled internal standards can be effective for targeted screening of fentanyl analogs and other drugs of abuse.

An ice sheet model validation framework for the Greenland ice sheet.

We propose a new ice sheet model validation framework - the Cryospheric Model Comparison Tool (CmCt) - that takes advantage of ice sheet altimetry and gravimetry observations collected over the past several decades and is applied here to modeling of the Greenland ice sheet. We use realistic simulations performed with the Community Ice Sheet Model (CISM) along with two idealized, non-dynamic models to demonstrate the framework and its use. Dynamic simulations with CISM are forced from 1991 to 2013 using combinations of reanalysis-based surface mass balance and observations of outlet glacier flux change. We propose and demonstrate qualitative and quantitative metrics for use in evaluating the different model simulations against the observations. We find that the altimetry observations used here are largely ambiguous in terms of their ability to distinguish one simulation from another. Based on basin- and whole-ice-sheet scale metrics, we find that simulations using both idealized conceptual models and dynamic, numerical models provide an equally reasonable representation of the ice sheet surface (mean elevation differences of <1 m). This is likely due to their short period of record, biases inherent to digital elevation models used for model initial conditions, and biases resulting from firn dynamics, which are not explicitly accounted for in the models or observations. On the other hand, we find that the gravimetry observations used here are able to unambiguously distinguish between simulations of varying complexity, and along with the CmCt, can provide a quantitative score for assessing a particular model and/or simulation. The new framework demonstrates that our proposed metrics can distinguish relatively better from relatively worse simulations and that dynamic ice sheet models, when appropriately initialized and forced with the right boundary conditions, demonstrate predictive skill with respect to observed dynamic changes occurring on Greenland over the past few decades. An extensible design will allow for continued use of the CmCt as future altimetry, gravimetry, and other remotely sensed data become available for use in ice sheet model validation.

Analysis of dried blood spots using DESI mass spectrometry.

Dried blood spot (DBS) analysis using mass spectrometry is an invaluable technique for examining blood markers of inborn metabolic diseases in clinical laboratories. Implementation of DBS sampling and analysis in pharmaceutical development have more recently gained traction due to the advantages of convenience in sample procurement and logistics, as well as the innate advantages associated with the collection of lower blood volumes. While there are several realized advantages of DBS, the bioanalytical laboratory is disadvantaged and burdened with additional preparative steps prior to analysis. Therefore, improvements in the laboratory workflow for DBS analysis are necessary. Here, we describe direct blood spot analysis using desorption electrospray ionization (DESI) mass spectrometry for quantitative determination of drugs in whole blood.

Ambient analysis of leachable compounds from single-use bioreactors with desorption electrospray ionization time-of-flight mass spectrometry.

RATIONALE: Trace levels of bis(2,4-di-tert-butylphenyl)phosphate (BdtbPP) leaching from single-use bioreactors (SUBs) were recently found to be highly detrimental to mammalian cell growth. The traditional approach to detect the leachable requires time-consuming solvent extraction of SUBs. To assist the qualification of SUBs and/or their manufacturing raw materials in biopharmaceutical development and manufacturing, it is essential to develop a rapid and sensitive analytical approach for detecting this leachable and related compounds, which is described in this study. METHODS: Native films from several commercially available SUBs were directly examined by desorption electrospray ionization (DESI) time-of-flight mass spectrometry (TOFMS) without sample preparation. As a comparison, the same SUBs were also analyzed by high-performance liquid chromatography (HPLC)/ultraviolet (UV) following the solvent extraction. RESULTS: With a suitable spray solvent selected in this study, DESI-TOFMS enabled rapid and sensitive detection of leachable compounds directly from SUBs. Accurate mass measurement from TOFMS allowed ready identification of BdtbPP, its parent analog compound, and other polymer components in the SUBs from their protonated surrogates. The relative abundances of BdtbPP in different SUBs measured by DESI-TOFMS exhibited good correlation with those from the traditional extraction-based approach with HPLC/UV. CONCLUSIONS: A rapid and sensitive approach was developed for direct detection of BdtbPP and other leachables from SUBs using DESI-TOFMS. The results are in high accordance with those from conventional approaches, which indicates the usefulness of the proposed method as a qualification tool for SUBs in biopharmaceutical development and also its great potential in the analysis of extractables/leachables in a wide variety of materials, process components, devices and containers used in the pharmaceutical industry.

Coupling desorption electrospray ionization with solid-phase microextraction for screening and quantitative analysis of drugs in urine.

Direct analysis of silica C(18)-coated solid-phase microextraction (SPME) fibers using desorption electrospray ionization mass spectrometry (DESI-MS) for the purpose of analyzing drugs from raw urine is presented. The method combines a simple, inexpensive, and solvent-less sample preparation technique with the specificity and speed of DESI-MS and MS/MS. Extraction of seven drugs from raw urine is performed using specially designed SPME fibers coated uniformly with silica-C(18) stationary phase. Each SPME device is inserted into unprocessed urine under gentle agitation and, then, removed, rinsed, and analyzed directly by DESI-MS (MS/MS). Rapid screening over a wide mass range is afforded by coupling the method with a time of flight (TOF) mass spectrometer while quantitative analysis is performed using selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer. The performance of the SPME DESI-MS/MS method was evaluated by preparing calibration standards and quality control (QC) samples of the seven drug compounds from urine over a range from 20 to 1000 ng/mL, with the exception of meprobamate which was prepared from 200 to 10000 ng/mL. The calibration curves constructed for each analyte had an R(2) > 0.99. The range of precision (%CV) and accuracy values (% bias) for low QC samples was 1-11% and 3-38%, respectively. Precision and accuracy values for high QC samples range from 0.9 to 8% and -31 to -8%. Results from urine specimens of actual exposure to drugs screened using the SPME DESI-MS/MS method showed good agreement with the conventional immunoassays and GC/MS analysis. Liquid desorption of the SPME fiber followed by LC/MS/MS also showed good agreement with the SPME DESI-MS/MS method.

Direct analysis of Salvia divinorum leaves for salvinorin A by thin layer chromatography and desorption electrospray ionization multi-stage tandem mass spectrometry.

Salvia divinorum is widely cultivated in the US, Mexico, Central and South America and Europe and is consumed for its ability to produce hallucinogenic effects similar to those of other scheduled hallucinogenic drugs, such as LSD. Salvinorin A (SA), a kappa opiod receptor agonist and psychoactive constituent, is found primarily in the leaves and to a lesser extent in the stems of the plant. Herein, the analysis of intact S. divinorum leaves for SA and of acetone extracts separated using thin layer chromatography (TLC) is demonstrated using desorption electrospray ionization (DESI) mass spectrometry. The detection of SA using DESI in the positive ion mode is characterized by several ions associated with the compound - [M+H](+), [M+NH(4)](+), [M+Na](+), [2M+NH(4)](+), and [2M+Na](+). Confirmation of the identity of these ions is provided through exact mass measurements using a time-of-flight (ToF) mass spectrometer. The presence of SA in the leaves was confirmed by multi-stage tandem mass spectrometry (MS(n)) of the [M+H](+) ion using a linear ion trap mass spectrometer. Direct analysis of the leaves revealed several species of salvinorin in addition to SA as confirmed by MS(n), including salvinorin B, C, D/E, and divinatorin B. Further, the results from DESI imaging of a TLC separation of a commercial leaf extract and an acetone extract of S. divinorum leaves were in concordance with the TLC/DESI-MS results of an authentic salvinorin A standard. The present study provides an example of both the direct analysis of intact plant materials for screening illicit substances and the coupling of TLC and DESI-MS as a simple method for the examination of natural products.

Direct analysis of dried blood spots utilizing desorption electrospray ionization (DESI) mass spectrometry.

A novel approach to the quantitative determination of xenobiotics in whole blood samples without sample preparation or chromatography is described. This method is based on direct analysis of microlitre volumes of blood which are spotted onto specialized paper cards and dried, with the resulting dried blood spots (DBS) analyzed directly via desorption electrospray ionization (DESI) mass spectrometry (MS). Using sitamaquine, terfenadine, and prazosin as model compounds with verapamil as a common internal standard, this methodology demonstrated detection of each compound down to 10 ng mL(-1) from DBS where standard calibration curves show linearity from 10-10,000 ng mL(-1) with r(2) > 0.99. Three (3) different untreated types of filter papers (Whatman 903 and 31ETF as well as Ahlstrom 237) and two (2) treated types of filter paper (Whatman FTA and FTA Elute) were examined and the effect of each surface on the recovery of each analyte was evaluated. The results show that the untreated papers provide the best substrates for DBS analysis by DESI. A more in depth study of the quantitation of sitamaquine on 31ETF paper stock provided bias and error measurements of less than 20%. The promising results shown in this study may have important implications in the areas of therapeutic drug monitoring (TDM), clinical and forensic toxicology, and pharmacology.

Evaluation and performance of desorption electrospray ionization using a triple quadrupole mass spectrometer for quantitation of pharmaceuticals in plasma.

The present work describes the methodology and investigates the performance of desorption electrospray ionization (DESI) combined with a triple quadrupole mass spectrometer for the quantitation of small drug molecules in human plasma. Amoxepine, atenolol, carbamazepine, clozapine, prazosin, propranolol and verapamil were selected as target analytes while terfenadine was selected as the internal standard common to each of the analytes. Protein precipitation of human plasma using acetonitrile was utilized for all samples. Limits of detection were determined for all analytes in plasma and shown to be in the range 0.2-40 ng/mL. Quantitative analysis of amoxepine, prazosin and verapamil was performed over the range 20-7400 ng/mL and shown to be linear in all cases with R(2) >0.99. In most cases, the precision (relative standard deviation) and accuracy (relative error) of each method were less than or equal to 20%, respectively. The performance of the combined techniques made it possible to analyze each sample in 15 s illustrating DESI tandem mass spectrometry (MS/MS) as powerful tool for the quantitation of analytes in deproteinized human plasma.

(S)-N-{3-[1-cyclopropyl-1-(2,4-difluoro-phenyl)-ethyl]-1H-indol-7-yl}-methanesulfonamide: a potent, nonsteroidal, functional antagonist of the mineralocorticoid receptor.

A novel, potent series of indole analogs were recently developed as MR antagonists, culminating in 14. This compound represents the first MR antagonist in this class of molecules, exhibiting picomolar binding affinity and in vivo blood pressure lowering at pharmaceutically relevant doses.

Enantiomeric separation of mineralocorticoid receptor (hMR) antagonists using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems.

This study demonstrates the increased versatility of the Chiralcel OJ-H stationary phase when using various alcohol/acetonitrile mobile phases. This chiral stationary phase has traditionally been employed in the normal phase mode and more recently with neat alcohols as eluents. Selected isomeric human mineralocorticoid receptor (hMR) antagonist pharmaceutical candidates and synthetic intermediates were separated using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems. The capacity factors, resolution, and selectivity of the chiral separations were assessed while varying the alcohol/acetonitrile composition and alcohol identity. The mixed polar eluents provide separations that are nearly always superior to both the traditional hexane-rich and single-alcohol "polar organic" eluents for the compounds tested in this article.

Biological evaluation of cryptophycin 52 fragment A analogues: effect of the multidrug resistance ATP binding cassette transporters on antitumor activity.

Cryptophycin 52 (LY355703) is a potent antiproliferative analogue of the marine natural product cryptophycin 1. It has been shown to have a broad range of antitumor activity against human tumor xenografts and murine tumors including tumors resistant to Taxol and Adriamycin. Its mechanism of action involves arresting cells in the G2-M phase of the cell cycle by binding to microtubules and suppressing their dynamics. This 16-membered depsipeptide can be divided into four major subunits or fragments (A-D). We reported previously on our synthetic efforts around fragment A and discovered that this region of the molecule was amenable to a structure-activity relationship study that resulted in highly active antiproliferative agents when evaluated in the CEM leukemia cell line. The synthetic analogues were designed to help improve the efficacy and aqueous solubility of the parent compound; therefore, many in this series contained ionizable functional groups such as an amino group, a hydroxy group, or a carboxylic acid. Although several of these analogues showed improvements in potency over cryptophycin 52 in drug-sensitive tumor xenograft models, many lost their activity against Adriamycin-resistant tumor lines. It was discovered on additional in vitro evaluation that these analogues became good substrates of the multidrug resistance transporter P-glycoprotein.

Solid-phase synthesis of constrained terminal and internal lactam peptidomimetics.

Lactams are key components of many peptidomimetic structures. Five- and six-membered lactam peptidomimetics with hydrogen or amino acid side chains at the alpha-position can be constructed from peptide precursors during a solid-phase synthesis. There is no significant racemization of remote stereocenters during synthesis.

A convergent approach to cryptophycin 52 analogues: synthesis and biological evaluation of a novel series of fragment a epoxides and chlorohydrins.

Cryptophycin 52 is a synthetic derivative of Cryptophycin 1, a potent antimicrotubule agent isolated from cyanobacteria. In an effort to increase the potency and water solubility of the molecule, a structure-activity relationship study (SAR) was initiated around the phenyl ring of fragment A. These Cryptophycin 52 analogues were accessed using a Wittig olefination reaction between various triphenylphosphonium salts and a key intermediate aldehyde prepared from Cryptophycin 53. Substitution on the phenyl ring of fragment A was well tolerated, and several of these analogues were equally or more potent than Cryptophycin 52 when evaluated in vitro in the CCRF-CEM leukemia cell line and in vivo against a murine pancreatic adenocarcinoma.