Development and validation of a high resolving power absolute quantitative per- and polyfluoroalkyl substances method incorporating Skyline data processing.

RATIONALE: The ability to perform absolute quantitation and non-targeted analysis on a single mass spectrometry instrument would be advantageous to many researchers studying per- and polyfluoroalkyl substances (PFAS). High-resolution accurate mass (HRAM) instrumentation (typically deployed for non-targeted work) carries several advantages over traditional triple quadrupole workflows when performing absolute quantitation. Processing this data using a vendor-neutral software would promote collaboration for these environmental studies. METHODS: LC-MS (Orbitrap Exploris 240) was used for absolute quantitation of 45 PFAS using precursor (MS1) peak areas for quantitation, whereas isotope pattern matching and fragmentation (MS2) pattern matching were used for qualitative identification. In addition, a fluorinated chromatographic column achieved superior separation compared to the typical C18 columns typically used in PFAS analyses. This method was validated across eight different chemical classes using recommended guidelines found in EPA Method 537.1 and Skyline data processing software. RESULTS: The validated limits of all 45 compounds, as well as metrics or accuracy and reproducibility, are reported. Most compounds achieved limits of quantitation in the range of 2-50 ng/L. Four newly released Chemours-specific compounds (PEPA, PFO3OA, PFO4DA, and PFO5DoA) were also validated. Aspects of data analysis specific to high resolving power absolute quantitation are reviewed as are the details of processing these data via Skyline. CONCLUSIONS: This method shows the feasibility of performing reproducible absolute quantitation of PFAS on an HRAM platform and does so using an open-source vendor-neutral data processing software to facilitate sharing of data across labs and institutions.

Understanding the electrospray ionization response factors of per- and poly-fluoroalkyl substances (PFAS).

Per- and polyfluoroalkyl substances (PFAS) are used extensively in commercial products. Their unusual solubility properties make them an ideal class of compounds for various applications. However, these same properties have led to significant contamination and bioaccumulation given their persistence in the environment. Development of analytical techniques to detect and quantify these compounds must take into account the potential for these properties to perturb these measurements, specifically the potential to bias the electrospray ionization (ESI) process. Direct injection ESI analysis of 23 different PFAS species revealed that hydrophobicity and PFAS class can predict the ESI overall response factors. In this study, a method for predicting the behavior of individual PFAS compounds, including relative retention order in chromatography, is presented which is simply based on the number of fluorine atoms in the molecule as well as the class of the compound (e.g., perfluroalkylcarboxylic acids) vs. computational estimations (e.g., non-polar surface area and logP).

Design, synthesis, and evaluation of substrate - analogue inhibitors of Trypanosoma cruzi ribose 5-phosphate isomerase type B.

Ribose 5-phosphate isomerase type B (RPI-B) is a key enzyme of the pentose phosphate pathway that catalyzes the isomerization of ribose 5-phosphate (R5P) and ribulose 5-phosphate (Ru5P). Trypanosoma cruzi RPI-B (TcRPI-B) appears to be a suitable drug-target mainly due to: (i) its essentiality (as previously shown in other trypanosomatids), (ii) it does not present a homologue in mammalian genomes sequenced thus far, and (iii) it participates in the production of NADPH and nucleotide/nucleic acid synthesis that are critical for parasite cell survival. In this survey, we report on the competitive inhibition of TcRPI-B by a substrate - analogue inhibitor, Compound B (Ki = 5.5 ± 0.1 µM), by the Dixon method. This compound has an iodoacetamide moiety that is susceptible to nucleophilic attack, particularly by the cysteine thiol group. Compound B was conceived to specifically target Cys-69, an important active site residue. By incubating TcRPI-B with Compound B, a trypsin digestion LC-MS/MS analysis revealed the identification of Compound B covalently bound to Cys-69. This inhibitor also exhibited notable in vitro trypanocidal activity against T. cruzi infective life-stages co-cultured in NIH-3T3 murine host cells (IC50 = 17.40 ± 1.055 µM). The study of Compound B served as a proof-of-concept so that next generation inhibitors can potentially be developed with a focus on using a prodrug group in replacement of the iodoacetamide moiety, thus representing an attractive starting point for the future treatment of Chagas' disease.

Silver nanoparticles alter epithelial basement membrane integrity, cell adhesion molecule expression, and TGF-ß1 secretion.

Silver nanoparticles (AgNPs) are widely used in consumer and pharmaceutical products due to their antipathogenic properties. However, safety concerns have been raised due to their bioactive properties. While reports have demonstrated AgNPs can embed within the extracellular matrix, their effects on basement membrane (BM) production, integrin engagement, and tissue-integrity are not well-defined. This study analyzed the effects of AgNPs on BM production, composition and integrin/focal adhesion interactions in representative lung, esophageal, breast and colorectal epithelia models. A multidisciplinary approach including focused proteomics, QPCR arrays, pathway analyses, and immune-based, structural and functional assays was used to identify molecular and physiological changes in cell adhesions and the BM induced by acute and chronic AgNP exposure. Dysregulated targets included CD44 and transforming growth factor-beta, two proteins frequently altered during pathogenesis. Results indicate AgNP exposure interferes with BM and cell adhesion dynamics, and provide insight into the mechanisms of AgNP-induced disruption of epithelial physiology.

Non-Targeted PFAS Suspect Screening and Quantification of Drinking Water Samples Collected through Community Engaged Research in North Carolina's Cape Fear River Basin.

A community engaged research (CER) approach was used to provide an exposure assessment of poly- and perfluorinated (PFAS) compounds in North Carolina residential drinking water. Working in concert with community partners, who acted as liaisons to local residents, samples were collected by North Carolina residents from three different locations along the Cape Fear River basin: upper, middle, and lower areas of the river. Residents collected either drinking water samples from their homes or recreational water samples from near their residence that were then submitted by the community partners for PFAS analysis. All samples were processed using weak anion exchange (WAX) solid phase extraction and analyzed using a non-targeted suspect screening approach as well as a quantitative approach that included a panel of 45 PFAS analytes, several of which are specific to chemical industries near the collection site locations. The non-targeted approach, which utilized a suspect screening list (obtained from EPA CompTox database) identified several PFAS compounds at a level two confidence rating (Schymanski scale); compounds identified included a fluorinated insecticide, a fluorinated herbicide, a PFAS used in polymer chemistry, and another that is used in battery production. Notably, at several locations, PFOA (39.8 ng/L) and PFOS (205.3 ng/L) were at levels that exceeded the mandatory EPA maximum contaminant level (MCL) of 4 ng/L. Additionally, several sites had detectable levels of PFAS that are unique to a local chemical manufacturer. These findings were communicated back to the community partners who then disseminated this information to the local residents to help empower and aid in making decisions for reducing their PFAS exposure.

A dual-column solid phase extraction strategy for online collection and preparation of continuously flowing effluent streams for mass spectrometry.

Current desalination techniques for mass spectrometry-based protocols are problematic for performing temporal response studies where increased temporal resolution requires small samples and faster sampling frequencies, which greatly increases the number of samples and sample preparation time. These challenges are pertinent to cellular dynamics experiments, where it is important to sample the biological system frequently and with as little sample waste as possible. To address these needs, we present a dual-column online solid phase extraction (SPE) approach capable of preconcentrating and preparing a constantly perfusing sample stream, with minimal to no sample loss. This strategy is evaluated for use in microfluidic bioreactor studies specifically aimed at characterizing suitable sample flow rates, temporal resolving power, and analyte concentrations. In this work, we demonstrate that this strategy may be used for flow rates as low as 500 nL/min, with temporal resolving power on the order of 3 min, with analyte loadings ranging from femtomoles to picomoles for metabolites. Under these conditions, recoveries of ca. 80% are obtained even at femtomole loadings.

Recovery of per- and polyfluoroalkyl substances after solvent evaporation.

Per- and polyfluoroalkyl substances (PFAS) are persistent, bioaccumulative chemicals that can be toxic at very low levels. Many of these compounds have unusual chemical properties that can have a large impact on analytical methods intended to quantitate them. When analyzing environmental samples, concentrating extraction eluents can greatly increase the sensitivity of PFAS extraction and analysis workflows. However, data on PFAS stability when evaporated under vacuum drying conditions are lacking. In this study two common sample preparation methods were replicated (methanol or methanolic ammonium hydroxide) to determine if PFAS material would undergo any observable loss during vacuum evaporation. Standards containing 49 different analytes from 7 different PFAS classes were evaporated to dryness under vacuum either with or without heat and reconstituted using one of two methods. It was found that recovery of some classes (e.g. PFSA, PFESA, FTS) was not greatly impacted by evaporation conditions or reconstitution method. Some analytes such as the very long chain PFCAs were not affected by evaporation conditions but saw drastic differences in recovery depending on the reconstitution method. Others analytes, for example PFSAms, experienced significant loss during evaporation that could not be mitigated by the chosen reconstitution method. This difference could be due to the number of fluorines present on the compound which correlated with a compound's hydrophobicity. Due to these findings, it is recommend that researchers consider PFAS class, chain length, and fluorine number when designing concentration and reconstitution protocols for PFAS to ensure conditions are optimal for the specific analytes of interest.

Monitoring decellularization via absorbance spectroscopy during the derivation of extracellular matrix scaffolds.

Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g. DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the standardization, cost-effectiveness, and scale-up process. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization systems to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor's effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allow for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulationin vitrosuggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.

Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

Glucuronidation controls androgen levels in the prostate and the dysregulation of enzymes in this pathway is associated with castration resistant prostate cancer. UDP-glucose dehydrogenase (UGDH) produces UDP-glucuronate, the essential precursor for glucuronidation, and its expression is elevated in prostate cancer. We compared protein and metabolite levels relevant to the glucuronidation pathway in five prostate cancer patient-derived xenograft models paired with their isogenic counterparts that were selected in vivo for castration resistant (CR) recurrence. All pairs showed changes in UGDH and associated enzymes and metabolites that were consistent with those we found in an isogenic androgen dependent (AD) and CR LNCaP prostate cancer model. Ectopic overexpression of UGDH in LNCaP AD cells blunted androgen-dependent gene expression, increased proteoglycan synthesis, significantly increased cell growth compared to controls, and eliminated dose responsive growth suppression with enzalutamide treatment. In contrast, the knockdown of UGDH diminished proteoglycans, suppressed androgen dependent growth irrespective of androgens, and restored androgen sensitivity in CR cells. Importantly, the knockdown of UGDH in both LNCaP AD and CR cells dramatically sensitized these cells to enzalutamide. These results support a role for UGDH in androgen responsiveness and a target for therapeutic strategies in advanced prostate cancer.

A Dilute and Shoot LC-MS/MS Method for Antipsychotics in Urine.

Adherence to prescribed antipsychotics is an ongoing problem. Traditionally, estimates of adherence have been made from patient interviews, pill counting and blood testing. A number of methods for the analysis of antipsychotics in blood have been reported for both therapeutic drug monitoring and postmortem testing for toxicity. This report details a dilute and shoot method for the analysis of 19 different antipsychotics and metabolites. The method takes advantage of earlier reports demonstrating unique, prevalent urine metabolites for aripiprazole, brexpiprazole, haloperidol and lurasidone to enhance sensitivity for these analytes. With a fast analysis time and minimal sample preparation, this method can be used for quantitation of antipsychotics in urine. Finally, this method has been used to test samples for over a year with the results summarized in this report. While further improvements are certainly possible, this method is selective and sensitive for this group of important compounds.

Fast Automated Approach for the Derivation of Acellular Extracellular Matrix Scaffolds from Porcine Soft Tissues.

Decellularized extracellular matrix (ECM) scaffolds derived from tissues and organs are complex biomaterials used in clinical and research applications. A number of decellularization protocols have been described for ECM biomaterials derivation, each adapted to a particular tissue and use, restricting comparisons among materials. One of the major sources of variability in ECM products comes from the tissue source and animal age. Although this variability could be minimized using established tissue sources, other sources arise from the decellularization process itself. Overall, current protocols require manual work and are poorly standardized with regard to the choice of reagents, the order by which they are added, and exposure times. The combination of these factors adds variability affecting the uniformity of the final product between batches. Furthermore, each protocol needs to be optimized for each tissue and tissue source making tissue-to-tissue comparisons difficult. Automation and standardization of ECM scaffold development constitute a significant improvement to current biomanufacturing techniques but remains poorly explored. This study aimed to develop a biofabrication method for fast and automated derivation of raw material for ECM hydrogel production while preserving ECM composition and controlling lot-to-lot variability. The main result was a closed semibatch bioreactor system with automated dosing of decellularization reagents capable of deriving ECM material from pretreated soft tissues. The ECM was further processed into hydrogels to demonstrate gelation and cytocompatibility. This work presents a versatile, scalable, and automated platform for the rapid production of ECM scaffolds.

Porcine Vocal Fold Lamina Propria-Derived Biomaterials Modulate TGF-ß1-Mediated Fibroblast Activation in Vitro.

The vocal fold lamina propria (VFLP), one of the outermost layers of the vocal fold (VF), is composed of tissue-specific extracellular matrix (ECM) proteins and is highly susceptible to injury. Various biomaterials have been clinically tested to treat voice disorders (e.g., hydrogels, fat, and hyaluronic acid), but satisfactory recovery of the VF functionality remains elusive. Fibrosis or scar formation in the VF is a major challenge, and the development and refinement of novel therapeutics that promote the healing and normal function of the VF are needed. Injectable hydrogels derived from native tissues have been previously reported with major advantages over synthetic hydrogels, including constructive tissue remodeling and reduced scar tissue formation. This study aims to characterize the composition of a decellularized porcine VFLP-ECM scaffold and the cytocompatibility and potential antifibrotic properties of a hydrogel derived from VFLP-ECM. In addition, we isolated potential matrix-bound vesicles (MBVs) and macromolecules from the VFLP-ECM that also downregulated smooth muscle actin ACTA2 under transforming growth factor-beta 1 (TGF-ß1) stimulation. The results provide evidence of the unique protein composition of the VFLP-ECM and the potential link between the components of the VFLP-ECM and the inhibition of TGF-ß1 signaling observed in vitro when transformed into injectable forms.

Chiral and structural analysis of biomolecules using mass spectrometry and ion mobility-mass spectrometry.

This report describes the strategies for gas-phase chiral and structural characterization of biomolecules using mass spectrometry (MS) and ion mobility-MS (IM-MS) techniques. Because both MS and IM-MS do not directly provide chiral selectivity, methodologies for adding a chiral selector are discussed in the context of (i) host-guest (H-G) associations, (ii) diastereomeric collision-induced dissociation (CID) methods, (iii) ion-molecule reactions, and (iv) the kinetic method. MS techniques for the analysis of proteins and protein complexes are briefly described. New advances in performing rapid 2D gas-phase separations on the basis of IM-MS are reviewed with a particular emphasis on the different forms of IM instrumentation and how they are used for chiral and/or structural biomolecular studies. This report is not intended to be a comprehensive review of the field, but rather to underscore the contemporary techniques that are commonly or increasingly being used to complement measurements performed by chiroptical methodologies.

Experimental data demonstrating the effects of silver nanoparticles on basement membrane gene and protein expression in cultured colon, mammary and bronchial epithelia.

This data article is related to the research article entitled "Silver nanoparticles alter epithelial basement membrane integrity, cell adhesion molecule expression and TGF-beta secretion", available in the journal Nanomedicine: Nanotechnology, Biology, and Medicine [1]. This Data in Brief consists of data that describe changes in the expression of basement membrane (BM)-associated genes and proteins in three non-transformed epithelial cell lines following acute (6 h) and chronic (24 h plus 7-day chase) exposure to silver nanoparticles (AgNPs). Human BEAS2B (lung), MCF10AI (breast), and CCD-18Co (colon) cultured epithelia were analyzed for protein expression by LC-MS/MS and for gene expression by pathway-focused QRT-PCR arrays of 168 focal adhesion, integrin, and extracellular matrix (ECM) genes known to be localized to the plasma membrane, the BM/ECM, or secreted into the extracellular space. Ingenuity pathway analysis (IPA) of combined gene and protein expression datasets was then used to predict canonical pathways affected by AgNP exposure.

Machine Learning Reveals Protein Signatures in CSF and Plasma Fluids of Clinical Value for ALS.

We use shotgun proteomics to identify biomarkers of diagnostic and prognostic value in individuals diagnosed with amyotrophic lateral sclerosis. Matched cerebrospinal and plasma fluids were subjected to abundant protein depletion and analyzed by nano-flow liquid chromatography high resolution tandem mass spectrometry. Label free quantitation was used to identify differential proteins between individuals with ALS (n = 33) and healthy controls (n = 30) in both fluids. In CSF, 118 (p-value < 0.05) and 27 proteins (q-value < 0.05) were identified as significantly altered between ALS and controls. In plasma, 20 (p-value < 0.05) and 0 (q-value < 0.05) proteins were identified as significantly altered between ALS and controls. Proteins involved in complement activation, acute phase response and retinoid signaling pathways were significantly enriched in the CSF from ALS patients. Subsequently various machine learning methods were evaluated for disease classification using a repeated Monte Carlo cross-validation approach. A linear discriminant analysis model achieved a median area under the receiver operating characteristic curve of 0.94 with an interquartile range of 0.88-1.0. Three proteins composed a prognostic model (p = 5e-4) that explained 49% of the variation in the ALS-FRS scores. Finally we investigated the specificity of two promising proteins from our discovery data set, chitinase-3 like 1 protein and alpha-1-antichymotrypsin, using targeted proteomics in a separate set of CSF samples derived from individuals diagnosed with ALS (n = 11) and other neurological diseases (n = 15). These results demonstrate the potential of a panel of targeted proteins for objective measurements of clinical value in ALS.

Impact of ß-Glucuronidase Mediated Hydrolysis on Haldol® Urinalysis.

Reports have suggested that patients with mental health disorders including major depressive disorder and schizophrenia have dramatically low adherence levels to prescribed medications. Patients on haloperidol (Haldol®) therapy, regardless of their disease, were found to have higher adherence levels-though still strikingly low. This work shows that high levels of the glucuronidated form of haloperidol are present in patient urine samples. Time-of-Flight (TOF) mass spectrometry experiments are consistent with both the presence of haloperidol glucuronide and that hydrolysis of haloperidol patient urine samples leads to significantly increased concentrations of free haloperidol. Urine samples collected from patients prescribed haloperidol were tested with and without hydrolysis revealing a significant increase in the number of patients testing positive when the samples were hydrolyzed before analysis. These data demonstrate that hydrolysis greatly improves the sensitivity and consistency of results for patients on haloperidol therapy resulting in positivity data that strongly correlates with the dosage form administered.

Analytical Considerations When Developing an LC-MS/MS Method for More than 30 Analytes.

BACKGROUND: While validation of analytical (LC-MS/MS) methods has been documented in any number of articles and reference texts, the optimal design and subsequent validation of a method for over 30 analytes presents special challenges. Conventional approaches to calibration curves, controls, and run time are not tenable in such methods. This report details the practical aspects of designing and implementing such a method in accordance with College of American Pathologists validation criteria. METHODS: Conventional criteria were followed in the design and validation of a method for 34 analytes and 15 internal standards by LC-MS/MS. These criteria are laid out in a standard operating procedure, which is followed without exception and is consistent with College of American Pathologists criteria. RESULTS: The method presented herein provides quality results and accurate medication monitoring. The method was optimized to negate interferences (both from within the method and from potential concomitant compounds), increase throughput, and provide reproducible quality quantification over relevant analyte concentrations ranges. CONCLUSIONS: The method was designed primarily with quality and accurate medication monitoring in mind. The method achieves these goals by use of novel approaches to calibration curves and controls that both improve performance and minimize risk (financial and operational). As automation and LC-MS/MS equipment continue to improve, it is expected that more methods like this one will be developed.

Normalizing Oral Fluid Hydrocodone Data Using Calculated Blood Volume.

Oral fluid testing to assist in the assessment of treatment adherence for chronic pain patients is attractive for a number of reasons. However, efforts focused on interpreting patient results have been modest when compared to urine drug testing. This work details a retrospective approach developed to transform and normalize oral fluid testing results to provide a historical picture of patient values in this important test fluid. Using this approach, a model was developed using data from 6,800 independent patients who were both prescribed hydrocodone and tested positive (with limitations: reporting cutoff < X < upper limit of quantitation) by liquid chromatography-mass spectrometry. Patient demographic data were used to calculate the relevant parameters (e.g., calculated blood volume (CBV)) used in the transformation and normalization of the oral fluid data. The crucial normalizing factor in oral fluids was found to be the CBV which parallels the use of creatinine to normalize drug concentration levels in urine and is consistent with the view that oral fluid samples reflect plasma concentrations of the respective drugs. The resulting near Gaussian distribution is dose independent and as such should be of value to physicians in quickly assessing whether their patient is consistent with this historical population in the broad terms of this model. While this comparison alone is not definitive for adherence with a treatment regimen, together with patient interviews, prescription history and other clinical criteria, it can add an idea of expected patient values from oral fluid testing.