Quantifying the bitter masking effect of drug-cyclodextrin complexation: NMR-ROESY mixing time approach.

Taste, especially unpleasant taste, can be key for patient compliance. In the formulation development process, drug-cyclodextrin (CD) inclusion complexes are often used to improve the solubility of a drug and/or mask its bitterness. This study aimed to evaluate the bitter masking effect of CDs on different drugs using NMR-ROESY analysis, human sensory tests, and e-tongue measurements. The strength of inclusion complex formation between drugs and CDs was investigated by NMR-ROSEY, and these results were compared to human sensory test results. In the sensory test, participants identified which drug-CD inclusion complexes were not bitter. NMR-ROSEY results aligned with the sensory tests; short magnetization transfer times corresponded to masked bitterness. The electrical tongue was not able to detect the taste of any of the drug-CD inclusion complexes. Additionally, we used NMR-ROSEY to determine which drug-CD inclusion complex formed in a system with multiple drug substances present. This research offers valuable insights into the bitter masking effect of CDs on different drugs and presents a comprehensive evaluation approach using various methods. This knowledge has significant implications for the pharmaceutical industry, clinical practice, and patient care, contributing to improved patient compliance and satisfaction with bitter medications.

Predicting Influenza and Rhinovirus Infections in Airway Cells Utilizing Volatile Emissions.

BACKGROUND: Respiratory viral infections are common and potentially devastating to patients with underlying lung disease. Diagnosing viral infections often requires invasive sampling, and interpretation often requires specialized laboratory equipment. Here, we test the hypothesis that a breath test could diagnose influenza and rhinovirus infections using an in vitro model of the human airway. METHODS: Cultured primary human tracheobronchial epithelial cells were infected with either influenza A H1N1 or rhinovirus 1B and compared with healthy control cells. Headspace volatile metabolite measurements of cell cultures were made at 12-hour time points postinfection using a thermal desorption-gas chromatography-mass spectrometry method. RESULTS: Based on 54 compounds, statistical models distinguished volatile organic compound profiles of influenza- and rhinovirus-infected cells from healthy counterparts. Area under the curve values were 0.94 for influenza, 0.90 for rhinovirus, and 0.75 for controls. Regression analysis predicted how many hours prior cells became infected with a root mean square error of 6.35 hours for influenza- and 3.32 hours for rhinovirus-infected cells. CONCLUSIONS: Volatile biomarkers released by bronchial epithelial cells could not only be used to diagnose whether cells were infected, but also the timing of infection. Our model supports the hypothesis that a breath test could serve to diagnose viral infections.

SPME-based mobile field device for active sampling of volatiles.

Monitoring plant volatile organic compound (VOC) profiles can reveal information regarding the health state of the plant, such as whether it is nutrient stressed or diseased. Typically, plant VOC sampling uses sampling enclosures. Enclosures require time and equipment which are not easily adapted to high throughput sampling in field environments. We have developed a new, easily assembled active sampling device using solid phase microextraction (SPME) that uses a commercial off the shelf (COTS) hand vacuum base to provide rapid and easy mobile plant VOC collection. Calibration curves for three representative plant VOCs (α-pinene, limonene, and ocimene) were developed to verify device functionality and enable the quantification of field-samples from a Meyer lemon tree. We saw that the active sampling allowed us to measure and quantify this chemical in an orchard setting. This device has the potential to be used for VOC sampling as a preliminary diagnostic in precision agriculture applications due to its ease of manufacturing, availability, and low cost of the COTS hand vacuum module.

Bacteria isolated from Bengal cat (Felis catus × Prionailurus bengalensis) anal sac secretions produce volatile compounds potentially associated with animal signaling.

In social animals, scent secretions and marking behaviors play critical roles in communication, including intraspecific signals, such as identifying individuals and group membership, as well as interspecific signaling. Anal sacs are an important odor producing organ found across the carnivorans (species in the mammalian Order Carnivora). Secretions from the anal sac may be used as chemical signals by animals for behaviors ranging from defense to species recognition to signaling reproductive status. In addition, a recent study suggests that domestic cats utilize short-chain free fatty acids in anal sac secretions for individual recognition. The fermentation hypothesis is the idea that symbiotic microorganisms living in association with animals contribute to odor profiles used in chemical communication and that variation in these chemical signals reflects variation in the microbial community. Here we examine the fermentation hypothesis by characterizing volatile organic compounds (VOC) and bacteria isolated from anal sac secretions collected from a male Bengal cat (Felis catus × Prionailurus bengalensis), a cross between the domestic cat and the leopard cat. Both left and right anal sacs of a male Bengal cat were manually expressed (emptied) and collected. Half of the material was used to culture bacteria or to extract bacterial DNA and the other half was used for VOC analysis. DNA was extracted from the anal sac secretions and used for a 16S rRNA gene PCR amplification and sequencing based characterization of the microbial community. Additionally, some of the material was plated out in order to isolate bacterial colonies. Three taxa (Bacteroides fragilis, Tessaracoccus, and Finegoldia magna) were relatively abundant in the 16S rRNA gene sequence data and also isolated by culturing. Using Solid Phase Microextraction (SPME) gas chromatography-mass spectrometry (GC-MS), we tentatively identified 52 compounds from the Bengal cat anal sac secretions and 67 compounds from cultures of the three bacterial isolates chosen for further analysis. Among 67 compounds tentatively identified from bacterial isolates, 51 were also found in the anal sac secretion. We show that the bacterial community in the anal sac consists primarily of only a few abundant taxa and that isolates of these taxa produce numerous volatiles that are found in the combined anal sac volatile profile. Several of these volatiles are found in anal sac secretions from other carnivorans, and are also associated with known bacterial biosynthesis pathways. This is consistent with the fermentation hypothesis and the possibility that the anal sac is maintained at least in part to house bacteria that produce volatiles for the host.

Volatile organic compound (VOC) emissions of CHO and T cells correlate to their expansion in bioreactors.

Volatile organic compound (VOC) emissions were measured from Chinese Hamster Ovary (CHO) cell and T cell bioreactor gas exhaust lines with the goal of non-invasively metabolically profiling the expansion process. Measurements of cellular 'breath' were made directly from the gas exhaust lines using polydimethylsiloxane (PDMS)-coated magnetic stir bars, which underwent subsequent thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) analysis. Baseline VOC profiles were observed from bioreactors filled with only liquid media. After inoculation, unique VOC profiles correlated to cell expansion over the course of 8 d. Partial least squares (PLS) regression models were built to predict cell culture density based on VOC profiles of CHO and T cells (R 2 = 0.671 and R 2 = 0.769, respectively, based on a validation data set). T cell runs resulted in 47 compounds relevant to expansion while CHO cell runs resulted in 45 compounds; the 20 most relevant compounds of each cell type were putatively identified. On the final experimental days, sorbent-covered stir bars were placed directly into cell-inoculated media and into media controls. Liquid-based measurements from spent media containing cells could be distinguished from media-only controls, indicating soluble VOCs excreted by the cells during expansion. A PLS-discriminate analysis (PLS-DA) was performed, and 96 compounds differed between T cell-inoculated media and media controls with 72 compounds for CHO cells; the 20 most relevant compounds of each cell line were putatively identified. This work demonstrates that the volatilome of cell cultures can be exploited by chemical detectors in bioreactor gas and liquid waste lines to non-invasively monitor cellular health and could possibly be used to optimize cell expansion conditions 'on-the-fly' with appropriate control loop systems. Although the basis for statistical models included compounds without certain identification, this work provides a foundation for future research of bioreactor emissions. Future studies must move towards identifying relevant compounds for understanding of underlying biochemistry.

Modeling cellular metabolomic effects of oxidative stress impacts from hydrogen peroxide and cigarette smoke on human lung epithelial cells.

The respiratory system is continuously exposed to variety of biological and chemical irritants that contain reactive oxygen species, and these are well known to cause oxidative stress responses in lung epithelial cells. There is a clinical need to identify biomarkers of oxidative stress which could potentially support early indicators of disease and health management. To identify volatile biomarkers of oxidative stress, we analyzed the headspace above human bronchial epithelial cell cultures (HBE1) before and after hydrogen peroxide (H2O2) and cigarette smoke extract (CSE) exposure. Using stir bar and headspace sorptive extraction-gas chromatography-mass spectrometry, we searched for volatile organic compounds (VOC) of these oxidative measures. In the H2O2 cell peroxidation experiments, four different H2O2 concentrations (0.1, 0.5, 10, 50 mM) were applied to the HBE1 cells, and VOCs were collected every 12 h over the time course of 48 h. In the CSE cell peroxidation experiments, four different smoke extract concentrations (0%, 10%, 30%, 60%) were applied to the cells, and VOCs were collected every 12 h over the time course of 48 h. We used partial-least squares (PLS) analysis to identify putative compounds from the mass spectrometry results that highly correlated with the known applied oxidative stress. We observed chemical emissions from the cells that related to both the intensity of the oxidative stress and followed distinct time courses. Additionally, some of these chemicals are aldehydes, which are thought to be non-invasive indicators of oxidative stress in exhaled human breath. Together, these results illustrate a powerful in situ cell culture model of oxidative stress that can be used to explore the putative biological genesis of exhaled breath biomarkers that are often observed in human clinical studies.

Headspace sorptive extraction-gas chromatography-mass spectrometry method to measure volatile emissions from human airway cell cultures.

The human respiratory tract releases volatile metabolites into exhaled breath that can be utilized for noninvasive health diagnostics. To understand the origin of this metabolic process, our group has previously analyzed the headspace above human epithelial cell cultures using solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). In the present work, we improve our model by employing sorbent-covered magnetic stir bars for headspace sorptive extraction (HSSE). Sorbent-coated stir bar analyte recovery increased by 52 times and captured 97 more compounds than SPME. Our data show that HSSE is preferred over liquid extraction via stir bar sorptive extraction (SBSE), which failed to distinguish volatiles unique to the cell samples compared against media controls. Two different cellular media were also compared, and we found that Opti-MEM® is preferred for volatile analysis. We optimized HSSE analytical parameters such as extraction time (24 h), desorption temperature (300 °C) and desorption time (7 min). Finally, we developed an internal standard for cell culture VOC studies by introducing 842 ng of deuterated decane per 5 mL of cell medium to account for error from extraction, desorption, chromatography and detection. This improved model will serve as a platform for future metabolic cell culture studies to examine changes in epithelial VOCs caused by perturbations such as viral or bacterial infections, opening opportunities for improved, noninvasive pulmonary diagnostics.

Effects of Phytophthora ramorum on volatile organic compound emissions of Rhododendron using gas chromatography-mass spectrometry.

Phytophthora ramorum is an invasive and devastating plant pathogen that causes sudden oak death in coastal forests in the western United States and ramorum blight in nursery ornamentals and native plants in various landscapes. As a broad host-range quarantine pest that can be asymptomatic in some hosts, P. ramorum presents significant challenges for regulatory efforts to detect and contain it, particularly in commercial nurseries. As part of a program to develop new detection methods for cryptic infections in nursery stock, we compared volatile emissions of P. ramorum-inoculated and noninoculated Rhododendron plants using three gas chromatography-mass spectrometry methods. The first used a branch enclosure combined with headspace sorptive extraction to measure plant volatiles in situ. Seventy-eight compounds were found in the general Rhododendron profile. The volatile profile of inoculated but asymptomatic plants (121 days post-inoculation) was distinguishable from the profile of the noninoculated controls. Three compounds were less abundant in inoculated Rhododendron plants relative to noninoculated and mock-inoculated control plants. A second method employed stir bar sorptive extraction to measure volatiles in vitro from leaf extractions in methanol; 114 volatiles were found in the overall profile with 30 compounds less abundant and one compound more abundant in inoculated Rhododendron plants relative to mock-inoculated plants. At 128 days post-inoculation, plants were asymptomatic and similar in appearance to the noninoculated controls, but their chemical profiles were different. In a third technique, volatiles from water runoff from the soil of potted healthy and inoculated Rhododendron plants were compared. Runoff from the inoculated plants contained four unique volatile compounds that never appeared in the runoff from mock-inoculated plants. These three volatile detection techniques could lead to innovative approaches that augment detection and diagnosis of P. ramorum and oomycete pathogens in nurseries and other settings. Graphical abstract Detection of volatile signatures may aid in discriminating healthy vs. infected but asymptomatic plants in nursery and greenhouse facilities.