An investigation of temperature and fever burdens in patients with sepsis admitted from the emergency department to the hospital.

Aim: We sought to collect granular data on temperature burden to further explore existing conflicting information on the relationship between temperature alterations and outcomes in patients with sepsis requiring hospital admission. Methods: This was a prospective cohort study that enrolled a convenience sample of patients with sepsis or septic shock admitted to the hospital from the emergency department (ED). A "unit of temperature burden (UTB)" was defined as >1°C (1.8°F) above or below 37°C (98.6°F) for 1 min. Fever burden was defined as the number of UTBs >38°C (100.4°F). The primary objective was to calculate the fever burden in patients with sepsis during their ED stay. This was analyzed for patients who present to triage febrile or hypothermic and also for those who developed temperature abnormalities during their ED stay. The secondary objectives were correlating fever and hypothermia burden with in-hospital mortality, Systemic Inflammatory Response Syndrome (SIRS) criteria, and the quick Sequential (Sepsis-Associated) Organ Failure Assessment (qSOFA) score and identification of patients who may benefit from early implementation of targeted temperature management. Results: A total of 256 patients met the inclusion criteria. The mean age of patients was 60.1 ± 18.4 years; 46% were female and 29.6% were black. The median (interquartile range [IQR]) fever burden for the fever in triage cohort (n = 99) was 364.6 (174.3-716.8) UTB and for the no fever in triage cohort (n = 157) was 179.3 (80.9-374.0) UTB (p = 0.005). The two groups had similar in-hospital mortality (6.1 vs 8.3%; p = 0.5). The median fever burden for the fever anytime cohort was 303.8 (IQR 138.8-607.9) UTB and they had lower mortality than the no fever anytime cohort (4.7% vs 11.2%; p = 0.052). Patients with fever at triage had higher mean SIRS criteria than those without (2.8 vs 2.0; p < 0.001) while qSOFA points were similar (p = 0.199). A total of 27 patients had hypothermia during their ED stay and these patients were older with higher mean SIRS criteria. Conclusions: Patients with sepsis and septic shock have a significant temperature burden in the ED. When comparing patients who had fever at any time during their ED stay with those who never had a fever, a trend toward an inverse relationship between fever burden and mortality was found.

A Diagnostic Nitrosamine Detection Approach for Pharmaceuticals by Using Tandem Mass Spectrometry Based on Diagnostic Gas-Phase Ion-Molecule Reactions.

N-Nitrosamines are strictly regulated in pharmaceutical products due to their carcinogenic nature. Therefore, the ability to rapidly and reliably identify the N-nitroso functionality is urgently needed. Unfortunately, not all ionized N-nitroso compounds produce diagnostic fragment ions and hence tandem mass spectrometry based on collision-activated dissociation (CAD) cannot be used to consistently identify the N-nitroso functionality. Therefore, a more reliable method was developed based on diagnostic functional-group selective ion-molecule reactions in a linear quadrupole ion trap mass spectrometer. 2-Methoxypropene (MOP) was identified as a reagent that reacts with protonated N-nitrosamines in a diagnostic manner by forming an adduct followed by the elimination of 2-propenol (CH3C(OH)═CH2]). From 18 protonated N-nitrosamine model compounds studied, 15 formed the diagnostic product ion. The lack of the diagnostic reaction for three of the N-nitrosamine model compounds was rationalized based on the presence of a pyridine ring that gets preferentially protonated instead of the N-nitroso functionality. These N-nitrosamines can be identified by subjecting a stable adduct formed upon ion-molecule reactions with MOP to CAD. Further, the ability to use ion-molecule reactions followed by CAD to differentiate protonated O-nitroso compounds with a pyridine ring from analogous N-nitrosamines was demonstrated This methodology is considered to be robust for the identification of the N-nitroso functionality in unknown analytes. Lastly, HPLC/MS2 experiments were performed to determine the detection limit for five FDA regulated N-nitrosamines.

Enabling online determination of the size-dependent RNA content of lipid nanoparticle-based RNA formulations.

The potential of lipid nanoparticles (LNPs) as nucleic acid delivery vehicles has been demonstrated in recent years, culminating in the emergency use approval of LNP-based mRNA SARS-CoV-2 vaccines in late 2020. The determination of RNA content relative to LNP size can be important to the understanding of efficacy and adverse effects. This work presents the first description of a facile and rapid analytical method for online, size-dependent RNA payload distribution measurement using data from multi-angle light scattering, ultraviolet and refractive index detectors following separation of the LNPs by size-exclusion chromatography. The analysis was validated by size-based fractionation of the LNPs with subsequent offline analysis of the fractions. Four LNPs formulated with different PEG-lipids and different lipid compositions were tested. Good agreement was observed between the online and offline size-based RNA distributions among all four LNPs, demonstrating the utility of the online method for LNP-encapsulated RNA in general, and suggesting a means for simplified biophysical quantitation of a dosing-related critical quality attribute.

MULTIMODAL IMAGING OF MACULAR CHOROIDAL MACROVESSEL: A REPORT OF TWO CASES.

PURPOSE: To report two cases of macular choroidal macrovessel with description of multimodal imaging and review of published cases. METHODS: Medical and imaging records were retrospectively reviewed. A literature review was performed to identify other cases of macular choroidal macrovessel published between 1990 and 2018. RESULTS: There were 2 patients referred for evaluation of a potential choroidal tumor, including a 55-year-old white woman with no visual symptoms and a 68-year-old white woman with blurred vision. Funduscopic examination in each case revealed a focal area of choroidal elevation in the temporal foveal area with a single, dilated, slightly tortuous choroidal vessel, estimated to measure 250 µm and 300 µm, respectively, at widest girth, and extending temporally toward the equator with a tapering width. Optical coherence tomography showed an optically hollow choroidal lesion elevating the retinal pigment epithelium with overlying ellipsoid zone mottling. Both cases demonstrate shallow subretinal fluid, sparing the foveola. Fluorescein angiography showed faint staining along the course of the vessel in both cases. Indocyanine green angiography demonstrated clear visualization of the ectatic vessel with early filling and late staining without leakage. Both patients retained visual acuity of 20/25 in the affected eye and did not require treatment. CONCLUSION: Macular choroidal macrovessel is a rare vascular anomaly presenting as a single, dilated, tortuous choroidal vessel, originating in the temporal foveal region and extending temporally to the equator. This finding can simulate a choroidal neoplasm or parasitic track. Optical coherence tomography and indocyanine green angiography have diagnostic value demonstrating the vascular nature of this lesion and delineating the vascular course.

Endoscopic endonasal resection of orbital schwannoma assisted with small-incision medial orbitotomy: case series and surgical technique.

PURPOSE: To describe a surgical approach for the resection of schwannomas occurring in the medial aspect of the orbit and to review a series of patients who underwent this novel technique. METHODS: This retrospective, non-comparative case series presents the surgical technique and outcomes of patients who underwent removal of a medial orbital schwannoma via an endoscopic endonasal approach combined with a small-incision medial orbitotomy by a team of two surgeons (BSB and SKF). Patient demographics, pre- and post-operative clinical examination findings, visual field testing, and radiographic studies were reviewed. Operative reports were reviewed for technical details and complications. RESULTS: The patients included a 12 year-old male, 73 year-old female and 8 year-old male. Indications for surgery included: decreased visual acuity, diplopia, proptosis and Humphrey visual field (HVF) deficit, in the presence of a medial orbital biopsy-proven schwannoma. The surgical approach in all three patients was primarily endoscopic endonasal. Additionally, two had transcaruncular orbitotomies and one had a small-incision medial lid crease orbitotomy to assist with lateral tumor dissection. Tumor resection was complete in one case and near-total in two cases. There were no intra-operative surgical complications. Average resected specimen volume was 3.41 cm3 ± 2.20. All patients had post-operative improvement in visual acuity (VA) and proptosis. Post-operative follow-up intervals were 27.5 months, 12.3 months and 3.5 months, respectively. CONCLUSION: Resection of orbital schwannomas using an endoscopic endonasal approach with small-incision medial transorbital assistance is a safe and effective option for a multidisciplinary surgical team.

Integration of Precipitation Kinetics From an In Vitro, Multicompartment Transfer System and Mechanistic Oral Absorption Modeling for Pharmacokinetic Prediction of Weakly Basic Drugs.

Solubility, dissolution, and precipitation in the gastrointestinal tract can be critical for the oral bioavailability of weakly basic drugs. To understand the dissolution and precipitation during the transfer out of the stomach into the intestine, a multicompartment transfer system was developed by modifying a conventional dissolution system. This transfer system included gastric, intestinal, sink and supersaturation, and reservoir compartments. Simulated gastric fluid and fasted state simulated intestinal fluid were used in the gastric and intestinal compartment, respectively, to mimic fasted condition. The new transfer system was evaluated based on 2 model weak bases, dipyridamole and ketoconazole. Traditional 2-stage dissolution using 250 mL of simulated gastric fluid media, followed by 250 mL of fasted state simulated intestinal fluid, was used as a reference methodology to compare dissolution and precipitation results. An in silico model was built using R software suite to simulate the in vitro time-dependent dissolution and precipitation process when formulations were tested using the transfer system. The precipitation rate estimated from the in vitro data was then used as the input for absorption and pharmacokinetic predictions using GastroPlus. The resultant simulated plasma concentration profiles were generally in good agreement with the observed clinical data, supporting the translatability of the transfer system in vitro precipitation kinetics to in vivo.

Botryoid Rhabdomyosarcoma of the Conjunctiva in a Young Boy.

Primary conjunctival rhabdomyosarcoma (RMS) is a rare entity that can present with or without papillomatous features. A 5-year-old Asian boy was referred for a rapidly growing conjunctival tumor in the superior fornix of the left eye. Surgical excision yielded a 28 mm multilobulated papillomatous specimen that exhibited histopathologic and immunohistochemical features consistent with embryonal (botryoid) RMS. Molecular analysis revealed the absence of the PAX3/FOXO1 fusion gene, indicating favorable prognosis. After surgery, he was promptly treated with systemic chemotherapy and proton beam radiotherapy.

Development of a directly correlated Raman and uHPLC-MS content uniformity method for dry powder inhalers through statistical design, chemometrics and mathematical modeling.

CONTEXT: Content uniformity (CU) is a critical quality attribute measured and monitored throughout the development and commercial supply of pharmaceutical products. Traditional high-performance liquid chromatography (HPLC) methods are time-consuming in both sample preparation and analysis. Thus, a rapid, nondestructive and preparation free spectroscopy based method such as Raman is preferred. OBJECTIVE: Multiple mathematical algorithms were used to establish robust and directly correlated Raman and ultra-HPLC-mass spectrometry (uHPLC-MS) CU methods for the rapid analysis of blends and agglomerates formulated for dry powder inhalers (DPIs). MATERIALS AND METHODS: Model samples included blends of caffeine and lactose; albuterol and lactose; and albuterol and lactose agglomerates. Design of experiments (DoE) was employed to optimize Raman spectra. Multivariate curve resolution (MCR) was leveraged to assess Raman method robustness. Mathematical modeling provided direct method to method correlation by allowing samples to be scanned first for Raman spectra and then dissolved for uHPLC-MS analysis. Several chemometric models were developed and evaluated for the quantitative analysis of CU. RESULTS: The DoE revealed Raman power and exposure time were negatively correlated when optimizing albuterol and caffeine spectra but positively correlated for lactose. MCR revealed regions in which small changes to power and time resulted in an 8-10% change in concentration predictions. A PCR model worked well for the analysis of caffeine blend samples and a PLS model worked best for both albuterol blends and agglomerates. DISCUSSION AND CONCLUSION: Utilization of DoE, chemometrics and mathematical modeling provided a robust and directly correlated CU method for DPIs.

A canine biorelevant dissolution method for predicting in vivo performance of orally administered sustained release matrix tablets.

Preclinical species are a crucial component of drug development, but critical differences in physiology and anatomy need to be taken into account when attempting to extrapolate to humans or between species. The same is true when trying to develop oral formulations for preclinical species, especially unconventional formulations, such as sustained release tablets. During the evaluation of such specialized dosage forms, dissolution can be a critical in vitro tool used to rank-order formulations and ultimately choose the desired release rate. Here, the development of a canine biorelevant dissolution method for the prediction of the in vivo performance of sustained release matrix tablets in beagle dogs is described. The method accounts for differences in physiology between humans and dogs such as gastrointestinal fluid composition, gastric emptying forces, and gastric residence time. The most critical dissolution method parameters were found to be the paddle speed used to simulate the gastric emptying forces as well as the time spent in simulated gastric fluid. The resulting differences in method conditions are further explored through in silico models of the hydrodynamic forces applied to a dosage form. Two case studies are reported showing that the method was able to obtain excellent in vitro-in vivo relationships (slopes ranging from 1.08-1.01) which are significantly (p < 0.01-0.05) improved compared to human biorelevant dissolution used to predict in vivo performance in humans (slopes ∼1.5-1.75). The quality of the method's predictive ability allows for it to help drive the development of matrix sustained release formulations intended for preclinical studies.

Development of an LC-MS method for ultra trace-level determination of 2,2,6,6-tetramethylpiperidine-1-oxl (TEMPO), a potential genotoxic impurity within active pharmaceutical ingredients.

TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable free radical which has been widely used for various research and industrial applications, including the manufacture of many active pharmaceutical ingredients. TEMPO has been identified as a potential genotoxic impurity resulting in the need for analytical methodology to accurately determine its level at several orders of magnitude less than typical impurity quantitation limits. TEMPO can undergo disproportionation to form both oxidized and reduced TEMPO, making individual determination unreliable. To overcome this challenge, all TEMPO related species were converted to the reduced form through reduction with sodium ascorbate. Given the ultra-trace (0.5 ppm) level requirements and the lack of UV response in the reduced form, a single quadrupole mass spectrometer (MS) was utilized. In order to implement a highly sensitive MS method in a GMP environment, several approaches were employed to optimize accuracy and robustness including: internal standard correction for drift elimination, six-level standard addition to reduce matrix effects, and weighted linear regression to cover a broad analytical range. The method was fully validated according to ICH guidelines. The method is specific, linear, accurate, precise, and robust within a range of 0.5-100 ppm.

A uHPLC-MS mathematical modeling approach to dry powder inhaler single agglomerate analysis.

Demonstration of content uniformity (CU) is critical toward the successful development of dry powder inhalers (DPIs). Methods for unit dose CU determination for DPI products are well-established within the field of respiratory science. Recent advances in the area include a uHPLC-MS method for high-throughput uniformity analysis, which allows for a greater understanding of blending operations as the industry transitions to a quality-by-design approach to development. Further enhancements to this uHPLC-MS method now enable it to determine CU and sample weight at the single agglomerate level, which is roughly 50× smaller than a unit dose. When coupled with optical microscopy-based agglomerate sizing, the enhanced uHPLC-MS method can also predict the density and porosity of individual agglomerates. Expanding analytical capabilities to the single agglomerate level provides greater insights and confidence in the DPI manufacturing process.

Development of a high-throughput UHPLC-MS-based content uniformity method as a tool for assessing dry powder inhalers.

BACKGROUND: Dry powder inhaler (DPI) product manufacturing requires the assessment of uniformity at various stages of the manufacturing process. RESULTS: To efficiently and precisely determine the uniformity of the small doses inherent to DPI technology, an ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS)-based content uniformity method was developed. Using mathematical modeling and proper selection of bracketing standards, a volumetric approximation of sample weight was utilized, eliminating the need for accurate sample weights and reducing sample preparation time. CONCLUSION: UHPLC-MS coupled with mathematical modeling makes high-throughput CU testing of DPI drug products possible which allows for an enhanced understanding of the manufacturing process.

Spray pattern and droplet size analyses for high-shear viscosity determination of aqueous suspension corticosteroid nasal sprays.

Aqueous suspension corticosteroid nasal sprays exhibit the rheological property of shear thinning, meaning they exhibit a decrease in viscosity upon application of shear. Most rheological methods are limited in the amount of shear that can be applied to samples (approximately 1,000 s(-1)) and thus can only approximate the viscosities at the high-shear conditions of nasal spray devices (approximately 10(5)-10(6) s(-1)). In the current work, spray area and droplet size were shown to demonstrate viscosity dependence. Three Newtonian fluids were used to determine equations to approximate viscosity at the spray nozzle from correlations to spray area and droplet size using a standard 100 microL Pfeiffer nasal spray pump. Several shear-thinning solutions, including four commercial aqueous suspension corticosteroid nasal sprays and three aqueous Avicel (1, 2, and 3%, wt/wt) samples, were analyzed to demonstrate the ability of spray area and droplet size analysis to estimate high-shear viscosities. The calculated viscosity values trend in accordance with the rheometer data along with the ability to distinguish differences between all samples analyzed.

Selective fluorogenic derivatization of 3-nitrotyrosine and 3,4-dihydroxyphenylalanine in peptides: a method designed for quantitative proteomic analysis.

There is a need for the selective derivatization and enrichment of posttranslational protein modifications from tissue samples. This chapter describes a method for the selective derivatization of 3-nitrotyrosine (after reduction to 3-amino-tyrosine) and 3,4-dihydroxyphenylalanine with benzylamine derivatives to yield 6-amino- and 6-benzylamine-substituted benzoxazoles, which display characteristic fluorescence properties. The methodology can be expanded to other substituted benzylamines, which carry functional groups for affinity enrichment.

Synthesis of sulfonamide- and sulfonyl-phenylboronic acid-modified silica phases for boronate affinity chromatography at physiological pH.

Two new types of boronate affinity solid phases were synthesized and characterized. The materials were prepared by silylation of porous silica gel with monochlorosilane derivatives containing synthetic sulfonyl- and sulfonamide-substituted phenylboronic acids. The new solid phases were evaluated for boronate affinity chromatography with aryl and alkyl cis-diol compounds and were found to be suitable for the retention of cis-diols under acidic conditions. Significant correlations between the retention factor (K) and the pH of the mobile phase demonstrate that the binding of cis-diols to the solid phases is best rationalized by chelation. Based on the lower pKa, caused by the electron-withdrawing effects of the sulfonyl and sulfonamide groups, these media display an enhanced affinity for cis-diols as compared with unsubstituted phenylboronic acid. Using isocratic elution, a mixture of various biologically relevant l-tyrosines, l-DOPA, and several catecholamines were resolved with a mobile phase composed of 0.05M phosphate buffer (pH 5.5). Mono-, di-, and triphosphates of adenosine were also separated at pH 6.0. Hence, the new boronate solid phase offers efficient affinity separation and purification of cis-diol-containing molecules under rather mild pH conditions.