Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry.

Analyzing coeluting impurities with similar masses in synthetic oligonucleotides by liquid chromatography-mass spectrometry (LC-MS) poses challenges due to inadequate separation in either dimension. Herein, we present a direct method employing fully resolved isotopic envelopes, enabled by high resolution mass spectrometry (HRMS), to identify and quantify isobaric impurity ions resulting from the deletion or addition of a uracil (U) or cytosine (C) nucleotide from or to the full-length sequence. These impurities may each encompass multiple sequence variants arising from various deletion or addition sites. The method utilizes a full or targeted MS analysis to measure accurate isotopic distributions that are chemical formula dependent but nucleotide sequence independent. This characteristic enables the quantification of isobaric impurity ions involving sequence variants, a capability typically unavailable in sequence-dependent MS/MS methods. Notably, this approach does not rely on standard curves to determine isobaric impurity compositions in test samples; instead, it utilizes the individual isotopic distributions measured for each impurity standard. Moreover, in cases where specific impurity standards are unavailable, the measured isotopic distributions can be adequately replaced with the theoretical distributions (calculated based on chemical formulas of standards) adjusted using experiment-specific correction factors. In summary, this streamlined approach overcomes the limitations of LC-MS analysis for coeluting isobaric impurity ions, offering a promising solution for the in-depth profiling of complex impurity mixtures in synthetic oligonucleotide therapeutics.

Scanning Electron-Raman Cryomicroscopy for Characterization of Nanoparticle-Albumin Drug Products.

Nanomaterials have expanded the use of active pharmaceutical ingredients by improving efficacy, decreasing toxicity, and facilitating targeted delivery. To systematically achieve this goal, nanomaterial-containing drugs need to be manufactured with precision in attributes such as size, morphology, surface chemistry, and composition. Their physicochemical characterization is essential as their attributes govern pharmacokinetics yet can be challenging due to the nature of many nanomaterial-based formulations unless advanced sample fixation and in vitro characterization methods are utilized. Here, different cryogenic and other fixation strategies were assessed, and a novel physicochemical characterization method was developed using scanning electron Raman cryo-microscopy (SERCM). A complex nanoparticle albumin bound paclitaxel (nab-paclitaxel) formulation was chosen as a model drug. Plunge freezing (PF), high pressure freezing (HPF), freeze substitution (FS), and membrane filtration were compared for their influence on size and morphology measurements, and formulation-based variations were quantified. SERCM was introduced as a multiattribute physicochemical characterization platform, and the composition of nanoparticles was confirmed as albumin-paclitaxel complexes. By coupling image-based quantitative analysis with chemical analysis, SERCM has the potential to pave the way for the development of comprehensive tools for assessing injectable and ophthalmic nanomaterial-containing drugs in their native-like state.

Old Polymorph, New Technique: Assessing Ritonavir Crystallinity Using Low-Frequency Raman Spectroscopy.

Two decades ago, postmarket discovery of a second crystal form of ritonavir with lower solubility had major implications for drug manufacturers and patients. Since then, ritonavir has been reformulated via the hot-melt-extrusion process in an amorphous form. Here, quantitative low- and mid-frequency Raman spectroscopy methods were developed to characterize polymorphs, form I and form II, in commercial ritonavir 100 mg oral tablets as an alternate analysis approach compared to X-ray powder diffraction (XRPD). Crystallization in three lots of ritonavir products obtained from four separate manufacturers was assessed after storage under accelerated conditions at 40 °C and 75% relative humidity (RH). Results were compared with quantitative XRPD methods developed and validated according to ICH Q2 (R1) guidelines. In a four-week open-dish study, form I crystallization occurred in two of the four products and form II crystallization was detected in another ritonavir product. The limits of detection for XRPD, low-frequency Raman (LFR), and mid-frequency Raman (MFR) were determined to be 0.7, 0.8, and 0.5% for form I and 0.6, 0.6, and 1% for form II, respectively. Root-mean-squared-error of predictions were 0.6-1.0 and 0.6-2.5% for LFR- and MFR-based partial least-squares models. Further, ritonavir polymorphs could also be identified and detected directly from ritonavir tablets using transmission LFR. In summary, LFR was applied for the assessment of polymorphism in real-world samples. While providing analytical performance similar to conventional techniques, LFR reduced the single measurement time from 66 min (XRPD) to 10 s (LFR) without the need for tedious sample preparation procedures.

Seroconversion and fever are dose-dependent in a nonhuman primate model of inhalational COVID-19.

While evidence exists supporting the potential for aerosol transmission of SARS-CoV-2, the infectious dose by inhalation remains unknown. In the present study, the probability of infection following inhalation of SARS-CoV-2 was dose-dependent in a nonhuman primate model of inhalational COVID-19. The median infectious dose, assessed by seroconversion, was 52 TCID50 (95% CI: 23-363 TCID50), and was significantly lower than the median dose for fever (256 TCID50, 95% CI: 102-603 TCID50), resulting in a group of animals that developed an immune response post-exposure but did not develop fever or other clinical signs of infection. In a subset of these animals, virus was detected in nasopharyngeal and/or oropharyngeal swabs, suggesting that infected animals without signs of disease are able to shed virus and may be infectious, which is consistent with reports of asymptomatic spread in human cases of COVID-19. These results suggest that differences in exposure dose may be a factor influencing disease presentation in humans, and reinforce the importance of public health measures that limit exposure dose, such as social distancing, masking, and increased ventilation. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, and, ultimately, mitigation strategies. Additionally, these data will be useful to inform dose selection in future studies examining the efficacy of therapeutics and vaccines against inhalational COVID-19, and as a baseline in healthy, young adult animals for assessment of the importance of other factors, such as age, comorbidities, and viral variant, on the infectious dose and disease presentation.

Inhaled gene therapy of preclinical muco-obstructive lung diseases by nanoparticles capable of breaching the airway mucus barrier.

INTRODUCTION: Inhaled gene therapy of muco-obstructive lung diseases requires a strategy to achieve therapeutically relevant gene transfer to airway epithelium covered by particularly dehydrated and condensed mucus gel layer. Here, we introduce a synthetic DNA-loaded mucus-penetrating particle (DNA-MPP) capable of providing safe, widespread and robust transgene expression in in vivo and in vitro models of muco-obstructive lung diseases. METHODS: We investigated the ability of DNA-MPP to mediate reporter and/or therapeutic transgene expression in lung airways of a transgenic mouse model of muco-obstructive lung diseases (ie, Scnn1b-Tg) and in air-liquid interface cultures of primary human bronchial epithelial cells harvested from an individual with cystic fibrosis. A plasmid designed to silence epithelial sodium channel (ENaC) hyperactivity, which causes airway surface dehydration and mucus stasis, was intratracheally administered via DNA-MPP to evaluate therapeutic effects in vivo with or without pretreatment with hypertonic saline, a clinically used mucus-rehydrating agent. RESULTS: DNA-MPP exhibited marked greater reporter transgene expression compared with a mucus-impermeable formulation in in vivo and in vitro models of muco-obstructive lung diseases. DNA-MPP carrying ENaC-silencing plasmids provided efficient downregulation of ENaC and reduction of mucus burden in the lungs of Scnn1b-Tg mice, and synergistic impacts on both gene transfer efficacy and therapeutic effects were achieved when DNA-MPP was adjuvanted with hypertonic saline. DISCUSSION: DNA-MPP constitutes one of the rare gene delivery systems providing therapeutically meaningful gene transfer efficacy in highly relevant in vivo and in vitro models of muco-obstructive lung diseases due to its unique ability to efficiently penetrate airway mucus.

Evaluation of the Physicochemical Properties of the Iron Nanoparticle Drug Products: Brand and Generic Sodium Ferric Gluconate.

Complex iron nanoparticle-based drugs are one of the oldest and most frequently administered classes of nanomedicines. In the US, there are seven FDA-approved iron nanoparticle reference drug products, of which one also has an approved generic drug product (i.e., sodium ferric gluconate (SFG)). These products are indicated for the treatment of iron deficiency anemia and are administered intravenously. On the molecular level, iron nanomedicines are colloids composed of an iron oxide core with a carbohydrate coating. This formulation makes nanomedicines more complex than conventional small molecule drugs. As such, these products are often referred to as nonbiological complex drugs (e.g., by the nonbiological complex drugs (NBCD) working group) or complex drug products (e.g., by the FDA). Herein, we report a comprehensive study of the physiochemical properties of the iron nanoparticle product SFG. SFG is the single drug for which both an innovator (Ferrlecit) and generic product are available in the US, allowing for comparative studies to be performed. Measurements focused on the iron core of SFG included optical spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), X-ray powder diffraction (XRPD), 57Fe Mössbauer spectroscopy, and X-ray absorbance spectroscopy (XAS). The analysis revealed similar ferric-iron-oxide structures. Measurements focused on the carbohydrate shell comprised of the gluconate ligands included forced acid degradation, dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and gel permeation chromatography (GPC). Such analysis revealed differences in composition for the innovator versus the generic SFG. These studies have the potential to contribute to future quality assessment of iron complex products and will inform on a pharmacokinetic study of two therapeutically equivalent iron gluconate products.

Comparison between minimal fluoroscopy and conventional approaches for visually guided laser balloon pulmonary vein isolation ablation.

INTRODUCTION: Although balloon-based techniques, such as the laser balloon (LB) ablation have simplified pulmonary vein isolation (PVI), procedural fluoroscopy usage remains higher in comparison to radiofrequency PVI approaches due to limited 3-dimensional mapping system integration. METHODS: In this prospective study, 50 consecutive patients were randomly assigned in alternating fashion to a low fluoroscopy group (LFG; n = 25) or conventional fluoroscopy group (CFG; n = 25) and underwent de novo PVI procedures using visually guided LB technique. RESULTS: There was no statistical difference in baseline characteristics or cross-overs between treatment groups. Acute PVI was accomplished in all patients. Mean follow up was 318 ± 69 days. Clinical recurrence of atrial fibrillation after PVI was similar between groups (CFG: 19% vs LFG: 15%; P = .72). Total fluoroscopy time was significantly lower in the LFG than the CFG (1.7 ± 1.4  vs 16.9 ± 5.9 minutes; P < .001) despite similar total procedure duration (143 ± 22 vs 148 ± 22 minutes; P = .42) and mean LA dwell time (63 ± 15 vs 59 ± 10 minutes; P = .28). Mean dose area product was significantly lower in the LFG (181 ± 125 vs 1980 ± 750 µGym2 ; P < .001). Fluoroscopy usage after transseptal access was substantially lower in the LFG (0.63 ± 0.43 vs 11.70 ± 4.32 minutes; P < .001). Complications rates were similar between both groups (4% vs 2%; P = .57). CONCLUSIONS: This study demonstrates that LB PVI can be safely achieved using a novel low fluoroscopy protocol while also substantially reducing fluoroscopy usage and radiation exposure in comparison to conventional approaches for LB ablation.

Raman mapping of fentanyl transdermal delivery systems with off-label modifications.

Raman mapping is a powerful and emerging tool in characterization of pharmaceuticals and provides non-destructive chemical and structural identification with minimal sample preparation. One pharmaceutical form that is suitable but has not been studied in-depth with Raman mapping is transdermal delivery systems (TDS). TDS are dosage forms designed to deliver a therapeutically effective amount of active pharmaceutical ingredient (API) across a patient's skin. To enhance drug delivery through the skin, the API in the formulation is often close to a saturated or supersaturated state. Thus, improper use or off-label modifications can lead to occurrence of unwanted API changes, specifically, crystallization over time. Here, off-label modifications were mimicked on a set of fentanyl drug-in-adhesive TDS sold on the U.S. market by four different manufacturers via die cutting, and then the die cut TDS were investigated through confocal Raman mapping for structural and chemical changes. Using Multivariate Curve Resolution (MCR), not only was morphological and chemical characterization of transdermal systems provided, but also fentanyl crystals in certain products due to off-label modifications were identified. The chemometric model used in analysis of Raman maps allowed precise identification of fentanyl as the crystalline material as confirmed by the hit-quality-index correlation of component spectra from the chemometric model with library spectra of a fentanyl reference standard. The results show that confocal Raman mapping with MCR can be utilized in assessing pharmaceutical quality of TDS. This method has the potential to be widely used in characterization of such systems as an alternative to existing techniques.

QCM Sensor Arrays, Electroanalytical Techniques and NIR Spectroscopy Coupled to Multivariate Analysis for Quality Assessment of Food Products, Raw Materials, Ingredients and Foodborne Pathogen Detection: Challenges and Breakthroughs.

Quality checks, assessments, and the assurance of food products, raw materials, and food ingredients is critically important to ensure the safeguard of foods of high quality for safety and public health. Nevertheless, quality checks, assessments, and the assurance of food products along distribution and supply chains is impacted by various challenges. For instance, the development of portable, sensitive, low-cost, and robust instrumentation that is capable of real-time, accurate, and sensitive analysis, quality checks, assessments, and the assurance of food products in the field and/or in the production line in a food manufacturing industry is a major technological and analytical challenge. Other significant challenges include analytical method development, method validation strategies, and the non-availability of reference materials and/or standards for emerging food contaminants. The simplicity, portability, non-invasive, non-destructive properties, and low-cost of NIR spectrometers, make them appealing and desirable instruments of choice for rapid quality checks, assessments and assurances of food products, raw materials, and ingredients. This review article surveys literature and examines current challenges and breakthroughs in quality checks and the assessment of a variety of food products, raw materials, and ingredients. Specifically, recent technological innovations and notable advances in quartz crystal microbalances (QCM), electroanalytical techniques, and near infrared (NIR) spectroscopic instrument development in the quality assessment of selected food products, and the analysis of food raw materials and ingredients for foodborne pathogen detection between January 2019 and July 2020 are highlighted. In addition, chemometric approaches and multivariate analyses of spectral data for NIR instrumental calibration and sample analyses for quality assessments and assurances of selected food products and electrochemical methods for foodborne pathogen detection are discussed. Moreover, this review provides insight into the future trajectory of innovative technological developments in QCM, electroanalytical techniques, NIR spectroscopy, and multivariate analyses relating to general applications for the quality assessment of food products.

An NMR Protocol for In Vitro Paclitaxel Release from an Albumin-Bound Nanoparticle Formulation.

The paclitaxel protein-bound particles for injectable suspension (marketed under the brand name Abraxane®) contains nanosized complexes of paclitaxel and albumin. The molecular interaction between paclitaxel and albumin within the higher-order nanostructure is analytically challenging to assess, as is any correlation of differences to differences in therapeutic effect. However, because the higher-order nanostructures may affect the paclitaxel release, a suitable in vitro assay to detect potential differences in paclitaxel release between comparator lots and products is desirable. Herein, solution NMR spectroscopy with a T2-filtering technique was developed to detect paclitaxel signal while suppressing albumin signals to follow the released paclitaxel in the NMR tube upon dilution. The non-invasive nature of NMR allows for precise measurement of a full range of dilution-induced drug release percentage from 14 to 92% without any sample extraction. The critical concentration of the drug product (DP) at 50% of release was 0.63 ± 0.04 mg/mL in PBS buffer. In addition, 2D diffusion ordered NMR spectroscopy (DOSY) results revealed that the released paclitaxel experiencing slightly slowed diffusion rates than free paclitaxel, which was attributed to paclitaxel in equilibrium with albumin-bound states. Collectively, the dilution-based NMR method offered an analytical approach to investigate physicochemical attributes of complex injectable products with minimal needed sample preparation and perturbation to nanoparticle formulation.

Raman Barcode for Counterfeit Drug Product Detection.

Potential infiltration of counterfeit drug products-containing the wrong or no active pharmaceutical ingredient (API)-into the bona fide drug supply poses a significant threat to consumers worldwide. Raman spectroscopy offers a rapid, nondestructive avenue to screen a high throughput of samples. Traditional qualitative Raman identification is typically done with spectral correlation methods that compare the spectrum of a reference sample to an unknown. This is often effective for pure materials but is quite challenging when dealing with drug products that contain different formulations of active and inactive ingredients. Typically, reliable identification of drug products using common spectral correlation algorithms can only be made if the specific product under study is present in the library of reference spectra, thereby limiting the scope of products that can be screened. In this paper, we introduce the concept of the Raman barcode for identification of drug products by comparing the known peaks in the API reference spectrum to the peaks present in the finished drug product under study. This method requires the transformation of the Raman spectra of both API and finished drug products into a barcode representation by assigning zero intensity to every spectral frequency except the frequencies that correspond to Raman peaks. By comparing the percentage of nonzero overlap between the expected API barcode and finished drug product barcode, the identity of API present can be confirmed. In this study, 18 approved finished drug products and nine simulated counterfeits were successfully identified with 100% accuracy utilizing this method.

Rapid screening of anti-infective drug products for counterfeits using Raman spectral library-based correlation methods.

A new spectral library-based approach that is capable of screening a diverse set of finished drug products using only an active pharmaceutical ingredient spectral library is described in this paper. This approach obviates the need for a comprehensive drug product library, thereby streamlining the use of spectral library-based tests for anti-counterfeiting efforts, specifically to target finished drug products containing the wrong active ingredient or no active ingredient at all. Both laboratory-based and portable spectrometers are used in the study to demonstrate the usefulness and transferability of the spectral correlation method for field screening. The spectral correlation between the active pharmaceutical ingredient and finished drug product spectra is calculated using both full spectral analysis and targeted spectral regions analysis of six types of antimalarial, antibiotic and antiviral products. The spectral regions were determined using a moving window spectral correlation algorithm, and the use of specific spectral regions is shown to be crucial in screening finished drug products using only the active pharmaceutical ingredient spectrum. This comprehensive screening spectral correlation method is tested on seven different validation samples from different manufacturers as those used to develop the method, as well as simulated counterfeits which were prepared to mimic falsified drugs containing no active ingredient. The spectral correlation method is successful in correctly identifying 100% of the authentic products and simulated counterfeit samples tested.

Convergent rhythm generation from divergent cellular mechanisms.

Different modulatory inputs commonly elicit distinct rhythmic motor patterns from a central pattern generator (CPG), but they can instead elicit the same pattern. We are determining the rhythm-generating mechanisms in this latter situation, using the gastric mill (chewing) CPG in the crab (Cancer borealis) stomatogastric ganglion, where stimulating the projection neuron MCN1 (modulatory commissural neuron 1) or bath applying CabPK (C. borealis pyrokinin) peptide elicits the same gastric mill motor pattern, despite configuring different gastric mill circuits. In both cases, the core rhythm generator includes the same reciprocally inhibitory neurons LG (lateral gastric) and Int1 (interneuron 1), but the pyloric (food-filtering) circuit pacemaker neuron AB (anterior burster) is additionally necessary only for CabPK rhythm generation. MCN1 drives this rhythm generator by activating in the LG neuron the modulator-activated inward current (IMI), which waxes and wanes periodically due to phasic feedback inhibition of MCN1 transmitter release. Each buildup of IMI enables the LG neuron to generate a self-terminating burst and thereby alternate with Int1 activity. Here we establish that CabPK drives gastric mill rhythm generation by activating in the LG neuron IMI plus a slowly activating transient, low-threshold inward current (ITrans-LTS) that is voltage, time, and Ca(2+) dependent. Unlike MCN1, CabPK maintains a steady IMI activation, causing a subthreshold depolarization in LG that facilitates a periodic postinhibitory rebound burst caused by the regular buildup and decay of the availability of ITrans-LTS. Thus, different modulatory inputs can use different rhythm-generating mechanisms to drive the same neuronal rhythm. Additionally, the same ionic current (IMI) can play different roles under these different conditions, while different currents (IMI, ITrans-LTS) can play the same role.

Qualitative and quantitative analysis of glutathione and related impurities in pharmaceuticals by qNMR.

In this study, an alternative method to compendial analytical procedures with enhanced detection and separation capabilities was validated for the quality assessment of glutathione (GSH) drug substance. The related impurities A, B, C, and D present in GSH drug substance were characterized using a one-dimension proton nuclear magnetic resonance (1D 1H NMR) method on a 600 MHz spectrometer equipped with a liquid nitrogen cryoprobe. Two sample preparations at different pH were optimized to ensure the unambiguous identification of different impurities in the GSH samples. Specifically, impurities A and C in a GSH sample can be tested at pH 3.0, while pH 7.4 is more suitable for testing impurities B and D. The quantitative NMR (qNMR) method was validated following International Council for Harmonisation (ICH) guidelines. The limit of detection (LOD) was less than 0.1% wt for an individual impurity, and the limit of quantitation (LOQ) ranged from 0.14 to 0.24% wt, using about 14 min experimental time per spectrum. Following validation, the qNMR method was applied to assess different commercial GSH bulk substance samples, an in-house compounded GSH drug product, and a GSH dietary supplement product. The method was also applied to monitor GSH degradation (hydrolysis and oxidation) over time to provide quantitative information on GSH degradation and stability. The results suggest that the qNMR method can serve as a highly specific and efficient orthogonal tool for assessing the quality of GSH pharmaceuticals, providing both qualitative and quantitative information on GSH and its related impurities A-D.

An isocratic HPLC-UV analytical procedure for assessment of glutathione and its related substances.

Reduced glutathione (GSH) is an endogenous tripeptide antioxidant which plays a crucial role in a variety of physiological and pathological activities. Although GSH is not present in any FDA-approved drug product, GSH dietary supplement products and compounded GSH drugs are available to patients in the US. Several incidents of toxicity have occurred in recent years due to endotoxin or otherwise contaminated GSH in compounded drugs. Efficient and sensitive analytical methods are needed for assessing and ensuring the quality of GSH substance and associated drug or dietary supplement products. Impurities A (L-cysteinylglycine), B (cysteine), C (oxidized L-glutathione) and D (γ-L-glutamyl-L-cysteine) are the main related impurities for GSH drug substance which have been detected and quantified by capillary electrophoresis and qNMR analytical procedures. However, there are no reported HPLC methods for detecting or quantifying the three main related impurities A, B and D even though numerous HPLC analytical methods have been reported for analyzing GSH and impurity C. In this report, an isocratic HPLC-UV analytical procedure was developed and validated for separating and identifying GSH and related impurities A-D as well as a newly identified degradant, L-pyroglutamic acid (pGlu), within 10 minutes with resolution (RS) more than 3. The LOD and LOQ were determined to be 0.02 % w/w and 0.05 % w/w, respectively, for impurities A-D and pGlu. Importantly, the optimized HPLC analytical procedure for GSH assay does not have interference from impurities A, B and D, providing highly specific results compared to the commonly used iodine titration method. The newly validated analytical procedure was applied to assess different commercial GSH bulk substance samples. The results suggest that the analytical procedure described in this work is suitable for quality assessment of GSH samples.

A top-down spectroscopic approach for correlating coating thickness distributions with the dissolution profiles of enterically coated pellets.

Pharmaceutical dosage forms such as tablets and capsules are often coated with a functional polymer to modify the drug release. To obtain the drug release profiles, ensure quality control and predict in-vivo performance, dissolution studies are performed. However, dissolution tests are time-consuming, sample destructive and do not readily allow for at-line or in-line characterization. Rapid assessment of functional coatings is essential for products where a single capsule is comprised of hundreds of functionally-coated pellets and the collective drug release kinetics of the entire capsule depends on contributions from each pellet. Here, single Raman measurements were used to evaluate the coating thickness distributions of a dosage form comprised of small, functionally-coated pellets in capsules. First, the composition and physicochemical properties of pellets were characterized by multivariate analysis assisted Raman mapping of pellet cross-sections. Second, a method of collecting single Raman spectrum with spectral contributions from the coating and API layers was developed and optimized to estimate the thickness of coatings. The coating thicknesses obtained from single Raman measurements of pellets in each capsule revealed thickness distributions that correlated with the dissolution profiles (capsules with one distribution had single stage release and capsules with two distributions had a two-stage release). Finally, an unsupervised multivariate analysis method was demonstrated as a rapid and efficient way to correlate dissolution profiles of enterically coated pellets. In summary, this study presents a non-destructive and rapid characterization method for assessing coating thickness and has the potential to be applied in process analytical technologies to ensure coating uniformity and predict product dissolution rate performance.

Development and validation of a quantitative proton NMR method for the analysis of pregnenolone.

Pregnenolone (PREG) is an endogenous steroid frequently sold as an over-the-counter dietary supplement touted to promote neurological and immunological health. While the PREG dietary supplement is added to the diet for health benefits, there are no FDA approved PREG drugs. However, compounded PREG drug products are available to U.S. patients. The FDA works with state regulatory authorities on the oversight of compounding activities, including developing 503A and 503B lists of bulk substances that compounders are permitted to use. PREG is one of the substances publicly nominated to be included on the 503B list. Compounded hormone therapies such as those using PREG are of interest given the lack of standardization in compounded drug products which may increase the possibility of underdosing, overdosing, or contamination. However, no USP monograph currently exists to evaluate the quality of PREG drug substance or product. To address knowledge gaps and assist in quality control, a simple and rapid quantitative proton nuclear magnetic resonance spectroscopy (qNMR) method for the identification and assay of PREG in different types of PREG products was developed and validated. PREG samples were characterized using 1D 1H and 2D 1H-13C HSQC NMR spectra. The qNMR assay method (taking approximately 10 min per NMR spectrum) was validated for precision, accuracy, specificity, robustness and linearity per ICH Q2(R1) guidance. The method was validated in a range from 0.032 to 3.2 mg/mL. As a proof of concept, seven PREG bulk substance samples, three tablet and two capsule PREG dietary supplements were assessed by the qNMR analytical procedure. NMR data from all tested samples met the expected criteria for identification and assay. The results demonstrate the potential of qNMR for the quality assessment of different types of PREG samples.

Performance Characteristics of Mass Spectrometry-Based Analytical Procedures for Quantitation of Nitrosamines in Pharmaceuticals: Insights from an Inter-laboratory Study.

With the discovery of carcinogenic nitrosamine impurities in pharmaceuticals in 2018 and subsequent regulatory requirements for risk assessment for nitrosamine formation during pharmaceutical manufacturing processes, storage or from contaminated supply chains, effective testing of nitrosamines has become essential to ensure the quality of drug substances and products. Mass spectrometry has been widely applied to detect and quantify trace amounts of nitrosamines in pharmaceuticals. As part of an effort by regulatory authorities to assess the measurement variation in the determination of nitrosamines, an inter-laboratory study was performed by the laboratories from six regulatory agencies with each of the participants using their own analytical procedures to determine the amounts of nitrosamines in a set of identical samples. The results demonstrated that accurate and precise quantitation of trace level nitrosamines can be achieved across multiple analytical procedures and provided insight into the performance characteristics of mass spectrometry-based analytical procedures in terms of accuracy, repeatability and reproducibility.

Complete genome sequence of Desulfurococcus fermentans, a hyperthermophilic cellulolytic crenarchaeon isolated from a freshwater hot spring in Kamchatka, Russia.

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.

Development of In Vitro Dissolution Testing Methods to Simulate Fed Conditions for Immediate Release Solid Oral Dosage Forms.

In vitro dissolution testing is widely used to mimic and predict in vivo performance of oral drug products in the gastrointestinal (GI) tract. This literature review assesses the current in vitro dissolution methodologies being employed to simulate and predict in vivo drug dissolution under fasted and fed conditions, with emphasis on immediate release (IR) solid oral dosage forms. Notable human GI physiological conditions under fasted and fed states have been reviewed and summarized. Literature results showed that dissolution media, mechanical forces, and transit times are key dissolution test parameters for simulating specific postprandial conditions. A number of biorelevant systems, including the fed stomach model (FSM), GastroDuo device, dynamic gastric model (DGM), simulated gastrointestinal tract models (TIM), and the human gastric simulator (HGS), have been developed to mimic the postprandial state of the stomach. While these models have assisted in expanding physiological relevance of in vitro dissolution tests, in general, these models lack the ability to fully replicate physiological conditions/processes. Furthermore, the translatability of in vitro data to an in vivo system remains challenging. Additionally, physiologically based pharmacokinetic (PBPK) modeling has been employed to evaluate the effect of food on drug bioavailability and bioequivalence. Here, we assess the current status of in vitro dissolution methodologies and absorption PBPK modeling approaches to identify knowledge gaps and facilitate further development of in vitro dissolution methods that factor in fasted and fed states. Prediction of in vivo drug performance under fasted and fed conditions via in vitro dissolution testing and modeling may potentially help efforts in harmonizing global regulatory recommendations regarding in vivo fasted and fed bioequivalence studies for solid oral IR products.