In vitro permeation testing (IVPT) is a method widely used to assess the skin permeation profiles of topical drug products for product development and regulatory evaluation. Various skin models serve as diffusion barriers in IVPT, including surgically excised human skin (EHS) and human cadaver skin (HCS). Limited EHS supply has led to the use of HCS in IVPT, but HCS shows variable skin permeation among donors, creating challenges in obtaining consistent results. To address these supply and variability issues, this study evaluated the potential of reconstructed human skin (RHS) as an alternative diffusion barrier for IVPT. Sunscreen (cream) permeation through four skin models were compared, including two HCS models and two RHS models with or without dermal layers. Among the tested skin models, EpiDermFT (RHS with dermal layers) exhibited consistent results for both intra-batch and inter-batch permeation. Additionally, EpiDermFT exhibited similar rank order of cumulative permeation compared to the HCS counterpart, whereas EpiDerm (RHS without dermal layers) exhibited greater sensitivity to pH changes in cream formulations than HCS models. These findings suggest that EpiDermFT has potential utility as a reliable alternative that provides comparable permeation results to HCS, whereas EpiDerm may serve as a sensitive tool to detect pH differences in topical formulations. However, to determine the broader applicability of RHS in IVPT, further investigations using diverse topical formulations across different dosage forms are necessary.

AIM:D-penicillamine (PSH) is an active pharmaceutical ingredient used for the treatment of various diseases, such as Wilson's disease, rheumatoid arthritis, cystinuria, and heavy metal poisoning. However, evaluating PSH in plasma poses substantial analytical challenges due to drug instability, thiol-disulfide exchange reactions with endogenous thiols, and the potential formation of multiple chemical forms of PSH. This study describes the development and validation of an analytical method to ensure the stability of PSH during analysis and enable accurate bioavailability measurements and bioequivalence assessments in human plasma. METHODS:An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated to simultaneously determine PSH and its two major metabolites, D-penicillamine disulfide and L-cysteine-D-penicillamine disulfide, in human plasma. PSH stability was investigated under various experimental conditions to identify the optimal sample preparation procedure. The method was then applied to conduct a 90-day stability study of PSH in human plasma stored at -80°C. RESULTS AND CONCLUSION:A sensitive and specific analytical method was developed and validated in accordance with the US FDA M10 guidance. PSH remained stable under optimized conditions for at least 90 days. This method provides pharmaceutical researchers with a standardized approach for PSH pharmacokinetic analysis and bioequivalence evaluations.

Understanding naloxone permeation is important for optimizing nasal delivery and supporting comparative assessment of nasal drug products. In this study, a stability-indicating LC-MS/MS method was developed and validated for the simultaneous quantification of naloxone and its related impurities, naloxone N-oxide and noroxymorphone, in in vitro permeation test receptor media. The validated method was applied to characterize naloxone permeation following cloud-based aerosol dosing across a synthetic Nuclepore Track-Etched membrane and a differentiated human EpiAirway™ mucociliary tissue model under finite-dose conditions. The analytical procedure demonstrated linearity over 0.25-20.0 ng/mL in Dulbecco's Phosphate-Buffered Saline and Krebs-Ringer Bicarbonate Buffer, with acceptable accuracy and precision. No degradation products or additional impurities were detected in permeation samples, confirming the stability-indicating capability of the method. Naloxone exhibited rapid early-time permeation across the synthetic membrane, whereas transport across the epithelial tissue model was attenuated and plateaued, reflecting physiological barrier function. Integration of cloud-based aerosol delivery with a validated LC-MS/MS platform enables mechanistic evaluation of nasal naloxone permeation and provides a supportive in vitro framework for formulation characterization and comparative assessments.

Liquid chromatography-mass spectrometry (LC-MS) is the standard instrumental procedure for quantitating nitrosamine drug substance-related impurities (NDSRIs) due to its superior specificity and sensitivity. Electrospray (ESI) is the most used ionization source in LC-MS. However, analytes can undergo fragmentation directly within the ESI source before reaching the collision cell. This phenomenon is known as in source fragmentation (ISF). To our knowledge, the impact of ISF on analytical procedure performance for NDSRI measurements has not been explored. Thus, here, we present a case study on an NDSRI (nitroso-bumetanide) to illustrate how efforts can be taken during analytical procedure development to minimize ISF while still achieving the analytical target profile (ATP) measurement goals. In addition, we share some thoughts about incorporating risk assessment and leveraging prior knowledge for analytical procedure development for NDSRI LC-MS technology-based testing purposes.

BACKGROUND:While Polyethylene glycol 3350 (PEG 3350) is approved by the USFDA for short term use by adults, it is commonly recommended for use in constipated children. Multiple reports of adverse events in children taking PEG 3350 raised safety concerns suggesting that low molecular weight species of PEG 3350 might be absorbed from the gut and cause side effects such as ethylene glycol (EG), diethylene glycol (DEG), and triethylene glycol (TEG). RESEARCH DESIGN AND METHODS:This article documents the development, validation, and application of analytical methods using GC-MS and GC-MS/MS for the quantitation of EG, DEG, and TEG in human plasma, serum, and urine. RESULTS:The analytical range for EG, DEG, and TEG was 2-20 µg/mL. The sample preparation process involves derivatization using N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane in each biological matrices. Deuterated internal standards for each of the analytes were included to provide accurate quantitation of the glycol analytes. CONCLUSIONS:The validated methods were applied to analyze samples a pilot study of children taking PEG 3350. DEG and TEG were detected at levels below the limit of quantitation. In summary, a platform of analytical methods was developed to evaluate glycol analogs in urine, serum, and plasma clinical samples.

Asymmetrical flow field-flow fractionation (AF4) with multi-detection has continued to gain wider acceptance for characterizing complex drug products. An important quality attribute for these products is the measurement of the particle size distribution (PSD). Current limitations of established procedures (e.g., dynamic light scattering) for accurately determining PSD can be overcome by AF4. However, while gaining acceptance this technique has not been fully adopted within the pharmaceutical industry. A technical understanding of fundamental operational factors is necessary for the successful application of utilizing any emerging technology. For example, recovery (R% = AS/AD*100, where AS and AD are the peak areas from the concentration detector with and without the crossflow field, respectively) is one factor that is used to assess the robustness during AF4 method development, but currently little is known about the interplay between analyte recovery and PSD. This work highlights factors that impact calculated AF4 recovery, and how differences in analyte and absolute recovery ultimately influence the PSD of nanoparticle size standards and complex drug product formulations such as emulsions and liposomes. Factors like ionic strength, buffer composition, and analyte chemistries, which are the most common factors associated with changes to R% in AF4, contributed to changes in AS. While AD is not typically examined in detail, the selection of the concentration detector (UV or dRI) along with their instrumental parameters (e.g., wavelength, attenuation value, linear range) and sample preparation was shown to under- or over-estimate AD thus changing R%. Examining both components of R% and their contributions to analyte and absolute recovery show that decreases in analyte recovery may not be exclusively due to sample loss but could be influenced by changes in analyte-membrane interactions or analyte instability. Because of this, four relationships between recovery and PSD were defined. While R% is used as a tool for assessing AF4 methodology, the factors investigated through this work warrant further considerations when establishing an appropriate R% threshold.

Data integrity is necessary to help ensure the accuracy, consistency, validity and completeness of data. Data can be untraceably manipulated when it lacks adequate integrity. The FDA identified data integrity concerns with the pharmacokinetic data of a bioequivalence (BE) study for rivaroxaban 20 mg tablets, which was conducted by a contract research organization (CRO) in support of an abbreviated new drug application (ANDA) submission. The hypothesis was that samples from the late cohort of the study might have been substituted or manipulated to allow an otherwise failing study to meet the BE endpoint. To test this hypothesis FDA investigators collected 2,392 plasma samples from the BE study at the CRO's clinical site. FDA laboratory then developed and validated a bioanalytical method and re-analyzed the BE study plasma samples. Comparison of the data generated by the CRO and FDA suggested that the study was manipulated by altering the volume of plasma samples used for bioanalysis. This manipulation was likely done to achieve a lower than actual maximum plasma concentration test/reference (Cmax T/R) ratio.

Ion mobility mass spectrometry is emerging as a useful tool to probe native protein structural information. Advance ion mobility methods like collision-induced unfolding (CIU) can be used to characterize proteins' conformational dynamics. The impact of instrument source conditions on the native protein conformations is not well characterized or standardized. High values of drying gas temperature and gas flow parameters on the Agilent IM-QTOF instrument were shown to apply collision-induced unfolding (CIU) effects on protein ions ionized from physiological solution condition. Ion conformation heat maps of model proteins ubiquitin, myoglobin, and bovine serum albumin were obtained using a novel CIU method utilizing high drying gas temperature and varying drying gas flow. Protein charge states also increased as drying gas flow was increased at high temperature indicating a thermal heating element. Overall, drying gas temperature and gas flow on IMQTOF and the associated impacts on ionic structure need to be considered when using ion mobility mass spectrometry technology to assess protein structure.

BackgroundHigh-throughput solid-phase extraction coupled with tandem mass spectrometry (HT-SPE-MS/MS) is an automated sample delivery system to mass spectrometry that operates without chromatographic separation. The typical analysis time per sample using this platform is 10-30 s. While the HT-SPE-MS/MS system has demonstrated efficacy for in vitro assays, its application to the analysis of biological samples from in vivo bioavailability and bioequivalence studies presents challenges due to the complexity of the sample matrix. Three critical issues - matrix effect, specificity, and carryover - have not been thoroughly evaluated in complex biological matrices such as plasma.Research design and methodsThis study assessed the feasibility of utilizing HT-SPE-MS/MS for the analysis of three metabolically related compounds (bupropion, hydroxybupropion, and threobupropion) in human plasma samples from a clinical bioequivalence study. Critical bioanalytical parameters, including matrix effect, specificity, accuracy, precision, and carryover, were systematically investigated.ResultsThese methods were subsequently applied to a bioequivalence study of bupropion. The HT-SPE-MS/MS approach achieved comparable accuracy, precision, linearity, and sensitivity to conventional ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) methods, while offering 20- to 30-fold higher analysis speeds.ConclusionThe results of this study indicate that the HT-SPE-MS/MS system shows potential for high-throughput in vivo bioanalysis, particularly in bioavailability and bioequivalence studies.

Aim: To improve the palatability and increase compliance in pediatric patients, different taste-masking technologies have been evaluated to support the NIH Pediatric Formulation Initiative. Methods: This bioavailability approach combined a juvenile porcine model which represented the pediatric population, and an advanced UHPLCMS/MS method. Juvenile pigs were administered with either commercial Tamiflu or its taste-masking formulation and plasma samples were obtained from 0 to 48 h. The mass spectrometer was operated in positive mode with electrospray ionization. Results: The bioavailability profiles were not significantly different between the two formulations which demonstrated that taste-masking by forming an ionic complex was a promising approach for formulation modification. Conclusion: The pre-clinical study revealed a promising model platform for developing and screening taste-masking formulations.

With the finalization of the ICH Q14 Analytical Procedure Development guideline, how to apply enhanced approaches (such as analytical quality by design (AQbD)) to develop an analytical procedure, and to propose Established Conditions (ECs) and corresponding reporting categories, is increasingly being discussed. To gain practical experience in applying an enhanced approach for method development and identifying ECs, we developed, validated, and implemented an analytical procedure for a nitrosamine drug substance-related impurity (NDSRI). Here, as an example of the application of Q12 Lifecycle Management guideline principles in regards to analytical procedures, we briefly elaborate how: 1) the principles documented in the ICH Q14 guideline for analytical procedure development were applied, with the focus on identifying an Analytical Target Profile (ATP), knowledge management and risk assessment; 2) analytical procedure robustness according to the recommendations in ICH Q2(R2) Validation of Analytical Procedure guideline and Q14, were evaluated; and 3) mass spectrometry ECs and associated proposed reporting categories were proposed.

Winlevi® (clascoterone) topical cream (1

The mitigation of nitrosamine formation in drug products has been studied and approaches such as using formulations with pH modifiers and antioxidants have been shown to decrease the formation of nitrosamines. However, more studies are needed to explore the effectivness of mitigation strategies with different drug models and formulations. The primary objective of this work was to assess the role of different antioxidants and pH modifiers in tablet formulations to mitigate the formation of NDMA, prepared in-house, using metformin hydrochloride as a model drug. A study design for manufacturing metformin hydrochloride formulations was created to evaluate potential mitigation stratigies. The formulations were prepared by wet granulation that included a sodium nitrite spike and various antioxidants such as ascorbic acid, caffeic acid and ferulic acid at various concentrations that may inhibit nitrosamine formation. The study design also included pH modifiers such as hydrochloric acid and sodium carbonate. The metformin hydrochloride formulations were placed under stability conditions that included humidity, temperature and time over a six month period. NDMA inhibition was found to be most effective in formulations with basic pH, followed by the addition of tested antioxidants with 0.1% concentrations in the formulations. All tested antioxidants showed complete mitigation in formulations with 0.5% and 1% concentrations. In summary, basic pH and the inclusion of antioxidants exhibited the potential to mitigate the formation of NDMA in metformin hydrochloride tablets.

Pharmaceutical manufacturers are working to mitigate the formation of nitrosamine impurities in drug products. The work herein describes the development and validation of a headspace GC-MS method according to ICH Q2(R1) guidelines for the detection and quantification of NDMA, NDEA, NDIPA, and NEIPA in drug products. The analytical procedure was further modified to include detection and quantitation of DMF due to the potential decomposition pathway of DMF to form dimethylamine, a known precursor for NDMA formation. The NDMA impurity was detected in the "sartan" class of drug products between 0.1 and 113 ppm. The validated analytical procedure was applied in an investigation of approaches to mitigate nitrosamine formation in metformin drug products. The developed analytical procedures provide another tool for pharmaceutical manufacturers to evaluate drug products for nitrosamine impurities.

Reformulation with addition of antioxidants is one potential mitigation strategy to prevent or reduce nitrosamine drug substance-related impurities (NDSRIs) in drug products. To explore whether there could be other approaches to demonstrate bioequivalence for a reformulated oral product, which typically needs in vivo bioequivalence studies to support the changes after approval, the effects of antioxidant on the in vitro permeability of BCS III model drug substances were investigated to see whether there could be any potential impact on drug absorption. Six antioxidants were screened and four (ascorbic acid, cysteine, α-tocopherol and propyl gallate) were selected based on their nitrosamine inhibition efficiencies. The study demonstrated that these four antioxidants, at the tested amounts, did not have observable impact on the in vitro permeability of the BCS III model drug substances across Caco-2 cell monolayers in the In Vitro Dissolution Absorption System (IDAS). An in vitro permeability study could be considered as part of one potential bioequivalence bridging approach for reformulated low-risk immediate release solid oral products and oral suspension products. Other factors such as the influence of antioxidants on intestinal transporter activities should be considered where appropriate.

The four most used antimicrobial preservatives in biopharmaceutical parenteral formulations are phenol, meta-cresol, chlorobutanol, and benzyl alcohol. Preservatives are included in various combinations in biopharmaceuticals highlighting the importance of an analytical method to quantify the four preservatives simultaneously. A headspace GC-MS method was developed to quantify phenol, chlorobutanol, meta-cresol, and benzyl alcohol. The method was validated according to USP <1225>. System suitability was conducted daily for retention time (%RSD < 2.0%), peak area (%RSD < 5.0%), USP tailing factor (< 2.0 and %RSD < 10.0%), and peak resolution (> 2.0). Analytical ranges were 1.5-90 μg/mL for phenol and meta-cresol, 30-240 μg/mL for benzyl alcohol, and 30-300 μg/mL for chlorobutanol. Method accuracy ranged from 94% to 108% and precision from 4% to 15 %RSD for all the tested preservatives. The method was applied to three marketed teriparatide drug products selected as a model. Preservative concentrations of the biopharmaceutical marketed products were determined and were found to be comparable with the labeled concentrations, except for an expired product with 2.5% of the label claim. The developed headspace GC-MS method can be used to evaluate the drug quality of the parenteral formulations and to support the assessment of biopharmaceutical peptide drug products.

A brompheniramine taste-masked pediatric formulation was developed as part of the National Institutes of Health Pediatric Formulation Initiative to help address low patient compliance caused by the bitter taste of many adult formulations. To confirm that the taste-masked formulation can provide a similar pharmacological effect to the previous marketed adult formulations, a juvenile porcine model was used to screen the model pediatric formulation to compare the bioavailability between the marketed brompheniramine maleate and the taste-masked maleate/tannate formulation. Pigs were dosed orally with both formulations and blood samples were obtained from 0 to 48 h. Plasma samples were prepared and extracted using solid-phase extraction. The mass spectrometer was operated under selected ion monitoring mode. The selected ion monitoring channels were set to m/z 319.1 for brompheniramine and m/z 275.2 for the internal standard chlorpheniramine. Calibration curves were linear over the analytical range 0.2-20 ng/ml (r2 > 0.995) for brompheniramine in plasma. The intra- and inter-day accuracies were between 98.0 and 105% with 5.73% RSD precision. The bioanalytical method was successfully applied to a preclinical bioavailability study. The bioavailability profiles were not significantly different between the two formulations, which demonstrates that taste-masking with tannic acid is a promising approach for formulation modification for pediatric patients.

Nitrosamine compounds are classified as potential human carcinogens, the origin of these impurities can be broadly classified in two categories, nitrosamine impurity found in drug products that are not associated with the Active Pharmaceutical Ingredient (API), such as N-nitrosodimethylamine (NDMA) or nitrosamine impurities associated with the API, such as nitrosamine drug substance-related impurities (NDSRIs). The mechanistic pathway for the formation of these two classes of impurities can be different and the approach to mitigate the risk should be tailored to address the specific concern. In the last couple of years number of NDSRIs have been reported for different drug products. Though, not the only contributing factor for the formation of NDSIRs, it is widely accepted that the presence of residual a nitrites/nitrates in the components used in the manufacturing of the drug products can be the primary contributor to the formation of NDSRIs. Approaches to mitigate the formation of NDSRIs in drug products include the use of antioxidants or pH modifiers in the formulation. The primary objective of this work was to evaluate the role of different inhibitors (antioxidants) and pH modifiers in tablet formulations prepared in-house using bumetanide (BMT) as a model drug to mitigate the formation of N-nitrosobumetanide (NBMT). A multi-factor study design was created, and several bumetanide formulations were prepared by wet granulation with and without sodium nitrite spike (100 ppm) and different antioxidants (ascorbic acid, ferulic acid or caffeic acid) at three concentrations (0.1%, 0.5% or 1% of the total tablet weight). Formulations with acidic and basic pH were also prepared using 0.1 N hydrochloric acid and 0.1 N sodium bicarbonate, respectively. The formulations were subjected to different storage (temperature and humidity) conditions over 6 months and stability data was collected. The rank order of N-nitrosobumetanide inhibition was highest with alkaline pH formulations, followed by formulations with ascorbic acid, caffeic acid or ferulic acid present. In summary, we hypothesize that maintaining a basic pH or the addition of an antioxidant in the drug product can mitigate the conversion of nitrite to nitrosating agent and thus reduce the formation of bumetanide nitrosamines.