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The development and expansion of analytical methods for per- and polyfluoroalkyl substances (PFAS) in food are essential for the continued monitoring of the United States (US) food supply and assessments of dietary exposure. In March 2022, the European Union Reference Laboratory for Halogenated Persistent Organic Pollutants in Feed and Food (EURL POPs) released a guidance document covering priority PFAS of interest, including analytical method parameters and limits of quantification (LOQs). As a result, the Food and Drug Administration (FDA) began method extension work to incorporate ten new additional analytes to method C-010.02 including long-chain perfluorosulfonic acids, fluorotelomer sulfonates, and perfluorooctane sulfonamide. Four long-chain carboxylic acids were also validated across all foods, which were previously added to C-010.02 but only validated in seafood. In December 2022, the European Union published Commission Regulation 2022/2388, establishing maximum levels for perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS) in certain foodstuffs, primarily fish, molluscs, crustaceans, and eggs. As a result, the FDA method was evaluated for performance in reaching LOQs defined in Commission Regulation (EU) 2022/1431. The FDA method was found to be able to reach all required LOQs for analytes in matrices with established maximum levels. Currently, method detection limits (MDLs), which are used by the FDA as the lower limit for reporting PFAS in surveillance samples, were in the same range as defined indicative levels. With further method modifications, required LOQs could be met in fruits, vegetables, and milk. Reaching the lower targeted LOQs for these food matrices will require moving the method to an instrument that can provide increased signal:noise gains at the lower limits of quantification. [ABSTRACT FROM AUTHOR]

Introduction: Azilsartan has been scientifically proven to be effective in treating hypertension. According to International Conference on Harmonization, the HPLC method was developed and validated to estimate the azilsartan in formulated solid lipid nanoparticles and marketed pharmaceutical formulations. Objectives: Develop and validate an HPLC method for analysing azilsartan in drugs and various formulations that demonstrated stability by ICH (International Conference on Harmonization) standards. Materials and Methods: The mobile phase uses methanol: phosphate buffer (0.1% orthophosphoric acid, pH 3.2) (70:30), having the chromatographic separator is an HPLC column C18 (4.6 mm X 250 mm) with a wavelength of 249 nm and a flow rate of 1 mL/min. Results: The developed method showed a correlation coefficient value is 0.999 and to be linear throughout a concentration range of 2-10 µg/mL. The proposed method was precise (percent RSD 2.0%), accurate (percent recovery 99-101%), and reliable. The detection and quantification limits for azilsartan were determined to be 0.01 µg/mL and 0.04 µg/mL, respectively. According to ICH criteria, the developed method was validated and a stress degradation study was conducted. The developed method was used to estimate azilsartan in solid lipid nanoparticles to determine the applicability of the developed method. Conclusion: A quick, accurate, simple and economical HPLC method was successfully developed and validated for the estimation of azilsartan in solid lipid nanoparticles and marketed formulation. [ABSTRACT FROM AUTHOR]

The current work comprises of development of a simple, rapid, and precise reverse phase‐high‐performance liquid chromatography method for simultaneous estimation of propyphenazone, flurbiprofen, and their mutual prodrug. Column C18 (Shimadzu Shim‐pack Gist, 250 × 4.6 mm, 5 μm) was employed as a stationary phase, and the ratio of acetonitrile:methanol:water (40:40:20 %v/v) was used as the mobile phase. The flow rate was held at 1 mL/min and detection was carried out at 245 nm using an ultraviolet detector. The retention time of propyphenazone, flurbiprofen, and their mutual prodrug was found to be 4.0, 2.5, and 10.0 min, respectively. The proposed method was validated according to the International Council on Harmonization Q2(R1) guideline in terms of accuracy, precision, linearity, limit of detection, limit of quantitation, and solution stability. Calibration plots were linear over the concentration ranges of 2–10 μg/mL (R2 = 0.9998, R2 = 0.9992 and R2 = 0.9994 for propyphenazone, flurbiprofen, and their mutual prodrug, respectively). Results of mean percentage recoveries were in the range of 99.6%–100.1% for flurbiprofen, 99.3%–100.2% for propyphenazone, and 99.9%–100.7% for prodrug, respectively. The developed method can be used for the assay of mutual prodrug and drug release study of the developed mutual prodrug also routine quality control evaluation of mutual prodrug in bulk form. [ABSTRACT FROM AUTHOR]

A rapid stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous determination of Rilpivirine (RIL) and Cabotegravir (CAB) combination in API and pharmaceutical dosage forms. An octadecylsilane HPLC Column (Ascentis C18) with a five-micron particle size of 150 mm length and 4.6 mm internal diameter is used for analysis. A mixture of orthophosphoric acid buffer (0.1% OPA) and acetonitrile (ACN) solvent in the ratio of 3:2 is chosen as the mobile phase at a flow rate of 1.0 mL/min. A photodiode array detector was used at 257 nm for the detection. RIL and CAB solutions were analyzed in the range of 37.5 - 225 µg/mL and 25 -150 µg/mL respectively and the peak area response versus concentration curve obtained is rectilinear. The selectivity, specificity, linearity, robustness, accuracy, and precision were determined. The intended method was successful in the validation of the simultaneous determination of RIL and CAB in the pharmaceutical dosage form. The performance of the proposed method was found to be rapid and economical and is suitable for the QC and QA analysis. [ABSTRACT FROM AUTHOR]

The bio-analytical method development of Maralixibat using Elobixibat as an internal standard is a convenient, fast, accurate, and consistent new LC-MS technique and was validated. The present work explains the development of the LC-MS/MS bio-analytical method by RP-18(150x4.6 mm, 3.5µ) column and a binary mixture(0.1% formic acid & methanol) of organic mobile phase in the ratio 60:40. By using liquid-liquid extraction process, these drugs are removed from rat plasma. The linearity in the standard curve was observed under the experimental concentration range. 10%-200% (6-12ng/ml) of Maralixibat. The calibration plots were linear with a regression coefficient of R2> 0.999. Precision, Accuracy, Stability results, and Matrix effect were observed within the acceptable limit. The method is more accessible and effective for analyzing the sample in the body fluids. The work represents that Specificity, Suitability, Accuracy, and linearity parameters ideally agree with the USFDA guidelines and are efficiently practiced in rat plasma for pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

Objectives: A straightforward, accurate, and precise reverse-phase high-performance liquid chromatography method was developed to determine the quantity of Lobeglitazone in both bulk and pharmaceutical dosage forms. Materials and Methods: The chromatographic separation was achieved on a Phenomenex Luna column with dimensions of 250 cm×4.6 mm×5 µm, and the mobile phase was a combination of potassium dihydrogen orthophosphate and acetonitrile in a 70:30 V/V ratio with a pH of 4.0, adjusted using orthophosphoric acid. The flow rate was set at 1.0 mL/min, and detection of the effluents occurred at 250 nm. Results: The retention time for Lobeglitazone was determined to be 2.157 min. The drug exhibited linearity within the concentration range of 10-60 µg/mL, the correlation coefficient was established to be 0.9996. The LOD, LOQ were found to be 0.8 µg/mL and 2.5 µg/mL. The accuracy of the method was considered satisfactory and the mean recovery percentage is found to be in the acceptable range of 99.78-101.31%. Conclusion: The HPLC method was successfully developed, validated as per ICH guidelines. The proposed method was simple, precise, sensitive, rapid, robust for the estimation of Lobeglitazone in both bulk and tablet dosage forms. [ABSTRACT FROM AUTHOR]

Background: The primary objective of this research was to establish a spectrophotometric technique to quantitatively analyze the concentration of favipiravir in both its pure form and pharmaceutical preparations using bromothymol blue reagent. Materials and Methods: In this method, a yellow-colored chromagen was developed when favipiravir reacted with bromothymol blue reagent. Acetonitrile was selected as a solvent and the colored complex was detected at a wavelength of 475 nm. Results: The validation of the developed method was conducted following the guidelines set by the International Council for Harmonisation (ICH). The results demonstrated a strong linear relationship within the concentration range of 10-50 µg/mL, exhibiting a correlation coefficient of 0.9995. Moreover, the developed method exhibited excellent accuracy, precision, specificity, and sensitivity Conclusion: For routine analysis purposes, this method can be readily utilized to determine the concentration of favipiravir in both bulk samples and pharmaceutical dosage forms. [ABSTRACT FROM AUTHOR]

The Chinese yam (Dioscorea polystachya, DP) is known for the nutritional value of its tuber. Nevertheless, DP also has promising pharmacological properties. Compared with the tuber, the leaves of DP are still very little studied. However, it may be possible to draw conclusions about the plant quality based on the coloration of the leaves. Magnesium, as a component of chlorophyll, seems to play a role. Therefore, the aim of this research work was to develop an atomic absorption spectrometry-based method for the analysis of magnesium (285.2125 nm) in leaf extracts of DP following the graphite furnace sub-technique. The optimization of the pyrolysis and atomization temperatures resulted in 1500 °C and 1800 °C, respectively. The general presence of flavonoids in the extracts was detected and could explain the high pyrolysis temperature due to the potential complexation of magnesium. The elaborated method had linearity in a range of 1–10 µg L−1 (R2 = 0.9975). The limits of detection and quantification amounted to 0.23 µg L−1 and 2.00 µg L−1, respectively. The characteristic mass was 0.027 pg, and the recovery was 96.7–102.0%. Finally, the method was applied to extracts prepared from differently colored leaves of DP. Similar magnesium contents were obtained for extracts made of dried and fresh leaves. It is often assumed that the yellowing of the leaves is associated with reduced magnesium content. However, the results indicated that yellow leaves are not due to lower magnesium levels. This stimulates the future analysis of DP leaves considering other essential minerals such as molybdenum or manganese. [ABSTRACT FROM AUTHOR]

The main objective of the present study was to develop and validate a simple, precise, sensitive, and accurate ultra-performance liquid chromatography (UPLC) method for the estimation of belzutifan in pure and dosage forms. The UPLC technique was developed by using an Acquity UPLC BEH Shield C8 (100 × 2.1 mm, 1.7 μm) column. The developed technique was validated in compliance with the guidelines of the International Conference on Harmonization (ICH). Belzutifan was separated chromatographically with good resolutions using the mobile phase acetonitrile: buffer (40:60 v/v) at a flow rate of 0.5 mL/min, injection volume of 5 μL, and a wavelength of 228 nm. The validated technique was found to be linear within the range 2.5–15 μg/mL. Belzutifan detection and quantification limits were determined to be 0.3 and 1 μg/mL respectively. The % RSD was not more than 2%, demonstrating the precision of the developed technique. Furthermore, the rate of recovery was close to 100%, confirming the method's accuracy. Minor changes in the chromatographic conditions revealed the method's robustness. The analytical method developed was precise, simple, reproducible, and sensitive. Hence, it can be used to estimate belzutifan. [ABSTRACT FROM AUTHOR]

A precise and specific reverse phase high-performance liquid chromatographic method has been developed and validated to quantify curcumin, ascorbic acid, and salicylic acid in both bulk and hydrogel. Utilizing a Hypersil BDS C18 column and an isocratic mode, the mobile phase comprised of a mixture of 0.1% orthophosphoric acid and acetonitrile (50:50 v/v). The calibration range spanned concentrations of 100 - 300 µg/mL for curcumin, 50 - 150 µg/mL for ascorbic acid and 50 - 150 µg/mL for salicylic acid. The specificity of the proposed method for estimating these compounds was established through chromatographic peak purity analysis. The limit of detection and the limit of quantification were found to be 18.54 µg/mL and 56.20 µg/mL for curcumin, 10.05 µg/mL and 30.46 µg/mL for ascorbic acid, and 11.39 µg/mL and 34.51 µg/mL for salicylic acid respectively. The accuracy of the method was demonstrated by recovering curcumin, ascorbic acid, and salicylic acid from the hydrogel formulation with a recovery rate exceeding 98%. This indicates the capability of the method to accurately estimate active pharmaceutical ingredients in hydrogel dosage form without interference from excipients. Validation results support the potential applicability of the proposed method for the quantitative estimation of these three drugs in hydrogel. [ABSTRACT FROM AUTHOR]

Principles and problems in the development of simultaneous liquid chromatography (LC) analytical methods for potent antioxidative molecules resveratrol, tocopherol, and coenzyme Q10 in capsules, have been investigated and systematically compared and summarized. For these purposes, experiments within the full polarity spectrum of LC techniques. were tested and recorded. The whole range of polarities included: Alkyl C18 bonded reversed phase, phenyl, cyanopropyl, diol, and the most polar base silica‐filled column matrixes have been used. The summarized results concluded that all mentioned LC techniques could be used for the determination of the mentioned group of the three analytes with different run characteristics and efficiency. These successes could be achieved after careful analyses of molecular physicochemical data of analytes. They are especially organic solubilities. The ultraviolet spectral absorption characteristics of each analyte and the mobile phase constituents for appropriate separation were very important to be known. The ultimate targets were the development method with the isocratic mode of separation yielding symmetrical peak shapes for the best sensitivity and accuracy, with the shortest run time and best reproducibility. From an analytical point of view important for LC, the three analytes have quite distinct characteristics that contribute to successful method development. These features are their organic solvent and water solubility, molecular polarities, and ultraviolet‐absorption characteristics, like spectra and absorptivities. All these mentioned parameters were taken into account for solving complications appearing in the development of rapid LC methods for the simultaneous determination of three antioxidant molecules. [ABSTRACT FROM AUTHOR]

A cost-effective and unique RP-HPLC technology has been developed to correctly and precisely quantify the amount of Metolazone in both bulk amounts and solid dosage forms. The separation procedure was performed with an Inertsil ODS-3 HPLC Column with a particle size of 5 μm and dimensions of 250 x 4.6 mm. The experiment used the isocratic method, with a mobile phase composed of an 80:20 (v/v) blend of Acetonitrile and methanol. A pump was used to introduce the mobile phase into the column at a flow rate of 1.0 mL min-1. The eluent from the column was identified using a UV detector configured to function at a wavelength of 295 nm. The experiment lasted for 8 minutes, during which the column was continually maintained at the surrounding temperature. The calculated retention time for Metolazone was 3.557 minutes. The standard curves demonstrated a consistent linear correlation over the concentration range of 02-10 μg/ml, with a very high coefficient of determination (R2 = 0.9997). The investigation yielded a range of percentage recoveries, spanning from 98% to 102%. In addition, the Relative Standard Deviation (RSD) was calculated to be 0.290%. The percentage content of a commercially available version of Metolazone was tested to be 100.18%. The technique was validated in compliance with the guidelines established by the International Council for Harmonisation of Technical guidelines for Pharmaceuticals for Human Use (ICH). The proposed RP-HPLC technology has undergone validation via study, demonstrating its attributes of simplicity, specificity, speed, reliability, and consistency. Hence, the methodology proposed in this research may be used for the routine examination of Metolazone in both its concentrated and solid forms, particularly with the objective of ensuring quality assurance. [ABSTRACT FROM AUTHOR]

A cost-effective and innovative reverse phase high performance liquid chromatography (RPHPLC) technique has been devised to accurately and precisely measure the quantity of Atomoxetine Hydrochloride in both large quantities and solid dosage forms. The separation process was conducted using an Xterra RP 18 column with dimensions of 250 mm × 4.6 mm and a particle size of 5 μ. The experiment used the isocratic technique, with a mobile phase consisting of a 70:30 (v/v) mixture of water and methanol. The mobile phase was injected into the column using a pump, with a flow rate of 1.0 mL min-1. The eluent from the column was detected using a UV detector set to operate at a wavelength of 280 nm. The experiment had a duration of 5 minutes, during which the column was consistently kept at the ambient temperature. The determined retention time for Atomoxetine Hydrochloride was 3.08 minutes. The standard curves exhibited a linear relationship throughout the concentration range of 02-10 μg/ml, with a high coefficient of determination (R2 = 0.9997). The study resulted in a variety of percentage recoveries, ranging from 99% to 101%. Moreover, the Relative Standard Deviation (RSD) was computed to be 0.290%. The measured percentage composition of a commercially available formulation of Atomoxetine Hydrochloride was determined to be 100.18%. The approach was verified in accordance with the requirements set out by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The suggested RP-HPLC technique has been validated via research, showcasing its characteristics of simplicity, specificity, speed, reliability, and uniformity. Therefore, the approach suggested in this study may be used for the regular analysis of Atomoxetine Hydrochloride in both its concentrated and solid states, specifically with the aim of assuring quality control. [ABSTRACT FROM AUTHOR]

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Development of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom described. Over the last twenty years we have been developing several new methods, such as in situ PLA, proxHCR, and MolBoolean, using oligonucleotide-conjugated antibodies to visualize protein-protein interactions. Herein, we describe the rationale behind the oligonucleotide systems of these methods. The main objective of this paper is to provide researchers with a description on how we thought when we designed those methods. We also describe in detail how the methods work and how one should interpret results. Understanding how the methods work is important in selecting an appropriate method for your experiments. We also hope that this paper may be an inspiration for young researchers to enter the field of method development. Seeing a problem is a motivation to develop a solution. [ABSTRACT FROM AUTHOR]

An innovative, cost-effective reverse phase high performance liquid chromatography (RPHPLC) approach has been devised to accurately and precisely determine the quantity of Nelaribine in both bulk and sterile dosage forms. The separation process was conducted using an Inertsil C18-ODS 3V column with dimensions of 250×4.6 mm and a particle size of 5 µm. Isocratic mode was employed, with a mobile phase consisting of a mixture of acetic acid pH-4.0 buffer and methanol in a ratio of 75:25 (v/v). The mobile phase was pumped into the column at a flow rate of 1.5 mL min-1. The eluent from the column was detected using a UV detector set at 260 nm. The whole duration of the experiment was 8 minutes, during which the column was kept at the ambient temperature. The observed retention time for Nelaribine was determined to be 3.99 minutes. The linearity of the standard curves was seen within the concentration range of 20-100 µg/ml, exhibiting a high coefficient of determination (R2 = 0.9998). The results of the experiment indicated a percentage recovery ranging from 100.0272% to 101.8%. Additionally, the relative standard deviation (RSD) was determined to be 0.3693%. The measured percentage content of a commercially available Nelaribine formulation was determined to be 100.20 %. The methodology was verified in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) recommendations. The suggested RP-HPLC technique has been validated by investigations, which have shown that it has characteristics of simplicity, specificity, rapidity, reliability, and reproducibility. Therefore, the approach presented in this study may be used for the regular analysis of Nelaribine in both bulk and injectable dose forms, specifically for quality control purposes. [ABSTRACT FROM AUTHOR]

A novel and economical reverse phase high performance liquid chromatography (RP-HPLC) method has been developed to reliably and precisely quantify the amount of Doravirine in both bulk and solid dose forms. The separation procedure was performed with a Dionex C18 column with dimensions of 250 x 4.6mm and a particle size of 5μ. The experiment used the isocratic method, using a mobile phase composed of a combination of Ortho phosphoric acid pH-6.0 buffer and methanol in a ratio of 60:40 (v/v). The mobile phase was introduced into the column by means of a pump, with a flow rate of 1.0 mL min-1. The eluent from the column was identified using a UV detector configured to operate at a wavelength of 316 nm. The experiment lasted for a total of 6 minutes, during which the column was maintained at the surrounding temperature. The measured retention period for Doravirine was found to be 2.20 minutes. The standard curves demonstrated linearity within the concentration range of 10-50 μg/ml, with a good coefficient of determination (R2 = 0.9997). The trial yielded a range of percentage recoveries, ranging from 102% to 98%. Furthermore, the RSD was calculated to be 0.3693%. The quantified percentage composition of a commercially accessible Doravirine formulation was found to be 100.30%. The technique was validated following the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The proposed RP-HPLC technology has undergone validation via research, demonstrating its attributes of simplicity, specificity, speed, dependability, and consistency. Hence, the methodology proposed in this research may be used for the routine examination of Doravirine in both its concentrated and solid forms, particularly for the goal of ensuring quality control. [ABSTRACT FROM AUTHOR]

Tricyclic antidepressants are commonly employed in the management of major depressive disorders. The present work describes two visible (VIS) spectrophotometric techniques that utilize the formation of charge transfer complexes between four antidepressant compounds, namely, amitriptyline hydrochloride (AMI), imipramine hydrochloride (IMI), clomipramine hydrochloride (CLO), and trimipramine maleate (TRI) acting as electron donors and two p-benzoquinones, namely, p-chloranilic acid (pCA) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), serving as electron acceptors. The stoichiometry of the compounds produced exhibited a consistent 1:1 ratio in all instances, as established by Job's method. Molar absorptivities, equilibrium association constants, and several other spectroscopic properties were determined for all complexes. The developed spectrophotometric techniques were validated intra-laboratory and successfully applied for quantitative assessment of the four antidepressant active ingredients in several commercial pharmaceutical formulations. The methods are relatively simple, fast, and use readily available laboratory instrumentation, making them easily applicable by most quality control laboratories worldwide. [ABSTRACT FROM AUTHOR]

Amphotericin B (AMB) is a polyene macrolide antifungal agent used for treating invasive fungal infections. Liposomal AMB is a lipid dosage form, available as AmBisome, which reduces the toxicity of the drug. A simple HPLC‐UV method was developed for the determination of AMB in plasma to study its pharmacokinetic profile in a critical patient receiving AmBisome and treated with extracorporeal replacement therapies. Sample preparation was performed using plasma deproteinization and drug release from liposome by the addition of acetonitrile (ACN)/zinc sulfate and ultrasonication. Chromatographic separation was performed using a C18 column and a mobile phase consisting of phosphate buffer (pH 3.0)/ACN (65/35, v/v). The UV detector was set at 407 nm. The total run time analysis was 23 min. The method was validated according to the standard guidelines and applied to study the pharmacokinetics of AMB in a critical patient. The total run time analysis obtained was shorter than that of the previously reported methods, being useful for therapeutic drug monitoring or pharmacokinetic profile research. [ABSTRACT FROM AUTHOR]

Determination of the chemical composition of waste Sm-Co magnets is required for their efficient recycling. The non-stereotypical composition of said magnets makes an analysis extremely challenging. X-ray fluorescence spectrometry is a promising analytical tool for this task. It offers high accuracy and simplicity of sample preparation as it does not require sample dissolution. However, a serious limitation of X-ray fluorescence analysis is the spectral interference of matrix elements and impurities. In this work, a two-stage technique has been developed for the determination of the main components (Sm, Co) and impurities (Fe, Cu, Zr, Hf, Ti, Ni, Mn, Cr) in samples of spent samarium–cobalt magnets using wavelength dispersive X-ray fluorescence spectrometry. In order to overcome the main limitation of the chosen method and to maximize its capabilities of qualitative and quantitative analysis, we propose an approach to the selection of analytical lines and experimental conditions, as well as a preparation method for the calibration standards. The obtained results have been shown to have a good correlation with ICP-OES. The limits of detection are in the range of 0.001–0.02 wt%, and the limits of quantification are 0.003–0.08 wt%. [ABSTRACT FROM AUTHOR]

Abstract Shilajit, along with Triphala, has been one of the most widely used herbal drug combinations for the symptomatic treatments of hyperthyroidism. However, the high polarity difference among active constituents proposes difficulties while analyzing its quality. In this study, two high-performance thin-layer chromatography methods were developed for chromatographic fingerprinting of the proposed herbal mixture of Triphala and Shilajit. A reliable HPTLC method effectively analyzed bioactive phytoconstituents such as quercetin, myricetin, gallic acid, ellagic acid, tannic acid, ascorbic acid, and humic acid. Method 1 showed good separation of quercetin, myricetin, and tannic acid, but separating gallic and ellagic acid proved difficult. To address this issue, the 2D-TLC approach involves a second mobile phase. Although humic acid and ascorbic acid were not detected due to their highly polar and non-polar nature, so a different HPTLC method 2 was developed. The successful method of development using the Quality by Design approach and validated. The developed method successfully analyzed the bioactive constituents of Shilajit, in combination with Triphala, and validation parameters such as linearity, accuracy, precision, range, specificity, robustness, the limit of detection (LOD), and limit of quantification (LOQ) agreed with given values. [ABSTRACT FROM AUTHOR]

Background: In line with the European Paediatric Regulation, the European Medicines Agency (EMA) asks for investigation of a medicine's acceptability in paediatric medicines development. A standardised acceptability testing method combining the outcome of "swallowability" and "palatability" assessments to a "composite endpoint on acceptability" was recently developed. Before this method's suitability for selection of the most acceptable drug formulation of a new medicine for children can be broadly recommended, the acceptance and relevance of such established acceptability needs the critical review and input from young patients with understanding of the medicines development methodology. The benefit of involving patients in drug product development, clinical research and innovation is well established. Methods: During a focus group meeting with the KIDS Barcelona (young people advisory group, age 16–23 years) the suitability of the "composite endpoint on acceptability" methodology was assessed. Via electronic questionnaires the importance of involving patients in the medicines development and in the acceptability method development was investigated. Questions on how best to determine palatability and swallowability were asked. The relevance of all EMA-listed acceptability elements was assessed via coloured and numbered stickers and questionnaires. Results: The results showed that the involvement of young people in the medicines and acceptability method development was rated high. The group worked out that a 5-point smiley Likert Scale is preferred for assessing acceptability by 6–11 year old patients, while a Visual Analogue Scale is preferred for collecting adolescents' opinion. The ranking of the EMA-listed acceptability elements showed that palatability and swallowability are the most relevant parameters, while colour of the medicine was rated as least relevant. These results, established face-to-face, were confirmed in a repeat of the ranking through an electronic questionnaire, completed by the participants individually and remotely, 5 weeks later. Conclusion: This work reinforced the need and value to involve young people in the medicines lifecycle, and specifically in this acceptability method development. As next step other focus group meetings with more young people from different European countries are planned. Plain English Summary: Before a new medicine is authorized, its acceptability by children must be investigated according to law. An acceptability testing method combining the outcomes of "swallowability" and "palatability" assessments was recently developed. During a focus group meeting with KIDS Barcelona (young people advisory group, age 16–23 years) their opinion on the suitability of the method and the relevance of patient engagement in the medicines development process were assessed with paper-based and electronic questionnaires. Questions on how best to determine palatability and swallowability were asked. The importance of different elements that typically affect acceptability was rated. The order of relevance of those listed acceptability elements was assessed using coloured and numbered stickers and questionnaires. The results showed that the involvement of young people in the medicines and acceptability method development was rated high. The group worked out that a 5-point smiley Likert Scale that allows for marking a choice between total agreement and total disagreement is preferred for assessing acceptability by 6–11 year old patients. A Visual Analogue Scale (scale consisting of a 10 cm long line on which a mark has to be placed at the desired position, between total agreement and total rejection) is preferred for collecting adolescents' (12–18 years) opinion. The ranking of acceptability elements showed that palatability and swallowability of a new medicine are the most relevant parameters, and colour the least. The clarity of the outcome reinforced the benefit of involving young people in the development of medicines relevant for children. [ABSTRACT FROM AUTHOR]

Herbal medicines are considered to be safe and efficient and are widely used for the prevention and treatment of chronic disorders. However, quality control of these formulations and their standardization in terms of quantities of biomarker compounds are extremely important. These tasks are quite challenging in case of polyherbal formulations as there are hundreds of phytoconstituents present at nanogram or picogram levels. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are useful tools in such cases as they are highly sensitive and specific. Majoon-e-Nisyan (MJN) is a polyherbal Unani formulation used in the treatment of amnesia. It contains five constituent herbs Kundur (Boswellia serrate Roxb.), Waj (Acorus calamus L.), Saad Koofi (Cyperus rotundus L.), FilfilSiyah (Piper nigrum L.), Zanjabeel (Zingiber officinale Rosc.) made in Asl-asli (honey). In this paper, we have identified five biomarker compounds, one from each constituent herb, viz., (α + β) boswellic acid, β-asarone, luteolin, 6-gingerol and piperine, and developed an LC-MS/MS method for their simultaneous determination in MJN formulation. Further, the developed method was validated for accuracy, precision, linearity, specificity and sensitivity according to ICH guidelines. We found that the linearity of each calibration curve displayed r2 value not less than 0.99, the intra- and interday precisions showed RSD in the range of 0.7–1.0 and 1.0–2.9%, respectively. We achieved recovery of each marker compound in the range of 87.5–94.1%. Moreover, the limit of detection and limit of quantification were found to be in the range of 0.3 to 5 ng/mL. Finally, the developed method was applied to quantify the marker compounds in MJN formulation. Our method successfully quantified (α + β) boswellic acid, β-asarone, luteolin, 6-gingerol and piperine as 0.973 ± 0.009, 0.093 ± 0.004, 0.003 ± 0.001, 0.0256 ± 0.002 and 0.0266 ± 0.008 mg/g of MJN formulation. To the best of our knowledge, this is the first reported method for the simultaneous determination of (α + β) boswellic acid, β-asarone, luteolin, 6-gingerol and piperine in MJN formulation. [ABSTRACT FROM AUTHOR]

A novel, simple, specific, rapid, enantioselective normal phase chiral high‐performance liquid chromatographic method with amylose‐based Chiral Pak IG‐3(250 × 4.6 mM) 3.0 μM column was developed and validated for separation and quantification of isomers and enantiomer of Valbenazine. The mobile phase composed of n‐Heptane, isopropyl alcohol, dichloromethane, ethanol, and diethylamine in the ratio of 70:10:15:5:0.1 (V/V/V/VV) with a gradient flow rate was applied. The injection volume was 10 μl, and detection was carried out using a photodiode array detector at 282 nM. The column compartment was set at 35°C. The resolution between the enantiomer and isomers was found to be more than 2.0. The method was linear over the concentration range of limit of quantitation to 250% for isomers and enantiomers. The method was found to be robust with column temperature. The proposed chiral method is applicable for the determination of isomers and enantiomer of Valibenazine and was successfully used in the quality control of bulk drug manufacturing and pharmaceuticals. [ABSTRACT FROM AUTHOR]

There is no stability-indicating specific related substances high-performance liquid chromatography (HPLC) method for anagliptin active pharmaceutical ingredients in the presence of its degradation products. Hence, there is a necessity to have a specific stability-indicating HPLC method for the quantification of anagliptin-related substances in the presence of its forced degradation products. The aim of the research work is to develop an innovative simple, accurate, precise, and selective HPLC method with the lesser use of organic solvents, for the quantification of anagliptin-related substances in the presence of its forced degradation products. The chromatographic separation was achieved on a YMC Pro C18 reversed-phase HPLC column (250 mm × 4.6 mm, 5 μ) with a runtime of 50 min. Mobile phase A and mobile phase B were chlorate buffer with pH 3.2 and acetonitrile respectively. The HPLC column oven temperature was set at 28°C and the photodiode array detector was set at 205 nm. The proposed test procedure has shown excellent linearity within the concentration range 0.081–6.296 μg/mL with correlation coefficient (r) about 0.9998. The limit of detection of anagliptin and related substances was observed within the range 0.052–0.106) μg/mL and the limit of quantification was observed within the range 0.156–0.317 μg/mL. The developed test procedure was successfully utilized for the quantification of related substances of anagliptin bulk drug without any interference with the degradation compounds. Hence, the test procedure can be utilized successfully in pharmaceutical organizations for the separation and quantification of anagliptin-related substances. [ABSTRACT FROM AUTHOR]

Pharmaceutical companies in Ukraine aspire to develop their innovative medicinal products and successfully introduce them to the global market. However, along with the prospects of increased usage of these pharmaceuticals, there arises a challenge of heightened waste production, making them a part of the over twenty million tons of PPCPs produced annually. Consequently, one of the tasks in producing new pharmaceuticals is the development of methodologies and approaches not only for quality control but also for their determination in the environment matrices. The aim. Develop and validate GC-FID chromatographic method for the simultaneous determination of Enisamium iodide and Tilorone dihydrochloride, evaluate their applicability, and compare their "greenness" with the previously developed HPLC method. Materials and methods. The determination of the Tilorone dihydrochloride and Enisamium iodide was carried out by gas chromatography with a flame ionization detector using the Rxi-5 ms (30 m long, 0.25 mm outer diameter and 0.25 μm liquid stationary phase thickness). Results. Chromatographic GC-FID methods have been developed for the simultaneous determination of Enisamium iodide and Tilorone dihydrochloride. Optimal sample preparation conditions were established, and a validation process was conducted. A comparison with the previously developed HPLC method was made regarding "greenness." Conclusions. The developed GC-FID methodology is accurate and more environmentally friendly compared to the previously established methods. It can be recommended to determine Enisamium iodide and Tilorone dihydrochloride in the environmental matrices. It is considered environmentally friendly based on the overall GREENness (AGREE) scale, scoring 0.73 (>0.70), which demonstrates the environmentally favourable nature of the proposed analytical approach. [ABSTRACT FROM AUTHOR]

Quality by Design (QbD) principles play an increasingly important role in the pharmaceutical industry. Here, we used an analytical QbD (AQbD) approach to develop a capillary electrophoresis sodium dodecyl sulfate under reducing conditions (rCE‐SDS), with the aim of replacing SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) as release and stability test method for a commercialized monoclonal antibody product. Method development started with defining analytical method performance requirements as part of an analytical target profile, followed by a systematic risk assessment of method input parameters and their relation to defined method outputs. Based on this, design of experiments studies were performed to identify a method operable design region (MODR). The MODR could be leveraged to improve method robustness. In a bridging study, it was demonstrated that the rCE‐SDS method is more sensitive than the legacy SDS‐PAGE method, and a conversion factor could be established to compensate for an off‐set due to the higher sensitivity, without losing the correlation to the historical data acquired with the former method. Overall, systematic application of analytical Quality by Design principles for designing and developing a new analytical method helped to elucidate the complex dependency of method outputs on its input parameters. The link of the method to product quality attributes and the definition of method performance requirements were found to be most relevant for derisking the analytical method switch, regarding impact on the control strategy. [ABSTRACT FROM AUTHOR]

Diabetes is a long-term metabolic disease epitomized by postprandial hyperglycemia. The prolonged use of synthetic drugs renders distinct side effects, necessitating the development of safe and cost-effective substitutes. The aim of the current study is to isolate, evaluate the antidiabetic potential and HPLC method development for simultaneous estimation of antidiabetic compounds from the leaves of Tradescantia pallida. The leaves were extracted, fractionated and subjected to column chromatography. The isolated compounds' antidiabetic potential was evaluated using α-amylase and glycosylation of hemoglobin assays. The study employed molecular docking to scrutinize interactions between antidiabetic compounds and human α-amylase and hemoglobin protein. Prime MM-GBSA calculations determined binding energies of ligand–protein complexes. Further analysis of morin and catechin involved exploring dynamic and thermodynamic constraints through molecular dynamics simulations under specific biological conditions. A rapid HPLC method was developed and validated for the simultaneous estimation of isolated compounds. The column chromatography culminated in the isolation of four antidiabetic compounds (syringic acid, catechin, p-coumaric acid and morin). The in vitro analyses revealed that morin and catechin exhibited 72.67 % and 78 % α-amylase inhibition and 67 % and 71.66 % inhibition of hemoglobin glycosylation, respectively. In silico studies substantiated the in vitro assay, confirming the stability of catechin and morin complexes via root mean square deviation analysis. Interactions, encompassing hydrophilic, hydrophobic, water bridges, and ionic interactions, identified key residues involved in these processes. The validated HPLC method exhibited excellent correlation coefficients ranged from 0.9909 to 0.9997. The antidiabetic compounds were quantified from the extract in the range of 0.072 – 0.160 µg/mL. The study concluded that the isolated compounds from Tradescantia pallida have remarkable antidiabetic activity, and the developed method can be successfully used for the identification and quantification of phenolic compounds in Tradescantia pallida and other plant-derived matrix.

Molecular biomonitoring programs increasingly use environmental DNA (eDNA) for detecting targeted species such as marine non-indigenous species (NIS) or endangered species. However, the current molecular detection workflow is cumbersome and time-demanding, and thereby can hinder management efforts and restrict the “opportunity window” for rapid management responses. Here, we describe a direct droplet digital PCR (direct-ddPCR) approach to detect species-specific free-floating extra-cellular eDNA (free-eDNA) signals, i.e., detection of species-specific eDNA without the need for filtration or DNA extraction, with seawater samples. This first proof-of-concept aquarium study was conducted with three distinct marine species: the Mediterranean fanworm Sabella spallanzanii, the ascidian clubbed tunicate Styela clava, and the brown bryozoan Bugula neritina to evaluate the detectability of free-eDNA in seawater. The detectability of targeted free-eDNA was assessed by directly analysing aquarium marine water samples using an optimized species-specific ddPCR assay. The results demonstrated the consistent detection of S. spallanzanii and B. neritina free-eDNA when these organisms were present in high abundance. Once organisms were removed, the free-eDNA signal exponentially declined, noting that free-eDNA persisted between 24–72 h. Results indicate that organism biomass, specimen characteristics (e.g., stress and viability), and species-specific biological differences may influence free-eDNA detectability. This study represents the first step in assessing the feasibility of direct-ddPCR technology for the detection of marine species. Our results provide information that could aid in the development of new technology, such as a field development of ddPCR systems, which could allow for automated continuous monitoring of targeted marine species, enabling point-of-need detection and rapid management responses.

Abstract Atomic force microscopy (AFM) is an analytical technique that is increasingly utilized to determine interaction forces on the colloidal and cellular level. Fluidic force microscopy, also called FluidFM, became a vital tool for biomedical applications. FluidFM combines AFM and nanofluidics by means of a microchanneled cantilever that bears an aperture instead of a tip at its end. Thereby, single colloids or cells can be aspirated and immobilized to the cantilever, for example, to determine adhesion forces. To allow for quantitative measurements, the so‐called (inverse) optical lever sensitivity (OLS and InvOLS, respectively) must be determined, which is typically done in a separate set of measurements on a hard, non‐deformable substrate. Here, we present a different approach that is entirely based on hydrodynamic principles and does make use of the internal microfluidic channel of a FluidFM‐cantilever and an external pressure control. Thereby, a contact‐free calibration of the (inverse) optical lever sensitivity (InvOLS) becomes possible in under a minute. A quantitative model based on the thrust equation, which is well‐known in avionics, and finite element simulations, is provided to describe the deflection of the cantilever as a function of the externally applied pressure. A comparison between the classical and the here‐presented hydrodynamic method demonstrates equal accuracy.

Differential scanning calorimetry (DSC) is one of the most widely developed thermal analysis methods for meat samples for halal authentication of food or processed products. Research on adulteration detection for various types of meat and its derivatives has been developed before and still requires organic solvents. Therefore, the concept of the "green method" is being tried to develop in this research. DSC analyses are performed in the same experimental conditions for all sample powder: sample mass 2 mg, temperature range 30–400 °C, and heating rate 20 °C min−1. The results showed there is a characteristic minor endothermic peak for each meat. Chemometric analysis was carried out using the principal component analysis (PCA) method to ensure that the thermal characteristics of each meat were utterly different in both pure and mixed meat. The results of this analysis indicate that each pure meat has a different score plot. Therefore, the developed thermal analysis method is quite reliable in determining the different types of meat based on the characteristic minor endothermic peak in the thermogram and the score plot from PCA analysis.

Abstract Background Veterinary drugs are widely used in animals to prevent diseases and are a complex set of drugs with very different chemical properties. Multiclass and multi-residue methods for simultaneous detection of residues from veterinary drugs and contaminants in urine are very rare or non-existent. Therefore, the aim of this study was to develop and validate a sensitive and reliable quantitative LC-MS/MS method for simultaneous determination of a wide range of veterinary drug and pesticide residues and mycotoxins in bovine urine. This involved 42 veterinary drug residues (4 thyreostats, 6 anabolic hormones, 2 lactones, 10 beta agonists, 15 antibiotics, 5 sulphonamides), 28 pesticides and 2 mycotoxins. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. Analysis was performed in both positive and negative ionization modes with multiple reaction monitoring transitions over a period of 12 min. Results The parameters validated included linearity, limit of detection (LOD), limit of quantification (LOQ), detection capability (CCβ), decision limit (CCα), stability, accuracy and precision. The process followed guidelines of the regulation 2021/808/EC. The calibration curves were linear with coefficient of correlation (R2) from 0.991 to 0.999. The LODs were from 0.01 to 2.71 µg/L, while the LOQs were from 0.05 to 7.52 µg/L. The CCα and CCβ were in range 0.05–12.11 µg/L and 0.08–15.16 µg/L. In addition, the average recoveries of the spiked urine samples were from 71.0 to 117.0% and coefficient of variation (CV)

Mass spectrometry (MS) is a highly selective and sensitive analytical tool with a myriad of applications, but such techniques are typically used in laboratory settings due to the handling and preparations that are necessary. The merging of two streams of robust research, portable MS systems and next-generation ambient ionization methods, now provides the ability to perform high-performance chemical screening in an on-site and on-demand manner, with natural applications in disciplines such as forensic science, where samples of interest are typically found in field environments (i.e., traffic stops, crime scenes, etc.). Correspondingly, investigations regarding the suitability and robustness of these methodologies when they are utilized for authentic forensic evidence processing are prudent. This work reports critical insights into the role that choice of spray solvent system plays regarding analytical performance of two spray-based ambient ionization sources, paper spray ionization (PSI) and filter cone spray ionization (FCSI), when employed for evidence types containing emerging synthetic cannabinoids. The systematic characterization studies reported herein show that the applied spray solvent can dramatically affect both spectral intensity and signal duration, and in some circumstances, yield deleterious false negative responses. Overall, acetonitrile-based systems are shown to strike a balance between analyte solubility concerns and spray ionization dynamics of the novel ion sources employed on portable MS systems.

Abstract Background Nateglinide and metformin HCl are used in combination for the treatment of type 2 diabetes. A simple, sensitive and reliable UPLC method was developed for simultaneous estimation of nateglinide and metformin HCl using Phenomenox C18 (50*2.1 mm, 3.5 µm) column at ambient temperature as stationary phase in addition to mobile phase containing 75 volumes of ammonium formate buffer (pH = 3) along with 25 volumes of acetonitrile with a flow rate of 0.2 mL/min with UV detection at 260 nm and a run time of 3 min. The developed method was validated as per ICH Q2(R1) guidelines. Results The separation of metformin HCl and nateglinide was done at retention times of 1.014 min and 1.435 min, respectively. The mean % recovery for nateglinide and metformin HCl in the accuracy study was observed to be 99.9% and 99.2%, respectively. LOD and LOQ values were determined considering the S/N ratio and were found to be 0.09 µg/mL and 0.3 µg/mL, respectively, for nateglinide and 0.75 µg/mL and 2.5 µg/mL, respectively, for metformin HCl. The method was found to be precise with % RSD values of 0.58 and 0.45, respectively, for repeatability and intermediate precision of nateglinide and 0.43 and 0.43, respectively, for repeatability and intermediate precision of metformin HCl which were within acceptance criteria. The method was found to be linear in the range of 7.5–45 µg/mL and 62.5–375 μg/mL for nateglinide and metformin HCl, respectively. The regression equations for nateglinide and metformin HCl were found to be y = 17377x + 6543.4 and y = 18439x + 43,537, respectively. The method was found to be robust by deliberate changes in the method parameters like flow rate and mobile phase composition. Forced degradation studies were performed as per ICH Q1A(R2) and Q1B guidelines, and peak purity was observed in all types of degradation studies for both the drugs. Conclusion The developed method was found to be satisfactory as it is simple, sensitive, accurate, precise, robust, rapid, economical and yet stability indicating and can be applied successfully in the routine laboratory analysis for the simultaneous estimation of nateglinide and metformin HCl in the bulk and pharmaceutical dosage forms.

The main objective of this study is to develop and validate an LC-MS/MS stability-indicating method for the isolation, identification, and characterization of forced degradation products of mobocertinib under various stress conditions, utilizing LC-MS/MS and NMR techniques. Four degradation products (DP1, DP2, DP3 and DP4) were isolated. Degradation under acidic and oxidative conditions provides DP1 and DP4, respectively. Both DP2 and DP3 were obtained under basic conditions. Chromatographic development and separation were achieved using an ODS C18 column (Chemsil) with a constant flow rate of 1.0 ml/min. The mobile phase consisted of 40 % water containing 0.1 % formic acid in methanol, and detection was performed at a wavelength of 225 nm. The application of LC-MS/MS technology was utilized to separate the above degradation products. The LC-MS/MS method has validated which includes specificity, linearity, LOD and LOQ, precision, accuracy and robustness. The chemical structure of the degradation products were characterized by 1H NMR and mass spectroscopy. The above listed degradation impurities are novel and not listed in the literature. In conclusion, this study provides vital information about mobocertinib’s chemical stability and helpful in further drug development process.

The goal of this study was to use High performance liquid chromatography (HPLC) with an Analytical Quality by Design (AQbD) approach to measure the amount of caffeine in selected coffee samples available in the local Jazan market in Saudi Arabia. The experiment was performed on a Waters HPLC system equipped with a Raptor C-18 column. To achieve chromatographic separation, a mobile phase of ammonium acetate buffer (10 mM; pH 4.0) and acetonitrile in a 90:10 v/v ratio was used with a flow rate of 1 mL/min for 5 minutes at 274 nm. AQbD was used to optimize chromatographic conditions using a central composite design (CCD). A factorial design of 23 (two factors and three responses) was used with Stat-Ease Inc.'s Design Expert 13.0.3.0 software, Minneapolis, USA. The HPLC method was validated using ICH and USP guidelines, and it met all acceptance criteria. In the dilution range of 2–28 µg/mL, the procedure was shown to be linear. The caffeine content of all five samples (5 g/100 mL) was found to be 20.59–25.38 mg, which was below the level of toxicological concern recommended by the USFDA.

Soil organic carbon (SOC) is in the focus of research due to its central role in regulating climate and maintaining fertility and resilience of soils. Methodologically, shifting from whole soil C measurements to specific SOC fractions increases possibility to detect small changes in the vast SOC storage, and enhances estimation of SOC stability. However, SOC fractionation schemes are numerous and variable. In this study, deionized water and sodium hexametaphosphate (SHMP) were compared in soil dispersion by separating soils into coarse (0.25–2 mm), medium (0.063–0.25 mm) and fine (

Organochlorine pesticides (OCPs) are persistent organic pollutants (POPs) widely used in agriculture and industry, causing serious health and ecological consequences upon exposure. This review offers a thorough overview of OCPs analysis emphasizing the necessity of ongoing work to enhance the identification and monitoring of these POPs in environmental and human samples. The benefits and drawbacks of the various OCPs analysis techniques including gas chromatography-mass spectrometry (GC-MS), gas chromatography-electron capture detector (GC-ECD), and liquid chromatography-mass spectrometry (LC-MS) are discussed. Challenges associated with validation and optimization criteria, including accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ), must be met for a method to be regarded as accurate and reliable. Suitable quality control measures, such as method blanks and procedural blanks, are emphasized. The LOD and LOQ are critical quality control measure for efficient quantification of these compounds, and researchers have explored various techniques for their calculation. Matrix interference, solubility, volatility, and partition coefficient influence OCPs occurrences and are discussed in this review. Validation experiments, as stated by European Commission in document SANTE/11813/2017, showed that the acceptance criteria for method validation of OCP analytes include ≤20 % for high precision, and 70–120 % for recovery. This may ultimately be vital for determining the human health risk effects of exposure to OCP and for formulating sensible environmental and public health regulations.

Objective: This study was performed to determine the effect of fasting on reproducibility of the glucose tolerance test. Due to individual variation in animal feeding behaviors, fasting animals prior to metabolic and behavioral experiments is widely held to reduce inter-subject variation in glucose and metabolic parameters of preclinical rodent models. Reducing variability is especially important for studies where initial metabolite levels can influence the magnitude of experimental interventions, but fasting also imposes stress that may distort the variables of interest. One such intervention is the glucose tolerance test (GTT) which measures the maximum response and recovery following a bolus of exogenous glucose. We sought to investigate how fasting affects the response of individual mice to a GTT. Methods: Using simultaneous continuous glucose monitoring (CGM) and indirect calorimetry, we quantified blood glucose, physical activity, body temperature, metabolic rates, and food consumption levels on a minute-to-minute basis in adult male mice for 4 weeks. We tested the effects of a 4-h or 18-h fast on the GTT to examine the effect of food withdrawal in light or dark photoperiods. Studies were also performed with 4-h fasting in additional mice without implanted CGM probes. Results: Contrary to our expectations, a 4-h fast during the light photoperiod promotes a paradoxical increase in inter-animal variation in metabolic rate, physical activity, body temperature, glycemia, and glucose tolerance. This hyperglycemic and hyper-metabolic phenotype promotes increased corticosterone levels and is consistent with a behavioral stress response to food deprivation, even in well-fed mice. We find that mice undergoing an 18-h fast entered torpor, a hibernation-like state. In addition to low body temperature and metabolic rate, torpor is also associated with glucose levels 56 mg/dl lower than those seen in mice with ad libitum access to food. Moreover, the time spent in torpor affects the response to a GTT. Conclusion: Our results suggest fasting mice before glucose tolerance testing, and perhaps other experiments, can have the opposite of the intended effect where fasting can increase, rather than decrease, experimental variability.