Bioanalysis[JOURNAL]
Sun
200 papers
MS-DIAL pre-processing for mining schistosomiasis metabolites in urine samples from the Okavango Delta of Botswana from a GC-MS dataset.
Tawana-Ndolo SM, Zachariah M, Phaladze NA … +1 more , Sichilongo KF
Bioanalysis · 2025 Nov · PMID 41170651 · Full text
BACKGROUND: Metabolites were re-mined using MS-DIAL from an SPME dataset preprocessed using AMDIS/Metab R in which data was acquired by GC-MS from schistosomiasis infected (positive) and non-infected (negative)/control s...
BACKGROUND: Metabolites were re-mined using MS-DIAL from an SPME dataset preprocessed using AMDIS/Metab R in which data was acquired by GC-MS from schistosomiasis infected (positive) and non-infected (negative)/control samples from school-going children aged between 9 and 12 years in the Okavango Delta region of Botswana. The remining was meant to exhaust scouring for metabolites that could have been overshadowed by Metab R. METHODS: In this study, MS-DIAL open-source freeware was used to preprocess the same dataset to increase the number of identified metabolites and to distinguish positive and control samples. RESULTS: Ninety-nine (99) metabolites were mined using MS-DIAL in contrast to thirteen identified putatively using Metab R. Both naphthalene and benzophenone, used as surrogate, and internal standards, had greater than 90% match factors using m/z ratios 128 and 105, respectively, for deconvolution with retention time similarity scores of 1000 in all the samples they were added to. CONCLUSION: More metabolites were mined using MS-DIAL compared to Metab R thus confirming the preprocessing power of the earlier compared to the latter.
Progress in monitoring molecular, cellular and metabolic dynamics using mass spectrometry imaging.
Gamcsik MP, Bruce ER, Muddiman DC
Bioanalysis · 2025 Nov · PMID 41164994 · Full text
Mass spectrometry imaging (MSI) enables the visualization of hundreds to thousands of analytes in biological tissues. MSI is also capable of mapping time-dependent processes that, combined with these static metabolite pr...
Mass spectrometry imaging (MSI) enables the visualization of hundreds to thousands of analytes in biological tissues. MSI is also capable of mapping time-dependent processes that, combined with these static metabolite profiles, provides a clearer picture of the molecular underpinnings of tissue function. This perspective is organized into sections demonstrating how MSI-based methods can provide unique functional data on systems ranging from single step enzyme-catalyzed transformations to complex metabolic network activities and cellular dynamics. This multisystem capability can be exploited to provide detailed descriptions of the molecular mechanisms contributing to tissue function. An aspect missing in many studies are corresponding maps of tissue microenvironments including oxygenation and pH which influence functional activities. Some progress has been made to map hypoxic and acidic tissue using MSI methods, but further development is needed. This includes pairing in vivo MSI functional studies to in vitro models. Additionally, integrating the capabilities of other imaging methods, such as magnetic resonance and vibrational spectroscopy, that are proven to detect tissue microenvironments, with dynamic MSI methods offer a route to match environment with functional activities. The combination of static molecular profiles, metabolic and cellular dynamics, and environmental mapping will provide the most detailed understanding of tissue function.
Development of a fit-for-purpose semi-homogeneous competitive ELISA for total PEG quantitation in a PEGylated molecule.
Yu N, Chang V, Nijem I … +1 more , Liu Y
Bioanalysis · 2025 Nov · PMID 41164983 · Full text
AIM: The therapeutic effectiveness of a drug could be significantly enhanced by prolonging its retention time. Polyethylene glycosylation (PEGylation) technology is a method to increase the half-life of protein therapeut...
AIM: The therapeutic effectiveness of a drug could be significantly enhanced by prolonging its retention time. Polyethylene glycosylation (PEGylation) technology is a method to increase the half-life of protein therapeutics. PEGylated anti-Factor D Fab (PEG-aFD) was developed as a potential therapeutic, targeting factor D to reduce the frequency of intravitreal injections in patients with geographic atrophy. To support a GLP Toxicology study, there was a need to develop a PEG assay to measure total PEG concentration. RESULT: PEG-aFD consists of Fabs conjugated to a multi-arm PEG molecule, which posed challenges for PEG assay development. A sensitive fit-for-purpose, semi-homogeneous competitive ELISA (Enzyme-Linked Immunosorbent Assay) using a biotinylated version of the therapeutic as the competing molecule was successfully developed and validated with LLOQ (lower limit of quantification) at 200 ng/mL. The assay was used to measure total PEG concentrations in cynomolgus monkey serum samples from the study. The results revealed measurable PEG levels (ranging from 0.468 μg/mL to 188 µg/mL) in all samples across all dosage levels, but no PEG-related toxicity was observed in all the animals. CONCLUSION: A competitive ELISA was successfully developed and validated to measure total PEG concentrations in cynomolgus monkey serum samples to support a GLP Toxicology study.
Correction.
Bioanalysis · 2025 Oct · PMID 41163339 · Full text
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Objective criteria for statistical assessments of cross validation of bioanalytical methods.
Yan D, Herrera M, Qian HR … +1 more , Brockus CL
Bioanalysis · 2025 Oct · PMID 41147683 · Full text
AIM: To improve the integrity and comparability of pharmacokinetic data in clinical trials by refining the statistical assessment methodology for cross validation of bioanalytical methods across multiple laboratories. MA...
AIM: To improve the integrity and comparability of pharmacokinetic data in clinical trials by refining the statistical assessment methodology for cross validation of bioanalytical methods across multiple laboratories. MATERIALS AND METHODS: Cross validation assessments were conducted using statistical tools recommended by International Council for Harmonization M10 guidance, including Bland-Altman plots, Deming regression, and Lin's Concordance. Recognizing limitations in Deming regression and Lin's Concordance for interpreting cross validation results, we introduced a combined approach: Bland-Altman plots with equivalence testing. The acceptance threshold was defined such that the 95% confidence interval of the mean log10 difference between laboratories must fall within boundaries based on method validation criteria. RESULTS: The proposed methodology was validated across diverse bioanalytical methods. Unlike conventional approaches that impose strict constraints on Deming regression parameters, our framework accommodates practical assay variability. This approach provided consistent and credible cross validation outcomes in real-world scenarios. CONCLUSIONS: Integrating Bland-Altman plots with equivalence boundaries offers a robust, statistically sound framework for cross validation in bioanalytical studies. This method enhances the quality and consistency of pharmacokinetic data, supporting more reliable clinical trial endpoints.
Significant matrix effect in fludarabine quantification in plasma: implications for CAR-T cell therapy monitoring.
De Gregori S, Capone M, Zecca M … +1 more , Albertini R
Bioanalysis · 2025 Oct · PMID 41120806 · Full text
BACKGROUND: Event-free survival (EFS) in patients with B-cell acute lymphoblastic leukemia (B-ALL) significantly improves with adequate exposure (as Area Under the Curve: AUC or AUC) to fludarabine (FLU) during lymphodep...
BACKGROUND: Event-free survival (EFS) in patients with B-cell acute lymphoblastic leukemia (B-ALL) significantly improves with adequate exposure (as Area Under the Curve: AUC or AUC) to fludarabine (FLU) during lymphodepletion prior to chimeric antigen receptor (CAR) T-cell infusion. A cumulative AUC of FLU exceeding 14 mgxh/L is considered optimal, making therapeutic drug monitoring (TDM) crucial for personalized dosing and prolonged CAR T-cell persistence. RESEARCH DESIGN AND METHODS: We developed and validated an European Medicines Agency (EMA)-compliant high-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) for FLU TDM, based on similar and published methods, to support clinicians during the patient conditioning phase. All EMA acceptance criteria were met. RESULTS: Analysis of real plasma samples revealed unexpectedly high FLU concentrations and, consequently, an excessively high AUC. Analysis of plasma samples from a child receiving FLU therapy highlighted the necessity for accurate drug response verification in real matrices. Preparation of quality controls in FLU-free plasma consistently failed without preliminary dilution. Analysis of internal standard range variation enabled more precise and accurate analyses. CONCLUSIONS: This experience highlights the importance of complementing standard validation tests with analyses in each patient's plasma, collected immediately before FLU infusion, to ensure reliable TDM to support CAR T-cell therapy.
Recent advances and clinical applications for the rapid detection of pathogenic bacteria in biological samples.
Liu J, Miao Z, Yan Y … +5 more , Deng L, Yang S, Li X, Liu W, Wang H
Bioanalysis · 2025 Oct · PMID 41108125 · Full text
Infectious diseases have a wide range of significant characteristics with complex clinical manifestations. The incidence rate and mortality rate remain high, seriously endangering human health. Therefore, rapid identific...
Infectious diseases have a wide range of significant characteristics with complex clinical manifestations. The incidence rate and mortality rate remain high, seriously endangering human health. Therefore, rapid identification and sensitive diagnosis of pathogens are crucial for patient recovery. In order to overcome the limitations of traditional cultivation methods, such as long detection time and limited microbial species, various new detection technologies have rapidly developed in recent years. This not only greatly shortens the detection time, but also effectively improves the accuracy and sensitivity of pathogen detection. This article reviews the rapid detection methods for pathogens that are currently in the research process or early application stage in recent years, including detection technologies in molecular biology, immunology, biosensors, and microfluidics. It focuses on summarizing the sample pretreatment method, detection principles, mechanisms, and advantages of various technologies, and analyzes the current application status of various technologies in pathogen detection in clinical biological samples, in order to provide technical references for rapid detection of different clinical biological samples.
Development and validation of an LC-MS/MS method for the quantification of novel therapeutic TT-478, a selective adenosine receptor 2B antagonist, for a phase I/II clinical trial.
Whitaker D, Francis L, Karaborni S … +5 more , Smith S, Craigen JL, Svetlik S, Barnett S, Veal GJ
Bioanalysis · 2025 Sep · PMID 41104540 · Full text
BACKGROUND: TT-478 is a novel adenosine receptor 2B antagonist, administered orally as a prodrug (TT-702) for the treatment of advanced metastatic prostate cancer in a Phase I/II clinical trial setting. A liquid chromato...
BACKGROUND: TT-478 is a novel adenosine receptor 2B antagonist, administered orally as a prodrug (TT-702) for the treatment of advanced metastatic prostate cancer in a Phase I/II clinical trial setting. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was required to quantify TT-478 in plasma samples obtained from patients recruited to the ongoing early-phase trial. METHODS AND RESULTS: An LC-MS/MS method has been developed and fully validated that allows the quantification of TT-478 in patient plasma samples following a simple extraction procedure using acetonitrile. The assay was shown to be sensitive and selective for TT-478, with an analytical range of 75-25,000 ng/mL, and exhibited excellent precision (coefficient of variation < 12%) and accuracy in the range of 96-107%. Consistently high recovery was achieved, and no matrix effect observed. Analysis of patient samples confirmed that TT-702 rapidly and completely hydrolyzes to TT-478 following administration. CONCLUSION: A novel robust method to quantify TT-478 in human plasma has been fully validated and is currently being utilized in an ongoing clinical trial. Analysis of TT-478 levels in plasma samples from these patients will provide first-in-human pharmacokinetic data for this novel compound. https://clinicaltrials.gov/study/NCT05272709?term=AREA%5BConditionSearch%5D(Advanced%20Prostate%20Cancer)%20AND%20AREA%5BBasicSearch%5D(phase%203%20drug)%20AND%20AREA%5BOverallStatus%5D(NOT_YET_RECRUITING%20OR%20RECRUITING%20OR%20ENROLLING_BY_INVITATION%20OR%20ACTIVE_NOT_RECRUITING)&rank=10 identifier is NCT05272709.
Metabolic alterations in the serum of patients with acute ischemic stroke.
Lee YR, Kim JG, Lee SJ … +2 more , Lee J, Kang HG
Bioanalysis · 2025 Oct · PMID 41089029 · Full text
AIM: Ischemic stroke (IS) occurs when a clot obstructs cerebral blood flow, leading to potentially irreversible damage. Metabolites, as the end products of cellular metabolism, can reflect pathological changes in the bod...
AIM: Ischemic stroke (IS) occurs when a clot obstructs cerebral blood flow, leading to potentially irreversible damage. Metabolites, as the end products of cellular metabolism, can reflect pathological changes in the body. This study aimed to characterize serum metabolic alterations associated with acute ischemic stroke. METHOD: Participants were divided into discovery and validation sets, each comprising healthy controls and IS patients. In the discovery set, untargeted serum metabolomic profiling was performed using liquid chromatography - tandem mass spectrometry. Metabolites showing significant intergroup differences were selected as biomarker candidates and quantitatively validated in the independent set. Diagnostic performance was assessed using receiver operating characteristic (ROC) curve analysis. RESULTS: Three metabolites - cytidine, decanoyl-L-carnitine, and 2-hydroxyestradiol - were identified as significantly altered in IS patients. Cytidine and 2-hydroxyestradiol demonstrated excellent diagnostic performance (AUCs of 0.968 and 0.929, respectively), while decanoyl-L-carnitine showed good performance (AUC = 0.808). CONCLUSION: The three metabolites exhibit significant alterations in acute ischemic stroke patients and provide insight into disease-associated metabolic changes. Their validation lays the groundwork for future exploratory studies investigating potential biomarkers.
Recent advances in the identification and quantification of xylazine and medetomidine in biological specimens.
Crews BO
Bioanalysis · 2025 Oct · PMID 41085046 · Full text
Xylazine is a veterinary sedative that is frequently detected in the illicit drug supply, often found mixed with illicitly manufactured fentanyl (IMF). It has been detected in the blood of overdose victims and patients w...
Xylazine is a veterinary sedative that is frequently detected in the illicit drug supply, often found mixed with illicitly manufactured fentanyl (IMF). It has been detected in the blood of overdose victims and patients who use illicit drugs. Xylazine is not approved for use in humans. It is an alpha-2-adrenergic receptor agonist that causes deep sedation that is non-responsive to naloxone, the antidote for opioid overdose. Chronic exposure to xylazine has been linked to severe wounds that can progress to amputation. Medetomidine is another related veterinary sedative that has more recently emerged as an adulterant in IMF. Medetomidine is also an alpha-2-adrenergic agonist, and in veterinary medicine it is known to be significantly more potent than xylazine. The mixture of these drugs with IMF complicates the treatment of patients exposed to these drugs. Wider availability of analytical methods to detect xylazine, and now medetomidine, is crucial for responding to these health threats and increasing knowledge on the harms and potential therapies for exposed patients. This review covers what is currently known about these drugs, including observed concentrations in various biospecimens, expected major metabolites and windows of detection, and available analytical approaches for detecting exposure.
Performance properties of filter-paper used in blood spot collection devices for quantitation of phenylalanine.
Rodham A, von Ruhland C, Carling R … +1 more , Moat SJ
Bioanalysis · 2025 Oct · PMID 41084969 · Full text
AIMS: Accurate and precise measurement of dried blood spot (DBS) phenylalanine (Phe) is vital for managing phenylketonuria (PKU). Standard DBS collection devices use grade-226 filter-paper, while the CapitainerB quantita...
AIMS: Accurate and precise measurement of dried blood spot (DBS) phenylalanine (Phe) is vital for managing phenylketonuria (PKU). Standard DBS collection devices use grade-226 filter-paper, while the CapitainerB quantitative device utilizes grade-222 filter-paper. Although grade-226 filter-paper performance is well characterized, data on grade-222 filter-paper are sparse. This study aimed to investigate the analytical properties of grade-222 and grade-226 filter-papers. MATERIALS AND METHODS: We compared grade-222 and grade-226 filter-papers for Phe measurement accuracy and imprecision in DBS generated using both filter-papers. Scanning electron microscopy (SEM) and slit lamp imaging were used to assess the physical properties of the filter-papers. RESULTS: Using an aqueous calibrator as reference, grade-222 exhibited a mean bias of -1.1%, the mean bias for grade-226 was -7.3%. Intra-assay imprecision was 2.3% for grade-222, versus 4.2% for grade-226. SEM revealed that fibers in grade-226 filter-paper are bonded by an amorphous material, which is absent in grade-222 filter-paper. Total error analysis indicated grade-222 filter-paper reduced uncertainty of Phe measurement compared to grade-226 filter-paper. CONCLUSIONS: Grade-222 filter-paper was proven to have superior analytical performance for Phe quantification, providing improved differentiation between safe and harmful Phe concentrations and offering more reliable PKU monitoring compared to traditional grade-226 filter-paper.
CRISPR-based platforms for detecting tumor-associated genetic materials in clinical samples.
Chen X, Ye Q, Liang Q … +7 more , Li J, Huang Y, Xia Q, Xiao J, Liao C, Lau CH, Zhu H
Bioanalysis · 2025 Oct · PMID 41064997 · Full text
Tumor-associated genetic markers are useful for early cancer screening, diagnosis, and treatment monitoring. However, traditional detection methods are complex in operation procedures, time-consuming, and the equipment c...
Tumor-associated genetic markers are useful for early cancer screening, diagnosis, and treatment monitoring. However, traditional detection methods are complex in operation procedures, time-consuming, and the equipment costs are expensive. CRISPR/Cas systems are becoming emerging detection tools for tumor detection due to their programmability, rapid reaction, high targeting specificity, and the ability to amplify the signals. CRISPR/Cas has made breakthroughs in the detection of tumor-associated genetic materials including gene mutations, DNA methylation, miRNA, lncRNA, and circRNA detection. Herein, we critically discuss these advancements and describe the key concepts of each CRISPR/Cas system for detecting tumor-associated genetic materials. The significance of these tumor-associated genetic materials in cancer diagnosis and prognosis is highlighted.
Patient centric blood sampling and analysis for diagnostics and laboratory medicine.
Spooner N, Baker D, Carling RS … +7 more , Collier BB, Gong P, Maroto-García J, Rayburn E, Rospo C, Ström M, Theodoridis G
Bioanalysis · 2025 Oct · PMID 41064852 · Full text
Blood sampling and diagnostic laboratory analysis are important aspects of our healthcare systems and patient management. However, the process by which the majority of blood specimens are currently collected, venipunctur...
Blood sampling and diagnostic laboratory analysis are important aspects of our healthcare systems and patient management. However, the process by which the majority of blood specimens are currently collected, venipuncture, does not put the needs of the patient at the center of the process. This article explores the potential utilization of patient centric sampling (PCS) for the collection of smaller blood volumes using technologies that can enable this sampling to take place at a time and location that is more comfortable and convenient for the patient, including self-sampling at home. We discuss the benefits of these technologies, where they are currently used (including case studies), what to consider when contemplating their use and the current regulatory environment. We then explore why the routine adoption of these technologies has been relatively slow and how this impasse may be overcome for the benefit of all patients. This article describes a viable alternative approach for the collection of diagnostic specimens that puts the requirements of the patient at the center. It provides an invaluable resource for those interested in learning about and potentially implementing this approach into their workflows and addresses the concerns that individuals and organizations may have when doing so.
Development of a UPLC-MS/MS method for simultaneous quantification of 7 bile acids in feces: application to UC mice.
Cao J, Chang Y, Li Y … +2 more , Li H, Tan Y
Bioanalysis · 2025 Oct · PMID 41055249 · Full text
AIM: This study aimed to establish a method for the simultaneous quantification of 7 bile acid components in mouse feces and to apply this method to investigate the variations in bile acid profiles among control, DSS, an...
AIM: This study aimed to establish a method for the simultaneous quantification of 7 bile acid components in mouse feces and to apply this method to investigate the variations in bile acid profiles among control, DSS, and mice treated with Alpiniae Oxyphyllae Fructus extract (AOE). The findings may provide insights into the potential regulatory effects of AOE on bile acid metabolism in ulcerative colitis (UC). METHODS: A murine model of UC was established by dextran sulfate sodium (DSS) induction. Following treatment with AOE, fecal samples were collected from each experimental group. Mouse fecal samples were extracted with methanol, and bile acid concentrations were determined using UPLC-MS/MS with chenodeoxycholic acid-d4 as the internal standard. Separation was performed on a Kinetex® 2.6 μm C18 column (50 mm × 2.1 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile. An electrospray ion source was employed to scan in multiple reaction monitoring mode in negative ion mode. RESULTS AND CONCLUSION: The method established by our research institute is simple to operate, with high accuracy, sensitivity, and strong specificity, enabling rapid quantification of seven bile acid components in mouse feces. Fecal analysis revealed that bile acid alterations are associated with UC progression and suggesting AOE's therapeutic potential.
The acute oral toxicity and pharmacokinetic determination of a novel chloroquinoline hybrid molecule by LC-MS/MS.
Labuschagne M, Lekhooa M, Moodley T … +2 more , Mashaba C, Tukulula M
Bioanalysis · 2025 Oct · PMID 41025902 · Full text
BACKGROUND: CB-1 is a novel compound evaluated in Sprague Dawley rats to assess its acute oral safety and pharmacokinetic disposition. OBJECTIVES: To determine the short-term oral toxicity of CB-1 and characterize its ph...
BACKGROUND: CB-1 is a novel compound evaluated in Sprague Dawley rats to assess its acute oral safety and pharmacokinetic disposition. OBJECTIVES: To determine the short-term oral toxicity of CB-1 and characterize its pharmacokinetic parameters. METHODS: Acute oral toxicity was tested at doses up to 100 mg/kg. For pharmacokinetics, six rats received a single 100 mg/kg oral dose. Plasma samples were analyzed using a polar C18 column and quantified by LC - MS/MS monitoring the 490.063→262.00 m/z transition. RESULTS: No mortality occurred at 100 mg/kg, indicating acceptable acute safety. CB-1 reached a peak plasma concentration (C) of 4324.24 ng/mL at 1 h (T). Pharmacokinetic analysis revealed an apparent clearance (Cl/F) of 3.6 L/h, elimination half-life (t) of 7.86 h, and volume of distribution (Vd/F) of 40 L. A secondary plasma concentration peak at 8 h (T) suggested enterohepatic recirculation. CONCLUSIONS: CB-1 demonstrated short-term oral safety with rapid absorption, relatively low clearance, and prolonged systemic exposure, likely influenced by enterohepatic recirculation.
A clever evaluation of the antidiabetic medications linagliptin and empagliflozin in bulk and spiked urine samples.
Abdel Sattar OI, Abuseada HHM, Emara MS … +2 more , Selim I, Abdelshafi NA
Bioanalysis · 2025 Sep · PMID 41021677 · Full text
BACKGROUND: The objective of this research is to provide differential pulse voltammetry (DPV) method that is an ingenious, simple, sensitive, and selective method for the quantitative analysis of linagliptin and empaglif...
BACKGROUND: The objective of this research is to provide differential pulse voltammetry (DPV) method that is an ingenious, simple, sensitive, and selective method for the quantitative analysis of linagliptin and empagliflozin in spiked human urine samples. Faster analysis times and the use of small sample volumes are great advantages of DPV. RESEARCH DESIGN AND METHODS: Before centrifuging the samples for 5 min at 3000 rpm, methanol was added to precipitate and remove any suspended compounds. As a working electrode, glassy carbon electrode (GCE) was employed. The auxiliary electrode was a platinum electrode, while the reference electrode was Ag|AgCl|KCl(sat.). To maximize the experimental conditions for simultaneous determination, the relationship between the current, pH and scan rate was examined. RESULTS: Optimal conditions for quantitative determination were obtained in a phosphate buffer (PB) at pH 7. Plotting LIN and EMP concentrations against the DPV peak height revealed good linearity. It was found that the linear ranges for LIN and EMP were 10-90 and 10-70 µM, respectively. CONCLUSIONS: This approach has been designed for effectively estimating the levels of examined drugs in human urine without matrix effect. The obtained results showed a good recovery values of both drugs.
Simultaneous estimation of finerenone and canagliflozin using HPLC: application in metabolic stability studies.
Rathaur S, Varshney P, Vishwakarma S … +3 more , Maity D, Khan SR, Gayen JR
Bioanalysis · 2025 Sep · PMID 41017576 · Full text
AIM: A robust, sensitive, and reliable method was developed and validated on HPLC for the simultaneous estimation of Finerenone (FNR) and Canagliflozin (CFZ). METHOD: The resolution was performed by using mobile-phase ac...
AIM: A robust, sensitive, and reliable method was developed and validated on HPLC for the simultaneous estimation of Finerenone (FNR) and Canagliflozin (CFZ). METHOD: The resolution was performed by using mobile-phase acetonitrile (ACN): Water (51:49) at a flow rate of 0.75 ml/min with the C18 analytical column Phenomenex Luna (250 mm, 4.6 mm, 5 µm). FNR and CFZ were estimated at a retention time of 6.33 and 8.26 min with 10.0 min analysis run time and detected by PDA detector at a λ 249 and 290 nm, respectively. The calibration curve was linear over the concentration range of 0.05-10 µg/ml with R 0.998 and 0.999 for FNR and CFZ, respectively. For FNR and CFZ, the limit of detection (LOD) was 0.53 and 0.36 μg/ml & limit of quantitation (LOQ) was 1.62 and 1.10 μg/ml, respectively. The method was validated using specificity, linearity, accuracy, precision, LOD, LOQ, robustness, and stability. CONCLUSION: According to the International Council for Harmonisation (ICH) guideline, the validation studies confirmed that the optimization method is specific, simple, highly sensitive, reliable, robust, and reproducible. The developed method was successfully applied for simultaneous estimation with applications in in-vitro metabolic stability studies of pooled microsomes of humans, monkeys, dogs, rabbits, rats and mice.
Recent advances in analytical separation techniques for therapeutic oligonucleotides.
Dérerová T, Vosáhlová Z, Kalíková K
Bioanalysis · 2025 Sep · PMID 41013891 · Full text
Therapeutic oligonucleotides are an emerging class of drugs designed for gene expression modulation. The increasing number of clinical trials and currently expanding market is facilitating further integration and accessi...
Therapeutic oligonucleotides are an emerging class of drugs designed for gene expression modulation. The increasing number of clinical trials and currently expanding market is facilitating further integration and accessibility of these therapeutics. A crucial step in drug development involves reliable analytical tools for characterization and quality control. For clinical applications, oligonucleotides must be separated and purified to ensure regulatory compliance. However, their analysis represents a complex bioanalytical challenge, grounded in their complex impurity profiles. Chemical stability and binding affinity of oligonucleotide-based therapeutics are enhanced during synthesis by extensive modifications, inducing formation of various synthesis failures or truncated sequences. Furthermore, meeting current guidelines or addressing manufacturing scale-up strategies remains challenging as each oligonucleotide typically necessitates a custom analytical protocol. Here, we provide an overview of the most recent advances in separation methods, including various chromatography methods and capillary electrophoresis for nucleic acid-based therapeutics.
Upgrading nucleic acid and antisense therapeutics: challenges, solutions, and future directions.
Zia A, Yokota T
Bioanalysis · 2025 Sep · PMID 40981427 · Full text
Only a small fraction of disease-modifying proteins present druggable pockets for conventional small-molecule or biologic therapies, underscoring the urgent need for innovative strategies such as nucleic acid-based antis...
Only a small fraction of disease-modifying proteins present druggable pockets for conventional small-molecule or biologic therapies, underscoring the urgent need for innovative strategies such as nucleic acid-based antisense therapeutics. Antisense approaches-including antisense oligonucleotides (ASOs), RNA interference (RNAi), and decoy oligodeoxynucleotides (ODNs)-offer powerful means to directly modulate gene expression at the RNA level. Over the past four decades, these modalities have advanced from early proof-of-concept studies to numerous FDA- and EMA-approved therapies for neuromuscular, metabolic, and neurodegenerative diseases. Despite these successes, critical barriers remain. Antisense drugs face challenges related to nuclease degradation, off-target binding, dose-dependent toxicities, limited tissue penetration, and inefficient endosomal escape. Addressing these limitations will require advances in nucleotide chemistry, conjugation strategies, and delivery platforms. Personalized "N-of-1" therapies further highlight the promise of customized oligonucleotides but also raise ethical and cost considerations. This review synthesizes the current state of antisense modalities, the obstacles impeding their broader application, and the innovative approaches needed to upgrade existing platforms and expand their therapeutic potential across a wider range of genetic and acquired diseases.
Correlation between LC-MS/MS and ELISA methods for quantitative analysis of desmosine-containing solutions.
Araki A, Takano M, Chick CN … +3 more , Yamazaki T, Nojima J, Usuki T
Bioanalysis · 2025 Sep · PMID 40947852 · Full text
BACKGROUND: Desmosine and isodesmosine are crosslinking amino acids found in extracellular matrix protein elastin, which imparts elasticity to tissues such as those of the lungs and arteries. These compounds are promisin...
BACKGROUND: Desmosine and isodesmosine are crosslinking amino acids found in extracellular matrix protein elastin, which imparts elasticity to tissues such as those of the lungs and arteries. These compounds are promising biomarkers for diseases involving elastin degradation, such as chronic obstructive pulmonary disease. RESEARCH DESIGN AND METHOD: This study examined the correlation between isotope-dilution LC-MS/MS and a newly established ELISA for in vitro diagnosis using a variety of samples. RESULTS: Results of ELISA and LC-MS/MS analyses exhibited a high correlation coefficient (0.9941). However, whereas the LC-MS/MS measurements deviated approximately 2-fold from the theoretical values, the ELISA measurements ranged from 0.83 to 1.06 (avg 0.94) times the theoretical values. Precise measurement of the absorbance of synthetic desmosine revealed a molar extinction coefficient of 2403, which differed markedly from the previously reported value of 4900 in 1963. Using this value to recalculate the amount of added desmosine, the LC-MS/MS measurements were 0.68 to 0.99 (avg 0.87) times the theoretical values. CONCLUSION: Thus, the developed ELISA enables highly accurate determination of desmosine concentrations, comparable to LC-MS/MS, suggesting that ELISA is a potentially useful in vitro diagnostic tool.
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