Journal Of Pharmaceutical And Biomedical Analysis[JOURNAL]
Sun
200 papers
An integrated strategy of UPLC-Q/TOF MS-based untargeted metabolomics and targeted separation for identifying chemical markers of Fritillaria ussuriensis Maxim from different sources.
Bi S, Liu Z, Wu H … +6 more , Yang D, Wu L, Wei M, Wang Z, Kuang H, Wang Z
J Pharm Biomed Anal · 2026 May · PMID 42247735 · Publisher ↗
Quality control of natural products is an important prerequisite for their formulation or clinical application. Defining the material basis and chemical markers of natural products is a core requirement for quality contr...
Quality control of natural products is an important prerequisite for their formulation or clinical application. Defining the material basis and chemical markers of natural products is a core requirement for quality control. Fritillaria ussuriensis Maxim (FU), as a natural product, is widely used as food and health supplements. However, the chemical composition and content of FU from different sources are still unclear, which poses a challenge to the quality control of FU. This study proposes a comprehensive analytical strategy for HPLC-ELSD to distinguish FU from different sources. First, untargeted metabolomics was combined with chemical profiling to identify biomarkers that distinguish FU from different sources. A total of 164 steroidal alkaloids were identified, of which 21 are potential chemical markers. Subsequently, three steroidal alkaloids were obtained as chemical markers using targeted separation technology. The contents of three steroidal alkaloids in FU from different sources were determined by HPLC-ELSD. The results showed that FU from different sources could be distinguished by detecting the content of three chemical markers using HPLC-ELSD. This study established a reliable method for distinguishing FU from different sources using HPLC-ELSD technology. This strategy can be extended to distinguish the origins of other natural products.
Detection of Artemisia mongolica floss adulteration in moxa floss: A strategy based on UPLC-Q/Orbitrap HRMS, chromatographic analysis, and machine learning.
Chen L, Huang H, Li Z
J Pharm Biomed Anal · 2026 May · PMID 42229064 · Publisher ↗
Detecting adulteration in closely related herbal medicines is a challenge for quality control and market standardization. This study aimed to identify whether Artemisia mongolica floss was adulterated into moxa floss. Fi...
Detecting adulteration in closely related herbal medicines is a challenge for quality control and market standardization. This study aimed to identify whether Artemisia mongolica floss was adulterated into moxa floss. First, ultra-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry was employed to analyze the chemical compositions of moxa floss and Artemisia mongolica floss. Combined with partial least squares discriminant analysis, four key differential biomarkers were identified. Subsequently, high-performance liquid chromatography (HPLC) methods were established for these biomarkers, providing the data foundation for subsequent adulteration discrimination model development. Moreover, four machine learning classification models were constructed: logistic regression, decision tree, random forest, and extreme gradient boosting to compare their ability to identify adulterated samples. The results indicated that the decision tree model exhibited strong generalization capability and stability. Based on the established HPLC method and decision tree model, the ratio range of peak areas of the biomarker 5-hydroxy-3',4',6,7-tetramethoxyflavone to eupatilin or jaceosidin can be used as an indicator to effectively identify adulterated samples. In summary, the study established an analytical strategy for identifying specific markers, thereby facilitating practical discrimination in cases of moxa floss adulteration.
Quality evaluation of Rhododendri Mollis Flos formula granules by chemical and dissolution fingerprinting using UPLC‑DAD and UPLC‑Q‑exactive orbitrap MS/MS.
Wang T, Hu H, Zhang C
J Pharm Biomed Anal · 2026 May · PMID 42225020 · Publisher ↗
To overcome the limitation of conventional fingerprint profiling in identifying specific compounds, an integrated analytical strategy was developed for comprehensive quality evaluation of Rhododendri Mollis Flos Formula...
To overcome the limitation of conventional fingerprint profiling in identifying specific compounds, an integrated analytical strategy was developed for comprehensive quality evaluation of Rhododendri Mollis Flos Formula Granules (RMFFG). This strategy is structured as a stepwise, logically connected chain: from detection to identification to marker screening to functional validation. UPLC chemical fingerprints of 28 batches of RMFFG were established, all showing similarity indices above 0.90. However, to transform these anonymous peaks into chemically meaningful information and resolve peak alignment challenges between UPLC‑DAD and UPLC‑Q‑Exactive Orbitrap MS/MS, a "Dual Standards and Single Mass" method was implemented. This approach uses neohesperidin as an exogenous internal standard to predict retention times of unknown peaks via a calibrated formula, achieving deviations of only 0.04-0.20 min, while authenticated external standards confirm compound identities. Together, they eliminate the need for a full set of expensive or commercially unavailable reference standards. Using this method, 26 common peaks in the chemical fingerprint were unambiguously identified. Based on these identified peaks, chemometric analysis (HCA, PCA, OPLS‑DA) classified the 28 batches into two groups and screened 13 critical quality markers (VIP > 1). As a quantitative validation, fourteen bioactive components were quantified with concentration ranges (e.g., catechin 0.0432-0.0746%, quercitrin 0.1570-0.1942%). Furthermore, because a consistent chemical fingerprint does not guarantee consistent drug release, dissolution fingerprinting in three media (pH 1.2, pH 6.8, and aqueous) was performed, also yielding similarity indices above 0.90. The combination of chemical fingerprinting, Dual Standards and Single Mass identification, and dissolution fingerprinting provides a practical, cost‑effective, and industrially applicable quality control framework that ensures batch‑to‑batch consistency of RMFFG, supporting its future industrial scale‑up and clinical application.
Integration of NIR spectroscopy and chemometrics for multiclass adulterant analysis in Rubus chingii Hu medicinal plant powders.
Wang Y, Wang Y, Yu Q … +2 more , Xie J, Chen Y
J Pharm Biomed Anal · 2026 May · PMID 42217949 · Publisher ↗
Powdered Rubus chingii Hu (RcH) is a high-value medicinal crop. Adulteration of RcH with low-value processing byproducts has become a common practice. This study combined near-infrared spectroscopy and chemometrics -part...
Powdered Rubus chingii Hu (RcH) is a high-value medicinal crop. Adulteration of RcH with low-value processing byproducts has become a common practice. This study combined near-infrared spectroscopy and chemometrics -partial least squares (PLS), support vector machine (SVM), random forest (RF), and backpropagation neural network (BPNN)-to establish discriminant models for RcH powder separate adulterations with powdered walnut shells (WS), tea seed shells (TS), and peanut shells (PS), as well as predictive models for adulterant concentrations. Two-dimensional correlation spectroscopy (2D-COS) analysis based on NIR and mid-infrared (MIR) spectra was used as a pre-screening tool to select the optimal spectral range required for model construction. In distinguishing specific adulterants (WS, TS, and PS) and their adulteration ratios, the SVM and BPNN models outperformed the PLS and RF models; notably, the PLS-DA model had the lowest accuracy (both training and test set accuracies were below 85%). Furthermore, this study utilized the CARS algorithm to extract feature wavelengths, thereby optimizing the model's computational efficiency and accuracy. Among the models established for predicting adulterant concentrations, the PLS, RF, and BPNN models demonstrated superior predictive performance compared to the SVM model. Considering both model performance and computational efficiency, SVM is suitable for RcH adulteration discrimination models, while PLS-R is suitable for adulteration concentration prediction. This bimodal approach provides an effective solution for combating economically driven adulteration of plant powders.
Identification of anti-NSCLC bioactive compounds from Euphorbia helioscopia L. through integrated pharmacological classification and metabolomics analysis.
Yu X, Bai C, Wang T … +7 more , Yang Y, Deng Y, Li C, Hu W, Li R, Yang J, Hu K
J Pharm Biomed Anal · 2026 Oct · PMID 42208414 · Publisher ↗
Euphorbia helioscopia L. (commonly known as Zeqi) has been reported to possess anti-tumor potential, however, its bioactive constituents and underlying molecular mechanisms against non-small cell lung cancer (NSCLC) rema...
Euphorbia helioscopia L. (commonly known as Zeqi) has been reported to possess anti-tumor potential, however, its bioactive constituents and underlying molecular mechanisms against non-small cell lung cancer (NSCLC) remain to be elucidated. This study aimed to systematically identify anti-NSCLC components and explore their associated mechanisms through integrated pharmacological classification and metabolomics analysis. Zeqi samples exhibiting distinct in vitro antiproliferative activities were first classified into relatively higher-activity (RHA) and relatively lower-activity (RLA) group, and then subjected to metabolomics profiling using ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) and subsequent univariate and multivariate statistical analysis. Four compounds (fraxetin, 2''-O-galloylhyperoside, isofraxidin and myricitrin 3-O-galactoside) were identified as candidate bioactive constituents associated with the antiproliferative activity of Zeqi. Among these, fraxetin exhibited the most potent inhibitory effect against NSCLC cells. Flow cytometric analysis revealed a dose-dependent increase in apoptotic rates. Transcriptomic analysis indicated that fraxetin regulates the expression of apoptosis-related genes and activates apoptotic pathways. Further reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analyses confirmed that fraxetin-induced apoptosis in NSCLC cells might be associated with its ability to upregulate Noxa and downregulate Mcl-1 at both transcriptional and translational levels. In conclusion, the successful application of integrated pharmacological classification and metabolomics analysis not only enabled the identification of anti-NSCLC active components in Zeqi but also provides a robust methodological framework for efficient discovery of bioactive lead compounds from complex traditional Chinese medicine (TCM) systems.
Assessment of heavy metal contamination and source apportionment in smokeless tobacco products: Implications for human exposure and health risk.
Bandal RD, Bodake S, Pulimamidi SS … +2 more , Suriyaprakash M, Borkar RM
J Pharm Biomed Anal · 2026 May · PMID 42208110 · Publisher ↗
Smokeless tobacco (SLT) is highly used and associated with negative health effects, and therefore, it has to be strictly regulated. This study assessed the levels of heavy metals and their associated health hazards in SL...
Smokeless tobacco (SLT) is highly used and associated with negative health effects, and therefore, it has to be strictly regulated. This study assessed the levels of heavy metals and their associated health hazards in SLT products found in India. SLT samples were analysed by inductively coupled plasma mass spectrometry to quantify the concentration of heavy metals, including Vanadium (V), Chromium (Cr), Cobalt (Co), Nickel (Ni), Arsenic (As), Cadmium (Cd) and Barium (Ba), Mercury (Hg), and Lead (Pb). The concentrations of heavy metals in the samples from Maharashtra, Assam, and Uttar Pradesh were 12.20, 15.74, and 18.62 mg/kg, with Uttar Pradesh having the highest overall burden. Correspondingly, the Estimated Daily Intake values were also highest in Uttar Pradesh, followed by Assam and Maharashtra, and ranged from 5.47 × 10 to 1.77 × 10. Positive Matrix Factorisation yielded four source factors: Factor 1 powerfully contributed to Cr and some of Cd, Factor 2 powerfully contributed to Hg, Factor 3 powerfully contributed to V, Ni, Co and As, and Factor 4 powerfully contributed to Pb and a little of Cd. The contribution patterns among factor-contribution profiles were comparable, indicating that V, Ni, Co, and As were largely derived from soil-related sources; Cr from natural sources; Cd from combined sources; Pb from handling and dust contamination; and Hg from processing conditions. The target hazard quotients and hazard indices for all metals were less than 1, indicating non-carcinogenic risks at insignificant levels in both men and women. Carcinogenic risk assessment revealed moderate and concerning risks associated with As, Pb, and Cr, with the mean Total Carcinogenic Risk ranging from 7.29 × 10⁵ to 1.75 × 10 in men and women, respectively, particularly among high consumers of SLT. These results underscore the pressing need for systematic observation, efficient implementation of regulations, and targeted awareness campaigns to mitigate the long-term adverse health effects of SLT use on the population.
Study on the correlation between external traits and intrinsic quality of Pseudostellariae Radix based on intelligent sensory evaluation and DESI-MSI technology.
Liu Z, Han T, Wu S … +6 more , Jin M, Shao J, Zhou Y, Zha T, Wu Y, Chen R
J Pharm Biomed Anal · 2026 Oct · PMID 42202756 · Publisher ↗
Currently, the quality analysis methods for Pseudostellariae Radix (PR) are often time-consuming, costly, and operationally complex, which hinders growers, distributors, and consumers from making rapid, preliminary judgm...
Currently, the quality analysis methods for Pseudostellariae Radix (PR) are often time-consuming, costly, and operationally complex, which hinders growers, distributors, and consumers from making rapid, preliminary judgments regarding its intrinsic quality. This study established methods for the objective quantification of external trait indicators of PR using an electronic tongue, a colorimeter, and an ultra-fast gas chromatography electronic nose (UF-GC-EN). The intrinsic quality of PR was evaluated using heterophyllin A (HA), heterophyllin B (HB), pseudostellarin G (PG), total polysaccharides (TPS), total saponins (TSS), and water-soluble extractives (WSE) as key indicators. The spatial distribution of cyclic peptide active ingredients and plant hormones in the swollen parts of PR was visualized using desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). The results showed that, from the 29 trait parameters, 12 key trait parameters with the potential for quality discrimination were identified: diameter at the thickest part (DTP), single root weight (SRW), number of roots per 50 g (NPG), saltiness, yellowness index (YI), lightness (L*), red-green axis (a*), yellow-blue axis (b*), chroma (C*), hue angle (h°), and peaks P4 and P12. Specifically, a deeper yellow color of PR was associated with a higher WSE content. The degree of root tuber swelling showed a significant positive correlation with TPS content, while exhibiting significant negative correlations with the contents of HB, HA, PG, and TSS. The saltiness value displayed highly significant positive correlations with HA and PG contents, but a highly significant negative correlation with WSE content. In the odor component fingerprint, two chromatographic peaks, P4 and P12, were both identified as 1-Hexen-3-ol, which possesses a grassy odor, and their peak areas showed highly significant negative correlations with the contents of both HA and PG. HA, HB, and PG were distinctly distributed in the phloem and periderm of the swollen parts of PR, while zeatin was concentrated in the cambium. By analyzing and validating the correlations between traits and quality, this study provides a statistical reference and demonstrates potential application prospects for the rapid assessment of the intrinsic quality of raw medicinal materials, including PR.
Stoichiometric simultaneous quantification of mycophenolate mofetil and mycophenolic acid in paired culture medium and cell extracts by common product ion LC-MS/MS.
Ishikawa UW, Kanoh HX, Irie K … +5 more , Ota Y, Yasuda T, Hirayama A, Mizuno T, Sasaki AT
J Pharm Biomed Anal · 2026 Oct · PMID 42202755 · Publisher ↗
Mycophenolate mofetil (MMF) is an immunosuppressive prodrug whose efficacy depends on conversion to mycophenolic acid (MPA). Quantitative, stoichiometry-ready measurement of MMF-to-MPA conversion in matched extracellular...
Mycophenolate mofetil (MMF) is an immunosuppressive prodrug whose efficacy depends on conversion to mycophenolic acid (MPA). Quantitative, stoichiometry-ready measurement of MMF-to-MPA conversion in matched extracellular and intracellular matrices remains limited, restricting interpretation of prodrug activation and cellular exposure beyond plasma MPA. A common expectation is that a lipophilic prodrug rapidly equilibrates across membranes; however, extracellular concentrations alone cannot resolve intracellular accumulation, activation, and retention. Here, we developed a common product ion (m/z 207.1) LC-MS/MS workflow that enables stoichiometrically comparable quantification of MMF and MPA across matched extracellular and intracellular matrices from microliter-scale samples and applied it to paired medium/cell sampling in murine Hepa1-6 cells as a proof-of-application system. In Hepa1-6 cells, intracellular MMF transiently exceeded extracellular MMF, peaking at ∼5.5 µM at 15 min (≈1.4-fold above the 4 µM dosing level); in contrast, with direct MPA dosing, intracellular MPA did not exceed extracellular MPA, consistent with equilibration. With carboxylesterase inhibition by bis-nitrophenyl phosphate (BNPP), intracellular MMF reached ∼9.2 µM and exceeded extracellular MMF by ∼2.4-fold at 1 h (∼9.2 vs ∼3.9 µM), consistent with intracellular accumulation and/or retention not readily explained by simple passive equilibration alone. Integrating the data with a mechanistic medium-cell model separated intracellular conversion from bidirectional exchange and parameterized these processes. This compartment-resolved workflow provides a proof-of-application framework for cellular MMF/MPA measurements in matched medium/cell samples.
UPLC-MS/MS method for quantifying non-stimulant ADHD medications in human breast milk and plasma: Application in a lactating patient receiving atomoxetine.
Aoyagi R, Furugen A, Nishimura A … +6 more , Umazume T, Ishikawa S, Narumi K, Okamoto K, Ueda H, Kobayashi M
J Pharm Biomed Anal · 2026 Oct · PMID 42202754 · Publisher ↗
Non-stimulant medications, such as atomoxetine (ATX) and guanfacine (GFC), are increasingly prescribed for attention-deficit/hyperactivity disorder (ADHD), including among women of childbearing age. However, experimental...
Non-stimulant medications, such as atomoxetine (ATX) and guanfacine (GFC), are increasingly prescribed for attention-deficit/hyperactivity disorder (ADHD), including among women of childbearing age. However, experimental data on the transfer of these drugs into human breast milk remain extremely limited. In this study, we developed and validated a highly sensitive and precise ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of ATX, its major metabolites (4-hydroxy ATX: 4-OH ATX and N-desmethyl ATX: N-DAT), and GFC in human plasma and breast milk. Stable isotope-labeled compounds were used as internal standards (IS). Plasma samples were prepared using liquid-liquid extraction with ethyl acetate, and breast milk samples were treated by protein precipitation with acetonitrile containing 0.5% formic acid. The method required only 100 µL of sample volume and showed good linearity, accuracy, and precision over the tested ranges (plasma: 0.5-50 ng/mL; breast milk: 0.8-50 ng/mL). Matrix effects were evaluated using six individual lots of plasma and breast milk, and acceptable variability was achieved. The validated method was applied to plasma and breast milk samples from an ATX-treated lactating woman, yielding milk-to-plasma ratios (M/P) of 0.089 for ATX and 1.17 for 4-OH ATX. The relative infant dose (RID) was estimated at 0.086% for ATX and 0.059% for 4-OH ATX, and the combined RID was estimated to be 0.145%. This study provides a robust and reliable tool for assessing the transfer of non-stimulant ADHD medications into human breast milk and strengthens the evidence base for safe pharmacotherapy during lactation.
Top-down measurement uncertainty for subvisible particles by light obscuration in injections - Uncertainties from precision and recovery studies.
Silva MB, Lourenço FR
J Pharm Biomed Anal · 2026 Oct · PMID 42202753 · Publisher ↗
The subvisible particle counting by light obscuration is a critical quality control test in injectable drug products. Incorrect conformity decisions, particularly false acceptance of non-compliant batches, may result in...
The subvisible particle counting by light obscuration is a critical quality control test in injectable drug products. Incorrect conformity decisions, particularly false acceptance of non-compliant batches, may result in significant health risks to patients. This study evaluates the measurement uncertainty associated with subvisible particle quantification using the light obscuration test, as described in USP < 788 > . A top-down approach was applied, integrating precision and recovery data. Four intravenous drug products (linezolid, fluconazole, metronidazole and ciprofloxacin) were analyzed under different sampling volumes and discard flow rates. Results demonstrated that measurement uncertainty is magnitude dependent. Particles count in the range < 10, 10-100, 100-1000, 1000-10000, and > 10000 presented relative uncertainty values of 275%, 53%, 21%, 16%, and 11% respectively. Precision uncertainty contributions dominate at low counts, while bias uncertainty contributions become progressively more influential at higher subvisible particle range. Uncertainty estimation integrated contributions from precision and trueness supported by recovery studies using an independent particle counter. The application of a top-down enhances the reliability of conformity decisions, reducing the risk of false acceptance or rejection of injectable drugs and supports robust pharmaceutical quality control practices.
Development and exploratory evaluation of a quantitative immunochromatographic assay for serum ACOX1 in intrahepatic cholestasis of pregnancy.
Gao J, Yang X, Shuang L … +4 more , Dong R, Wang G, Wang R, Zhang T
J Pharm Biomed Anal · 2026 Oct · PMID 42190405 · Publisher ↗
Serum biomarkers play a key role in the early diagnosis and risk stratification of intrahepatic cholestasis of pregnancy (ICP). Our previous study showed that acyl-CoA oxidase 1 (ACOX1) was elevated in the serum and plac...
Serum biomarkers play a key role in the early diagnosis and risk stratification of intrahepatic cholestasis of pregnancy (ICP). Our previous study showed that acyl-CoA oxidase 1 (ACOX1) was elevated in the serum and placenta of ICP patients. In this study, a pair of commercially available anti-ACOX1 antibodies was purchased and used to develop a double-antibody sandwich time-resolved fluorescent nanomicrosphere immunochromatographic (TRFNI) strip for the quantitative detection of ACOX1 in serum. Methodological evaluation showed that the strip exhibited good linearity over a calibration set consisting of a blank and standards from 0.25 to 20 ng/mL, with intra-assay and inter-assay coefficients of variation (CVs) below 10% and 15%, respectively. The buffer-based LOD was 0.19 ng/mL, and the lower limit of the reportable quantitative range should not be set below the calculated LOQ of 0.29 ng/mL. In a preliminary accelerated storage assessment, the T/C ratio at 1 ng/mL remained relatively stable during 20 days at 37 °C. The TRFNI assay also showed correlation with a commercial ELISA kit and exhibited recovery rates of 85.5-100.5%. Furthermore, ACOX1 demonstrated an area under the receiver operating characteristic curve (AUC) of 0.731 for distinguishing severe from mild ICP and an AUC of 0.700 for discriminating adverse perinatal outcomes. This assay may warrant further evaluation as an exploratory adjunctive approach for measuring serum ACOX1 in ICP, pending prospective validation against disease-control cohorts and full serum-matrix analytical characterisation.
Integration of multidimensional data acquisition and data processing strategy for comprehensive characterization of chemical constituents in Qingre Sanjie capsule by UPLC-TWIMS-QTOF-MS and molecular networking.
Zhang S, Ma S, Chen X … +6 more , Hu R, Zhang J, Zhao Y, Xu X, Zhang W, Ye J
J Pharm Biomed Anal · 2026 Oct · PMID 42190404 · Publisher ↗
Molecular networking (MN) is widely utilized in the compositional analysis of herbal medicines and complex formulations. While the technique effectively links compounds through shared MS fragmentation patterns and struct...
Molecular networking (MN) is widely utilized in the compositional analysis of herbal medicines and complex formulations. While the technique effectively links compounds through shared MS fragmentation patterns and structural homology, it is often compromised by dispersed in-source fragment ions and adduct ions, which weaken spectral correlations and introduce redundant nodes. To address these limitations, we propose a novel integrated strategy combining Ion Identity Molecular Networking (IIMN) and Feature-Based Molecular Networking (FBMN), aiming to reduce interference from co-eluted ions and to enhance isomers discrimination, respectively. Evaluation based on precursor ion node counts, edge connectivity, and cluster topology confirmed IIMN and FBMN as complementary methods for identifying non-isomers and isomers, respectively. Importantly, IIMN significantly strengthens network connectivity, reduces data redundancy, and improves annotation reliability across diverse ion species. To demonstrate the applicability of this strategy, we employed Qingre Sanjie Capsule (QSC), a traditional Chinese medicine prescription for treating upper respiratory tract infections and allergic conjunctivitis, as a case study and performed systematic chemical identification using ultra-high performance liquid chromatography coupled with traveling wave ion mobility quadrupole time-of-flight mass spectrometry (UPLC-TWIMS-QTOF-MS). Two hybrid scan modes, Data-Dependent Acquisition (DDA) and high-definition DDA, were utilized to acquire both precursor and fragment ion data. A total of 143 compounds were unambiguously identified or tentatively characterized, including three novel constituents first reported. This strategy effectively minimizes interference from redundant nodes, increases the capacity for novel compound discovery, and provides a valuable approach for the systematic analysis of complex systems.
Rapid subunit-level multi-attribute monitoring of succinimide and other PTMs for enhanced quality control of therapeutics antibodies.
Xu J, Xu C, Wu G … +22 more , Li J, Zhou A, Chen Q, Wu G, Li N, Xu S, Yu W, Liu B, Zheng C, Xu M, Liu T, Zhou C, Fan W, Wang C, Wang X, Guo Q, Zhang D, Wang H, Li J, Sun Z, Yu C, Guo H
J Pharm Biomed Anal · 2026 Oct · PMID 42190403 · Publisher ↗
Succinimide (Asu), a critical intermediate in asparagine deamidation and aspartic acid isomerization, represents a potential critical quality attribute (CQA) of therapeutic antibodies but remains challenging to accuratel...
Succinimide (Asu), a critical intermediate in asparagine deamidation and aspartic acid isomerization, represents a potential critical quality attribute (CQA) of therapeutic antibodies but remains challenging to accurately quantify due to its pH-dependent instability during conventional peptide mapping (PM)-based analysis. To address this limitation, we developed a novel rapid subunit-based multi-attribute method (MAM) that combines IdeS-mediated antibody cleavage with liquid chromatography-mass spectrometry (LC-MS) for rapid, simultaneous monitoring of Asu and other post-translational modifications (PTMs), including oxidation and glycosylation. This approach significantly reduces sample processing time and minimizes Asu degradation by operating under optimized pH conditions (pH 6.5). The method was rigorously validated, demonstrating excellent specificity, precision and accuracy across multiple antibody isotypes. Forced oxidation studies further confirmed strong correlation with traditional PM results, underscoring the method's reliability for comprehensive PTM profiling. As the first reported rapid subunit-based MAM specifically optimized for Asu analysis, this strategy provides a rapid, accurate, and comprehensive (RAC) tool for enhanced quality control and stability monitoring of therapeutic antibodies, thereby supporting improved biopharmaceutical production efficiency and product safety.
Development of rapid diagnostic kit against sepsis: Barcode-based semi-quantitative measurement of C-reactive protein and lactate.
Savas EG, Tinaz E, Coskun F … +1 more , Ayar Kayali H
J Pharm Biomed Anal · 2026 Oct · PMID 42190402 · Publisher ↗
This study reports the development of a point-of-care (POC) detection platform integrating lateral flow immunoassays (LFIAs) and paper-based enzymatic sensors for the semi-quantitative measurement of C-reactive protein (...
This study reports the development of a point-of-care (POC) detection platform integrating lateral flow immunoassays (LFIAs) and paper-based enzymatic sensors for the semi-quantitative measurement of C-reactive protein (CRP) and lactate, two key biomarkers strongly associated with inflammatory conditions and sepsis. This approach relies on a visual barcode-like gradient readout that enables instrument-free analysis for rapid clinical evaluation. A lateral flow assay employing gold nanoparticles (AuNPs) conjugated to anti-CRP antibodies was designed for CRP detection, while lactate levels were assessed using multi-dilution lactate dehydrogenase (LDH)-embedded paper sensors coupled to a redox dye system. Characterization of AuNP-antibody conjugates by UV-Vis spectroscopy, salt-induced aggregation, and electron microscopy confirmed stable surface functionalization. The LFIA test strips provided a three-line gradient response, enabling semi-quantitative interpretation of CRP levels across clinically relevant thresholds, demonstrating a linear response between 0.2 and 12.5 mg/L (R = 0.95) with a visual cut-off of 10 mg/L in clinical samples. The lactate paper sensors, employing 2-fold, 5-fold, and 10-fold dilution strategies, generated distinguishable color gradients for various concentrations within a quantitative range of 0.39-9 mM with a coefficient of determination greater than 0.95. For visual interpretation, quantitative image analysis based on pixel intensity was employed to improve the accuracy of paper sensor. The results from the LFIA and paper-based sensors showed a strong correlation with reference laboratory methods, specifically ELISA for CRP and the spectroscopic method for lactate, respectively. Furthermore, stability studies indicated that the paper-based sensors retain over 90% of their initial activity for 16 days at 4 °C, while the LFIA strips remain stable for up to one year at room temperature. The combined biosensor platform provided a gradient-based readout for both biomarkers, enabling rapid, semi-quantitative discrimination across clinically relevant concentration ranges without the need for specialized instrumentation. Overall, this work demonstrates the development of a rapid diagnostic kit capable of detecting blood lactate and CRP levels within a short time and with clinically relevant accuracy.
MicroLC-HRMS selective quantification of a biotherapeutic fusion protein in plasma and cerebrospinal fluid of patients with Lafora disease using a linker-containing signature peptide.
Esposito E, Caravelli A, Muccioli L … +10 more , Cancellerini C, Tappatà M, Pizzi E, Minardi R, DEFEAT-LD Study Group, Carelli V, Vignatelli L, Michelucci R, Bisulli F, Fiori J
J Pharm Biomed Anal · 2026 Oct · PMID 42190401 · Publisher ↗
Although mass spectrometry-based approaches are increasingly used for protein biotherapeutic bioanalysis, validated workflows remain limited for fusion proteins across multiple clinical matrices, including cerebrospinal...
Although mass spectrometry-based approaches are increasingly used for protein biotherapeutic bioanalysis, validated workflows remain limited for fusion proteins across multiple clinical matrices, including cerebrospinal fluid (CSF). We developed a microflow liquid chromatography-high-resolution mass spectrometry (HRMS) bottom-up proteomics assay for VAL-1221, an investigational fusion protein (cell-penetrating antibody Fab linked to recombinant human acid α-glucosidase). HRMS data were acquired in data-dependent acquisition (DDA) mode to support untargeted characterization of the digest and select a linker-containing signature peptide. Quantification was performed by extracted-ion chromatogram (XIC) integration of a quantifier signal, with confirmation by a qualifier signal. The method was validated in plasma and cerebrospinal fluid, including assessment of selectivity, linearity, precision and accuracy, matrix effects, recovery, and stability. The assay was linear over 68-1700 nM in plasma and 6.8-1700 nM in CSF (coefficients of determination ≥ 0.998), with lower limits of quantification of 55.8 and 5.2 nM, respectively, and precision and accuracy were within 15%. The matrix factor was 80.5% in plasma and 102.4% in cerebrospinal fluid. Applied to compassionate-use samples from five patients with Lafora disease receiving intravenous VAL-1221 (20 mg/kg every other week), VAL-1221 was detected in plasma up to 4 h post-infusion, whereas cerebrospinal fluid concentrations were below the method detection limit under the tested regimen. Overall, this workflow provides a validated, sequence-specific approach for exposure and biodistribution assessment of fusion-protein biotherapeutics in human matrices.
Investigation of the on-column oxidative formation of impurity D during amlodipine impurities profiling by UHPLC.
Douša M, Kalášek S
J Pharm Biomed Anal · 2026 Oct · PMID 42190400 · Publisher ↗
The determination of related substances in amlodipine salts is a critical aspect of pharmaceutical quality control. This study investigated the unexpected formation of impurity D, a substituted pyridine derivative, obser...
The determination of related substances in amlodipine salts is a critical aspect of pharmaceutical quality control. This study investigated the unexpected formation of impurity D, a substituted pyridine derivative, observed as an on-column analytical artifact during UHPLC analysis. Although the initial impurity level was low (<0.01%), a pronounced time-dependent increase up to 1.0% was observed during routine analytical sequences. Systematic experiments demonstrated that the increase did not originate from intrinsic sample instability. Instead, it was triggered by a synergistic effect of the mobile phase composition, the pH environment, and stationary phase aging. In particular, the use of MS-compatible ammonium formate buffers or mildly acidic conditions (pH 6.4) accelerated the on-column oxidation process. The results further indicate that the phenomenon is associated with the accumulation of reactive sites or contaminants on column hardware. The effect became evident after approximately 400 injections of samples and reference solutions. A regeneration procedure based on a multicomponent organic solvent mixture was subsequently developed. This treatment effectively restored column performance and reduced impurity D levels to approximately 0.01%. These findings provide guidance for distinguishing true degradation from analytical artifacts in amlodipine impurity profiling and contribute to the reliability of stability-indicating methods.
Systematic development and optimization of a cIEF method for the charge variants analysis of moderately basic to basic monoclonal antibodies using the Analytical Quality by Design strategy.
Floris L, Pasquini B, Orlandini S … +4 more , Luciani F, Massolini G, Furlanetto S, Gotti R
J Pharm Biomed Anal · 2026 Oct · PMID 42190399 · Publisher ↗
This study proposes a Capillary IsoElectric Focusing Analytical Procedure Platform for the charge heterogeneity characterization of monoclonal antibodies. The Analytical Quality by Design approach was implemented to defi...
This study proposes a Capillary IsoElectric Focusing Analytical Procedure Platform for the charge heterogeneity characterization of monoclonal antibodies. The Analytical Quality by Design approach was implemented to define a robust Method Operable Design Region using Infliximab as the primary model compound. The Analytical Target Profile consisted in a robust Analytical Procedure Platform able to measure the monoclonal antibody isoforms pI values with a bias lower than 4%. Risk Management tools were used to evaluate and control the risk of Analytical Target Profile failure. Risk Analysis identified several potential Critical Procedure Parameters, including urea and methyl cellulose concentrations, cathodic/anodic stabilizer levels, carrier ampholytes composition. The carrier ampholytes composition was optimized utilizing a mixture design. A Scheffé mixture model allowed the selection of experiments, and the Partial Least Squares regression provided high predictive power models for the three defined responses, i.e. the calibration biases at the bracketing markers of Infliximab (pI 9.5 and pI 7.0) and the overall fit of the pI vs. migration time relationship. Within the Method Operable Design Region, the optimized conditions were achieved using as the specific carrier ampholyte a blend constituted of 1.5% at pH 3-10, 1.5% at pH 5-8, and 2.0% at pH 8-10.5. The validation results demonstrated the robustness of the Analytical Procedure Platform, with a bias in pI measurement lower than 0.3% for the markers bracketing the Infliximab isoforms. In addition, the method was validated for linearity, trueness and precision as degree of repeatability in the quantification of the charge variants of Infliximab. Under the same optimized experimental conditions, the method was applied to successfully profile Bevacizumab, Daratumumab, Trastuzumab and Durvalumab. By including molecules exhibiting a broader range of physicochemical properties compared to the Infliximab model, the study confirms the versatility of the proposed Capillary IsoElectric Focusing method for moderately basic to basic antibodies. This work provides a refined, regulatory-compliant conventional electrokinetic methodology for biopharmaceutical quality control.
Determination of five antihypertensives in dried capillary plasma spots by LC-MS/MS: A novel method for monitoring pharmacotherapy adherence.
Oliveira DRM, Fischer Costa F, Duarte JH … +6 more , Roieski E, Augustin CM, Linden R, Charão MF, Betti AH, Antunes MV
J Pharm Biomed Anal · 2026 Oct · PMID 42177855 · Publisher ↗
Adherence to hypertension treatment is crucial for therapeutic success, as noncompliance adversely affects quality of life and healthcare costs. Assessing adherence directly through plasma drug concentrations is particul...
Adherence to hypertension treatment is crucial for therapeutic success, as noncompliance adversely affects quality of life and healthcare costs. Assessing adherence directly through plasma drug concentrations is particularly effective for patients with complex medication regimens. This study aimed to develop and validate an LC-MS/MS method for quantifying five antihypertensive drugs in dried capillary plasma obtained via a plasma separation device. The process involved incubating three 6 mm discs in 150 µL of 0.1% bovine serum albumin, followed by chloride analysis of a 20 µL aliquot after 45 min. The residual volume was precipitated using 25% trichloroacetic acid, then mixed with 500 µL methanol containing internal standards (losartan-D4 and atenolol-D7). The extract was dried, reconstituted with methanol, and combined with the supernatant from protein precipitation for analysis. An octadecylsilic column and gradient elution with 0.1% formic acid in water and acetonitrile were used, with positive electrospray ionization except for hydrochlorothiazide. The analytical run time was eight minutes, exhibiting linear ranges from 0.5 to 200 ng/mL for amlodipine and losartan and 5-2000 ng/mL for atenolol, hydrochlorothiazide, and losartan, with r² > 0.99 and 1/x weighting. Precision and accuracy were acceptable, with CV% ranging from 4.3% to 14.5% and accuracy from 86% to 112%, while extraction yields were between 75% and 101%. The analytes were stable at 23 ºC for 21 days in DPS samples. Comparison of drug concentrations in DPS and fresh plasma samples from 72 patients demonstrated high correlation (r = 0.93-0.98), indicating reliability in assessing therapy adherence. Lower concentrations of amlodipine, hydrochlorothiazide, and losartan were observed in patients exhibiting poor adherence (n = 21) compared to adherent patients (n = 51) with statistical significance (p < 0.05). The method is promising for assessing antihypertensive adherence using dried plasma spots.
Analytical quality by design-based development and validation of a robust impurity analysis method for albumin-bound paclitaxel nanoparticles.
Jeong H, Gil MC, Cho YH … +1 more , Lee GW
J Pharm Biomed Anal · 2026 Oct · PMID 42176526 · Publisher ↗
Analytical Quality by Design (AQbD) was applied to develop a robust impurity (IMP) analysis method for albumin-bound paclitaxel nanoparticles (Nab-PTX). Preliminary studies revealed that the USP method could induce epime...
Analytical Quality by Design (AQbD) was applied to develop a robust impurity (IMP) analysis method for albumin-bound paclitaxel nanoparticles (Nab-PTX). Preliminary studies revealed that the USP method could induce epimerization, increasing IMP VI (7-epi-paclitaxel) formation. Risk assessment (Ishikawa/FMEA) identified four critical method parameters: flow rate, acetonitrile ratio (ACN), column temperature, and formic acid (FA) content. A 27-run Box-Behnken design (BBD) and response surface modeling (R² = 0.8688-0.9950) revealed complex nonlinear relationships. Specifically, FA content suppressed the dissociation of carboxylic acid groups in early-eluting impurities, significantly improving peak symmetry. To overcome standard software limits, a multidimensional Method Operable Design Region (MODR) was constructed using Monte Carlo simulation (n = 50,000) with a custom Python-based script. Optimal conditions (1.1 mL/min, 40.5% ACN, 25 °C, 0.025% FA) were identified via desirability function (D=0.9589). Experimental verification showed high predictive accuracy (Bias -1.4% to +1.0%). The method was validated per ICH Q2(R1) guidelines, showing excellent linearity (R≥0.9985), accuracy (87.8-106.2%), and precision (RSD≤0.59%). Notably, acidified sample preparation reduced method-induced IMP VI formation by > 75%, preventing false-positives. This study provides a systematic AQbD framework with computational integration for protein-bound nanomedicines, ensuring superior resolution and 33% faster analysis than compendial methods.
Interference of target protein translation in multivalent mRNA vaccine in cell-based assay.
Stiving AQ, Dornadula G, Roose BW … +5 more , Gonzalez A, Whiteman MC, Schuessler HA, Li HH, Li X
J Pharm Biomed Anal · 2026 Oct · PMID 42173746 · Publisher ↗
Messenger RNA (mRNA) vaccines are highly effective against infectious diseases, but viral diversity has underscored the necessity for multivalent and combination vaccines. We hypothesize there could be interference of ta...
Messenger RNA (mRNA) vaccines are highly effective against infectious diseases, but viral diversity has underscored the necessity for multivalent and combination vaccines. We hypothesize there could be interference of target protein translation in multivalent mRNA vaccines in cell-based assays used for potency testing. Following transfection into human cells, we observed that antigen protein translation is inhibited in multivalent formulations compared to their dose-matched monovalent counterparts using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Inhibited expression was further validated using Western blot and intra-cellular immunostaining with type-specific antibodies. Beyond direct antigen-specific analysis, LC-MS/MS proteomics data from the same assay without additional sample preparation provided a broad view of host cell responses, revealing proteome-level changes indicative of impaired global translation capacity in cells transfected with high dose mRNA(s). To the best of our knowledge, we report for the first time in literature that antagonistic interactions among mRNA constructs can reduce protein expression in multivalent drug products. Our study also demonstrates that LC-MS/MS is a viable alternative for potency testing without dependence on antibodies. These findings highlight the need for careful cell-based assay design, multivalent drug product reference standards for potency assessment, and deliberate mRNA dose optimization in multivalent formulations.
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