Journal Of Pharmaceutical And Biomedical Analysis[JOURNAL]
Sun
200 papers
Analyzing the impact of ionizable lipid identity, purity, and stability on lipid nanoparticle performance.
Rubio-Prego I, Beck-Broichsitter M, Samaridou E … +2 more , Alonso MJ, Simon J
J Pharm Biomed Anal · 2026 Jun · PMID 42385332 · Publisher ↗
Lipid nanoparticles (LNP) have become the leading platform for nucleic acid delivery. However, achieving reproducible and efficacious LNP formulations remains a challenge. Although ionizable lipids are recognized as key...
Lipid nanoparticles (LNP) have become the leading platform for nucleic acid delivery. However, achieving reproducible and efficacious LNP formulations remains a challenge. Although ionizable lipids are recognized as key determinant for LNP performance, the factors governing efficient RNA delivery are still not fully understood. In this work, we systematically investigated the influence of ionizable lipid identity, purity, and stability on the performance of messenger RNA (mRNA)-loaded LNP. Four structurally different ionizable lipids were evaluated, revealing that transfection efficiency is strongly lipid structure- and cell type-dependent, emphasizing the need of tailoring lipids and, thus, LNP to specific therapeutic applications. Furthermore, batch-to-batch variability and stability studies showed that lipid purity and chemical integrity are critical for LNP's in vitro potency. Lipids containing synthesis- or degradation-derived impurities exhibited markedly reduced mRNA expression in cell culture, despite no apparent differences in "conventional" physicochemical characteristics such as particle size distribution or encapsulation efficiency. Advanced analytical methods, including high performance liquid chromatography with a charged aerosol detector (HPLC-CAD) and liquid chromatography-high resolution mass spectrometry (LC-HRMS), enabled precise detection and quantification of impurities, directly correlating their presence with loss in transfection potency. Overall, this work provides valuable insights into the quality-performance relationships of ionizable lipids utilized in LNP formulations. It underscores the importance of rigorous raw material characterization and stability testing to ensure the reliability of LNP delivery technology.
Application of two-dimensional liquid chromatography as a complementary technique to circular dichroism spectroscopy and high-resolution mass spectrometry for the characterization of GalNAc-siRNA conjugates.
da Silva KM, Lippens JL, Vries R … +3 more , Ryvers K, de Vijlder T, Carloni LE
J Pharm Biomed Anal · 2026 Jun · PMID 42385331 · Publisher ↗
Synthetic small interfering RNA (siRNA) is an emerging class of oligo nucleotide therapeutics. In the last seven years, the approval of new siRNA therapies, particularly those conjugated with N-acetylgalactosamine (GalNA...
Synthetic small interfering RNA (siRNA) is an emerging class of oligo nucleotide therapeutics. In the last seven years, the approval of new siRNA therapies, particularly those conjugated with N-acetylgalactosamine (GalNAc) for targeted delivery to hepatocyte asialoglycoprotein receptor, has highlighted the growing importance of research into the analytical characterization of modified double-stranded oligonucleotides. Due to the complexity of GalNac-siRNA conjugates, complementary analytical techniques are required to gather comprehensive information to support various stages of drug development. In this work, a workflow for the characterization of GalNac-siRNA conjugates using high-resolution mass spectrometry (HRMS), circular dichroism (CD) spectroscopy and heart-cutting 2D-liquid chromatography (LC) was introduced. First, HRMS was used to confirm the individual strands via accurate mass measurements. Second, CD and melting temperature (Tm) analysis were used to confirm duplex formation. Finally, optimization of stationary-phase assisted modulation (SPAM) using different trap columns and conditions was performed to couple a non-denaturing size-exclusion chromatography (SEC) method with a denaturing ion-pair reversed-phase (IPRP) using UV and MS detection. The 2D-LC-UV-MS method was successfully applied to study a synthetically modified GalNAc-siRNA. This study presented a novel approach to incorporating (multiple) heart-cut 2D-LC for the characterization of siRNA conjugates.
The transfer of per- and polyfluoroalkyl substances (PFAS) from mother to child: Comparison between maternal and cord blood in an Italian cohort.
Fornasari A, Sech M, Piva E … +8 more , Morini L, Vaiano F, Seravalli V, Seidenari A, Lenzi J, Simonazzi G, Fais P, Pascali JP
J Pharm Biomed Anal · 2026 Jun · PMID 42385330 · Publisher ↗
Per- and polyfluoroalkyl substances (PFAS) are persistent synthetic pollutants associated with multisystemic toxicity, including endocrine disruption and developmental risks. While maternal-fetal transmission has been do...
Per- and polyfluoroalkyl substances (PFAS) are persistent synthetic pollutants associated with multisystemic toxicity, including endocrine disruption and developmental risks. While maternal-fetal transmission has been documented, significant gaps remain regarding the fetal burden of emerging and short-chain alternatives. This study quantified 23 PFAS in paired maternal and cord blood samples from 120 women recruited in Italy, between 2024 and 2025 during the ELENA project (Early Life Exposure to per- and polyfluoroalkyl substaNces (PFAS) and HeAlth risks). Analysis was performed using LC-HRMS to evaluate the single and total PFAS burden after solid-phase extraction. PFAS were detected in 99.04% of maternal and 84.8% of cord blood specimens. Legacy compounds, PFOS (99.0%), PFOA (83.7%), and PFHxS (78.8%), were the most prevalent in maternal samples. Significant associations were found between the regular use of processed food and higher levels of maternal 6:2FTS and PFHpS. While the median sums of PFAS in maternal and cord blood were 1.19 ng/mL and 0.49 ng/mL respectively, nearly 30% of women and 3% of cord blood exceeded the 2 ng/mL NASEM threshold associated with potential health risks. In general, when comparing the observation frequencies and concentration of the compounds in paired samples, the kinetic barrier of placenta has been confirmed, with no selective accumulation. The calculated Transplacental Transfer Efficiency (TTE) values suggested that chemical structure significantly dictates fetal burden. These results highlight the urgent need for biomonitoring approaches and targeted strategies to reduce maternal exposure to legacy and emerging PFAS to protect fetal development.
UHPLC/Q-TOF-MS-based blood-component profiling and multi-omics analysis reveal potential protective mechanisms of Shenzhuo Formula against diabetic kidney disease.
Zhang L, Zhu W, Wei Y … +5 more , Ma J, Huang Y, Li X, Zhao L, Tong X
J Pharm Biomed Anal · 2026 Jun · PMID 42385329 · Publisher ↗
Diabetic kidney disease (DKD) is a common microvascular complication of diabetes, with no definitive treatment currently available for DKD-related kidney injury. Podocyte injury, apoptosis, and microcirculation dysfuncti...
Diabetic kidney disease (DKD) is a common microvascular complication of diabetes, with no definitive treatment currently available for DKD-related kidney injury. Podocyte injury, apoptosis, and microcirculation dysfunction play critical roles in the pathogenesis and progression of DKD. Shenzhuo Formula (SZF), a traditional Chinese herbal formulation, has shown clinical potential in reducing proteinuria, but its chemical basis and protective mechanisms remain unclear. This study combined Ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC/Q-TOF-MS) analysis, network pharmacology, renal proteomics, metabolomics, single-nucleus RNA sequencing (snRNA-seq), and in vivo validation to explore the potential mechanisms of SZF against DKD-related renal injury. UHPLC/Q-TOF-MS analysis annotated 27 SZF-related serum constituents, including 19 putative prototype constituents and 8 putative metabolites. Network pharmacology predicted 87 overlapping targets between SZF-related constituents and DKD, mainly enriched in apoptosis, inflammatory responses, focal adhesion, and metabolic regulation. In db/db mice, SZF reduced urinary albumin-to-creatinine ratio and renal injury biomarkers, including NGAL, CysC, and KIM-1, increased estimated glomerular filtration rate, and alleviated glomerular basement membrane thickening, mesangial matrix expansion, inflammatory infiltration, and collagen deposition. Proteomic and metabolomic analyses suggested that SZF regulated pathways related to necroptosis, complement and coagulation cascades, platelet activation, amino acid metabolism, and the citrate cycle. snRNA-seq and histological validation indicated that SZF reduced apoptosis-related gene signatures and podocyte injury. Western blot analysis showed increased BCL-2 expression and decreased cleaved Caspase-3 and cleaved PARP1 expression after SZF treatment. In zebrafish models, SZF improved microcirculatory phenotypes by increasing vessel diameter, cardiac output, and blood-flow velocity, and by reducing thrombus incidence. These hypothesis-generating findings suggest that SZF warrants further investigation as a potential modulator of podocyte apoptosis and microcirculatory dysfunction in DKD.
Multi-center study of the recombinant cascade reagent (kinetic chromogenic assay) as an alternative method for bacterial endotoxin testing: Method validation, product suitability, and consistency evaluation with limulus amebocyte lysate.
Pei Y, Cai T, Liu H … +13 more , Chen W, He H, Liu Y, Lu H, Lv X, Sang J, Wan L, Wang T, Yang H, Zhu Q, Chen C, Huang W, Zhao Y
J Pharm Biomed Anal · 2026 Jun · PMID 42385328 · Publisher ↗
Given the scarcity of global Limulus resources, developing animal-free alternatives for bacterial endotoxin testing is imperative. This study evaluated the Recombinant Cascade Reagent (rCR) kinetic chromogenic assay as a...
Given the scarcity of global Limulus resources, developing animal-free alternatives for bacterial endotoxin testing is imperative. This study evaluated the Recombinant Cascade Reagent (rCR) kinetic chromogenic assay as a sustainable alternative to traditional Limulus Amebocyte Lysate (LAL). Eleven laboratories collaborated to conduct a comprehensive validation of five commercial rCRs, assessing methodological performance, product suitability across 34 diverse pharmaceutical products (105 batches), and consistency with LAL using 82 naturally contaminated water samples. Results showed that rCRs met pharmacopoeial acceptance criteria for accuracy (50%-200% recovery), precision (RSD < 32%), and linearity (|r| > 0.980), with a limit of detection at 0.005 EU/mL. Interference testing demonstrated a 97.1% overall product suitability rate, including for complex biologics. Consistency evaluation revealed a significant positive correlation between rCR and LAL (Spearman's r > 0.7889), with no statistically significant differences in endotoxin quantification (Wilcoxon p > 0.05). Furthermore, rCR exhibited diagnostic accuracy equivalent to or higher than LAL (Youden's Index: 0.811-0.904 vs. baseline 0.535). In conclusion, the rCR kinetic chromogenic assay demonstrates excellent precision, broad applicability, and high consistency with LAL, proving to be a reliable and standardized alternative for industrial endotoxin testing.
Simultaneous enantioselective separation of 2-, 3- and 4-chloromethcathinones using supercritical fluid chromatography-tandem mass spectrometry and its application to human oral fluid samples.
Jorbenadze S, Tchelidze A, Sprega G … +4 more , Tkemaladze V, Farkas T, Busardo FP, Chankvetadze B
J Pharm Biomed Anal · 2026 Jun · PMID 42378913 · Publisher ↗
In this study, the analytical capabilities of supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) were investigated for the simultaneous chemo- and enantio-selective separation of chloromethcathinone...
In this study, the analytical capabilities of supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) were investigated for the simultaneous chemo- and enantio-selective separation of chloromethcathinone (CMC) isomers. Following extensive screening of polysaccharide-based chiral columns and optimization of mobile phase conditions, baseline separation of all three positional isomers and their corresponding enantiomers was achieved within less than 10 min. The optimized method was applied to the analysis of authentic oral fluid samples collected during roadside drug testing, enabling the effective identification of the individual isomers present in the samples. The SFC-MS/MS method demonstrated acceptable selectivity and in part enhanced MS sensitivity.
Untargeted metabolomics, GC-MS and intelligent sensory analysis reveal wine-thermal induced alterations in chemical, sensory and metabolic bioactivities of Curculigo rhizome.
Pan MH, Liu SF, Yu JL … +2 more , Ai YJ, Li F
J Pharm Biomed Anal · 2026 Jun · PMID 42378912 · Publisher ↗
Curculigo rhizome (CR) a phenolic acid-rich plant, is widely utilized as a raw material for health products, traditional Chinese medicine (TCM) preparations, and as a flavoring agent in the food industry. In the present...
Curculigo rhizome (CR) a phenolic acid-rich plant, is widely utilized as a raw material for health products, traditional Chinese medicine (TCM) preparations, and as a flavoring agent in the food industry. In the present study, a comprehensive multi-technique approach combining UPLC-Q-TOF-MS/MS, GC-MS, intelligent sensory technology (electronic nose and electronic tongue), and metabolic analysis was employed to investigate the effects of yellow rice wine processing on the chemical composition, bioactive metabolic properties, and sensory characteristics of CR. Wine-processed Curculigo rhizome (WCR) exhibited significantly decreased L* and b* values alongside reduced sweetness, whereas the a* value and total color difference (E) markedly increased compared to raw Curculigo rhizome (RCR). Notably, enhanced sensory responses to nitrogen-containing compounds, particularly amines, were observed following wine processing. Metabolic assessments across bacterial, cellular, and mouse models demonstrated improved bioactivity in WCR. Non-targeted metabolomics analysis identified 84 compounds in total, with 15 differential metabolites (VIP > 1, fold change ≥ 1, P < 0.05) discriminating RCR and WCR. Post-processing reductions were observed for orcinol, palmitic acid, and oleic acid, while orcinol glucoside (SAK), orcinol gentiobioside (ANA), and curculigoside B showed elevated levels. Pearson correlation analysis revealed that downregulated compounds were negatively correlated with a* values, bioactivity-related metabolic parameters, and electronic nose sensors (EN1-EN18) as well as electronic tongue sensors (ET1, ET4, ET7, ET10, ET13); conversely, upregulated compounds displayed positive correlations with these indices. These findings elucidate the critical role of yellow rice wine processing in modulating the sensory attributes, bioactivity profile, and chemical composition of CR, thereby providing scientific foundations for establishing standardized processing protocols and offering practical guidance for industrial-scale production.
A single bioanalytical LC-MS/MS method for the quantification of pelitinib across biological matrices: An insight into in vitro metabolic stability, in vivo pharmacokinetics, and IVIVE.
Bhalerao HA, Dhurjad P, Bharate A … +1 more , Sonti R
J Pharm Biomed Anal · 2026 Jun · PMID 42378911 · Publisher ↗
Pelitinib's (PTB) strong, irreversible inhibition of EGFR continues to pique scientific curiosity and warrants investigation of potential therapeutic uses, necessitating reliable analytical methods for its pharmacokineti...
Pelitinib's (PTB) strong, irreversible inhibition of EGFR continues to pique scientific curiosity and warrants investigation of potential therapeutic uses, necessitating reliable analytical methods for its pharmacokinetic and metabolic assessment. Here, we have developed and validated a single, sensitive, and reliable LC-MS/MS (liquid chromatography-tandem mass spectrometry) method in accordance with USFDA guidelines for the quantification of PTB in plasma, human, and rat liver microsomes, where greenness assessment by (Analytical GREEnness metric approach and software) AGREE and (Methodological Overall Green Analytical Procedure Index) MoGAPI tools exhibited a good to moderate environmental performance. Chromatographic separation was achieved on an Agilent Eclipse Plus C18 column using a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile. Pelitinib exhibited dose-dependent systemic exposure and slow systemic elimination in vivo, along with low intrinsic clearance in microsomal incubations. Hepatic clearance was underestimated relative to the observed pharmacokinetic clearance, according to in vitro-in vivo extrapolation (IVIVE). Further, SwissADME (Swiss Absorption, Distribution, Metabolism, and Excretion) provided additional insights regarding the pharmacokinetic characteristics using in silico predictions.
Multimodal analytical strategy for revealing the related compounds of color variation in Salvia miltiorrhiza after sweating.
Wu H, Wang R, Yuan C … +9 more , Shao M, Wang S, Zhao Y, Tang X, Zhang S, Peng D, Chen Y, Chen W, Wang L
J Pharm Biomed Anal · 2026 Jun · PMID 42372342 · Publisher ↗
This study investigated the color-related compounds of Salvia miltiorrhiza after sweating. Stereomicroscopy and Electronic eye system were employed to characterize color variation. UPLC-Q-Orbitrap-MS and HPLC identified...
This study investigated the color-related compounds of Salvia miltiorrhiza after sweating. Stereomicroscopy and Electronic eye system were employed to characterize color variation. UPLC-Q-Orbitrap-MS and HPLC identified differential compounds. Furthermore, μ-FTIR and MALDI-MSI visualized the spatial distribution of compounds, and the compounds distribution was confirmed via HPLC. Result showed that after sweating, the appearance characteristics of Salvia miltiorrhiza changed significantly, exhibiting a reddish-brown epidermis and purple-brown inner tissue. Among 49 significantly altered compounds, tanshinones accounted for 58%. HPLC further indicated that the content of tanshinones showed a significant increase. Integrated μ-FTIR and MALDI-MSI demonstrated tanshinones increased after sweating and accumulated in the epidermis. Further confirmation of the increase in tanshinones was achieved by HPLC analysis of epidermis and cortex isolated. These results reveal tanshinones as the color-related compounds and clarify their spatial distribution, providing insights into the chemical changes during the traditional sweating process of Salvia miltiorrhiza.
Development and validation of a UPLC-MS/MS method for real-time neuropharmacokinetic monitoring of iboga alkaloids in rat brain.
McIntosh S, Maldonado I, Smith NC … +5 more , Gadepalli RS, Rimoldi JM, Havel V, Sames D, Hemby SE
J Pharm Biomed Anal · 2026 Jun · PMID 42364550 · Publisher ↗
Iboga alkaloids, including ibogaine, its primary metabolite noribogaine, and the naturally occurring congener ibogamine, have shown promise for the treatment of substance use and other neuropsychiatric disorders but are...
Iboga alkaloids, including ibogaine, its primary metabolite noribogaine, and the naturally occurring congener ibogamine, have shown promise for the treatment of substance use and other neuropsychiatric disorders but are limited by cardiotoxic effects. Safer synthetic analogs, such as oxa-noribogaine, are under active development; however, validated analytical methods for quantifying these compounds in brain extracellular fluid are lacking. Existing approaches that rely on tissue homogenate analysis reflect total tissue drug content, encompassing intracellular, protein-bound, and vascular compartments, and cannot directly measure pharmacologically active, unbound drug at the site of receptor interaction. Here, we report the development and validation of a sensitive UPLC-MS/MS method capable of quantifying ibogaine, noribogaine, ibogamine, and oxa-noribogaine in rat brain microdialysate. Using D2-noribogaine, a custom-synthesized isotopically labeled internal standard, the method achieved lower limits of quantification of 0.78-1.56 ng/mL with a 6-minute run time. Calibration curves were linear over 0.78-75 ng/mL for ibogamine and 1.56-75 ng/mL for ibogaine, noribogaine, and oxa-noribogaine, and intra- and inter-day accuracy and precision met acceptance criteria for all analytes. Analytes were stable under freeze-thaw, long-term storage (-80°C), and autosampler conditions. The method was successfully applied to measure noribogaine levels in the nucleus accumbens following intraperitoneal administration (10 mg/kg) in rats (n = 4), yielding a recovery-corrected peak unbound extracellular fluid (ECF) concentration (C) of 292 ± 68 ng/mL (∼0.98 µM) at a mean T of 50 min, demonstrating the method's suitability for real-time neuropharmacokinetic profiling of iboga alkaloids in brain ECF. This validated method provides an essential preclinical tool for pharmacokinetic-pharmacodynamic characterization of iboga alkaloids at their sites of therapeutic action, supporting the development of safer iboga-derived therapeutics.
QC friendly polysorbate quantification in high concentration protein samples.
Dillard KE, Witmer C, Tran HV … +5 more , Yang RS, Martinez-Fonts K, Niedringhaus T, MacNeill M, Newman A
J Pharm Biomed Anal · 2026 Jun · PMID 42361487 · Publisher ↗
Polysorbate is a surfactant that serves an important role in stabilizing and solubilizing biologic drug products to prevent aggregation and generation of subvisible particles. Monitoring this low concentration excipient...
Polysorbate is a surfactant that serves an important role in stabilizing and solubilizing biologic drug products to prevent aggregation and generation of subvisible particles. Monitoring this low concentration excipient in complex formulations with high protein concentrations is required to ensure that the quality of the product is maintained, but interference from the protein poses a complex analytical problem. Many methods to monitor polysorbate content have been developed, but isolating this low concentration heterogeneous excipient from high protein concentration matrices continues to pose a problem in the pharmaceutical space. Here, we show a method for monitoring polysorbate concentration in high protein concentration-containing samples (e.g., > 100 mg/mL) using an HPLC-CAD. We confirm the method works for both PS-20 and PS-80 formulated biologics (mAbs, bioconjugates, antibody drug conjugates (ADCs)) and show the method tracks degradation of polysorbate in a formulated sample containing a high concentration monoclonal antibody. Finally, we show this method can be validated and transferred to other sites for testing and monitoring polysorbate concentration.
Corrigendum to "Aggregation-induced luminescence probe based lateral flow immunoassay for the simultaneous quantitative detection of IL-6/PCT" [J. Pharm. Biomed. Anal. 273 (2026) 117393].
He R, Zhu Y, Zhou W … +7 more , Nawaz MAH, Li Y, Zhu J, Feng H, Nowicka AM, Han W, Yu C
J Pharm Biomed Anal · 2026 Jun · PMID 42361486 · Publisher ↗
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Quantitative analysis of five components of Sargentodoxae caulis-Radix alangii drug pair using UPLC-MS/MS in rat plasma and RAW264.7 cells for comparative pharmacokinetics in normal and diseased states.
Zhou Y, Peng Y, Huang Z … +3 more , Zhou D, Jin F, Zhou Z
J Pharm Biomed Anal · 2026 Jun · PMID 42349094 · Publisher ↗
The Sargentodoxae caulis-Radix alangii (SR) drug pair has been traditionally used by the Hmong for treating rheumatoid arthritis (RA). However, its pharmacokinetic behavior under disease conditions remains largely unexpl...
The Sargentodoxae caulis-Radix alangii (SR) drug pair has been traditionally used by the Hmong for treating rheumatoid arthritis (RA). However, its pharmacokinetic behavior under disease conditions remains largely unexplored. To address this gap, a UPLC-MS/MS method was established to determine the concentrations of salidroside, loganate, vanillic acid, anabasine, and protocatechuic acid, key bioactive components of SR in rat plasma and RAW264.7 cells. This method aimed to investigate the effects of disease states on the pharmacokinetic profiles of these components both in vitro and in vivo. The components were detected using multiple reaction monitoring (MRM) in both positive and negative ion modes. Each calibration curve exhibited a high correlation coefficient (R > 0.99). The intra-and inter-day precisions for these analyses were all below 15%, with accuracy ranging from 85% to 115%. The relative standard deviation (RSD) values for stability, recovery, matrix effect, and reproducibility remained below 15.0%. This method was successfully applied to investigate the pharmacokinetics of these components in rat plasma and RAW264.7 cells after administration of SR. In rat pharmacokinetic experiments, significant differences were observed in the AUC, MRT, and Cl values of salidroside, loganate, vanillic acid, anabasine, and protocatechuic acid between adjuvant-induced arthritis (AA) rats and normal rats. In cellular pharmacokinetic experiments, compared to the normal group, the AUC and MRT of these components were increased in the LPS-induced inflammatory cell model, while Cl was decreased, consistent with the in vivo findings. These results suggest that the absorption rate of these components in SR was enhanced in AA rats and inflammatory cells, while their elimination rate was reduced. This indicates a potential alteration in the rate and extent of drug metabolism. This study provides a theoretical reference for further clarifying the pharmacodynamic basis of SR, particularly in the context of inflammatory conditions. Future research should focus on elucidating the specific mechanisms underlying these pharmacokinetic changes and exploring their clinical implications.
Tissue-specific phenolic profiling of Castanopsis sieboldii: Validated quantification, metabolite annotation, and chemometric discrimination.
Bae JY, Avula B, Tatapudi KK … +4 more , Kang YS, Katragunta K, Lee SH, Khan IA
J Pharm Biomed Anal · 2026 Jun · PMID 42349093 · Publisher ↗
Castanopsis sieboldii is a phenolic-rich evergreen species in the family Fagaceae, yet comprehensive quantitative and tissue-specific metabolite profiling remains limited. In this study, an integrated analytical workflow...
Castanopsis sieboldii is a phenolic-rich evergreen species in the family Fagaceae, yet comprehensive quantitative and tissue-specific metabolite profiling remains limited. In this study, an integrated analytical workflow comprising UHPLC-PDA quantification, LC-QToF-MS identification, and chemometric analysis was developed to characterize phenolic constituents in leaves, flowers, fruits, and stems. A validated UHPLC-PDA method enabled the simultaneous quantification of six major phenolics, with limits of detection ranging from 0.01 to 0.05 μg/mL and limits of quantification from 0.025 to 0.1 μg/mL. Among all tissues, 3‑O‑galloylshikimic acid (Compound 1) was the predominant metabolite (0.1-204 mg/g), followed by caffeoylquinic acids (Compounds 2-3), ellagic acid (Compound 4), and flavonoid glycosides (Compounds 5-6). LC-QToF-MS analysis facilitated the tentative annotation of 185 metabolites, including phenolic acids, ellagitannins, galloylshikimic acids, and flavonoid glycosides, based on accurate mass measurements and characteristic MS/MS fragmentation patterns. Chemometric evaluation using principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and hierarchical clustering analysis (HCA) revealed clear tissue-specific clustering, with leaves exhibiting the highest chemical diversity and phenolic abundance, whereas fruits showed minimal levels. PCA captured 80% of total variance in the first two components, and the PLS-DA model showed strong predictive performance (R²Y ≈ 1.00; Q² ≈ 0.98) although interpretation should consider the limited sample size. This study provides a comprehensive, tissue-resolved phenolic profile of C. sieboldii, establishing a robust chemical foundation for future pharmacological, ecological, and quality-control applications.
Addressing the elephant in the room: A comprehensive framework to resolve selectivity, multicollinearity, and scalability challenges in Raman chemometric models for mammalian cell culture applications.
Rapala S, Kuang B, Doddridge GD … +7 more , Shah K, Kwok A, Xia L, Anantharaman S, Lowry B, Harcum SW, Abu-Absi N
J Pharm Biomed Anal · 2026 Jun · PMID 42341650 · Publisher ↗
Raman spectroscopy offers significant potential for real-time bioprocess monitoring, but adoption in biologics manufacturing remains limited due to challenges in developing robust chemometric models. Key barriers include...
Raman spectroscopy offers significant potential for real-time bioprocess monitoring, but adoption in biologics manufacturing remains limited due to challenges in developing robust chemometric models. Key barriers include poor selectivity between structurally similar metabolites, multicollinearity among correlated analytes that confound model predictions, and insufficient training data ranges for critical quality attributes (CQAs). To overcome these barriers, a novel workflow called Spiking and Pure Analyte Characterization for Raman Chemometrics (SPARC) was developed. SPARC addresses these challenges through three innovations: using pure analytes to identify signature spectral regions for enhanced selectivity, spiking pure analytes into cell culture samples to mitigate multicollinearity, and using purified and enriched CQA material to spike into cell culture samples to broaden the CQA training data range. These protocols were implemented on the ambr250 high-throughput (HT) system, using an integrated liquid handler for automated spiked sample preparation. SPARC successfully modeled six analytes: glucose, lactate, glutamine, glutamate, monoclonal antibody (mAb), and CQA: High Molecular Weight (HMW) species. Pure analyte characterization identified 8-22 signature spectral regions per analyte using Variable Importance in Projection (VIP) scores. Cross-scale validation demonstrated successful model transfer from the ambr250 system (single-flow-cell probe) to 3 L bioreactors (dedicated in-situ immersion probe). SPARC consistently outperformed baseline methods with significant reductions in prediction errors: glucose (48%), lactate (49%), glutamine (69%), glutamate (57%), mAb (77%) and uniquely enabled modeling of HMW species where baseline methods failed. SPARC provides a systematic workflow for implementing Raman chemometrics in cell culture that overcomes technical barriers, automates data generation, and accelerates model development.
Oxidative degradation of melatonin in solution and solid-state pharmaceutical blends: HPLC-HRMS/MS degradant characterization, metal-mediated acceleration and EDTA mitigation.
Salles AG, Coelho RL, Rodrigues MT … +1 more , Guidotti BB
J Pharm Biomed Anal · 2026 Jun · PMID 42341649 · Publisher ↗
Melatonin (MLT) is used in pharmaceutical products, yet its oxidative degradation profile remains only partially defined, especially under formulation-relevant solid-state conditions. This study combined HPLC-PDA and HPL...
Melatonin (MLT) is used in pharmaceutical products, yet its oxidative degradation profile remains only partially defined, especially under formulation-relevant solid-state conditions. This study combined HPLC-PDA and HPLC-HRMS/MS to characterize MLT degradation in solution and in excipient-containing solid blends. Solution-phase forced degradation was performed with hydrogen peroxide, ACVA, and Cu(II), while solid-state stress studies were carried out in capsule-equivalent blends containing hydrogen peroxide and Cu(II), with EDTA used as a mechanistic mitigation probe. Five degradation products were detected overall and assigned at different confidence levels. Hydrogen peroxide generated DP-1, DP-2, and DP-3, whereas ACVA generated DP-4 and DP-5; Cu(II) selectively favored DP-4 in solution, indicating stress-dependent oxidative pathways. The chromatographic method resolved MLT from all observed degradation products and satisfactory mass balance was obtained under all tested solution stresses. In solid blends, MLT remained essentially stable under thermal control, degraded moderately in the presence of peroxide alone, and degraded substantially faster when peroxide and Cu(II) were combined. This acceleration was markedly attenuated by EDTA, supporting a metal-mediated catalytic contribution. The solid-state study should be interpreted as an accelerated worst-case mechanistic model rather than a predictive real-time stability study. A complementary computational toxicological screen did not indicate a strong overall mutagenic concern for the identified products, although AFMK (DP-3) may warrant targeted experimental clarification if required. The results expand the described oxidative degradation map of MLT and link degradant formation to microenvironmental factors relevant to impurity control and formulation-oriented stabilization strategies.
Processing-dependent hepatic risk and benefit of Polygonum Multiflorum Radix: Integrated chemical profiling, in vivo validation and candidate quality control markers.
Zhang H, Zhao X, Wang F … +7 more , Liu M, Qin X, Sun H, Liang Y, Ren X, Liu Y, Pu W
J Pharm Biomed Anal · 2026 Jun · PMID 42341648 · Publisher ↗
To clarify how steaming and sun-drying cycles affect the hepatic risk-benefit profile of Polygoni Multiflori Radix (PMR), the herb was processed with black bean decoction for 1-9 cycles following the traditional "nine st...
To clarify how steaming and sun-drying cycles affect the hepatic risk-benefit profile of Polygoni Multiflori Radix (PMR), the herb was processed with black bean decoction for 1-9 cycles following the traditional "nine steaming and nine sun-drying" protocol. UPLC-MS analysis showed that anthraquinones and trans-THSG progressively decreased with increasing cycles and stabilized after five cycles, whereas cassialactone-8-O-β-D-glucoside and hyperoside emerged as new components during processing. In a low-dose LPS-sensitized mouse model mimicking immune-idiosyncratic liver injury, PMR processed for three or more cycles significantly alleviated liver damage. In an acetaminophen (APAP)-induced acute liver injury model, PMR processed for 5-7 cycles exhibited the strongest hepatoprotective effects. Spectrum-effect correlation analysis identified twelve constituents (e.g., emodin-8-O-β-D-glucoside) associated with liver injury, involving targets related to drug metabolism (CYP3A4), membrane transporters (ABCB1, SLC2A1), and oxidative stress (SOD1). Five components (e.g., cassialactone-8-O-β-D-glucoside) were linked to hepatoprotection, potentially through modulation of synthetic function (ALB) and inflammatory response (TNF, EGFR, HSP90AA1). The Cellular Thermal Shift Assay (CETSA) was employed to validate the molecular docking results, demonstrating that emodin-8-O-β-D-glucopyranoside and physcion-8-O-β-D-glucoside significantly enhanced the thermal stability of TP53. In parallel, emodin and hyperoside were confirmed to increase the thermal stability of albumin (ALB). Overall, processing PMR with black bean decoction for 5-7 cycles is optimal for reducing hepatic risk while preserving or enhancing hepatoprotective activity. These findings support a risk-benefit-oriented processing standard and propose candidate quality markers for standardized manufacturing, including trans-THSG, emodin, aloe-emodin, cassialactone-8-O-β-D-glucoside, and hyperoside.
Dissection of potential mechanism of Bao-Yuan tablet against Spleen-Qi Deficiency by UPLC-Q/TOF MS and network pharmacology.
Chen ZH, He JL, Yang YD … +12 more , Xie TY, Xu SL, He Y, Zhang Y, Liang WT, Zhao ZQ, Hu HP, Yang YY, Yang X, Zhang FX, Liang H, Chen SY
J Pharm Biomed Anal · 2026 Jun · PMID 42341647 · Publisher ↗
Bao-Yuan tablet (BYT), a modern formulation derived from the classic Bao-Yuan decoction (BYD) first documented in the Ming Dynasty of China, is used as a therapeutic agent for Spleen-Qi Deficiency (SQD). However, its pha...
Bao-Yuan tablet (BYT), a modern formulation derived from the classic Bao-Yuan decoction (BYD) first documented in the Ming Dynasty of China, is used as a therapeutic agent for Spleen-Qi Deficiency (SQD). However, its pharmacodynamic effects and underlying mechanisms remain unclear. This study aimed to investigate its therapeutic effects and mechanism against SQD induced by a multifactorial method (cold-purging, fatigue, and irregular diet). BYT treatment significantly improved behavioral symptoms, increased thymus index, reduced serum IgA/IgG levels, elevated D-xylose content, and suppressed TNF-α levels in SQD mice. A total of 141 compounds were identified in BYT by UPLC-Q/TOF MS, including five previously undescribed compounds. Based on these identified components, network pharmacology predicted that TNF, IL-1β, and TLR4 are core targets of BYT against SQD, involving Toll-like receptor and TNF signaling pathways. Western blot validation confirmed that BYT dynamically regulated the splenic TLR4/NF-κB pathway: suppressing TLR4 and p65 phosphorylation at day 7, while restoring TLR4 and reducing elevated total p65 at day 14. This study provides the first experimental evidence for BYT against SQD and integrates chemical profiling, network prediction, and experimental validation of its multi-target mechanism, providing a scientific basis for further quality control and clinical application of BYT.
Quantification of isavuconazole in dried blood spots by LC-MS/MS and clinical application for therapeutic drug monitoring.
Liu X, Liu F, Dai H … +13 more , Tang F, Liu L, Gao J, Chen J, Qu Q, Xue M, Zhou H, Cai W, Liu Y, Xie Y, Huang C, Xu Y, Miao L
J Pharm Biomed Anal · 2026 Jun · PMID 42341646 · Publisher ↗
Invasive fungal diseases pose a significant threat to human health. Therapeutic drug monitoring (TDM) of isavuconazole, a new-generation triazole antifungal agent, is recommended; however, conventional cubital venous blo...
Invasive fungal diseases pose a significant threat to human health. Therapeutic drug monitoring (TDM) of isavuconazole, a new-generation triazole antifungal agent, is recommended; however, conventional cubital venous blood collection is invasive and requires large blood volumes as well as sample transportation and storage. Quantitative dried blood spot (qDBS) sampling can overcome these limitations and may provide better analytical accuracy than conventional dried blood spot assays. This study developed and validated an ultra-performance liquid chromatography-tandem mass spectrometry method to determine isavuconazole in qDBS. Samples were extracted using an acetonitrile-water solution (70:30, v/v). Chromatographic separation was achieved on an Acquity UPLC® BEH C column (100 × 2.1 mm, 1.7 µm). The mobile phase consisted of water (0.02% formic acid) and acetonitrile at a flow rate of 0.6 mL/min, with a total runtime of 2.5 min. The method was linear over 0.1-50 µg/mL and demonstrated good precision (maximum coefficient of variation, 9.9%) and accuracy. Mean recovery was 96.9%. Hematocrit values ranging from 0.2 to 0.65 did not significantly affect the analysis results. qDBS samples were stable for 17 days at room temperature and for 22 days at -20 °C. Reconstituted samples in the autosampler (4 °C) were stable for at least 24 h. Plasma and fingerprick qDBS concentrations from 20 patients were compared using Passing-Bablok regression and Bland-Altman analyses, confirming good agreement. Overall, these findings support qDBS sampling for isavuconazole TDM and suggest that it may replace or supplement conventional sampling in hematology patients (HCT range 0.15-0.40).
Unveiling the comprehensive alterations in the in vivo components, tissue distribution profiles, and pharmacokinetic characteristics between Jiawei Erzhi formula and Erzhi formula.
Jing Q, Zou YD, Ma LL … +7 more , Yao YQ, Zhang M, Liu XN, Gao XM, Li X, Liu EW, Yang WZ
J Pharm Biomed Anal · 2026 Jun · PMID 42335580 · Publisher ↗
Jiawei formula holds a prevalent position in clinical application of Chinese medicine. However, there is a notable lack of methodologies capable of effectively revealing the metabolic discrepancies between the base formu...
Jiawei formula holds a prevalent position in clinical application of Chinese medicine. However, there is a notable lack of methodologies capable of effectively revealing the metabolic discrepancies between the base formula and its Jiawei-modified counterpart. Erzhi formula (EZF) is a tonic formulation, and incorporation of Spatholobi Caulis and Achyranthis Bidentatae Radix yields Jiawei Erzhi formula (JWEZF), which exhibits augmented pharmacological efficacy. This study was devised to formulate a strategy facilitating a thorough comparison of the in vivo components, tissue distribution patterns, and pharmacokinetic behaviors of JWEZF and EZF in rats. Specifically, comprehensive in vivo components analysis was executed utilizing ion mobility liquid chromatography/mass spectrometry, complemented by automated peaks annotation workflows. The distribution of primary components in rat's heart, liver, kidney, and spleen was visualized through desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). Additionally, pharmacokinetic variations in rat plasma were elucidated employing scheduled multiple reaction monitoring (sMRM). Overall, we successfully identified 129 prototype compounds (128 in JWEZF and 89 in EZF) along with 140 metabolites (41 unique for JWEZF). Moreover, DESI-MSI detected differences in the in situ tissue distribution of seven components between EZF and JWEZF, with JWEZF showing a likely higher tissue exposure. Pharmacokinetic analysis of 21 prototypes and six associated metabolites indicated analogous pharmacokinetic profiles between EZF and JWEZF; however, the plasma exposure of isochlorogenic acid B, neochlorogenic acid, luteolin-7-O-glucoside, and its metabolite was significantly higher in the JWEZF group. In summary, we have established a methodology to delve into the distinctions between the original formula and its Jiawei-modified variant.
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