During routine accelerated stability testing of assay and related compounds for a marketed hydrogel formulation by ultra-high pressure liquid chromatography (UHPLC), two new peaks were detected at or near the ICH Q3B(R2) identification threshold. Preliminary examination of the extracted UV spectra for these peaks by the Quality Control laboratory showed that they contained an aromatic moiety (absorbance ∼255 nm), which the active component and its related compounds do not have. However, a preservative (benzyl alcohol) is present in the formulation and showed a similar extracted UV spectra as the unknown peaks. Analysis by liquid chromatography-mass spectrometry (LC-MS) determined that these peaks were isomeric monobenzyl-citrate esters. Synthesis, isolation, purification by fraction collection and analysis by benchtop nuclear magnetic resonance (NMR) determined the identity of each of the ester isomers seen in the stability testing.
Personalized chemotherapy requires precise compounding and reliable quality control to assure therapeutic efficacy and safety. Raman spectroscopy offers a non-destructive and non-invasive method for the identification and quantitation of cytostatic drug substances, which could improve current quality assurance processes in hospital or community pharmacies. In this study, a dedicated Raman detector was developed that enables direct identification and measurement of cytostatic drug concentrations in aqueous liquids through closed primary packaging. Measurements were performed for cyclophosphamide and gemcitabine in clinically relevant concentrations (1 - 45 mg mL-1) using sealed infusion glass vials, filled syringes for bolus injections and infusion bags. Each concentration was analyzed at eight different positions. These measurement positions were chosen randomly to account for signal variability arising from local material variations, including inhomogeneities in wall thickness and curvature. For all tested drug substances and packaging types and materials, relative prediction errors remained within ±10% across the clinically relevant concentration range, with a RMSECV of 1.07 mg mL⁻¹ for cyclophosphamide and 0.54 mg mL⁻¹ for gemcitabine. As routine quality control of individually compounded antineoplastic formulations lacks standardized final-product verification, non-invasive through-container spectroscopic analysis offers the possibility to add identity and concentration confirmation without opening or sampling the CMR-classified formulation. This method preserves the integrity of the final drug product while reducing costs and the risk of occupational exposure for pharmacy personnel.
α-Amylase (α-Amy) is a clinically important biomarker for the diagnosis of acute pancreatitis and related disorders. However, currently available methods for α-Amy detection often require sophisticated instrumentation, labor-intensive procedures, and prolonged assay times, thereby limiting their applicability in rapid and point-of-care analysis. Herein, we developed a simple, sensitive, and rapid nanozyme-based colorimetric assay for the detection of α-Amy. This sensing strategy is based on a substrate-shielding and enzymatic deprotection mechanism, in which γ-cyclodextrin (γ-CD) serves as both a crosslinking agent and a catalytic site blocker for the Fe₃Ni-MOF-NH₂ (FNMN) nanozyme, forming the composite Fe₃Ni-MOF-NH₂@γ-CD (FNMNCD). The specific hydrolysis of γ-CD by α-Amy induces the disassembly of the aggregated FNMNCD structure and restores the peroxidase-like activity of the nanozyme, thereby modulating the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in a concentration-dependent manner. Under optimal conditions, the proposed assay exhibited a wide linear detection range from 2 to 800 U/L, with a low detection limit of 1.1 U/L and relative standard deviation below 4.21%. Furthermore, the method was successfully applied to the determination of α-Amy activity in urine samples from healthy individuals and patients with pancreatitis. Therefore, we believe that the γ-CD-crosslinked FNMN nanozyme system could be applicable for the sensitive and quantitative detection of α-Amy in complex biological samples.
Cinnamomum tamala (Indian bay leaf) is a widely used spice whose essential oils exhibit several pharmacological activities and the presence of several chemotypes, including eugenol, linalool, and cinnamaldehyde types. However, its quality and authenticity are frequently compromised by adulteration and compositional variability. Though comprehensive NMR-based profiling of these chemotypes remains limited, Nuclear Magnetic Resonance (NMR)-based phytometabolomics offer a robust platform for identifying and quantifying natural compounds in plant extracts including essential oils. Unlike mass spectrometry, which may not distinguish structural and configurational isomers, NMR enables precise compound characterization and quantitation even without authentic reference standards. This study aimed to apply NMR spectroscopy-based metabolomics for qualitative and quantitative profiling of C. tamala essential oils to identify chemotypic variations and to evaluate the antibacterial potential of these different chemotypes. Essential oils were extracted from C. tamala leaves collected from northern and eastern India via hydro-distillation. Quantitative 1H NMR (qNMR) was used to determine the concentrations of major volatile compounds. Two-dimensional (2D) heteronuclear NMR and Principal Component Analysis (PCA) were employed for structural elucidation and chemotype discrimination. Sixteen volatiles were quantified, including eugenol, cinnamaldehyde, and linalool, which defined distinct chemotypes. PCA effectively differentiated oils based on metabolic fingerprints. Furthermore, minimum inhibitory concentration (MIC) of cinnamaldehyde, eugenol and linalool and their specific chemotypes were assessed using the broth microdilution method against Gram negative and Gram positive ATCC strains. Antibacterial assays revealed inhibitory potential of specific oil constituents. Taken together integration of 1H qNMR with PCA offers a reliable approach for distinguishing regional and commercial variations and assessing safety, while antibacterial screening highlights the potential specific chemotype driven antibacterial activity and our data also suggest a cautionary note to select right chemotype of essential oils for intended applications.
Taurine is an abundant endogenous amino acid-related compound involved in several physiological processes, including osmoregulation, antioxidative defense, and cytoprotection. Its quantification in biological matrices is analytically challenging due to its high polarity, endogenous background levels, and the absence of a true blank biological matrix. In addition, taurine has emerged as a potential endogenous biomarker for renal transporter phenotyping, particularly for organic anion transporters 1 and 3 (OAT1/3). In this study, a rapid and robust LC-MS/MS method was developed and validated for the quantification of endogenous taurine in human plasma and urine using surrogate matrix calibration combined with background subtraction. Chromatographic separation was achieved on a Luna NH₂ 100 Å column (5 µm, 150 × 4.6 mm) with a total run time of 5.5 min. Calibration curves prepared in 2% bovine serum albumin and diluted urine were linear over concentration ranges of 0.05-50 µg/mL and 0.05-100 µg/mL, respectively (r² > 0.99). Precision and accuracy met regulatory acceptance criteria (≤15%, or ≤20% at the lower limit of quantification). No significant matrix effects were observed across independent plasma and urine sources, including lipemic and hemolyzed plasma samples. Matrix and dilutional parallelism were confirmed, and taurine remained stable under all tested conditions. The method was successfully applied to clinical plasma and urine samples, enabling taurine pharmacokinetic profiling and renal clearance estimation. This validated LC-MS/MS assay provides a reliable analytical platform for taurine quantification in biological matrices and supports future pharmacokinetic, biomarker, nutritional, and transporter phenotyping studies.
d-Amino acids are the enantiomers of l-amino acids that occur in minimal concentrations within organisms and exert significant effects on physiological regulation and pathological progression. Abnormal d-amino acid levels are closely associated with neurological disorders and metabolic disturbances. The present research is dedicated to establishing a highly sensitive and reliable quantitative detection approach for evaluating metabolic changes in d-amino acids linked to kidney disease, aiming to assist clinical diagnosis. Samples were processed by precipitating proteins, drying them with nitrogen, and resolubilizing them in water. Finally, l-FDLA (Nα-(5-fluoro-2,4-dinitrophenyl)-l-leucinamide) functioned as a reagent for derivatizing samples of plasma, urine, and cyst fluid. For the calibration curve, activated carbon treatment was used to prepare a blank matrix, which was then tested for separation and detection using reverse-phase chromatography and tandem mass spectrometry. The analysis of biological fluid samples obtained from patients with chronic kidney disease (CKD) and autosomal dominant polycystic kidney disease (ADPKD) was carried out following method validation. A progressive increase in plasma d-serine levels, accompanied by a marked reduction in urinary d-serine, was observed during the course of kidney disease, indicating that D-serine may represent a potential metabolic biomarker of kidney dysfunction. Furthermore, higher levels of d-serine were found in the cyst fluid of an ADPKD patient. Based on the quantitative method used in this study, it was found that it exhibits high sensitivity and selectivity and that it was effective in identifying the metabolic characteristic changes that occur in d-amino acids during kidney disease. Overall, patterns of d-amino acid distribution and their dynamic alterations were strongly associated with the initiation and progression of the disease. The detection of these markers holds promise for providing novel biochemical indicators applicable to the early diagnosis and assessment of kidney disease progression. In terms of methodology, this study extended the use of l-FDLA derivatization to other biological matrices besides human blood, and applied the matrix stripping method for the method validation, thus improving the accuracy of endogenous amino acid quantification. In terms of method application, a focus on amino acid trends in various biofluids of ADPKD patients is offered, within the larger group of kidney dysfunction patients.
Antibody‑drug conjugate (ADC) off‑target toxicity is largely driven by exposure to payloads and related conjugated catabolites. Determining whether to measure free payload or conjugated linker‑payload catabolites for exposure assessment is critical but not straightforward. Typically, only free payload is measured for ADCs with cleavable linkers, whereas conjugated linker‑payload catabolites are monitored for ADCs with non‑cleavable linkers. For ADC‑1, which contains a cleavable linker, both free Payload-1 and the cysteine‑conjugated linker‑payload catabolite (CLP) were detected, adding complexity to the bioanalysis. Triple‑quadrupole LC‑MS/MS and high‑resolution mass spectrometry were used to confirm the identity of CLP. Temperature‑dependent in‑source fragmentation of CLP and its contribution to the Payload-1 chromatographic signal were investigated. Additionally, the interconversion of the major linear CLP to a cyclized form during sample handling was characterized. The inclusion of 500 mM ascorbic acid during sample processing effectively stabilized the linear form. The linear CLP exhibited pharmacokinetics closely matching those of the Payload-1. Overall, this work improves the bioanalytical understanding of cleavable‑linker ADCs and provides a quantitation strategy to support the routine monitoring of CLP alongside free payload in safety and pharmacokinetic assessments.
Depression is characterized by a dysregulated brain-gut axis. Xiaoyao San (XYS), a classic Traditional Chinese Medicine formula for soothing Liver and strengthening Spleen, is clinically effective in alleviating depression. However, the systems-level mechanisms by which XYS coordinates gut-brain communication to exert its antidepressant effects remain insufficiently understood. This study aimed to systematically elucidate the antidepressant mechanisms of XYS, with a focus on identifying a key gut-derived metabolic pathway that modulates prefrontal cortex (PFC) function. A mouse model of depression was established using isolated housing combined with chronic unpredictable mild stress (CUMS). Mice were treated with XYS at low, medium, and high doses or paroxetine. We employed a multimodal approach, integrating behavioral tests, resting-state functional magnetic resonance imaging (rs-fMRI), gut microbiota profiling (16S rRNA sequencing), serum metabolomics and PFC transcriptomics. To establish causal evidence, pseudo-germ-free mice received fecal microbiota transplantation (FMT) from donor mice treated with XYS, followed by comprehensive behavioral and biochemical assessments. XYS treatment significantly ameliorated depressive-like behaviors and restored functional connectivity within emotion-regulation brain networks. Multi-omics integration revealed that XYS reshaped the gut microbiota, which was associated with a reduction in systemic levels of kynurenine (KYN), a key tryptophan-derived metabolite. In the PFC, this decrease in KYN was accompanied by the normalization of aryl hydrocarbon receptor (AhR) signaling activity. Furthermore, GABAergic neurotransmission, mediated by γ-aminobutyric acid (GABA), was enhanced, as evidenced by upregulated expression of glutamate decarboxylase 1 (Gad1), gamma-aminobutyric acid type A receptor subunit alpha1 (Gabra1), increased GABA content, and elevated levels of key synaptic plasticity-related molecules, including brain-derived neurotrophic factor (BDNF), postsynaptic density protein-95 (PSD-95), and synaptophysin (SYN). Critically, FMT from XYS-treated donors recapitulated the antidepressant phenotype in recipient mice, directly implicating the gut microbiota in these therapeutic effects. This study demonstrates that XYS alleviates depression by orchestrating a gut-brain signaling cascade that converges on the PFC to enhance inhibitory synaptic transmission. These findings provide novel and causal mechanistic insights into the brain-gut modulatory action of XYS, offering a comprehensive framework for its therapeutic potential in treating depression.
4-Methyl-N-ethylcathinone (4-MEC) is a novel psychoactive substance and its in vivo exposure characterization remains unknown. In this study, a sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the simultaneous quantification of 4-MEC and its two major metabolites in rat whole blood. Then it was applied to pharmacokinetic studies of 4-MEC in rats by intravenous and intragastric administration, characterizing the concentration-time profiles of the parent compound and its metabolites. The results revealed distinct exposure patterns between 4-MEC and its metabolites. Notably, M2 (N-deethylated metabolite) exhibited a prolonged detection window compared with 4-MEC, supporting metabolite-based monitoring strategies and thereby extending its application in forensic toxicology and drug abuse identification.
Complex natural product processing involves dynamic compositional changes, yet rapid and standardized process characterization remains difficult when relying mainly on off-line laboratory analysis and sensory-based empirical judgment. To address this analytical need, Fuzi (Aconiti Lateralis Radix Praeparata), the processed lateral root of Aconitum carmichaelii Debx., and Si Ni Tang, a classical Chinese herbal formula containing Fuzi, were used as representative single-herb and formula decoction systems. An analytical framework was established in which high-performance liquid chromatography provided reference values for calibration and validation, the electronic tongue provided objective and reproducible digital responses related to sensory-based empirical evaluation, and mid-infrared spectroscopy supplied rapid molecular information on toxicity-related constituents. High-performance liquid chromatography was used to quantify six Aconitum alkaloids in the decoction samples, while electronic tongue responses and mid-infrared spectra were collected in parallel and modeled using chemometric methods. The decoction process showed a clear time-dependent conversion of diester diterpenoid alkaloids into the corresponding less toxic monoester diterpenoid alkaloids. Based on the chromatographic reference values, mid-infrared models provided reliable quantitative prediction of the target alkaloids, and the electronic tongue differentiated samples collected at different decoction time points according to their overall multi-sensor response patterns, thereby providing complementary information for rapid characterization of process states. Rather than replacing high-performance liquid chromatography for endpoint confirmation, this framework retains high-performance liquid chromatography as the reference method and extends evaluation from off-line endpoint testing to rapid process-oriented characterization of toxicity-related alkaloid transformation, providing a complementary analytical route for safety-related quality control of complex herbal decoctions.
Chronic Kidney Disease (CKD) is a progressive disorder requiring strategies for early detection and disease stratification. In this context, volatilomics represents a promising analytical approach for investigating metabolic alterations through the profiling of volatile organic compounds (VOCs) in biological matrices. In this study, headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC/MS) was applied to characterize VOC profiles in blood and urine samples collected from healthy controls and CKD patients at different disease stages. The analytical workflow combined headspace extraction, SPME-based pre-concentration, non-target GC/MS analysis, matrix-matched semi-quantification, and chemometric data analysis to evaluate matrix-specific VOC signatures associated with renal dysfunction. Blood and urine VOC datasets were analysed in parallel to compare their discriminatory information and complementary contribution to CKD-related volatilomic characterization. Univariate analysis revealed VOCs significantly differing among study groups (p < 0.05), while multivariate Partial Least Squares-Discriminant Analysis (PLS-DA) demonstrated clear separation of the investigated groups according to disease status. Comparative analysis indicated that blood- and urine-derived VOC patterns provide matrix-dependent and complementary information. Correlation analysis showed significant associations between selected VOCs and conventional clinical parameters, including hemoglobin, serum creatinine, urea, and estimated glomerular filtration rate (eGFR). Regression models based on VOC profiles indicated potential for estimating key indicators of renal function. Overall, these findings demonstrate the applicability of HS-SPME-GC/MS-based volatilomic profiling to complex biological samples and support blood and urine VOC signatures as complementary analytical markers of renal dysfunction. The parallel evaluation of biological matrices supports method-driven biomarker discovery in clinical and biological chemistry.
Additives are essential to plastic materials, to obtain specific physico-chemical properties depending on intended applications. However, some of them are considered as toxic, such as bis(2-ethylhexyl)-phthalate (DEHP). This is a matter of concern, since plastic additives can migrate from plastic medical devices to contact matrices and then be harmful to patients. Therefore, extracting and analyzing compounds that can migrate from plastic materials is an essential step to evaluate the risk. The advantages of online supercritical fluid extraction and chromatography coupled to mass spectrometry (SFE-SFC-MS) were demonstrated previously, notably to avoid laboratory contamination. Here, this method is applied to the characterization of 24 plastic additives in medical devices used to infuse medical solutions to patients. The target compounds included plasticizers (phthalates and non-phthalates) such as DEHP or its substitutes (like TOTM, ATBC, DEHA, DINCH), antioxidants (Irganox 1076, Irganox PS800) and lubricants (erucamide and oleamide). First, the polymers were characterized using Fourier-Transform Infrared spectroscopy (FTIR). Then, plastic additives were characterized with SFE-SFC-MS. The proportion of DEHP and its substitutes varied greatly from one sample to another, depending on the plastic material nature, and its manufacturing date. Furthermore, the method was validated for DEHP using accuracy profiles. The validated method allowed quantifying DEHP from 5 to 35 ng, with average RSD values of 9% and 15% obtained for the calibration and validation series respectively. Finally, the greenness of the method was favorably evaluated with AGREEprep green score (0.74), especially when compared to traditional extraction methods used for plastic additives.
This work focused on N-nitroso-fluoxetine, a N-nitrosamine drug substance-related impurity (NDSRI) formed in fluoxetine-containing products via nitrosation pathways. N-nitroso-fluoxetine was previously detected above the established safety threshold of 100 ng/day, leading to temporary withdrawal of a marketed product in Belgium. An Analytical Quality by Design (AQbD) approach supported the development of a reversed-phase LC-(ESI+)-MS/MS method. Design of experiments identified aqueous mobile phase pH and gradient time as critical method parameters affecting detection or quantification limits and chromatographic separation performance, respectively. Adequate separation was achieved using Acquity® HSS T3 guard-column and column with a run time of 22 min. The method was validated according to ICH Q2(R2) guideline using a combined approach based on total error. It enables accurate and precise quantification of N-nitroso-fluoxetine from 0.09 to 4.75 ppm (injected concentration in ng/mL) while simultaneously screening for other small-molecule N-nitrosamines in both drug substance and finished products. The validated method was applied to assess risk mitigation measures implemented by the manufacturer, including the use of low-nitrite excipients, desiccant-integrated aluminum/aluminum blister packaging, and reduced shelf-life. In the updated formulation, N-nitroso-fluoxetine contents were 0.58 ppm at release and increased to 1.15 ppm after 21 months of storage at 25 °C/60% RH, remaining below the safety threshold of 1.67 ppm calculated according to the EMA "Call for Review" procedure. Together, these modifications have effectively controlled N-nitroso-fluoxetine formation. Therefore, the present study demonstrates a robust AQbD-guided LC-MS/MS method, contributes to NDSRI analysis, and supports science- and risk-based control strategies for these emerging impurities.
Characterizing fecal molecular perturbations associated with diagnosis and treatment may facilitate the development of novel non-invasive biomarkers for better clinical management of inflammatory bowel disease (IBD). Here, we employed rapid untargeted lipidomics, combined with various lipid data mining methods, to investigate alterations in the fecal lipidome associated with IBD, disease activity, and body mass index (BMI) levels. Machine learning modeling with a robust nested cross-validation procedure was employed to identify fecal-based potential biomarkers across three clinically meaningful scenarios. A total of 719 fecal lipids of 65 treated IBD patients were annotated, with lipid alterations analyzed at different levels, from subclasses to individual species. Univariate analysis revealed significant differences in the abundances of ceramides, fatty acids, triglycerides (TGs), ether-linked diacylglycerols (DG(O-)), and microbiota-associated lipids - fatty acid esters of hydroxy fatty acids (FAHFAs), between ulcerative colitis (UC) and Crohn's disease (CD). Significant alterations in lipid-related gene functions between UC and CD were linked to the sphingolipid signaling pathway, ether lipid metabolism, sphingolipid metabolism, and cholesterol metabolism. Interestingly, no significant fecal lipidome alterations were associated with disease activity, fecal calprotectin levels, or BMI. Partial least squares-discriminant analysis and support vector machine models achieved satisfactory classification performance, with an area under the receiving operating characteristic curve ≥ 0.80. FAHFA, TG, and DG(O-) were consistently identified as the top potential predictors to differentiate between UC and CD. Altogether, our findings provide better insights into the lipidome of IBD undertreatment and suggest that fecal lipids are potential non-invasive biomarkers to aid in IBD diagnosis.
Human carboxylesterase 1 (hCE1) is highly expressed in the liver and can be released into the bloodstream upon hepatocellular injury, suggesting its potential as a biomarker for liver damage. However, current methods for measuring hCE1 activity suffer from poor selectivity and insufficient sensitivity and are easily interfered with by serum albumin (HSA), butyrocholinesterase (BChE), and paraoxonase 1 (PON1). In this study, a novel fluorescent probe, NCMNe, was designed and synthesized, and an ultrasensitive liquid chromatography-fluorescence detection (LC-FD) method was established for quantifying plasma hCE1 activity and evaluating its feasibility as a biomarker of liver injury. Specificity studies showed that NCMNe was selectively hydrolyzed by hCE1 without interference from HSA, PON1, or BChE. The developed LC-FD method achieved a lower limit of quantification of 0.0001 μM, representing the most sensitive method reported to date for hCE1 activity determination. Clinical sample analysis revealed that plasma hCE1 activity in patients with hepatitis B was significantly higher than that in healthy controls and was strongly correlated with aspartate transaminase (AST)and alanine transaminase (ALT). Receiver operating characteristic (ROC) curve analysis demonstrated that plasma hCE1 activity measurement was effective for detecting liver pathology (AUC = 0.901, p < 0.0001), with diagnostic performance comparable to that of ALT and AST. Collectively, the proposed LC-FD method enables highly sensitive and selective determination of plasma hCE1 activity and supports hCE1 as a promising serum biomarker for liver injury, providing a new approach for early diagnosis and disease assessment.
Prefilled syringes (PFS) increasingly serve as standalone injection systems and as critical components in autoinjectors, where device performance and patient safety depend on tightly controlled component attributes. Silicone oil (SO) is widely used as a lubricant on syringe barrels, plungers, needles, and elastomeric needle shields to reduce frictional forces; however, accurately quantifying the microgram‑level SO present on individual elastomeric components remains analytically challenging. Here, we present a rapid and sensitive proton nuclear magnetic resonance (¹H NMR) method for direct measurement of SO amount on single elastomeric needle shields. A calibration curve constructed from SO standard demonstrated excellent linearity (R² = 0.999), and the method achieved a limit of detection of approximately 0.2 µg SO per component. Applied to two elastomeric needle shield batches with vendor‑controlled siliconization, the method successfully distinguished between low‑ and high‑SO conditions. In a separate PFS batch with known variability in pull‑off force (POF), an inverse relationship was observed between SO mass and POF: syringes with lower POF exhibited higher SO levels, whereas those with higher POF contained less SO. Overall, this ¹H NMR approach enables fast (<10 min per sample), specific, and quantification of SO on single elastomeric needle shields, providing a practical tool for routine quality control and for correlating siliconization levels with critical device performance attributes.
Futibatinib (TAS-120) is a next-generation covalent inhibitor of fibroblast growth factor receptor (FGFR) 1-4 that has been authorized for the treatment of patients with previously treated intrahepatic cholangiocarcinoma carrying FGFR2 fusions or rearrangements. However, information regarding its degradation behavior and impurity profile remains limited. The stability behavior of futibatinib under a series of forced degradation conditions was comprehensively evaluated using an HPLC-based analytical approach. Forced degradation studies demonstrated that futibatinib was relatively stable under oxidative, thermal, and photolytic conditions, whereas significant degradation occurred under alkaline conditions. Two previously unreported degradation-related products, designated as Fut-1 and Fut-2, were generated under intensified alkaline conditions and subsequently isolated by preparative liquid chromatography. Fut-1 and Fut-2 were characterized using high-resolution mass spectrometry, nuclear magnetic resonance and heteronuclear multiple bond correlation spectroscopy. Based on comprehensive spectral analyses, Fut-1 was identified as (S)-3-((3,5-dimethoxyphenyl)ethynyl)-1-(pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine, while Fut-2 was identified as (S)-1-(3-(4-amino-3-((3,5-dimethoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)-3-methoxypropan-1-one. Structural analysis suggested that degradation primarily involved transformation of the acrylamide side chain, whereas the pyrazolo[3,4-d]pyrimidine core and the 3,5-dimethoxyphenyl-ethynyl moiety remained largely intact. In vitro cytotoxicity studies against five human tumor cell lines revealed that Fut-1 retained considerable cytotoxic activity and exhibited stronger inhibitory effects than futibatinib in several cell lines, whereas Fut-2 showed reduced activity. The results provide valuable insights into the structural transformation behavior, impurity profiling, and quality control considerations of futibatinib and may contribute to future investigations of structure-activity relationships of futibatinib-related compounds.
Depression is a complex disorder involving multiple pathological processes, and the identification of bioactive candidates from traditional Chinese medicine (TCM) remains challenging. This study established an integrated network pharmacology (NP)-cell membrane chromatography (CMC)-high-performance liquid chromatography (HPLC) strategy (NP-CMC-HPLC) for activity-guided screening of potential bioactive compounds and activity-associated quality characterization of the dried tuberous root of Curcuma wenyujin Y. H. Chen & C. Ling (WYJ). The chemical profile of WYJ was comprehensively characterized using ultra-performance liquid chromatography coupled with quadrupole Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS), combined with headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) and headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS). A total of 184 compounds were identified, and potential bioactive compounds were prioritized through network pharmacology analysis. Multi-target cell membrane chromatography (CMC) systems based on 5-hydroxytryptamine 1 A receptor (5-HT1A), 5-hydroxytryptamine 2 A receptor (5-HT2A), dopamine D1 receptor (DRD1), dopamine D2 receptor (DRD2), and monoamine oxidase A (MAO-A) were subsequently established, enabling the identification of six compounds exhibiting target-associated retention behavior. Molecular docking and a corticosterone-induced PC12 cell injury model provided complementary computational and cellular evidence supporting compound selection. Finally, a parallel reaction monitoring (PRM)-based method was developed for the quantitative analysis of four non-volatile CMC-prioritized compounds. The proposed strategy integrates chemical profiling, target affinity screening, cellular evaluation, and quantitative analysis to facilitate the discovery of bioactive candidates and activity-associated quality characterization of complex herbal medicines.
A rapid and highly sensitive LC-MS/MS method was developed and validated for the determination of the nitrosamine drug substance-related impurity N-nitroso-desmethyl sumatriptan (NNDS) in sumatriptan succinate injection. Separation was achieved on an Inertsil ODS-3 column using gradient elution with 0.1% formic acid in water and acetonitrile, followed by positive electrospray ionization in multiple reactions monitoring mode. The method was validated according to ICH Q2(R2) and USP <1225> guidelines, demonstrating excellent specificity, linearity (R² = 1.0000), precision, accuracy, robustness, and solution stability. The limits of detection and quantification were 0.05 ppm and 0.15 ppm, respectively, providing sensitivity well below the regulatory specification limit for NNDS. Analysis of commercial injection batches confirmed NNDS levels within acceptable limits. The validated method is suitable for routine quality control, stability assessment, and regulatory monitoring of nitrosamine impurities in pharmaceutical products.
