FIREFISH was a multicentre, open-label, two-part, phase 2 trial that assessed the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of risdiplam in children with type 1 spinal muscular atrophy and two SMN2 copies, aged 1-7 months at enrolment. Part 1 assessed safety and determined the dose for part 2. In part 2, treatment with risdiplam for 24 months resulted in continual improvements in motor function and achievement of developmental motor milestones. The aim of the 3-year open-label extension reported here was to assess long-term safety and efficacy of risdiplam. The 3-year extension of the FIREFISH trial was a multicentre, open-label study in 17 centres across 12 countries (in Asia, Europe, Brazil, and the USA). Children with a confirmed genetic diagnosis of spinal muscular atrophy and two copies of the SMN2 gene who had completed 2 years of risdiplam treatment were eligible to continue in the open-label extension study, during which risdiplam was administered once daily by oral syringe or feeding tube at the pivotal dose of 0·20 mg/kg for infants younger than 2 years, at 0·25 mg/kg for children aged 2 years and older with bodyweight below 20 kg, and at 5 mg for children aged 2 years and older with bodyweight of 20 kg or more. Adverse events were assessed as part of physical assessments every 13 weeks. More comprehensive assessments every 26 weeks included level of respiratory support and efficacy assessments of motor function in addition to safety and physical assessments. Outcomes assessed during the open-label extension study included adverse events, survival and event-free survival (defined as being alive with no permanent ventilation), growth measures, swallowing, feeding, and motor function. Motor function was assessed with the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND), the gross motor subscale of the Bayley Scales of Infant and Toddler Development, third edition (BSID-III), and the Hammersmith Infant Neurological Examination, Module 2 (HINE-2). For all children, safety data including adverse events, serious and non-serious adverse events of special interest; ophthalmology assessments; laboratory assessments; vital signs; ECGs; and other protocol-specified tests deemed critical to the safety evaluation of the study, were collected up to 30 days after the last dose. Survival and event-free survival are reported at year 5 for all enrolled children. Adverse events are reported at year 5 for all children who received at least one dose of risdiplam (safety population). All other efficacy endpoints are reported at year 5 for the population who received the pivotal dose. FIREFISH is registered with ClinicalTrials.gov, NCT02913482. Between Dec 23, 2016, and Nov 19, 2018, 62 children aged 1-7 months were enrolled in FIREFISH parts 1 and 2. After 2 years in the study (parts 1 and 2), 55 children continued risdiplam treatment into the open-label extension for a further 3 years. The study was completed on Dec 22, 2023, at which time 52 children (84%) had completed 5 years of risdiplam treatment. After 5 years of risdiplam treatment, 56 children (90%) were alive, and 50 (80%) were alive without the need for permanent ventilation. In the pivotal-dose population (n=58), 17 children (29%) did not require invasive or non-invasive respiratory support at year 5 and 13 (22%) did not require hospitalisations during the study. Upward trajectories for motor function responses were observed between years 1 and 5. The number of children who could sit without support for at least 5 s and 30 s was maintained during the 3-year extension, with 36 children (62%) and 34 children (59%), respectively, doing so by year 5. Similarly, for HINE-2, eight children (14%) reached the stable sit milestone and 26 (45%) met pivots at year 5. By year 5, four children (7%) could stand without support as assessed by both the BSID-III and HINE-2. No children could walk independently, but six (10%) met the HINE-2 cruising milestone. The mean HINE-2 total score increased from baseline (0·93 [90% CI 0·72-1·14]) to year 4 (14·22 [12·66-15·79]), remaining stable thereafter. Mean CHOP-INTEND scores increased from 22·47 at baseline (90% CI 20·97-23·96) and stabilised at around 50 at year 5, with scores of ≥40 for 38 children (66%), ≥50 for 29 children (50%), and ≥60 for 11 children (19%) at year 5. Most children maintained oral feeding (n=42; 72%) and swallowing (n=46; 79%) abilities, and growth was consistent with typical patterns seen in type 1 spinal muscular atrophy. Safety findings over 5 years were consistent with the results from the primary analysis. Most adverse events were mild or moderate. The most frequently reported adverse events were pyrexia (65% [n=40]), upper respiratory tract infection (63% [n=39]), and pneumonia (50% [n=31]). Pneumonia was the most frequently reported serious adverse event in 28 children (45%). No new safety findings were reported and no treatment-related events led to withdrawal from study treatment. Unlike the natural history of children with type 1 spinal muscular atrophy, after 5 years of risdiplam treatment, most children in FIREFISH were alive without needing permanent ventilation, were able to swallow and feed orally, and had reached motor milestones not typically observed in untreated patients. These results show long-term continuous efficacy and safety of risdiplam in children with type 1 spinal muscular atrophy. F Hoffmann-La Roche.
This study aims to analyze the differences in chemical constituents before and after the ginger-processing of Anemarrhenae Rhizoma(AR), screen for key differential components, and elucidate their anti-inflammatory mechanisms. Furthermore, it seeks to clarify the principles of processing and the scientific rationale behind the synergistic enhancement achieved through ginger processing. This research provides scientific support for the preservation of traditional processing techniques and the rational clinical application of ginger-processed Anemarrhenae Rhizoma (GAR). The Ultra Performance Liquid Chromatography-Quadrupole-Time of Flight-Tandem Mass Spectrometry (UPLC-Q-TOF-MS/MS) technology was employed for qualitative analysis of the chemical constituents present in AR and GAR in both positive and negative ion modes. By comparing the variations in constituent levels before and after ginger-processing, as well as identifying newly emerged characteristic peaks, we prioritized novel and upregulated differential components with well-documented pharmacological activities as target compounds. The key differential components were subsequently validated quantitatively using High Performance Liquid Chromatography (HPLC) technology. Furthermore, molecular docking technology was employed to predict the binding affinity of these key differential components with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and cyclooxygenase-2 (COX-2) proteins. An inflammatory model was established utilizing lipopolysaccharide (LPS)-induced RAW264.7 macrophages, and the CCK-8 (Cell Counting Kit-8) assay was performed to determine the optimal concentrations of the key differential components and LPS. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cell supernatants were measured using ELISA, while the expression levels of IκBα (Inhibitor of nuclear factor kappa B alpha), NF-κB, phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-κB), nuclear NF-κB, and COX-2 proteins were assessed through Western blot (WB) analysis. UPLC-Q-TOF-MS/MS analysis identified 40 compounds in both AR and GAR under both positive and negative ion modes, predominantly comprising saponins. Key differential components (mangiferin, timosaponin BⅡ, 6-gingerol) were screened based on changes in the chemical profile before and after ginger-processing, as well as pharmacodynamic literature. HPLC quantitative analysis confirmed that the components timosaponin BⅡ and mangiferin were increased following ginger-processing, while 6-gingerol was identified as a unique component in GAR, a novel compound generated during ginger-processing. Molecular docking results indicated a strong binding affinity of these components with NF-κB and COX-2 proteins. CCK-8 assays demonstrated that at an LPS concentration of 1 µg/mL, mangiferin at 100 μmol/L, timosaponin BⅡ at 2 μmol/L, and 6-gingerol at 20 μmol/L, the cell viability exceeded 90%, with no significant cytotoxicity observed. ELISA results indicated that compared to the model group, the treatment groups treated with timosaponin BⅡ, mangiferin, and 6-gingerol significantly inhibited the release of TNF-α and IL-6 (P < 0.05). Furthermore, Western blot analysis confirmed that these compounds markedly upregulated the protein expression of IκBα while significantly downregulating the expression levels of NF-κB, p-NF-κB, nuclear NF-κB, and COX-2 (P < 0.05). This study demonstrates that GAR introduces 6-gingerol, a key anti-inflammatory component found in ginger, while also increasing the levels of mangiferin and timosaponin BⅡ. These differential components may enhance the anti-inflammatory efficacy of GAR by inhibiting the activation of the NF-κB signaling pathway and downregulating COX-2 protein expression, which in turn reduces the release of pro-inflammatory mediators such as IL-6 and TNF-α. By following the sequence of "component variation-enhanced efficacy-mechanism clarification," this study systematically elucidates the material basis, action mechanisms, and processing rationale that underlie the enhanced anti-inflammatory effects of GAR. Furthermore, it provides scientific support for the preservation of traditional processing techniques of GAR and its clinical applications.
"Fujv" (a traditional Chinese medicine processing method of chopping herbs into small pieces for convenient decoction and administration) compressed decoction pieces (FP) is a new type of improved processing technology that compresses leaf-type herbs into dense blocks. To compare the quality of pharmacopoeia-specified decoction pieces (YP) and FP of Isatidis Folium (IF), this study conducted analyses from both physical and chemical perspectives. First, a modified drainage method and a tilting method were employed to compare the volume and flowability of YP and FP. Subsequently, the UPLC-Q-Orbitrap-MS was used for qualitative identification of chemical components in IF. HPLC was then applied to establish chromatographic fingerprints for YP and FP. Based on the identification of 18 common peaks in fingerprints, the chemometric methods were employed to evaluate the overall quality of YP and FP. In combination with the variable importance for projection (VIP) value and the indicator components recorded in the Chinese Pharmacopoeia, the contents of isovitexin, indirubin, and total amount of indigo and indirubin were determined. Furthermore, the extraction rate and indirubin content of the standard decoctions prepared from YP and FP were compared. The results showed that the volume and flowability of FP were significantly superior to those of YP. At the chemical level, the overall quality difference between YP and FP was minimal. Regarding the content of key components in decoction pieces and standard decoctions, FP made from IF originating from Gansu and Henan provinces exhibited better quality than their YP counterparts. These findings support the potential of FP as a space-saving and flowability-improved alternative to the YP of IF, providing a basis for further process standardization and quality evaluation.
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.
Psoralea fructus (PF) is a well-documented traditional Chinese medicinal herb with potent tonic effects and has been widely used in clinical practice for centuries. This study systematically characterized the in vivo metabolic profiles of the ethanol extract of Psoralea fructus (PFE) in Sprague-Dawley (SD) rats. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to identify both prototype components and metabolites in rat plasma and major organ tissues. First, we performed comprehensive chemical profiling of PFE using UPLC-QTOF-MS, which enabled the tentative identification of 132 chemical constituents. Following oral administration of PFE, we further characterized the systemically absorbed prototype components in plasma and various tissues. In total, 85 prototypical compounds were identified across all biological samples: 56 prototype compounds were identified in plasma, primarily consisting of coumarins, flavonoids, and phenolics. For major organs, 57, 71, 43, 36, 48, and 26 prototype compounds were found in the heart, liver, spleen, lung, kidney, and brain, respectively, with all tissues dominated by the same classes of chemical components. Subsequently, metabolite profiling was performed to elucidate the biotransformation characteristics of PFE. Metabolites were putatively annotated based on public databases and previously reported literature. A total of 83 metabolites were identified in plasma, and 63, 79, 54, 50, 59, and 21 metabolites were detected in the heart, liver, spleen, lung, kidney, and brain, respectively. The major metabolic pathways of PFE included Phase I reactions (oxidation, reduction, hydrolysis, and hydroxylation) and Phase II conjugation reactions (sulfation and glucuronidation). Consistent with the selective permeability of the blood-brain barrier, the brain tissue exhibited the lowest abundance of prototype components and metabolites. In conclusion, this systematic investigation of the in vivo metabolic characteristics of PFE provides a solid scientific foundation for guiding the rational clinical application of PF.
Oxylipins derived from n-6 and n-3 polyunsaturated fatty acids (PUFAs) are crucial signaling molecules involved in various physiological and pathological processes. However, the quantification of these metabolites remains challenging due to their low abundance, high structural similarity, and poor electrospray ionization efficiency. Furthermore, existing methods often suffer from complex sample preparation and the lack of cost-effective, reliable internal standards. Therefore, establishing a highly sensitive, automated, and standardized analytical platform for comprehensive oxylipin profiling is the prerequisite for elucidating their complex roles in disease pathogenesis. To address these challenges, we developed a highly sensitive and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, incorporating automated sample preparation through magnetic solid-phase extraction and chemical isotope derivatization using 4- (aminomethyl)-N,N-dimethylaniline-d0/d6 (4-AND-d0/d6) reagents. This approach enables the quantification of 69 oxylipins derived from n-6 and n-3 PUFAs, achieving a remarkable 55- to 1000- fold increase in MS detection sensitivity compared to non-derivatized analysis. Additionally, a retention index (RI)-based predictive model was established to facilitate the screening and identification of unknown regio-isomers. As a proof of concept, this method was applied to quantify oxylipins in serum from a neonatal hypoxic-ischemic encephalopathy (HIE) rat model and to evaluate its applicability in complex biological matrices. Our method offers a sensitive, automated, and reliable tool for oxylipin profiling, with potential applications in biomarker discovery and lipid mediator research. This work presents the first integrated LC-MS/MS strategy combining automated magnetic-bead-assisted extraction and chemical isotopic derivatization for oxylipin analysis, enabling the highly sensitive quantification of 69 target analytes. This study is significant for establishing a robust retention index-based predictive model that facilitates the screening and identification of unknown regio-isomers, offering a reliable and high-throughput analytical solution for clinical biomarker discovery and lipid mediator profiling.
Unregistered and excessive use of restricted anti-dandruff agents and the illegal addition of prohibited antibiotics in hair care products pose growing concerns, but monitoring is complicated by lack of integrated screening workflows, as well as the presence of surfactant-rich matrices and metal-chelating properties of some anti-dandruff agents like zinc pyrithione (ZnPT) and piroctone olamine (PO). This study presents a high-throughput "profile-and-quantify" strategy combining direct analysis in real-time high-resolution mass spectrometry (DART-HRMS) with high-performance liquid chromatography (HPLC) to monitor eight targets in shampoos and conditioners. After acetonitrile-methanol ultrasonic extraction, samples were screened using DART-HRMS under optimized parameters. Samples containing the target analytes were subsequently quantified using high-performance liquid chromatography with diode array detector (HPLC-DAD). Validation of this dual-platform approach demonstrated limits of detection ranging from 0.1 to 4 μg/g for the target analytes. For quantitative analysis, the HPLC-DAD assay exhibited good linearity (r² ≥ 0.9995). Spiked recoveries ranged from 96.1% to 107.2% with precision (RSD) ≤ 3.22%. This integrated strategy was applied to a longitudinal survey of 150 commercial products collected from 2022 to 2024. Screening revealed a widespread occurrence of ZnPT, salicylic acid, climbazole, and PO, while also identifying illicit additions of antifungal drugs including miconazole, elubiol, ketoconazole, and clotrimazole in cosmetic formulations. Featuring a rapid screening step of under 2 min per sample prior to targeted quantification, this integrated workflow offers a highly efficient, sensitive, and eco-friendly solution for large-scale regulatory monitoring of cosmetic safety and compliance.
The coronavirus disease 2019 (COVID-19) pandemic highlighted the influence of behavioral and environmental factors on the risk of infection, as well as on disease progression and severity. Therefore, it is critical to improve public health knowledge regarding the factors influencing disease outcomes. This study describes the development and validation of the "Cuban population environmental and lifestyle factors Questionnaire" (CELF-Q), a tool designed to evaluate the impact of environmental and lifestyle factors on COVID-19 outcomes in the Cuban population. Based on an existing lifestyle questionnaire developed for the Latin American population, a thorough process of modification, cross-cultural adaptation, and iterative desk review was conducted to generate the CELF-Q. Pre-test analysis was performed to assess face and content validity. To evaluate content validity, a panel of 15 experts was selected. Test-retest reliability was assessed in 60 participants, while internal consistency was evaluated by administering the questionnaire to 309 individuals with SARS-CoV-2 infection. Confirmatory factor analysis (CFA) was performed to assess the fit of the CELF-Q's factor structure. The content validity of the questionnaire was deemed "acceptable" across several dimensions, based on the results of the content validity ratio (CVR), content validity index (CVI), and Aiken's validity (V). Only a few items were identified for revision using these quantitative measures. Additionally, the experts supplemented the evaluations with qualitative comments for the modification of the questions. The test-retest reliability analysis showed an overall mean kappa coefficient of 0.89 [standard deviation (SD): 0.21] and a mean overall correlation coefficient of 0.99 (0.02), indicating high to almost perfect agreement. An internal consistency analysis showed that the majority of the dimensions had acceptable Cronbach's alpha values. In particular, the self-care behavior, socioeconomic restrictions, and diet and nutritional habits dimensions achieved respectable Cronbach's alpha values (between 0.7 and 0.8). The generated CELF-Q is a comprehensive, valid, and reliable tool for obtaining information on environmental/lifestyle factors associated with SARS-CoV-2 infection, as well as the development and severity of COVID-19, in the Cuban population.
Ophiopogon japonicus fibrous roots have long been deemed to have secondary medicinal and economic value, remaining substantially underutilized in clinical and industrial settings, with a critical lack of comprehensive chemical characterization despite their official approval as a novel food material in China. This study systematically compared the chemical profiles between the medicinal tuberous roots and fibrous roots of Chuan Maidong, the geo-authentic medicinal material of Ophiopogon japonicus (L. f.) Ker-Gawl. exclusively produced in Santai County, Mianyang, Sichuan Province, China (hereafter uniformly abbreviated as CMD). We aimed to identify characteristic markers for reliable differentiation between the two root parts, and evaluate the underexplored compositional profile and application potential of CMD fibrous roots. We performed chemical profiling of 28 batch-paired CMD samples via ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), screened differential markers using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), then conducted targeted quantification of these markers and core bioactive constituents via UHPLC triple quadrupole mass spectrometry (UHPLC-QQQ-MS). A total of 66 compounds were annotated, revealing distinct chemical profiles between the two root parts, with 6 flavonoid-predominant differential markers identified. Targeted quantification of 8 representative compounds confirmed that key bioactive components including ophiopogonin D and methylophiopogonanone A were significantly enriched in CMD fibrous roots. These findings clarify the chemical basis of compositional divergence between CMD root parts, validate the high medicinal and edible value of CMD fibrous roots, and provide robust scientific support for sustainable full-plant utilization of CMD.
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 (Eab*) 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.
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.
The 2019 medication regimen complexity-intensive care unit (MRC-ICU) score is associated with patient outcomes, ICU complications, and critical care pharmacist workload. This score was developed using heuristic component selection and validated in a single-center cohort of 130 ICU patients. We sought to apply data-driven reweighting methodology in a large, multicenter cohort of ICU adults to improve the predictive capabilities of MRC-ICU. This was a retrospective, observational cohort study of adults admitted to an ICU between 2015 and 2023 at two academic health systems. Machine learning-based methods, including Principal Component Analysis and Random Forest, were used to create an updated MRC-ICU score optimized to predict three outcomes: hospital mortality, ICU fluid overload (FO) occurrence, and invasive mechanical ventilation (IMV) use. MRC-ICU 2.1 used average mortality, FO, and IMV use; MRC-ICU 2.2 used average mortality and FO and adjusted for prolonged IMV use. Data from one center were used for training and testing, and data from the other for validation. The predictive abilities of MRC-ICU 2.1 and 2.2 for each outcome were compared to MRC-ICU 1.0 and to severity of illness scores (i.e., Acute Physiology and Chronic Health Evaluation [APACHE] II and Sequential Organ Failure Assessment [SOFA]). A total of 19,117 patients across training, testing, and validation datasets were included. MRC-ICU 2.0 scores outperformed MRC-ICU 1.0 for predicting most outcomes, with improvements in Area Under the Receiver Operating Characteristic (AUROC) ranging from +0.03 to +0.08 across datasets. MRC-ICU 2.1 and 2.2 did not consistently outperform APACHE II and SOFA in predicting mortality. The addition of MRC-ICU 2.0 scores to models including APACHE II or SOFA resulted in statistically significant improvements in discrimination in several settings (DeLong p < 0.05), with AUROC increases generally ranging from approximately +0.01 to +0.13 depending on outcome and dataset. The updated MRC-ICU 2.0 score (MRC-ICU 2.1 and 2.2) demonstrated consistently improved discrimination compared with the original MRC-ICU 1.0 across outcomes and datasets. The performance of MRC-ICU 2.0 (MRC-ICU 2.1 and 2.2) was generally comparable to established severity-of-illness scores (SOFA and APACHE II), although it did not consistently outperform these measures. When incorporated into combined models, MRC-ICU 2.0 provided additional predictive value, indicating that it captures information complementary to traditional severity-of-illness scores. Overall, these findings suggest that MRC-ICU 2.0 represents an improved and clinically interpretable measure of medication regimen complexity that is useful as a complementary predictor.
A new and precise single-isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous analysis of Temozolomide (TMZ) and Alpha-lipoic acid (ALA) in bulk and lipidic nanovesicles. The chromatographic separation of both drugs was carried out on an Eclipse Plus (C18) column (250 ×4.6 mm, 5 μm). An acetonitrile: acetate buffer (pH 4.5) mixture (60:40% v/v) was used as the mobile phase at a flow rate of 0.7 mL/min in isocratic mode. The eluted compounds were detected with a diode array detector at 328 nm (TMZ) and 332 nm (ALA). The retention times for TMZ and ALA were recorded as 3.63 min and 5.59 min, respectively. The developed method was subsequently validated for various parameters in accordance with ICH Q2(R1). The method showed linearity within the concentration range of 2-60 μg/mL for TMZ (r2 = 0.9998) and 15-750 μg/mL for ALA (r2 = 0.9999). The % recovery for both drugs was within acceptable limits with minimal variability. Moreover, the limit of detection and limit of quantitation were 1.0 µg/mL and 2.9 µg/mL for TMZ, and 5.0 µg/mL and 15.1 µg/mL for ALA, respectively. The validated method was found to be adequately sensitive and specific in simultaneously quantifying TMZ and ALA entrapped in liposomes [73.01 ± 3.96% entrapment efficiency for TMZ and 79.63 ± 0.24% for ALA (average ± SD, n = 3)]. Therefore, the proposed RP-HPLC method was found to be quick, precise, and specific for the simultaneous analysis of TMZ and ALA in bulk and in lipidic nanovesicles.
The on-column hydrolytic degradation of metamizole to impurity C in aqueous mobile phases represents a major source of analytical errors and false-positive results. Accurate and precise determination of impurity C is critical because this analyte is a direct precursor to a monitored genotoxic nitrosamine impurity. To resolve this issue, a novel isocratic non-aqueous hydrophilic interaction liquid chromatography method was developed on a triazole-bonded stationary phase. The separation was performed on Cosmosil HILIC column column (150 × 3.0 mm, 2.5 µm). The mobile phase was composed of 10 mM ammonium formate and 5 mM formic acid in a mixture of methanol and acetonitrile (3:97, v/v). An isocratic flow rate of 0.8 mL/min was utilized at a column temperature of 10 °C. A short analysis time of 3 min was achieved under optimized conditions. Impurity C exhibited minimal retention (k = 0.4), whereas metamizole was strongly retained on the column (k > 100). Due to this distinct selectivity, on-column artifact formation was completely prevented throughout the chromatographic run. To manage metamizole accumulation on the stationary phase, a periodic column regeneration step was introduced after every 30 sample injections. The method was coupled with mass spectrometric detection in positive electrospray ionization mode. Quadrupole mass analyser was employed in selected ion monitoring mode using a target ion at m/z = 218 as [M+H]+. A low limit of quantification of 2 ng/mL (2 ppm) and excellent linearity (r = 0.9991) were obtained. The calculated back-fit bias remained between -2.6% and 2.3% across the entire calibrated range. Critical verification parameters demonstrated full compliance with International Council for Harmonisation guidelines. The practical utility of the procedure was confirmed through the analysis of two production batches of metamizole.
L-ornithine-L-aspartate (LOLA) is a commonly used drug for the treatment of hepatic encephalopathy. Its commercially available preparations mainly include injections, powders for injection, and granules. Due to the amphoteric properties of amino acids, matrix differences caused by different dosage forms can lead to changes in their ionization forms, thus posing challenges to the development of a universal content method for the three dosage forms of this drug. This paper established a universal quantitative nuclear magnetic resonance spectroscopy (qNMR) technique by optimizing parameters such as buffer pH and ionic strength, and collecting one-dimensional hydrogen spectra using the zgpr pulse sequence. Using 0.2 mol/L NaH2PO4 (pH 4.0) as the buffer solution for the test solution overcame matrix interference among the different dosage forms. The validation results showed that the RSDs of precision, intermediate precision, repeatability, and robustness were all less than 1.0%. The average recoveries of preparations were 99.50-99.90%. Compared with conventional HPLC, the proposed qNMR method demonstrates superior universality and robustness, together with distinct merits in sustainability and minimal solvent consumption. Significantly, the method achieved a high AGREEprep greenness score of 0.72, which is considerably better than the HPLC score of 0.38, highlighting its outstanding environmental performance. This qNMR approach enables simultaneous determination of active ingredient content and amino acid ratio across three different LOLA dosage forms with excellent compatibility, speed, and accuracy. By offering a unified, green, and versatile analytical solution, this work could further extend the application of qNMR to the multicomponent quality assessment of multi‑dosage‑form pharmaceutical preparations.
A unique combination of properties, such as heat resistance, transparency, and high deformability, has led to wide adoption of silicone rubber as a material of construction in medical devices. Potential toxicity of medical devices and their components are assessed by exhaustively extracting, identifying, and semi-quantifying extractable compounds in accordance with ISO 10993-18:2020. However, solvent compatibility with silicones can present analytical challenges, as non-polar solvents may cause swelling in excess of 200% of the original volume and mass and elicit degradation. This investigation therefore explores methods to assess degradation relevant to chemical characterization, quantifies the extraction efficiency of semi-polar solvents relative to non-polar solvents, and elucidates the extraction kinetics of common extractable species. Degradation of silicone rubber was evaluated by measuring the swelling ratio, and vibrational spectroscopy was used to monitor polymer network integrity. However, the Hansen solubility parameter and dielectric constant were shown to be a more effective indicator of polymer network deformation than the Snyder polarity index when samples were exposed to 20 solvents of varying polarities. A direct comparison of extractions of silicone tubing in ethanol and cyclohexane revealed the extraction efficiency of ethanol was greater than 70% effective as compared to cyclohexane. The extraction kinetics of silicone samples suggests chemicals were almost exhaustively extracted in 10 h.
Traditional processing of Asini Corii Colla (Ejiao) involves stir-frying with clam powder, during which the duration of heating plays a critical role in determining product quality. This study systematically investigated the dynamic changes in characteristic polypeptides during the processing of Ejiao beads and proposed an integrated quality evaluation strategy combining machine vision with Fourier-transform near-infrared (FT-NIR) spectroscopy. The results demonstrated that appropriate spectral preprocessing was critical for classification performance, with BPNN achieving the most accurate and stable discrimination of processing degrees when combined with MSC or SNV + 1D, outperforming SVM and RF models. For quantitative analysis, the PLSR model for predicting the total content of donkey-derived polypeptides exhibited excellent performance (R²c = 0.9942, R²p = 0.9624), indicating strong linearity and high predictive accuracy. Overall, FT-NIR combined with chemometric modeling allowed precise identification of processing stages and accurate quantification of key polypeptide, providing an efficient and non-destructive method for quality control and standardization of animal-derived traditional medicines.
The increasing demand for Cannabis-derived medicinal products in Brazil, coupled with the implementation of RDC 1013/2026 (ANVISA), necessitates robust analytical frameworks for pharmacological and forensic purposes. This study is part of the "Paracelsus Project," a feasibility study evaluating the production of plant-based active pharmaceutical ingredients (API) from seized Cannabis sativa L. (marijuana), aiming to repurpose material that is currently incinerated. Preliminary data suggests this seized material contained a total of roughly 25 tons of CBD. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of twelve phytocannabinoids (CBD, D9-THC, D8-THC, CBC, CBL, CBN, CBG, THCV, CBDV, CBDVA, CBGA, and THCA). The methodology utilized a triple quadrupole (QQQ) analyzer in positive ionization mode, designed to overcome the challenges of complex plant matrices and chemical isomers. The method achieved successful baseline separation of critical pairs, such as CBD/CBG and D9-THC/D8-THC, in under eight minutes. This analytical workflow was successfully applied to monitor cannabinoid concentrations during the extraction and purification processes from seized marijuana by Brazilian Federal Police. The validated LC-MS/MS method provides the necessary sensitivity and specificity for both quality control in API production and forensic standardization. By establishing a technical foundation for characterizing medicinal extracts and ensuring compliance with the 0.3% THC statutory threshold, this research supports public health safety and the potential for a sustainable pharmaceutical supply chain from seized materials in Brazil.
Rupestonic acid, a bioactive sesquiterpenoid from Artemisia rupestris L., has demonstrated notable anti-inflammatory and anti-allergic effects. However, the metabolic fate of rupestonic acid following intranasal administration remains unknown. Therefore, the present study aimed to systematically characterize the in vitro and in vivo metabolism of rupestonic acid after intranasal administration and to evaluate its metabolic stability. An integrated analytical strategy was employed to systematically investigate the in vitro and in vivo metabolism of rupestonic acid. A UHPLC-Q/TOF-MS method was established to identify metabolites in rat liver microsomes, plasma, bile, urine, and feces, and their structures were tentatively characterized based on fragmentation patterns. The results revealed that rupestonic acid undergoes multiple biotransformation pathways, including hydroxylation, oxidation, hydrogenation, and glucuronidation, forming a relatively complex metabolic network involving both phase I and phase II reactions. In addition, a validated UHPLC-MS/MS method was developed for the quantitative determination of rupestonic acid in rat liver microsomal incubation samples, meeting the requirements of the U.S. Food and Drug Administration. Metabolic stability studies demonstrated that rupestonic acid exhibits a low metabolic rate in rat liver microsomes, with a relatively long half-life and low intrinsic clearance, indicating favorable metabolic stability. These findings provide new insights into the disposition characteristics of rupestonic acid after intranasal administration and offer a foundation for the future development and pharmacological evaluation of intranasal formulations.
Acute lung injury (ALI) is a life‑threatening respiratory disease characterized by excessive inflammation, oxidative stress, and autophagic dysregulation. Traditional Chinese medicine has emerged as a promising strategy for managing respiratory inflammatory conditions. Ficus tikoua Bur. (FT), a well‑known ethnomedicinal prescription used for pneumonia in Guizhou, China, warrants investigation into its effects and mechanisms in ALI. This study integrated UHPLC‑Q‑TOF‑MS/MS chemical profiling, network pharmacology, molecular docking, and lipopolysaccharide‑challenged mouse model to characterize FT's bioactive constituents and therapeutic mechanisms. FT extract (FTE) significantly attenuated lung injury, reduced inflammatory cytokines (TNF‑α and IL‑6), and decreased oxidative stress markers (MPO and MDA). Network pharmacology and molecular docking identified stable interactions between core FT compounds and five key targets (mTOR, AKT1, PIK3CA, PIK3CB, and PIK3CD). Experimental validation further revealed that FTE inhibited PI3K/AKT overactivation, as evidenced by reduced p‑PI3K and AKT expression, while concurrently restoring autophagic homeostasis through downregulation of Beclin‑1 and LC3‑II/I and upregulation of p62. These findings demonstrate that FTE alleviates ALI through coordinated regulation of the PI3K/AKT pathway and autophagic homeostasis. Collectively, this study provides a mechanistic foundation for FT as a multi‑component therapeutic candidate against ALI and offers new perspectives for natural product‑based drug discovery.