Polyendocrine metabolic ovarian syndrome (PMOS), formerly known as polycystic ovary syndrome (PCOS), is characterized by dysregulation of multiple steroid hormones. Accurate quantification of these analytes is essential for laboratory evaluation; however, currently may involve complex sample-preparation workflows or provide insufficient analytical coverage, particularly for dehydroepiandrosterone sulfate (DHEAS). A liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was established for the simultaneous measurement of six steroid hormones associated with PMOS. Sample preparation combined liquid-liquid extraction with protein precipitation. Chromatographic separation was completed within 6 min. Validation followed international recommendations and included linearity, sensitivity, precision, accuracy, matrix effects, selectivity, and specificity. All analytes showed excellent linearity (R = 0.9959-0.9995), with intra- and inter-day CVs ≤ 5.6%. Spike recovery ranged from 85.2% to 114.9%. Accuracy was further supported by third-party QC materials and successful participation in an external quality assessment program. The assay provided an expanded linear range for DHEAS (20-20,000 ng/mL). This validated LC-MS/MS assay provides rapid, sensitive, and reproducible quantification of six PCOS-related steroid hormones and is suitable for routine laboratory implementation.
Daphnoretin (DAP) has various pharmacological activities, but its in vivo disposition after nanomicellar formulation remains unclear. This study compared the pharmacokinetics and tissue distribution of free DAP and two polymeric nanomicellar formulations, PP-DAP and GA-DAP, following intravenous administration. Plasma DAP concentrations in rats were determined by UPLC-MS/MS, and DAP concentrations in mouse tissues were determined by LC-MS/MS. Compared with free DAP, PP-DAP and GA-DAP showed higher systemic exposure, longer apparent elimination half-lives, and lower apparent clearance. The AUC0-∞ values of DAP, PP-DAP, and GA-DAP were 5474.14, 11,211.04, and 15,019.86 h · ng/mL, respectively, and the corresponding T1/2 values were 6.84, 9.24, and 9.90 h. DAP was detected in the heart, liver, spleen, lung, and kidney, with the nanomicellar formulations showing altered tissue distribution and GA-DAP exhibiting relatively sustained hepatic distribution. These findings suggest that nanomicellar formulation alters the in vivo disposition of DAP. However, free and micelle-associated DAP were not separately quantified, precluding direct characterization of in vivo drug release, and the validation range of the tissue quantification method was limited. Further studies are warranted to evaluate the in vivo behavior and liver-directed delivery potential of GA-DAP.
Breviscapine injection (BI), a standardized botanical drug for cardiovascular and cerebrovascular diseases, lacks adequate characterization of its pharmacokinetics and tissue distribution. A UPLC-MS/MS method was developed and validated for simultaneous quantification of scutellarin and its metabolite iso-scutellarin in rat plasma and tissues after intravenous BI administration. Sample preparation used methanol protein precipitation, with detection via positive electrospray ionization MRM. Validation followed the Chinese Pharmacopoeia and bioanalytical PK guidance. The assay showed LLQs of 10.0 ng/mL (scutellarin) and 5.0 ng/mL (iso-scutellarin), good linearity (r2 ≥ 0.990), precision (RSD ≤ 11.1%), accuracy (91.7%-106.8%), and acceptable recovery and matrix effects. PK analysis revealed moderate-to-rapid systemic elimination of scutellarin. Both analytes distributed to tissues within 0.25 h postdose, with exposure ranking: bladder > small intestine > kidney > small intestine > kidney > stomach > liver > plasma > heart > skeletal muscle > pancreas > skin > fat > lung > ovary > testis > adrenal > heart > skeletal muscle > pancreas > thymus > spleen > brain. The method is suitable for BI studies. The limited brain penetration suggests formulation optimization to prolong plasma exposure may be worth exploring, and the relatively high hepatic distribution points to a potential need for monitoring liver and kidney function.
This study aims to elucidate the chemical composition of the dichloromethane extract from the aerial parts of Ferula ferulaeoides (DEAFF) and to explore its biological activities. The composition of DEAFF was analyzed by UHPLC-Q-Orbitrap-MS/MS. Then, the carrageenan-induced tail thrombosis model, xylene-induced ear swelling model, and acetic acid-induced writhing model were established to evaluate the relevant activities of DEAFF. Network pharmacology research and molecular docking were conducted to elucidate the pathways and key targets underlying the multiple activities of DEAFF. A total of 30 chemical constituents were tentatively identified in DEAFF based on UHPLC-Q-Orbitrap-MS/MS analysis. Pharmacodynamic results showed that DEAFF significantly reduced the proportion of thrombus in the tail of mice, effectively alleviated ear swelling, improved the degree of ear tissue lesions, and significantly reduced the number of writhing movements in mice, indicating that it has multiple pharmacological effects such as antithrombosis, anti-inflammation, and analgesia. Network pharmacology studies have shown that multiple pathways such as the PI3K-Akt signaling pathway and the MAPK signaling pathway play a crucial role in the pharmacological effects of DEAFF. In this study, the chemical components of DEAFF were preliminarily identified, and its effects on thrombosis prevention, anti-inflammation, and analgesia were systematically investigated.
Sucralose, an artificial sweetener widely used in sugar-free foods and beverages, raises human exposure and potential accumulation in tissues and organs. Given the profound long-term impact of maternal diet on offspring, its safety during pregnancy is of particular concern. Existing detection methods primarily target food matrices and lack reliable, efficient approaches for biological sample analysis. To address this gap, we developed an ultrahigh-performance liquid chromatography-multiple reaction monitoring tandem mass spectrometry (UHPLC-MS/MS) method based on existing food detection methods to quantify sucralose in mouse serum and placenta. Method validation showed excellent linearity in the range of 20-1250 nmol/L, with limit of detection (LOD) of 4.88 nmol/L and limit of quantification (LOQ) of 9.77 nmol/L. The precision, accuracy, matrix effects, and freeze-thaw stability were all within acceptable limits. Using the method described above, we detected sucralose in both serum and placenta of mice following subchronic exposure, confirming its bioaccumulation, systemic absorption, and transplacental transfer, suggesting the need for further reproductive toxicity studies. In summary, this study developed and preliminarily applied a method for determining sucralose in mouse serum and placenta, providing a methodological reference for the toxicological evaluation and safety assessment of sucralose, and aids in improving food safety risk systems and the formulation of evidence-based public health policies.
Morindae Officinalis Radix (MOR), the dried root of Morinda officinalis How, contains abundant saccharides and is used clinically after different processing procedures. However, the anti-inflammatory material basis of MOR and the chemical consequences of Lycium barbarum processing remain insufficiently defined. In this study, high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) was developed for fingerprint analysis of 15 batches of raw MOR and the corresponding L. barbarum-processed MOR (LB-MOR). Seventeen common peaks were resolved, and 11 saccharides were assigned by comparison with reference standards. Anti-inflammatory activity was evaluated in LPS-stimulated RAW 264.7 macrophages by quantifying nitric oxide (NO), TNF-α, IL-1β, and IL-6. Grey relational analysis and partial least squares regression linked the HPLC fingerprints with bioactivity and identified glucose, sucrose, kestose, and nystose as the principal activity-associated saccharides. Subsequent validation showed that these saccharides inhibited inflammatory mediator release, with kestose and nystose showing the strongest effects. Western blotting further indicated that kestose and nystose restored IκBα expression and decreased COX-2 expression and the p-p65/p65 ratio, consistent with suppression of sustained NF-κB signaling. This separation-based fingerprint and spectrum-effect workflow identify anti-inflammatory oligosaccharides in MOR and provides candidate quality markers for raw and processed MOR products.
RU 58841 is a nonsteroidal antiandrogen, developed for the treatment of androgen-dependent conditions. This study aimed to characterize the human metabolism of RU 58841 for the first time and to assess its systemic exposure following topical administration. In an initial in vitro metabolism study, six potential metabolites were identified using liquid chromatography-high resolution (tandem) mass spectrometry (LC-HRMS/MS). Subsequently, an in vivo administration study was conducted with a RU 58841-containing hair treatment product, applied once to the scalp of six healthy male volunteers. Dried blood spots (DBS) and urine samples were collected and analyzed by LC-low resolution MS/MS. Five in vivo-derived metabolites of RU 58841 were detected in human urine, including products formed via oxidation, N-dealkylation, and glucuronidation of the parent compound and its phase I metabolites. In DBS, the parent compound and the oxidized metabolite were detected, indicating considerable systemic exposure to topically applied RU 58841. The steroid profile (SP) as part of the Athlete Biological Passport (ABP) was not affected by the administration. These findings provide information about the metabolism and the systemic exposure of RU 58841, and further indicate that a single topical dose of 30 mg does not interfere with SP parameter determinations in routine doping controls.
The rise of triple-negative breast cancer (TNBC) warrants the urgent need to identify novel therapies. Herein, untargeted metabolomics-guided isolation revealed the metabolome fingerprints of Polygonum bistorta (PB) and Polygonum viviparum (PV) in correlation with their TNBC inhibitory activity. Phytochemical analysis annotated 209 metabolites, with flavonoids (45 compounds) as the major class, followed by phenolic acids (25), tannins (24), and hydroxycinnamic acids (18). Multivariate data analysis identified chlorogenic acid, 6-O-galloylarbutin, and procyanidin B1 as key markers of PV, whereas naringenin-O-glucoside, catechin, and crataegunin D characterized PB. Among different polar extracts, ethyl acetate extract of PB (PB-EA) and the n-butanol extract of PV (PV-NB) exhibited the strongest cytotoxicity against 4T1 cells, with IC50 values of 23.28 ± 0.90 and 39.11 ± 2.36 μg/mL, respectively. Subsequently, the same extracts significantly suppressed tumor growth in the 4T1-Luc orthotopic xenograft mouse model compared with their respective model groups (p < 0.001, p < 0.01). Bioassay-guided isolation yielded 8 compounds, including crataegunin D, which isolated from Polygonaceae plants for the first time, displaying IC50 values of 24.91 ± 2.43 and 20.65 ± 1.77 μM against 4T1 and MDA-MB-231 cells, respectively. Molecular docking revealed binding free energies (ΔG) ranging from -52 to -48 kcal/mol targeting EGFR and CDK receptors.
Coccidiosis is a protozoan parasitic disease that necessitates sustainable therapeutic alternatives due to increasing resistance to conventional coccidiostats. This study evaluated the anticoccidial efficacy of Coriandrum sativum methanolic extract (CME) against Eimeria papillata infection in mice. Phytochemical profiling of CME was conducted using liquid chromatography-mass spectrometry (LC-MS), and identified compounds were subjected to in silico molecular docking. Twenty-five male Swiss albino mice were divided into the following five groups as follows: Group 1: noninfected, nontreated (control), Group 2: CME administered control, Group 3: infected with E. papillata, Group 4: infected+CME treated group (300 mg/kg), and Group 5: infected+amprolium treated group (120 mg/kg). Mice were orally infected with 1 × 103 sporulated oocysts and treated for 5 days. Docking analysis revealed strong binding affinities of several chromatographic CME constituents toward elongation factor Tu (tufA), with docking scores exceeded amprolium's binding affinity. In vivo, CME significantly restored goblet cell counts and upregulated MUC-2 expression. It reduced pathogenic Salmonella typhi and Staphylococcus aureus colonization, downregulated inducible nitric oxide synthase and CD4 expression, enhanced catalase activity, as well as suppressed pro-inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). These findings demonstrate that CME exerts anticoccidial, antioxidant, anti-inflammatory, and culture-based bacterial effects, highlighting its potential as a natural therapeutic agent against murine coccidiosis.
Deoxyuridine (dUrd) is often misincorporated into genomic DNA in cancer patients receiving antimetabolite chemotherapy. This activates base excision repair and recruits ataxia telangiectasia-mutated and Rad3-related (ATR), which coordinates DNA repair with nucleotide metabolism by facilitating ribonucleotide reductase (RNR) activity. Ceralasertib (AZD6738) is the most clinically advanced ATR inhibitor (ATRi) and may abrogate this regulatory pathway and diminish RNR-mediated deoxynucleotide triphosphate synthesis. To further understand ATRi-induced dUrd misincorporation, we developed a highly sensitive LC-MS/MS method to quantitate: deoxyuridine, uridine, guanosine, adenosine, cytidine, thymidine, deoxyguanosine, deoxyadenosine, and deoxycytidine from digested genomic DNA. Assay application was demonstrated with genomic DNA digested from multiple murine cell lines treated with ceralasertib. Chromatographic separation was achieved with an Inertsil ODS-3 (150 × 2.1 mm, 3 μm) column and a gradient elution program of 0.1% formic acid in water and 0.1% formic acid in methanol over an 18-min run time. Detection was performed on a SCIEX 6500+ tandem mass spectrometer. The method proved to be accurate (90.8%-114.2%) and precise (< 7.73% CV) across analytes. Freeze-thaw stability (106.4%-114.3%), stability for 12 months at -80°C (88.7%-113.7%), and stability for 4 h at room temperature (104.1%-114.0%) were acceptable across QCs.
Nitrosamine drug substance-related impurities (NDSRIs) pose a significant safety risk due to their mutagenic and carcinogenic potential, prompting strict regulatory oversight under ICH M7. The propensity for in situ formation of NDSRIs during analytical procedures, particularly in active pharmaceutical ingredients containing reactive amines, emphasizes the need for highly accurate and scrupulously validated methodologies. Melatonin, containing a vulnerable amine, was chosen as a model drug product. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantifying N-Nitroso melatonin. To prevent in situ formation, a scavenger was incorporated during sample preparation. Validation followed regulatory standards, assessing specificity, linearity, accuracy, precision, sensitivity, and robustness. The method showed excellent linearity (1.8-22.5 ng/mL, R2 = 0.9983), sensitivity with LOD of 0.9 ng/mL and LOQ of 1.8 ng/mL, and accuracy with recoveries between 85.3% and 95.9%. Precision was confirmed with %RSD below 2%, whereas robustness testing demonstrated consistent performance under varied conditions. Crucially, the scavenger eliminated false positives by suppressing in situ nitrosamine formation, ensuring data integrity. This validated approach strengthens analytical accuracy and provides a transferable framework for monitoring NDSRIs in other drug products containing reactive amines, supporting regulatory compliance and safeguarding patient health.
Epimedii Folium has been widely used to treat Alzheimer's disease (AD) in China. However, the potential active components of Epimedii Folium and its mechanism against AD are still not clear. In this study, an ultra-high performance liquid chromatography coupled to quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-OrbitrapHRMS) method was applied to screen the ingredients of Epimedii Folium. Network pharmacology was utilized to explore the potential active components and pharmacological mechanisms of Epimedii Folium against AD. The binding affinity and conformation of key active ingredients and core targets were performed by molecular docking. Consequently, 72 chemical constituents were identified in Epimedii Folium, including four phenolic acids, two quinones, three 9,10-dihydrophenanthrenes, and 63 flavonoids. Network pharmacology analysis revealed that quercetin, apigenin, kaempferol, and luteolin exhibit favorable pharmacological activities. Molecular docking demonstrated that apigenin-TNF, kaempferol-TNF, kaempferol-TP53, and quercetin-TP53 represent compound-target pairs with strong binding affinities. These findings help elucidate the material basis and underlying mechanisms of Epimedii Folium against AD and offer valuable evidence supporting the further development and clinical application of Epimedii Folium.
Fangji Dihuang Decoction (FJDH), a classic Chinese herbal formula, has historically been used to treat mental disorders. However, current research primarily focuses on clinical applications without analyzing the active components or elucidating modern pharmacological mechanisms for specific indications. This study aimed to discovery potential indications of FJDH and research on the components and mechanism of FJDH in treating ischemic stroke. Molecular network combined with LC-MS was used to annotate FJDH's component structures, while AI platforms enabled functional analysis of component target pathways for indication prediction. Additionally, network pharmacology revealed FJDH's therapeutic mechanisms and scientific implications in treating ischemic stroke. TargetNet-predicted disease directions aligned well with reported FJDH indications. A total of 116 structures were annotated, with main components including terpenoids, fatty acids, flavonoids, phosphocholines, and alkaloids. The extracts of Gancao and Guizhi rich in flavonoids have demonstrated neuroprotective effects, with EC50 values 18.68 ± 1.271 and 33.63 ± 1.527 μg/mL, respectively. Ninety-eight overlapping therapeutic targets were identified, such as key biomarkers AKT1, PTGS2, IL-1B, IL-6, and TNF. Via GO enrichment analysis and KEGG pathway mapping, the study clarified FJDH's therapeutic mechanisms, which is significant for explaining "homotherapy for heteropathy" of traditional Chinese medicine (TCM) and promoting secondary development of TCM.
Phytochemicals have become the central focus of scientific research in recent times due to their vast applicability and comparatively low side effects. Gallic acid (GA), quercetin (QUE), luteolin (LUT), and apigenin (API) are natural antioxidants that are ubiquitous and co-exist in numerous medicinal plants and have been reported to have therapeutic potential in various diseases. The present study aims to develop and validate a simple, robust analytical method for the simultaneous estimation of these phytoconstituents in various plant extracts and pharmaceutical formulations. The UV and RP-HPLC methods were developed and validated in accordance with ICH Q2 guidelines. The UV method demonstrated good linearity and was found to be robust for the analysis of individual drugs, whereas the gradient HPLC method was suitable for the simultaneous estimation of these drugs. All method validation parameters were within acceptable limits; however, the HPLC method showed slight sensitivity to changes in method parameters, such as flow rate and wavelength. The developed UV spectrophotometric and gradient RP-HPLC methods for the simultaneous estimation of GA, QUE, LUT, and API were validated in accordance with the ICH guidelines. These analytical methods can facilitate simultaneous identification and quantification of phytoconstituents in crude extracts, pharmaceutical formulations, and biological samples.
Polycystic ovary syndrome (PCOS) is a hormonal disorder marked by irregular menstrual cycles, elevated androgen levels, ovarian cysts, hirsutism, acne and other symptoms. While conventional medications such as Metformin and Spironolactone are commonly prescribed, they are often associated with undesirable side effects. As a result, there is growing interest in alternative treatments, particularly those involving medicinal plants. Vitex negundo L., a member of the Lamiaceae family, has demonstrated promising therapeutic effects against PCOS. However, its precise molecular mechanism of action remains unclear. To explore this, we conducted an untargeted metabolomics analysis using UPLC-MS/MS to identify bioactive compounds, followed by network pharmacology to elucidate potential mechanisms. Metabolite fingerprinting revealed 186 metabolites, among which 122 were identified as secondary metabolites. Network pharmacology analysis uncovered 910 potential targets associated with the identified compounds and 297 known PCOS-related disease targets, with 50 overlapping targets between the two datasets. Key hub targets identified included P53, ESR1, AKT1, STAT3, CTNNB1, ERBB2, BCL2, EGFR, MTOR and IL6. Furthermore, molecular docking highlighted several bioactive constituents-syringin, 4-(3,4-dihydroxyphenyl)-6,7-dihydroxynaphthalene-2-carboxylic acid, and Isovitexin-as potential lead compounds for PCOS treatment. Further evaluation of lead compounds can be conducted through in vitro and in vivo studies.
Liquid chromatography (LC) has become indispensable for characterizing nasal and inhalation drug products, yet a critical gap persists in integrating chromatographic data with particle size metrics and in vitro performance outcomes to predict in vivo behavior. This review highlights recent advances in LC strategies from reversed-phase high-performance liquid chromatography to ultrahigh-performance liquid chromatography-tandem mass spectrometry; across three interconnected domains: formulation physicochemical characterization, aerodynamic particle size distribution determination, and comprehensive in vitro performance testing. We critically evaluated how LC-based quantification enables simultaneous active pharmaceutical ingredient and impurity profiling, dissolution kinetics assessment, and regional deposition mapping, with method validation data demonstrating linearity spanning three to four orders of magnitude and limits of detection as low as 5 pg/mL. Notably, we highlighted the mechanistic discrimination of erosion-vs. diffusion-controlled release enabled by simultaneous active pharmaceutical ingredient-polymer dissolution profiling and established quantitative correlations between particle size and systemic bioavailability. The review identified persistent challenges in biorelevant dissolution standardization and device-formulation interaction modeling while proposing integrated frameworks for bioequivalence prediction. This comprehensive perspective positions LC not merely as an analytical tool but as the central bridge connecting formulation properties, aerosol performance, and therapeutic outcomes, offering actionable insights for regulatory evaluation in respiratory drug delivery.
Cystic fibrosis, a genetic disorder caused by mutations in the CFTR gene, is commonly treated using combination therapy with IVA and LUMA. Accurate quantification of these drugs in human plasma is essential for therapeutic monitoring; however, conventional analytical methods often lack sufficient sensitivity and robustness. In this study, a QbD-based RP-UPLC method was developed and validated for the simultaneous estimation of IVA and LUMA in human plasma. The primary objective was to achieve efficient separation of analytes from plasma interferences with reduced run time and consistent analytical performance. Chromatographic separation was carried out using a Waters Acquity UPLC system equipped with a CSH C18 column (100 mm × 2.1 mm, 1.8 μm). Method optimization was performed using a Central Composite Design model. The mobile phase is 0.01 N ammonium acetate and acetonitrile (60:40, v/v) at a flow rate of 0.3 mL/min, with the column maintained at 30°C. EMT was used as the internal standard, and detection was conducted at 260 nm. The retention times were 1.45, 1.77, and 1.99 min for EMT, IVA, and LUMA, respectively. The method is reliable, rapid and selective, exhibiting excellent linearity, precision, and accuracy, complying with USFDA guidelines.
Cyclophosphamide (CP) is a common alkylating anticancer drug. Artemisinin (ART), an antimalarial agent metabolized by hepatic CYP450s, exhibits promising antitumor activity. This study established a rapid and accurate LC-MS/MS method for the quantitative analysis of CP and its metabolite in mouse plasma, as well as to apply this method in assessing how ART influences the pharmacokinetics of CP. Given the instability of 4-OH-CP, 4-keto-CP was selected as a surrogate metabolite for detection. The effect of ART on the mRNA expression and activity of CYP450 enzymes were assessed by RT-qPCR and substrate cocktail assay. The results indicated that CP and 4-keto-CP were linear in the range of 1-2000 and 2-2000 ng/mL, respectively. The accuracy, precision, extraction recovery, stability, and matrix effect of CP and 4-keto-CP are complied with the Bioanalytical Method Validation Guidelines. ART significantly reduced the AUC0-t of CP by 26.6%, while increasing CL/F by 1.35-fold. Concurrently, the AUC0-t and Cmax of 4-keto-CP increased by 1.42- and 1.45-fold, respectively. Furthermore, ART can markedly upregulate the mRNA expression and activity of CAR/CYP2B6. The results showed that pretreatment with ART could induce CP metabolism to its active metabolite. This study provides implications for the rational use of the ART-CP combination in clinical practice.
Colorectal cancer (CRC) arises via the stepwise adenoma-carcinoma sequence (ACS). Gut microbial dysbiosis and host metabolic reprogramming jointly correlate with CRC onset and advancement, yet their stage-specific crosstalk across ACS remains largely unclear. Limited multi-omics research on microbial-metabolic interactions throughout ACS hinders the development of early diagnostic biomarkers and preventive strategies. Here, we combined untargeted mucosal metabolomics and fecal shotgun metagenomic sequencing in 36 participants, covering healthy controls, ACS, and CRC patients. We systematically analyzed microbial composition, functions, differential metabolites, and enriched pathways and integrated multi-omics data to screen stage-specific signatures. Distinct gut microbial profiles and progressive functional shifts toward pathogenicity and abnormal carbohydrate metabolism were observed along ACS. Mucosal metabolism was continuously disrupted, with prominent alterations in taurine-hypotaurine, sphingolipid, and bile acid pathways. Core differential metabolites showed excellent diagnostic performance. Microbe-metabolite interactions were progressively enhanced to form a concerted pro-tumor axis. This study characterizes unique ACS-stage microbial-metabolic features. Dysregulated metabolic pathways and key microbe-metabolite crosstalk are closely associated with CRC progression, offering novel non-invasive biomarkers and premalignant intervention targets.
A stability-indicating reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for quantitative estimation of satranidazole in bulk drug and tablet dosage form, followed by LC-MS characterization of stress-induced degradation products. Chromatographic separation was achieved on a HiQ Sil C18 column (250 × 4.6 mm, 5 μm) using methanol:0.1% formic acid in water (60:40, v/v) as mobile phase at a flow rate of 1.0 mL/min with UV detection at 317 nm. The method exhibited satisfactory linearity over 5-30 μg/mL with a correlation coefficient of 0.999. Stress-induced degradation studies performed under acidic, alkaline, oxidative, thermal, and photolytic conditions demonstrated significant degradation under alkaline and photolytic stress. The developed method successfully resolved satranidazole from its degradation products. LC-MS analysis enabled tentative characterization of major degradants and facilitated the proposal of probable degradation pathways. The developed method is suitable for routine stability assessment and pharmaceutical quality control of satranidazole formulations.
