Isomeric and isobaric fentanyl analogs can share precursor- and product-ion signals, limiting candidate discrimination by mass spectrometry (MS) in non-targeted screening. Retention time (RT) provides orthogonal chromatographic information, but measured reference RTs are unavailable for many candidate structures. We therefore developed a structure-based RT predictor for fentanyl analogs analyzed by liquid chromatography–tandem mass spectrometry using serially coupled cyano and phenyl-hexyl columns (SCC-LC-MS/MS). Ninety-four curated structure–RT records were represented by 75 selected Mordred descriptors and paired with five regression algorithms. The resulting pipelines were evaluated using repeated nested cross-validation (CV), leave-one-out cross-validation (LOOCV), y-scrambling, and k-nearest-neighbor applicability domain (AD) analysis. Mean CV R2 values were closely clustered (0.887–0.906), with the Mordred × Gaussian process regression pipeline (radial basis function kernel) ranking highest. In LOOCV, the selected pipeline achieved a mean absolute error (MAE) of 1.15 min, and 85.1
Reliable quantification of finasteride (FNS) in small-volume plasma samples is essential for preclinical pharmacokinetic studies but requires analytical methods that combine adequate sensitivity with practical sample preparation. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method coupled with liquid–liquid extraction (LLE) was developed and validated for the determination of FNS in rat plasma. Sample preparation was systematically optimized by evaluating plasma volume and extraction solvent composition. The optimized procedure required only 50 μL of plasma and employed methyl tert-butyl ether/dichloromethane (70:30, v/v) as the extraction solvent with dutasteride as a structurally related surrogate internal standard. The method exhibited excellent linearity over the validated concentration range of 1–500 ng/mL (R2 = 0.99963 ± 0.00075), with acceptable accuracy and precision, consistent recovery, and limited matrix effects. The validated method was successfully applied to the determination of FNS in rat plasma samples collected during a pharmacokinetic study, demonstrating its suitability for routine bioanalysis. Compared with a previously reported LC–MS/MS method for FNS in rat plasma, the proposed assay requires substantially less plasma while providing a lower limit of quantification, a broader validated working range, and a simple LLE procedure. Overall, the proposed method provides a practical balance between analytical performance, minimal sample consumption, and operational simplicity, making it a useful analytical tool for routine determination of FNS in rat plasma and future preclinical pharmacokinetic studies.
High-flux electron counting with event-driven detectors is fundamentally limited by pixel-level deadtime, resulting in nonlinear count loss, variance suppression, and distortion of spatial intensity profiles. We introduce a monitor-free, self-calibrated deadtime correction method based on the invariance of the long-time Poisson tail of inter-arrival time distributions. Under stationary Poisson illumination, the long-time inter-arrival tail provides an internal statistical estimate of the incident arrival rate, independent of external flux measurements. The approach is demonstrated using high-flux transmission electron microscopy beam-profile measurements, where spatial Fano-factor analysis confirms operation in the deadtime-limited regime. By calibrating an effective extended-deadtime model with the tail-derived rate, the corrected peak count rate increases from approximately 78 kHz to 131 kHz, corresponding to a 67
Abstract Cerebral infarction remains a leading cause of long-term disability and mortality, necessitating sensitive and reliable methods for early diagnosis. Herein, we develop a novel biosensing platform based on a primer exchange reaction (PER)-transcribed DNAzyme-catalyzed DNA walker coupled with inductively coupled plasma mass spectrometry (ICP-MS) for the detection of cerebral infarction-related microRNA-124. The target miRNA triggers split DNAzyme assembly, which cleaves a dumbbell-structured tracker to release a PER template. The PER process generates numerous DNAzyme strands that cyclically cleave Pt-labeled substrates, releasing platinum for ICP-MS quantification. This cascade amplification strategy achieves a detection limit as low as 0.11 fM with a linear range from 1 fM to 100 pM. The method exhibits excellent single-nucleotide discrimination capability, satisfactory recovery (94.1%–102.4%) in spiked serum samples, and high consistency with qPCR (correlation coefficient 0.9943). When applied to clinical serum samples, the platform successfully distinguishes cerebral infarction patients from healthy controls by detecting significantly elevated miRNA-124 levels. This PER-transcribed DNAzyme walker platform offers a highly sensitive, specific, and robust tool for early diagnosis and prognostic monitoring of cerebral infarction, and can be readily adapted for other low-abundance disease biomarkers.
Abstract Traditional immunoassays based on mouse or rabbit immunoglobulin G (IgG) often suffer from poor specificity in Staphylococcus aureus ( S. aureus ) detection, which is primarily due to the interference from protein G-producing Streptococcus species. This study aimed to propose a sensing platform for detecting S. aureus using feline IgG immobilized on magnetic beads (MBs) as capture agents, given its high affinity for staphylococcal protein A (SPA) via its Fc region and negligible affinity for protein G to achieve specific capture of S. aureus. Horseradish peroxidase (HRP)-conjugated chicken anti-protein A immunoglobulin Y (HRP-IgY) was used as the detection antibody, which formed a dual-recognition sandwich structure (MBs-feline IgG/ S. aureus /HRP-IgY). A colorimetric signal was generated through the HRP-catalyzed oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), enabling the quantitative detection of S. aureus . The proposed method displayed favorable linearity in the range of 6.3 × 10 2 to 8.0 × 10 4 colony-forming unit (CFU)/mL under optimized conditions, with a low detection limit of 5.3 × 10 2 CFU/mL. The entire detection procedure could be completed within 90 min. Specificity tests confirmed negative responses for common non-target bacteria. Furthermore, the method was successfully applied for S. aureus detection in milk, tap water, and apple juice with satisfactory detection limit. This platform exhibited excellent expansibility and can be further integrated with microfluidic devices, holding immense potential for point-of-care testing applications.
