Current Issue : January-March Volume : 2025 Issue Number : 1 Articles : 5 Articles
Background/Objectives: This study aimed to develop a fully validated HPLC-MS/MS method for quantifying total and unbound lenalidomide concentrations in human plasma. Methods: Unbound concentrations were measured using plasma ultrafiltrate prepared with Amicon® Centrifugal Filters. Lenalidomide and lenalidomide-d5 (internal standard) were extracted from 50 μL of human plasma using liquid–liquid extraction. Chromatography was conducted with a Halo® C18 column using 0.1% formic acid and methanol (20:80, v/v) as the mobile phase. The mass spectrometer was operated in a positive ion mode with an electrospray ionization interface and multiple reaction monitoring modes. Results: Calibration curves were linear over the range of 5 to 1000 ng/mL (r2 > 0.996) for both the total and unbound lenalidomide. For total lenalidomide concentrations, between-run precision (coefficients of variation) and accuracy were 1.70–7.65% and 94.45–101.10%, respectively. For unbound concentrations, inter-day precision and accuracy were 1.98–10.55% and 93.95–98.48%, respectively. Conclusions: We developed a highly reproducible, sensitive, and efficient bioanalytical method using a smaller volume of plasma sample (50 μL) with a relatively short run time (2.5 min). The proposed analytical method was successfully applied to measure total and unbound lenalidomide concentrations at various time points in multiple myeloma patients with renal impairment....
A highly accurate, precise, and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for ketotifen (KTF) estimation from Beagle dog plasma was developed and validated, with ketotifen-d3 (KTF-d3) as the internal standard (IS). KTF and IS were detected on an API 4000 mass spectrometer in multiple reaction monitoring (MRM) mode in electrospray ionization (ESI) positive ionization mode. The transitions were monitored at m/z 310.2 → 96.0 for KTF and m/z 313.2 → 99.1 for IS. KTF and IS were extracted from plasma using liquid-liquid extraction with methyl tertiary-butyl ether and then analyzed for 3 min with extracted samples (7 μL) into the LC– MS/MS system. Analytes were separated on a Luna® Hilic column (50 × 2.0 mm i.d., 3 μm) using the Nexera X2 HPLC. The mobile phase A consisted of 10 mmol/L ammonium formate (pH 3.0), while mobile phase B consisted of 0.05% formic acid in acetonitrile. The ratio of mobile phase was 5:95 (v/v) at a flow rate of 0.2 mL/min. The method has been thoroughly validated in accordance with the bioanalytical method validation guidelines established by the Ministry of Food and Drug Safety in Korea and the U.S. Food and Drug Administration, addressing selectivity, lower limit of quantification, linearity, carryover, precision, accuracy, recovery, matrix effect, and stability. The developed LC–MS/MS method was effectively utilized for the bioequivalence assessment of ketotifen in Beagle dog plasma following the oral administration of ketotifen syrup....
Nitrite/nitrate poisoning is an emerging problem, with an ongoing escalation of reported self-administration with suicidal intent in several countries. Nitrites toxicity mainly consists of their interaction with hemoglobin (Hb), causing its oxidization to methemoglobin (MetHb). In order to give support to the correct procedures for the analysis of these cases, this study aims to evaluate spontaneous sample degradation and consequent MetHb formation in the typical storage conditions of a forensic toxicology laboratory. Two different types of samples have been used in this study: the first stage of our study consisted of a retrospective analysis of blood samples obtained by judicial autopsies already stored in the toxicology laboratory, collected over four years (2018–2021), while the samples used for the second stage were appositely collected during judicial autopsies. The data obtained by the application of a derivative spectrophotometry method on these samples suggest that there seems not to be a maximum threshold for MetHb formation within which it is possible to state with a sufficient grade of certainty that the concentration of MetHb found is consistent with an ante-mortem formation and is not the result of an artifact due to sample degradation and storage conditions. On the other hand, the results suggest that MetHb formation depends on the time passed between sample collection and analysis, so that a tempestive sample processing, performed as soon as the samples are received in the laboratory, is crucial to obtain the maximum reliability and diagnostic values from the data when MetHb quantitation is necessary....
Immunodetection of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) in blood samples is widely used for the diagnosis of acute myocardial infarction. The cardiac troponin complex (ITC-complex), comprising cTnI, cTnT, and troponin C (TnC), makes up a large portion of troponins released into the bloodstream after the necrosis of cardiomyocytes. However, the stability of the ITC-complex has not been fully investigated. This study aimed to investigate the stability of the ITC-complex in blood samples. A native ITC-complex was incubated in buffer solutions, serum, and citrate, heparin, or EDTA plasma at various temperatures. Western blotting and gel filtration were performed, and troponins were detected using specific monoclonal antibodies. The ITC-complex dissociated at 37 ◦C in buffers with or without anticoagulants, in citrate, heparin, and EDTA plasmas, and in serum, into a binary cTnI-TnC complex (IC-complex) and free cTnT. In plasma containing heparin and EDTA, the IC-complex further dissociated into free TnC and cTnI. No dissociation was found at 4 ◦C or at room temperature (RT) in all matrices within 24 h except for EDTA plasma. After incubation at 37 ◦C in EDTA plasma and serum, dissociation was accompanied by proteolytic degradation of both cTnI and cTnT. The presence of anti-troponin autoantibodies in the sample impeded dissociation of the ITC-complex. The ITC-complex dissociates in vitro to form the IC-complex and free cTnT at 37 ◦C but is mostly stable at 4 ◦C or RT. Further dissociation of the IC-complex occurs at 37 ◦C in plasmas containing heparin and EDTA....
Background/Objectives: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a type 2 inflammatory disease often resistant to standard treatments. Dupilumab, a monoclonal antibody targeting the IL-4α receptor, has shown efficacy in CRSwNP, but a significant subset of patients do not respond to this therapy. This study aims to investigate pretreatment complete blood count (CBC)-based inflammatory biomarkers as predictors of response to dupilumab in patients with CRSwNP. Methods: This mono-centric, retrospective, single-arm longitudinal cohort study included 80 patients with uncontrolled CRSwNP who received dupilumab treatment at the Medical University of Graz. Patients were classified into responder and non-responder groups based on a reduction of >1 in nasal polyp score (NPS) and a sinonasal outcome test-22 (SNOT-22) score <40 points at six months. Pretreatment CBC-derived biomarkers, including eosinophil count, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and systemic inflammation indices including the aggregate inflammation systemic index (AISI), systemic inflammation index (SII), and systemic inflammation response index (SIRI), were analyzed for their predictive value. Results: Of the 80 patients, 72.5% were classified as responders, while 27.5% were non-responders. A significant positive correlation was found between baseline eosinophil count and NPS reduction (p = 0.027), suggesting that higher eosinophil levels may predict higher NPS reduction in dupilumab treatment. However, no significant associations were observed between NLR, PLR, and systemic inflammation indices with treatment outcomes. Conclusions: Pretreatment eosinophil count may serve as a potential biomarker for predicting nasal polyp reduction in dupilumab treatment of CRSwNP. Other CBC-based inflammatory markers did not show significant predictive value. Further prospective studies are needed to validate these findings and explore additional, reliable biomarkers to optimize treatment outcomes for CRSwNP patients....
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