Bioanalysis最新文献

Aim: We aim to establish a rapid and sensitive UPLC-MS/MS method to analyze the pharmacokinetics of oral anisodine hydrobromide (AH) tablets.

Methods: Comparison of pharmacokinetic differences between AH (present in vivo as anisodine) and its metabolite NOAT3 in two groups of rats after administration of AH at doses of 5, 10, and 20 mg/kg. In addition, plasma concentrations of AH were used as a pharmacokinetic parameter, and interleukin-1β (IL-1β) and lactic dehydrogenase (LDH) were selected as pharmacodynamic indicators to establish a pharmacokinetics-pharmacodynamics (PK-PD) combined model in physiological and CIRI model rats, to analyze the protective effect of AH against CIRI.

Results & conclusion: The study demonstrated that AH could be rapidly and extensively distributed to various tissues in rats. AH is a promising drug for the treatment of rat models of CIRI.

Background: Desmosine and isodesmosine are crosslinking amino acids found in extracellular matrix protein elastin, which imparts elasticity to tissues such as those of the lungs and arteries. These compounds are promising biomarkers for diseases involving elastin degradation, such as chronic obstructive pulmonary disease.

Research design and method: This study examined the correlation between isotope-dilution LC-MS/MS and a newly established ELISA for in vitro diagnosis using a variety of samples.

Results: Results of ELISA and LC-MS/MS analyses exhibited a high correlation coefficient (0.9941). However, whereas the LC-MS/MS measurements deviated approximately 2-fold from the theoretical values, the ELISA measurements ranged from 0.83 to 1.06 (avg 0.94) times the theoretical values. Precise measurement of the absorbance of synthetic desmosine revealed a molar extinction coefficient of 2403, which differed markedly from the previously reported value of 4900 in 1963. Using this value to recalculate the amount of added desmosine, the LC-MS/MS measurements were 0.68 to 0.99 (avg 0.87) times the theoretical values.

Conclusion: Thus, the developed ELISA enables highly accurate determination of desmosine concentrations, comparable to LC-MS/MS, suggesting that ELISA is a potentially useful in vitro diagnostic tool.

Background: TT-478 is a novel adenosine receptor 2B antagonist, administered orally as a prodrug (TT-702) for the treatment of advanced metastatic prostate cancer in a Phase I/II clinical trial setting. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was required to quantify TT-478 in plasma samples obtained from patients recruited to the ongoing early-phase trial.

Methods and results: An LC-MS/MS method has been developed and fully validated that allows the quantification of TT-478 in patient plasma samples following a simple extraction procedure using acetonitrile. The assay was shown to be sensitive and selective for TT-478, with an analytical range of 75-25,000 ng/mL, and exhibited excellent precision (coefficient of variation < 12%) and accuracy in the range of 96-107%. Consistently high recovery was achieved, and no matrix effect observed. Analysis of patient samples confirmed that TT-702 rapidly and completely hydrolyzes to TT-478 following administration.

Conclusion: A novel robust method to quantify TT-478 in human plasma has been fully validated and is currently being utilized in an ongoing clinical trial. Analysis of TT-478 levels in plasma samples from these patients will provide first-in-human pharmacokinetic data for this novel compound.Clinical trial registration: https://clinicaltrials.gov/study/NCT05272709?term=AREA%5BConditionSearch%5D(Advanced%20Prostate%20Cancer)%20AND%20AREA%5BBasicSearch%5D(phase%203%20drug)%20AND%20AREA%5BOverallStatus%5D(NOT_YET_RECRUITING%20OR%20RECRUITING%20OR%20ENROLLING_BY_INVITATION%20OR%20ACTIVE_NOT_RECRUITING)&rank=10 identifier is NCT05272709.

Aim: A robust, sensitive, and reliable method was developed and validated on HPLC for the simultaneous estimation of Finerenone (FNR) and Canagliflozin (CFZ).

Method: The resolution was performed by using mobile-phase acetonitrile (ACN): Water (51:49) at a flow rate of 0.75 ml/min with the C18 analytical column Phenomenex Luna (250 mm, 4.6 mm, 5 µm). FNR and CFZ were estimated at a retention time of 6.33 and 8.26 min with 10.0 min analysis run time and detected by PDA detector at a λ_max 249 and 290 nm, respectively. The calibration curve was linear over the concentration range of 0.05-10 µg/ml with R² 0.998 and 0.999 for FNR and CFZ, respectively. For FNR and CFZ, the limit of detection (LOD) was 0.53 and 0.36 μg/ml & limit of quantitation (LOQ) was 1.62 and 1.10 μg/ml, respectively. The method was validated using specificity, linearity, accuracy, precision, LOD, LOQ, robustness, and stability.

Conclusion: According to the International Council for Harmonisation (ICH) guideline, the validation studies confirmed that the optimization method is specific, simple, highly sensitive, reliable, robust, and reproducible. The developed method was successfully applied for simultaneous estimation with applications in in-vitro metabolic stability studies of pooled microsomes of humans, monkeys, dogs, rabbits, rats and mice.

Background: Volumetric Absorptive Microsampling (VAMS) is a blood sample collection method proposed as an alternative to venipuncture for metals/elements biomonitoring. However, the microsampler background concentration of metals and small blood volume remains critical limitations, particularly for metals or trace element analysis in environmental health and epidemiological research.

Materials & methods: Trace element analysis was performed to measure metal concentration in blood samples collected via VAMS and venipuncture using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). VAMS blanks showed elevated background concentration, and cleaning was attempted using a multi-step cleaning protocol. Detection limits and efficacy of background concentration reduction were evaluated.

Results: Initial analysis showed elevated background metal concentrations in the VAMS blank samplers, at or exceeding levels found in venous blood samples. VAMS blank with background concentration post-cleaning result indicated reductions in concentration for some metals; however, the concentration for most detected metals remained persistent.

Conclusions: The efficacy of VAMS in environmental health and epidemiological biomarker research demonstrates both the promising potential and limitations, and the effectiveness of a rigorous cleaning protocol in reducing or eliminating background metal concentrations on microsampler tips.

Background: The objective of this research is to provide differential pulse voltammetry (DPV) method that is an ingenious, simple, sensitive, and selective method for the quantitative analysis of linagliptin and empagliflozin in spiked human urine samples. Faster analysis times and the use of small sample volumes are great advantages of DPV.

Research design and methods: Before centrifuging the samples for 5 min at 3000 rpm, methanol was added to precipitate and remove any suspended compounds. As a working electrode, glassy carbon electrode (GCE) was employed. The auxiliary electrode was a platinum electrode, while the reference electrode was Ag|AgCl|KCl(sat.). To maximize the experimental conditions for simultaneous determination, the relationship between the current, pH and scan rate was examined.

Results: Optimal conditions for quantitative determination were obtained in a phosphate buffer (PB) at pH 7. Plotting LIN and EMP concentrations against the DPV peak height revealed good linearity. It was found that the linear ranges for LIN and EMP were 10-90 and 10-70 µM, respectively.

Conclusions: This approach has been designed for effectively estimating the levels of examined drugs in human urine without matrix effect. The obtained results showed a good recovery values of both drugs.

Aims: Risdiplam is a small molecule approved for the treatment of spinal muscular atrophy (SMA). The drug and its major metabolite had to be measured in plasma and tissue from several animal species and in human plasma and urine. Bioanalytical challenges including light sensitivity, instability, carryover, nonspecific binding, and complex tissue analysis, had to be overcome.

Materials & methods: Liquid chromatography tandem mass spectrometry with reversed-phase separation after protein precipitation/dilution was applied. Ascorbic acid was used as a stabilizer to mitigate degradation of the metabolite, and a surfactant additive prevented nonspecific binding in urine. Tissues were efficiently homogenized by bead beating and matrix-matched with plasma.

Results and conclusions: The above challenges were successfully addressed with bioanalytical methods tailored to study needs. Validations and regulatory analyses met requirements of current guidelines, including successful incurred sample reanalysis (ISR) in GLP and clinical studies. The 3R principles (Replacement, Reduction, Refinement) were applied in animal studies to minimize the use of real matrices. Pediatric studies were supported with rapid analysis and microsampling. Bioanalysis supported patient-centric approaches in dose finding and sampling and was key in answering important questions to enable risdiplam to the market.

Background: High-throughput solid-phase extraction coupled with tandem mass spectrometry (HT-SPE-MS/MS) is an automated sample delivery system to mass spectrometry that operates without chromatographic separation. The typical analysis time per sample using this platform is 10-30 s. While the HT-SPE-MS/MS system has demonstrated efficacy for in vitro assays, its application to the analysis of biological samples from in vivo bioavailability and bioequivalence studies presents challenges due to the complexity of the sample matrix. Three critical issues - matrix effect, specificity, and carryover - have not been thoroughly evaluated in complex biological matrices such as plasma.

Research design and methods: This study assessed the feasibility of utilizing HT-SPE-MS/MS for the analysis of three metabolically related compounds (bupropion, hydroxybupropion, and threobupropion) in human plasma samples from a clinical bioequivalence study. Critical bioanalytical parameters, including matrix effect, specificity, accuracy, precision, and carryover, were systematically investigated.

Results: These methods were subsequently applied to a bioequivalence study of bupropion. The HT-SPE-MS/MS approach achieved comparable accuracy, precision, linearity, and sensitivity to conventional ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) methods, while offering 20- to 30-fold higher analysis speeds.

Conclusion: The results of this study indicate that the HT-SPE-MS/MS system shows potential for high-throughput in vivo bioanalysis, particularly in bioavailability and bioequivalence studies.

Antibody-drug conjugates (ADCs) represent a rapidly advancing class of biotherapeutics for oncology and immunological indications. Comprehensive pharmacokinetic (PK) characterization is critical for assessing ADCs efficacy, safety, and overall therapeutic performance. Ligand binding assays (LBAs) are widely employed in both academic and industrial settings for the quantitative and semi-quantitative analysis of biologics. These assays rely on specific molecular interactions - commonly between antigens and antibodies or ligands and receptors - and offer high sensitivity, robustness, and cost-efficiency. In ADC bioanalysis, LBAs are utilized to quantify multiple types of analytes, including total antibody and antibody-drug conjugate. However, the development of LBA methods for ADCs is challenged by the structural heterogeneity of these molecules, analyte instability, and the need for high selectivity and sensitivity. This review summarizes the application of LBAs in ADC PK studies, outlines common methodological challenges, and discusses strategic considerations for assay development to ensure accurate and reliable bioanalytical measurements.