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Novel LC-MS/MS assay to quantify D,L-alpha-difluoromethylornithine (DFMO) in mouse plasma
Matthew A Swanson1, Carlye Szarowicz1, Schuyler T Pike1
1Department of Biological Sciences, College of Arts and Sciences, Ferris State University, Big Rapids, MI, United States.
Abstract:
D,L-alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC) that is being investigated to treat cancers such as pediatric neuroblastoma. A novel and sensitive LC-MS/MS assay was developed and validated to quantify DFMO concentrations in support of pre-clinical pharmacokinetic studies in mice. The study was performed using a Shimadzu triple quad LC-MS/MS equipped with an Atlantis HILIC Silica 3 µm 2.1 × 100 mm column, and an isocratic mobile phase (75:25 acetonitrile and 0.2 % formic acid) at a flow rate of 0.5 mL/min. Multiple Reaction Monitoring (MRM) was used to identify the precursor ion (183 m/z) with quantification of daughter ions at transitions of 183 > 120.10, 183 > 166.10, and 183 > 80.05. Plasma standards and quality control samples (20 µL) were processed using protein precipitation with cold acetonitrile. The lower limit of detection (LLOQ) was 5 ng/mL. Assay performance was determined from multiple runs (n = 10) with standards ranging from 250-50,000 ng/mL and three levels of quality control (500, 4000, and 40,000 ng/mL). Standard curves were linear with r2 values between 0.9960 and 0.9999. Quality control samples were stable and exhibited maximum inter-day % bias of ≤3 % and CV% of ≤0.7 %. The assay was successfully applied to an in vivo study to determine the pharmacokinetics of DFMO in athymic nu/nu mice.
Insights
A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay accurately measures D,L-alpha-difluoromethylornithine (DFMO) in mouse plasma. This method supports preclinical pharmacokinetic studies for DFMO, a potential cancer therapeutic.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Biochemistry
Background:
- D,L-alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC).
- DFMO is under investigation for treating cancers, including pediatric neuroblastoma.
- Accurate quantification of DFMO is crucial for preclinical pharmacokinetic studies.
Purpose of the Study:
- To develop and validate a novel and sensitive LC-MS/MS assay.
- To quantify DFMO concentrations in mouse plasma for pharmacokinetic analysis.
- To support the investigation of DFMO as a potential anti-cancer agent.
Main Methods:
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Shimadzu triple quad system.
- HILIC Silica column with an isocratic mobile phase of acetonitrile and formic acid.
- Multiple Reaction Monitoring (MRM) for precursor and daughter ion quantification; protein precipitation for sample preparation.
Main Results:
- The assay demonstrated high sensitivity with a lower limit of detection (LLOQ) of 5 ng/mL.
- Linear standard curves (r² values 0.9960–0.9999) and excellent quality control stability (% bias ≤3%, CV% ≤0.7%).
- The validated assay was successfully applied to determine DFMO pharmacokinetics in vivo.
Conclusions:
- A robust and sensitive LC-MS/MS assay for DFMO quantification in mouse plasma has been developed.
- This assay is suitable for supporting preclinical pharmacokinetic studies of DFMO.
- The validated method facilitates further research into DFMO's therapeutic potential.
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