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A stability indicating LC-MS/MS method for quantification of a NOX Inhibitor R14 in its bisulfite adduct form for
Yang Wang1, Jing Ma1, Shiaw-Yih Lin2
1Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Texas Southern University, Houston, TX 77004, USA.
Abstract:
R14, also known as NOX Inhibitor VII, is a potent inhibitor of NADPH oxidases (NOX) which has recently been identified as a novel agent targeting to triple-negative breast cancer. It is also rapidly degraded in collected pharmacokinetic plasma and blood samples even stored under - 70 °C. The purpose of this study was to develop a stability indicating LC-MS/MS assay that would be suitable for quantification of R14 in plasma and blood. In the presence of sodium sulfite under acidic pH, R14, an aryl lactam compound which is not a typically reactive compound for bisulfite addition, readily and completely converted to R14 bisulfite adduct, which was more stable in plasma and blood. The adduct has MRM transition at m/z 340.1-127.0 in negative mode and showed high sensitivity in LC-MS/MS quantification. Thus, monitoring the adduct provided a suitable way of quantitating R14 concentrations in mouse whole blood. The reacting conditions were optimized based on detecting R14 bisulfite adduct, and the assay was established and validated on a SCIEX 6500+ Triple Quad LC-MS/MS System. The method was then successfully adapted to pharmacokinetic studies after oral administration of R14 to mice.
Insights
R14, a novel triple-negative breast cancer treatment, is unstable in samples. A new LC-MS/MS assay quantifies R14 by converting it to a stable bisulfite adduct, enabling pharmacokinetic studies.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Biochemistry
Background:
- R14 (NOX Inhibitor VII) is a promising agent for triple-negative breast cancer.
- R14 exhibits rapid degradation in plasma and blood samples, even at -70°C.
- Accurate quantification of R14 is crucial for pharmacokinetic studies.
Purpose of the Study:
- To develop a stability-indicating LC-MS/MS assay for quantifying R14 in plasma and blood.
- To overcome the instability of R14 in biological samples.
Main Methods:
- R14 was converted to a more stable bisulfite adduct under acidic conditions with sodium sulfite.
- A sensitive LC-MS/MS method was developed and validated using the R14 bisulfite adduct.
- The assay utilized MRM transition m/z 340.1-127.0 in negative mode on a SCIEX 6500+ Triple Quad LC-MS/MS System.
Main Results:
- R14 readily formed a stable bisulfite adduct, facilitating its detection.
- The developed LC-MS/MS assay demonstrated high sensitivity and specificity for the adduct.
- The assay was successfully validated and applied to quantify R14 in mouse whole blood.
Conclusions:
- The bisulfite adduct formation provides a robust method for quantifying R14 in biological matrices.
- This validated assay is suitable for pharmacokinetic studies of R14.
- The method enables further investigation into R14's therapeutic potential in triple-negative breast cancer.
