Method development and validation for measuring o⁶-methylguanine in dried blood spot using ultra high-performance liquid chromatography tandem mass spectrometry

Background: Cyclophosphamide is a nitrogen mustard chemotherapy drug that damages DNA through alkylation in the DNA base and produces DNA adducts. Alkylation that occurs in the N7 position of guanine base has a cytotoxic effect which is useful for cancer therapy. However, the alkylation that occurs in the O6 position of guanine bases can have mutagenic and carcinogenic effects that can trigger secondary cancer. This carcinogenic compound can be found in very low concentrations in cancer patients who had been receiving alkylating agents as their anticancer therapy. Analysis of O⁶-methylguanine can be one of the ways of therapeutic drug monitoring to avoid secondary cancer risk. This study aims to develop a sensitive, selective, and validated analytical method using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). Methods: Analysis of O⁶-methylguanine was done in Dried Blood Spot (DBS) and using allopurinol as an internal standard. The optimal analysis conditions were obtained using a C18 Acquity® Bridged Ethylene Hybrid (BEH) column (1.7 µm, 100 mm x 2.1 mm); mobile phase was 0.05% formic acid-acetonitrile (95:5 v/v); flow rate 0.1 mL/minute; gradient elution for 6 minutes; and detection at m/z 165.95 > 149 for O⁶-methylguanine and m/z 136.9 > 110 for allopurinol. Results: The present study has fulfilled the FDA validation parameter requirements. The method provides rapid, sensitive, and selective analysis of O⁶-methylguanine using UPLC-MS/MS with a linear concentration range between 0.5–20 ng/mL.