A cost-efficient internal standard approach for busulfan quantification: MS-based investigation of adduct formation in therapeutic drug monitoring.

Abstract

Background: Busulfan plays a central role in conditioning regimens for hematopoietic stem cell transplantation (HSCT). However, its narrow therapeutic index and substantial interpatient pharmacokinetic variability necessitate precise therapeutic drug monitoring (TDM). Because busulfan is highly lipophilic, many LC-MS/MS assays rely on costly stable-isotope-labeled internal standards (e.g., busulfan-d8), which limits their routine application in resource-constrained settings.

Methods: This study aimed to synthesize cost-effective structural analogs of busulfan and evaluate their chromatographic behavior and cation-adduct formation as potential alternative internal standards. Four dimethanesulfonate analogs were synthesized from symmetrical n-diols (n = 3-6) under alkaline conditions (47-55% yields). The synthesized analogs were evaluated as candidate internal standards by investigating their cation-adduct formation behavior using liquid chromatography-electrospray ionization-quadrupole time-of-flight high-resolution mass spectrometry (LC-ESI-qTOF-HRMS).

Results: Each analog formed distinct cation adducts (H⁺, Li⁺, Na⁺, K⁺, and NH4⁺), with sodium adducts demonstrating the greatest stability in mixed-ion environments. Among the synthesized compounds, 1,6-hexanediol dimethanesulfonate (compound 4) exhibited the most favorable chromatographic retention and adduct stability, supporting its suitability as a structural internal-standard candidate.

Conclusion: These findings provide mechanistic and analytical evidence to support the selection of a cost-efficient internal standard for busulfan quantification. However, full bioanalytical validation in biological matrices will be required before routine clinical implementation.