Nuclear Magnetic Resonance, Mass Spectrometer and HPLC Based Identification and Characterization of an Unidentified Impurity in Busulfan

Abstract

Introduction: Busulfan is an alkylating representative that is used as an antineoplastic /cytotoxic chemotherapeutic drug. The safety and effectiveness of busulfan are directly influenced by its quality.

Objectives: The main focus of this work was to identify and characterise an unidentified impurity that consistently eluted in HPLC analysis at a relative retention time period of 0.34 after the predominant peak, busulfan.

Methods: The unknown busulfan impurity isolation was accomplished using a Luna column C18 (length of 250 mm, identification of 50 mm, particle size of 10 µm). 10 mM ammonium acetate (solvent I) and acetonitrile (solvent II) were mixed to make the gradient mode mobile phase. LC-MS analysis of unknown RRTP 0.34 impurity isolated was accomplished using a Luna column C8 (length of 50 mm, identification of 2 mm, particle size of 3 µm). Ammonium formate (10 mM) and formic acid (0.1%) was used as mobile phase I. Acetonitrile containing 0.1% formic acid was used as solvent mobile phase II.  The structure of unknown busulfan impurity was confirmed using ¹HNMR, ¹³CNMR, and DEPT techniques.

Results: The new impurity at retention period near to 5.384 min was realized to be an unknown compound and we labelled it as unknown RRTP 0.34 impurity. A quantity of 20.0 mg of the unknown RRTP 0.34 impurity was extracted. The mass investigation of unknown RRTP 0.34 impurity revealed a molecular ion peak at m/z 264.2 [M+H]⁺.  The ¹H NMR of the isolated unknown RRTP 0.34 impurity reveals 14 protons, while the ¹³C NMR reveals 7 carbons, comprising 2 methyl, 4 secondary, and 1 quaternary carbon, as confirmed by DEPT.

Conclusions: Based on obtained spectrum facts, the molecular chemical formula of unknown RRTP 0.34 impurity is C₇H₁₄O₅S and the consequent structure was regarded as 4 (methylsulfonyloxy) butyl acetate.