Validated LC–MS/MS method for the simultaneous determination of chlorambucil and its prodrug in mouse plasma and brain, and application to pharmacokinetics

• Simultaneous determination of chlorambucil and its prodrug.
• A simple precipitation is needed.
• Excellent chromatographic resolution with a short run time.
• Successful application to pharmacokinetic studies.

A simple, sensitive and rapid LC–MS/MS method was developed and validated for the simultaneous determination of chlorambucil (CHL) and the prodrug of chlorambucil (CHLS) in mouse plasma and brain tissue. Detection was performed on a Diamonsil ODS chromatography column using gradient elution with a mobile phase of 0.2% aqueous formic acid and acetonitrile. Mass spectrometry was carried out in multiple reaction monitoring mode using a positive electrospray ionization interface. Good linearity was found for CHLS and CHL in plasma and brain tissue in different linear ranges (r > 0.9996). Intra-day and inter-day precision was within 9.11% and accuracy was not more than 11.07%. The validated method was successfully applied to the pharmacokinetic study of CHLS and CHL in mice after intravenous administration.

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