LC-MS/MS Method for the Quantitative Determination of Tanespimycin and its Active Metabolite in Human Plasma: Method Validation and Overcoming an Insidious APCI Source Phenomenon

OBJECTIVES: To develop a LC-MS/MS method for simultaneous measurement of Tanespimycin (17-(allylamino)-17-demethoxygeldanamycin, BMS-722782, 17-AAG) and its active metabolite 17-(amino)-17-demethoxygeldanamycin (17-AG) in human plasma. METHODS: The samples were extracted by protein precipitation and analyzed on an LC-MS/MS system using reversed phase chromatography. Ionization was carried out using an Atmospheric Pressure Chemical Ionization (APCI) source. RESULTS: A sensitive method was developed and validated for the measurement of tanespimycin and its active 17-AG in human plasma using tanespimycin -13C315N as an internal standard. The assay was validated over the concentration range of 10.0 to 2500 ng/mL for tanespimycin and 5.00 to 1250 ng/mL for 17-AG. During method development, an internal standard variability due to an in-source reduction of the quinone moiety during ionization was observed. The in-source reduction was mitigated by selection of appropriate mobile phases, internal standard concentration, injection volume, source temperature, and continuous maintenance of the source between runs. CONCLUSION: This paper describes a validated method to simultaneously measure tanespimycin and its active metabolite in human plasma by LC-MS/ MS. In addition, this paper describes various approaches to mitigate the impact of in-source reduction during APCI ionization.