Determination of crenolanib in human serum and cerebrospinal fluid by liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS)

• A novel method for the determination of crenolanib by LC/MS/MS is described.
• The method was fully validated in human serum and partially validated in CSF.
• The curve ranges were 5–1000 ng/mL and 0.5–1000 ng/mL for human serum and CSF respectively.
• The method was applied to a clinical pharmacokinetic study of children treated with crenolanib.

A LC–ESI-MS/MS method for the determination of crenolanib (CP-868,596) in human serum was developed and validated employing d4-CP-868,596 as an internal standard (ISTD). In addition to human serum, the method was also partially validated for crenolanib determination in human cerebrospinal fluid (CSF) samples. Sample aliquots (50 μl of serum or CSF) were prepared for analysis using liquid–liquid extraction (LLE) with tert-butyl methyl ether. Chromatography was performed using a phenomenex Gemini C18 column (3 μm, 100 mm × 4.6 mm I.D.) in a column heater set at 50 °C and an isocratic mobile phase (methanol/water/formic acid at a volume ratio of 25/25/0.15, v/v/v). The flow rate was 0.45 mL/min, and the retention time for both analyte and ISTD was less than 3.5 min. Samples were analyzed with an API-5500 LC–MS/MS system (ESI) in positive ionization mode coupled to a Shimadzu HPLC system. The ion transitions monitored were m/z 444.4 → 373.1 and m/z 448.2 → 374.2 for crenolanib and ISTD, respectively. The method was linear over the range of 5–1000 ng/mL for serum and 0.5–1000 ng/mL for CSF. For human serum, both intra-day and inter-day precision were <4%, while intra-day and inter-day accuracy were within 8% of nominal values. Recovery was greater than 50% for both the analyte and ISTD. For CSF samples, both intra-day and inter-day precision were <9% except at the lower limit of quantification (LLOQ) which was <17%. The intra-day and inter-day accuracy were within 11% of the nominal CSF concentrations. After validation, this method was successfully applied to the analysis of serial pharmacokinetic samples obtained from a child treated with oral crenolanib.