An Automated HPLC Method to Determine Intracellular Vincristine Concentrations in Mononuclear Cells of Children with Acute Lymphoblastic Leukemia

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Abstract
A method to determine intracellular vincristine concentrations in vivo in leukemic cells of patients is useful to investigate mechanisms of vincristine resistance. We developed a high-performance liquid chromatographic (HPLC) method to measure vincristine concentrations in human mononuclear cells (MNC). This method, with on-line column solid-phase extraction and electrochemical detection, previously developed for determination of vincristine concentrations in human plasma, was validated for determination of intracellular vincristine concentrations over a range of 1.17 to 50.8 μg/L with a lower limit of quantitation (LOQ) of 1.17 μg/L. Linearity of the relationship between concentration and response was characterized by a slope (sd) of 0.0683 (0.0008), an intercept (sd) of 0.0004 μg/L (0.0085), and a regression coefficient of 0.997 (P = 0.05). Maximum bias and within-day and between-day coefficients of variation (CV) were 14.2%, 4.8%, and 5.7%, respectively. The HPLC detector response did not interfere with the vincristine peaks of five independent MNC sources. The method was used successfully to measure intracellular vincristine concentrations in 8 of 35 bone marrow MNC samples of children newly diagnosed with acute lymphoblastic leukemia (ALL), 3 days after the first injection of 1.5 mg/m2 vincristine. Vincristine concentrations were in the range of 4.0 to 26.4 μg/L, which was 5 to 20 times the bone marrow plasma concentration. The described HPLC method is accurate and precise and is suitable for detection of intracellular vincristine concentrations in MNC of children with ALL. The results confirm that in vivo vincristine accumulates in bone marrow MNC of patients.