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Topic Area(s): Tox / TDM / Endocrine
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Poster #41a View Map
This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.
INTRODUCTION
Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for a variety of malignant and nonmalignant disorders, especially leukemias and lymphomas. Prior to HCT, the diseased bone marrow must be ablated using a conditioning regimen containing a combination of different agents, most of which are either DNA alkylating agents or inhibitors of DNA synthesis. Various conditioning regimens exist, however, combinations containing busulfan (Bu), melphalan (Mel), fludarabine (Flu) and clofarabine (Clo) are most commonly used. For Bu, therapeutic ranges have been well-established and TDM is widely used. However, for Mel, Flu and Clo little pharmacokinetic data exists to enable optimal dosing.
OBJECTIVES
The objective was to develop a rapid and simple assay for the simultaneous measurement of Bu, Clo, Flu and Mel in plasma by LC-MS/MS. This assay will be used to support clinical trials and pharmacokinetic studies to better define optimal dosing to reduce toxicity and improve overall outcomes.
METHODS
Bu, Clo, Flu and Mel were isolated from plasma samples (100 mcL) after protein precipitation with methanol containing internal standards (IS). 2-chloroadenosine was the IS for Clo and Flu, and deuterated analogs (Bu-d8 and Mel-d8) were the IS for Bu and Mel. Following centrifugation the supernatant was injected into the liquid chromatography system followed by electrospray positive ionization tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a Thermo Scientific TLX-2 HPLC system interfaced to a TSQ Vantage mass spectrometer operated in positive ion ESI mode. MRM transitions were as follows: Busulfan 264.0>151.1 and 264.0>55.1 m/z; Clofarabine 304.0>170.0 and 304.0>134.0; Fludarabine 286.1>154.0 and 286.1>134.0; and Melphalan 305.1>246.1 and 305.1>288.0 m/z. The results were quantified using a six-point calibration curve. Assay accuracy was determined through recovery studies. Within day imprecision was evaluated by analysing 10 specimens in a single day, and between-day imprecision was evaluated by running two batches in duplicate over 20 days.
RESULTS
The LOQs of Bu, Clo, Flu and Mel were 5 ng/mL; the CVs were <20% over 20 days. For all compounds the calibration curves were linear over the analytical measurement range (AMR) with correlation coefficients (R2) ≥0.995. Dilutions were validated providing a final clinical reportable range of 5 to 25,000 ng/mL. Within day and between-day imprecision (CVs) at concentrations spanning the AMR were less than 7% for all analytes. Bu, Clo, Flu and Mel were sufficiently stable when placed on ice and under all relevant analytical conditions. No significant matrix effects were observed during the method validation. Recoveries ranged from 95.6-106.2% for three controls spanning the AMR for all compounds.
CONCLUSION
We have developed an accurate and sensitive LC-MS/MS method for the simultaneous quantification of busulfan, clofarabine, fludarabine and melphalan in human plasma. The method has been fully validated for imprecision, accuracy, linearity, recovery, carryover, specificity and matrix effects. This is the first reported method to measure all four compounds simultaneously and due to the assay’s performance it will be used to support clinical trials and pharmacokinetic studies.
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= Discovery stage. (19.79%, 2022)
= Translation stage. (37.97%, 2022)
= Clinically available. (42.25%, 2022)
