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Simple method based on fluorescent detection for the determination of 4-hydroxycyclophosphamide in plasma
Laimonas Griskevicius1, Lennart Meurling, Moustapha Hassan
1Department of Medicine, Division of Hematology, Laboratory of Hematology, KFC Novum, Karolinska Institutet, Huddinge University Hospital, 141 86 Stockholm, Sweden.
Therapeutic Drug Monitoring
|May 22, 2002
Summary
This study developed a new method to measure 4-hydroxycyclophosphamide, the active form of cyclophosphamide, in patient plasma. The validated assay provides accurate and precise quantification for chemotherapy monitoring.
Area of Science:
- Pharmacology
- Analytical Chemistry
- Clinical Chemistry
Background:
- Cyclophosphamide is a crucial prodrug in chemotherapy, requiring conversion to its active metabolite, 4-hydroxycyclophosphamide, by cytochrome P450 enzymes.
- Accurate measurement of 4-hydroxycyclophosphamide in plasma is essential for therapeutic drug monitoring and understanding treatment efficacy.
Purpose of the Study:
- To develop and validate a sensitive liquid chromatography method for quantifying 4-hydroxycyclophosphamide in human plasma.
- To establish a reliable analytical technique for assessing cyclophosphamide metabolite levels in patients undergoing chemotherapy.
Main Methods:
- A novel one-step liquid-liquid extraction and protein precipitation technique was employed for sample preparation.
- 4-hydroxycyclophosphamide was derivatized to a fluorescent dansylhydrazone derivative.
- Quantification was performed using reverse-phase liquid chromatography with spectrofluorometric detection (Ex: 350 nm, Em: 550 nm).
Main Results:
- The developed method achieved a limit of quantitation of 60 ng/mL.
- Excellent between-day accuracy and precision were demonstrated, with values less than 9%.
- The method was successfully applied to analyze plasma samples from patients receiving intravenous cyclophosphamide (1 g/m²).
Conclusions:
- A robust and sensitive analytical method for 4-hydroxycyclophosphamide in plasma has been established.
- This validated assay is suitable for clinical applications, including therapeutic drug monitoring of cyclophosphamide therapy.
- The method facilitates pharmacokinetic studies and optimization of cyclophosphamide treatment regimens.