Abstract
Objective: To develop the method for the simultaneous analysis of cyclophosphamide and 4-hydroxycyclophosphamide (4-OHCP) in Dried Blood Spot (DBS) using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) and its application in breast cancer patients for therapeutic drug monitoring.
 Methods: Sample preparation used protein precipitation with methanol and acetonitrile (2:1 v/v). The separation was conducted using 1.7μm (2.1 x 100 mm) Waters AcquityTM UPLC C18 column; mobile phase consists of 0.01% formic acid and methanol (50:50 v/v) with isocratic elution, column temperature 30 °C, flow rate 0.3 ml/min and hexamethylphosphoramide (HMP) used as an internal standard. Analysis was performed by a triple quadrupole mass spectrometry with a positive ion mode of Electrospray Ionization. Cyclophosphamide was detected at m/z 260.968>139.978, 4-OHCP at m/z 338.011>224.979, and HMP at m/z 180.17>92.08. The method was applied to quantify cyclophosphamide and 4-OHCP in DBS of breast cancer patients. Blood samples were collected at 2 and 4 h after cyclophosphamide administration for therapeutic drug monitoring.
 Results: The method was linear in the range of 50–30.000 ng/ml for cyclophosphamide and 10–1000 ng/ml for 4-OHCP. Lower Limit of Quantification (LLOQ) concentration of cyclophosphamide was 50 ng/ml and 4-OHCP was 10 ng/ml. Accuracy and precision within-run and between-run met the requirements with % diff and CV, not exceeding ±15% and not more than ±20% for LLOQ concentration. The results from DBS samples of cancer patients showed that the level of cyclophosphamide was in the range of 6045.980 ng/ml to 37024.403 ng/ml and 4-OHCP was in the range 33.155 ng/ml to 246.362 ng/ml.
 Conclusion: The developed method met the requirements of all validation parameters under the Guideline on Bioanalytical Method Validation by the European Medicines Agency in 2011. Method can be applied on DBS of cancer patients and the results showed that cyclophosphamide and 4-OHCP was detected on 17 samples of breast cancer patients. This can be one of the parameters for therapeutic drug monitoring.
Highlights
Cyclophosphamide is an anticancer drug that acts as an alkylating agent and is used singly, as well as being a regimen in multiple types of cancer therapies such as non-Hodgkin's lymphoma, sarcoma, and breast cancer [1]
10 μl of aliquot was injected into the chromatography system
The results show that the interference or impurity on the retention time of analytes and internal standard is still meet the criteria
Summary
Cyclophosphamide is an anticancer drug that acts as an alkylating agent and is used singly, as well as being a regimen in multiple types of cancer therapies such as non-Hodgkin's lymphoma, sarcoma, and breast cancer [1]. Cyclophosphamide is a prodrug that requires activation by the cytochrome P450 enzyme to convert into its active metabolite, 4-OHCP. This metabolite is in balance with its tautomeric form, aldophosphamide, which will become its active form, phosphoramide mustard and acrolein [2]. The number of enzymes that play a role in the metabolism of cyclophosphamide can cause various expressions and activities among patients, contributing to the metabolism, efficacy and toxicity of this drug [3]. Quantitative analytical methods of cyclophosphamide and 4-OHCP in DBS simultaneously using UPLC-MS/MS have never been done before. UPLC is a technical advancement of liquid chromatography where there are innovations in instrumentation and particle size to improve the resolution, speed, and sensitivity of the method [8]
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