Objective: To obtain optimum and validated method for simultaneous analysis of cyclophosphamide and 4-hydroxycyclophosphamide in Dried Blood Spot using Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry and its application in breast cancer patients for therapeutic drug monitoring. Methods: The separation was conducted using 1.7 μm (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 30oC, flow rate 0.3 mL/min and hexamethylphosphoramide used as internal standard. Analysis was performed by a triple quadrupole mass spectrometry with positive ion mode of Electrospray Ionization. Cyclophosphamide was detected at m/z 260,968>139,978, 4-hydoxycyclophosphamide at m/z 338,011>224,979, and hexamethylphosphoramide at m/z 180,17>92,08. Optimum sample preparation method was protein precipitation using methanol and acetonitrile (2:1 v/v). The method was validated accordance to EMEA Guidelines and the method was applied to quantify cyclophosphamide and 4-hydroxycyclophosphamide in Dried Blood Spot of breast cancer patients. Blood samples were collected 2 and 4 hours 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-hydroxycyclophosphamide. Lower Limit of Quantification (LLOQ) concentration of cyclophosphamide was 50 ng/mL and 4-hydroxycyclophosphamide 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 Lower Limit of Quantification (LLOQ) concentration. The results from dried blood spot 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-hydroxycyclophosphamide 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 dried blood spot of cancer patients and the results showed that cyclophosphamide and 4-hydroxycyclophosphamide was detected on 17 samples of breast cancer patients. This can be one of parameter for therapeutic drug monitoring.

Keywords: cyclophosphamide, 4-hydroxycyclophosphamide, UPLC-MS/MS, dried blood spot, validation, cancer