Development and Validation Method of Cyclophosphamide and 4-Hydroxycyclophosphamide with 4-Hydroxycyclophosphamide-d4 as Internal Standard in Dried Blood Spots Using UPLC-MS/MS
Background and Objective: Cyclophosphamide (CP) is an anticancer of alkylating agent (nitrogen mustard) and a prodrug which will be metabolized into an active metabolite form, 4-hydroxycyclophosphamide (4-OHCP). Therefore, the effectiveness of therapy with CP is determined by its metabolites concentration. The purpose of this study was to obtain a validated analytical method of CP and 4-OHCP simultaneously and sensitively in dried blood spots with SIL (Stable Isotope Labeled) 4-OHCP-d4 as the internal standard using liquid chromatography – tandem mass spectrometry, so optimization and full validation are conducted in this research. Materials and Methods: A simpler analytical method was developed and validated to quantify CP and 4-OHCP in DBS samples using a Ultra-High-Performance Liquid Chromatography- Tandem Mass Spectrometry (UPLC-MS/MS). A linear regression was used as the statistical analysis method. Sample preparation was performed by protein precipitation using methanol. The separation was performed on UPLC H-Class BEH C18 column using formic acid 0.01% - acetonitrile as the mobile phase in gradient mode at 0.2 mL/minute. The mass detection was performed on Waters Xevo TQD using ESI+ for CP, 4-OHCP-SCZ, and IS 4-OHCP-d4-SCZ with m/z value: 261.03 > 140.16 ; 334.10 > 221.04 ; and 338.10 > 225.06.
Results: This method was linear within the range of 10 – 40,000 ng/mL for CP and 5 – 4,000 ng/mL for 4-OHCP. Lower Limit of Quantification (LLOQ) concentration of CP was 10 ng/ml and 4-OHCP was 5 ng/ml.
Conclusion: This method has successfully fulfilled the validation requirement referring to the 2011 EMA and 2018 FDA guidelines.
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