Development and Validation Method of Cyclophosphamide and 4-Hydroxycyclophosphamide with 4-Hydroxycyclophosphamide-d4 as Internal Standard in Dried Blood Spots Using UPLC-MS/MS

  • Yahdiana Harahap Faculty of Pharmacy Universitas Indonesia
  • Yesi Ihdina Fityatal Hasanah

Abstract

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.

Keywords: 4-hydroxycyclophosphamide (4-OHCP), cyclophosphamide (CP), dried blood spots (DBS), SIL 4-hydroxycyclophosphamide-d4

References

REFERENCES
[1] International Agency for Research on Cancer (IARC), 2012. IARC monographs - 100A: Cyclophosphamide. IARC-WHO, Paris.
[2] Ekhart, C., V.D. Doodeman, S. Rodenhuis, P.H. Smits, J.H. Beijnen and A.D.R. Huitema, 2008. Influence of polymorphisms of drug metabolizing enzymes (CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1) on the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide. Pharmacogenet Genom, 18 (6): 515-523.
[3] Helsby, N.A., M. Yong, M. van Kan, J.R. de Zoysa and K.E. Burns, 2019. The importance of both CYP2C19 and CYP2B6 germline variations in cyclophosphamide pharmacokinetics and clinical outcomes. Br J Clin Pharmacol, 1–10.
[4] Ekhart, C., A. Gebretensae, H. Rosing, S. Rodenhuis, J.H. Beijnen and A.D.R. Huitema, 2007, Simultaneous quantification of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC–SM/SM). J Chromatogr B Biomed Appl, 854: 345–349.
[5] Harahap, Y., C. Samuel, R. Andalusia and N.F. Syafhan, 2016, Analysis of 4-hydroxycyclophosphamide in cancer patients plasma for therapeutic drug monitoring of cyclophosphamide. Int J Pharm Pharm Sci, 8 (9): 194-200.
[6] Harahap, Y., N. Gustiyanti, A.E.M. Sunanto, Harmita, and D.J. Purwanto, 2020. Method development on analysis cyclophosphamide and 4-hydroxycyclophophamide in dried blood spot and its application in breast cancer patients. Int J App Pharm, 12 (4): 119-125.
[7] Shu W., Wang X., Yang X., Liang L., Li J., Chen Z., Chen X., Jin J., Li R., Zhao L. and Huang M., 2011. Simultaneous determination of cyclophosphamide and 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry – application to Chinese systemic lupus erythematosus patients, Clin Chem Lab Med, 49 (12), 2029–2037.
[8] F.A. de Castro, S. Scatena Gdos, O.P. Rocha, M.P. Marques, Q.B. Cass, B.P. Simoes,V.L. Lanchote, 2016, Enantio selective analysis of 4-hydroxycyclophosphamide in human plasma with application to a clinical pharmacokinetic study, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 1011, 53–61.
[9] Hall, O.M.H., Peer, C.J., Fitzhugh, C.D., and Figg, W.D., 2018, A sensitive and rapid ultra high - performance liquid chromatography with tandem mass spectrometric assay for the simultaneous quantitation of cyclophosphamide and the 4-hydroxy-cyclophosphamide metabolite in human plasma, Journal of Chromatography B, 1086, 56–62.
[10] Hong P.S. and Chan K.K., 1989. Analysis of 4-hydroxycyclophosphamide by gas chromatography–mass spectrometry in plasma. J. Chromatogr, 495: 131–138.
[11] Anderson, L.W., S.M. Ludeman, O.M. Colvin, L.B. Grochow and J.M. Strong,1995. Quantitationof4-hydroxycyclophosphamide/aldophosphamide in whole blood.J Chromatogr B Biomed Appl. 667 (1995) 247–257.
[12] Baumann, F., C. Lorenz, U. Jaehde and R. Preiss, 1999. Determination ofcyclophosphamideand its metabolites in human plasma byhigh-performanceliquid chromatography–mass spectrometry. J ChromatogrB Biomed Sci Appl, 729: 297–305.
[13] Food and Drug Administration (FDA), 2018. Guidance for industry bioanalytical method validation.FDA, Rockville.
[14] European Medicines Agency (EMEA), 2011. Guideline on bioanalytical method validation. EMEA, London.
[15] Waters, 2016. Controlling contamination in LC/MS systems. Waters Coprporation.
[16] Lee Chuin Chen, 2019. High-temperature liquid chromatography and the hyphenation with mass spectrometry using high-pressure electrospray ionization. Mass Spectrom, 8. S0079.
[17] Dong, M.W., 2019. HPLC and UHPLC for practicing scientist. Wiley, USA.
[18] Bose, Anirban deep, 2014. HPLC calibration process parameters in terms of system suitability test. Austin Chromatogr, 1 (2): 1-4.
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Harahap, Y., & Yesi Ihdina Fityatal Hasanah. (2021). Development and Validation Method of Cyclophosphamide and 4-Hydroxycyclophosphamide with 4-Hydroxycyclophosphamide-d4 as Internal Standard in Dried Blood Spots Using UPLC-MS/MS. International Journal of Applied Pharmaceutics, 13(2). Retrieved from https://innovareacademics.in/journals/index.php/ijap/article/view/39590
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