DEVELOPMENT AND VALIDATION METHOD OF CYCLOPHOSPHAMIDE AND 4-HYDROXYCYCLOPHOSPHAMIDE WITH 4-HYDROXYCYCLOPHOSPHAMIDE-D4 AS INTERNAL STANDARD IN DRIED BLOOD SPOTS USING UPLC-MS/MS

Authors

  • YESI IHDINA FITYATAL HASANAH Bioavailability and Bioequivalence Laboratory, Faculty of Pharmacy, Universitas Indonesia, UI Depok Campus, 16424 Depok, Indonesia
  • YAHDIANA HARAHAP Bioavailability and Bioequivalence Laboratory, Faculty of Pharmacy, Universitas Indonesia, UI Depok Campus, 16424 Depok, Indonesia, Indonesia Defense University, Bogor, 16810, West Java, Indonesia
  • HERMAN SURYADI Bioavailability and Bioequivalence Laboratory, Faculty of Pharmacy, Universitas Indonesia, UI Depok Campus, 16424 Depok, Indonesia

DOI:

https://doi.org/10.22159/ijap.2021v13i2.39590

Keywords:

4-hydroxycyclophosphamide (4-OHCP), Cyclophosphamide (CP), Dried blood spots (DBS), SIL 4-hydroxycyclophosphamide-d4

Abstract

Objective: Cyclophosphamide (CP) is anticancer of the 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.

Methods: A simpler analytical method was developed and validated to quantify CP and 4-OHCP in DBS samples using an 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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References

International Agency for Research on Cancer (IARC). IARC monographs-100A: Cyclophosphamide. IARC-WHO, Paris; 2012.

Ekhart C, VD Doodeman, S Rodenhuis, PH Smits, JH Beijnen, ADR Huitema. Influence of polymorphisms of drug-metabolizing enzymes (CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1) on the pharmacokinetics of cyclophosphamide and 4-hydroxy-cyclophosphamide. Pharmacogenet Genom 2008;18:515-23.

Helsby NA, M Yong, M Van Kan, JR de Zoysa, KE Burns. The importance of both CYP2C19 and CYP2B6 germline variations in cyclophosphamide pharmacokinetics and clinical outcomes. Br J Clin Pharmacol 2019;85:1–10.

Ekhart C, A Gebretensae, H Rosing, S Rodenhuis, JH Beijnen, ADR Huitema. 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 2007;854:345–9.

Harahap Y, C Samuel, R Andalusia, NF Syafhan. Analysis of 4-hydroxycyclophosphamide in cancer patients plasma for therapeutic drug monitoring of cyclophosphamide. Int J Pharm Pharm Sci 2016;8:194-200.

Harahap Y, N Gustiyanti, AEM Sunanto, Harmita, DJ Purwanto. Method development on analysis cyclophosphamide and 4-hydroxycyclophophamide in dried blood spot and its application in breast cancer patients. Int J Appl Pharm 2020;12:119-25.

Shu W, Wang X, Yang X, Liang L, Li J, Chen Z, et al. 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 2011;49:2029–37.

FA de Castro, S Scatena Gdos, OP Rocha, MP Marques, QB Cass, BP Simoes, et al. Enantioselective analysis of 4-hydroxycyclophosphamide in human plasma with application to a clinical pharmacokinetic study. J Chromatogr B: Anal Technol Biomed Life Sci 2015;1011:53–61.

Hall OMH, Peer CJ, Fitzhugh CD, Figg WD. 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. J Chromatography B 2018;1086:56–62.

Hong PS, Chan KK. Analysis of 4-hydroxycyclophosphamide by gas chromatography-mass spectrometry in plasma. J Chromatogr 1989;495:131–8.

Anderson LW, SM Ludeman, OM Colvin, LB Grochow, JM Strong. Quantitationof4-hydroxycyclophosphamide/aldophosphamide in whole blood. J Chromatogr Biomed Appl 1995;667:247–57.

Baumann F, C Lorenz, U Jaehde, R Preiss. Determination of cyclophosphamide and its metabolites in human plasma by high-performance liquid chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl 1999;729:297–305.

Food and Drug Administration (FDA). Guidance for industry bioanalytical method validation. FDA, Rockville; 2018.

European Medicines Agency (EMEA). Guideline on bioanalytical method validation. EMEA, London; 2011.

Waters. Controlling contamination in LC/MS systems. Waters Coprporation; 2016.

Lee Chuin Chen. High-temperature liquid chromatography and the hyphenation with mass spectrometry using high-pressure electrospray ionization. Mass Spectrom 2019;8:S0079.

Dong MW. HPLC and UHPLC for practicing scientists. Wiley, USA; 2019.

Bose, Anirban Deep. HPLC calibration process parameters in terms of the system suitability test. Austin Chromatogr 2014;1:1-4.

Published

07-03-2021

How to Cite

HASANAH, Y. I. F., HARAHAP, Y., & SURYADI, H. (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), 148–152. https://doi.org/10.22159/ijap.2021v13i2.39590

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