Environmental Science and Pollution Research

, Volume 23, Issue 11, pp 11209–11223 | Cite as

Human metabolites and transformation products of cyclophosphamide and ifosfamide: analysis, occurrence and formation during abiotic treatments

  • Marjeta Česen
  • Tina Kosjek
  • Francesco Busetti
  • Boris Kompare
  • Ester HeathEmail author
Research Article


This study describes a gas chromatography-mass spectrometry analytical method for the analysis of cytostatic cyclophosphamide (CP), ifosfamide (IF) and their selected metabolites/transformation products (TPs): carboxy-cyclophosphamide (carboxy-CP), keto-cyclophosphamide (keto-CP) and 3-dechloroethyl-ifosfamide/N-dechloroethyl-cyclophosphamide (N-decl-CP) in wastewater (WW). Keto-cyclophosphamide, CP and IF were extracted with Oasis HLB and N-decl-CP and carboxy-CP with Isolute ENV+ cartridges. Analyte derivatization was performed by silylation (metabolites/TPs) and acetylation (CP and IF). The recoveries and LOQs of the developed method were 58, 87 and 103 % and 77.7, 43.7 and 6.7 ng L−1 for carboxy-CP, keto-CP and N-decl-CP, respectively. After validation, the analytical method was applied to hospital WW and influent and effluent samples of a receiving WW treatment plant. In hospital WW, levels up to 2690, 47.0, 13,200, 2100 and 178 ng L−1 were detected for CP, IF, carboxy-CP, N-decl-CP and keto-CP, respectively, while in influent and effluent samples concentrations were below LOQs. The formation of TPs during abiotic treatments was also studied. Liquid chromatography-high-resolution mass spectrometry was used to identify CP and IF TPs in ultrapure water, treated with UV and UV/H2O2. UV treatment produced four CP TPs and four IF TPs, while UV/H2O2 resulted in five CPs and four IF TPs. Besides already known TPs, three novel TPs (CP-TP138a, imino-ifosfamide and IF-TP138) have been tentatively identified. In hospital WW treated by UV/O3/H2O2, none of the target metabolites/TPs resulted above LOQs.


Cyclophosphamide Ifosfamide Cytostatic Metabolites Transformation products Occurrence Wastewater Abiotic treatment 



This work was financially supported by the EU through the EU FP7 project CytoThreat (fate and effects of cytostatic pharmaceuticals in the environment and the identification of biomarkers for an improved risk assessment on environmental exposure (Grant Agreement No.: 265264), by the Slovenian Research Agency (Program Groups P1-0143, Project L1-5457, Project J1-6744 and Young Researcher Grant to M. Č.) and the Slovene Human Resources Development and Scholarship Fund (Ad futura). The authors would like to thank the staff at the hospital and the WWTP involved in the study for their collaboration and help during sampling campaign.

Supplementary material

11356_2016_6321_MOESM1_ESM.docx (1 mb)
ESM 1 (DOCX 1053 kb)


  1. Besse J-P, Latour J-F, Garric J (2012) Anticancer drugs in surface waters: what can we say about the occurrence and environmental significance of cytotoxic, cytostatic and endocrine therapy drugs? Environ Int 39:73–86CrossRefGoogle Scholar
  2. Booker V, Halsall C, Llewellyn N, Johnson A, Williams R (2014) Prioritising anticancer drugs for environmental monitoring and risk assessment purposes. Sci Total Environ 473–474:159–170CrossRefGoogle Scholar
  3. Buerge IJ, Buser H-R, Poiger T, Müller MD (2006) Occurrence and fate of the cytostatic drugs cyclophosphamide and ifosfamide in wastewater and surface waters. Environ Sci Technol 40:7242–7250CrossRefGoogle Scholar
  4. Busetti F, Heitz A, Cuomo M, Badoer S, Traverso P (2006) Determination of sixteen polycyclic aromatic hydrocarbons in aqueous and solid samples from an Italian wastewater treatment plant. J Chromatogr A 1102:104–115CrossRefGoogle Scholar
  5. Busetti F, Linge KL, Heitz A (2009) Analysis of pharmaceuticals in indirect potable reuse systems using solid-phase extraction and liquid chromatography–tandem mass spectrometry. J Chromatogr A 1216:5807–5818Google Scholar
  6. Busetti F, Ruff M, Linge KL (2015) Target screening of chemicals of concern in recycled water. Environ Sci: Water Res Technol 1:659–667Google Scholar
  7. Castiglioni S, Bagnati R, Calamari D, Fanelli R, Zuccato E (2005) A multiresidue analytical method using solid-phase extraction and high-pressure liquid chromatography tandem mass spectrometry to measure pharmaceuticals of different therapeutic classes in urban wastewaters. J Chromatogr A 1092:206–215Google Scholar
  8. Česen M, Kosjek T, Laimou-Geraniou M, Kompare B, Širok B, Lambropolou D, Heath E (2015) Occurrence of cyclophosphamide and ifosfamide in aqueous environment and their removal by biological and abiotic wastewater treatment processes. Sci Total Environ 527–528:465–473Google Scholar
  9. Česen M, Eleršek T, Novak M, Žegura B, Kosjek T, Filipič M, Heath E (2016) Ecotoxicity and genotoxicity of cyclophospamide, ifosfamide, their metabolites/transformation products and their mixtures. Environ Pollut 210:192–201CrossRefGoogle Scholar
  10. Chen Z, Park G, Herckes P, Westerhoff P (2008) Physicochemical treatment of three chemotherapy drugs: irinotecan, tamoxifen, and cyclophosphamide. J Adv Oxid Technol 11:254–260Google Scholar
  11. Delgado LF, Faucet-Marquis V, Pfohl-Leszkowicz A, Dorandeu C, Marion B, Schetrite S, Albasi C (2011) Cytotoxicity micropollutant removal in a crossflow membrane bioreactor. Bioresour Technol 102:4395–4401CrossRefGoogle Scholar
  12. Eitel A, Scherrer M, Kümmerer K (2000) Handling cytostatic drugs: a practical guide (monograph).
  13. Farré M l, Pérez S, Kantiani L, Barceló D (2008) Fate and toxicity of emerging pollutants, their metabolites and transformation products in the aquatic environment. TrAC Trends Anal Chem 27:991–1007CrossRefGoogle Scholar
  14. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F (2015) Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 136:E359–E386Google Scholar
  15. Fernández LA, Hernández C, Bataller M, Véliz E, López A, Ledea O, Padrón S (2010) Cyclophosphamide degradation by advanced oxidation processes. Water Environ J 24:174–180CrossRefGoogle Scholar
  16. Ferrando-Climent L, Rodriguez-Mozaz S, Barceló D (2013) Development of a UPLC-MS/MS method for the determination of ten anticancer drugs in hospital and urban wastewaters, and its application for the screening of human metabolites assisted by information-dependent acquisition tool (IDA) in sewage samples. Anal Bioanal Chem 405:5937–5952CrossRefGoogle Scholar
  17. Franquet-Griell H, Gómez-Canela C, Ventura F, Lacorte S (2015) Predicting concentrations of cytostatic drugs in sewage effluents and surface waters of Catalonia (NE Spain). Environ Res 138:161–172CrossRefGoogle Scholar
  18. Garcia-Ac A, Broséus R, Vincent S, Barbeau B, Prévost M, Sauvé S (2010) Oxidation kinetics of cyclophosphamide and methotrexate by ozone in drinking water. Chemosphere 79:1056–1063CrossRefGoogle Scholar
  19. Gilard V, Malet-Martino MC, de Forni M, Niemeyer U, Ader JC, Martino R (1993) Determination of the urinary excretion of ifosfamide and its phosphorated metabolites by phosphorus-31 nuclear magnetic resonance spectroscopy. Cancer Chemother Pharmacol 31:387–394CrossRefGoogle Scholar
  20. Gómez-Canela C, Cortés-Francisco N, Oliva X, Pujol C, Ventura F, Lacorte S, Caixach J (2012) Occurrence of cyclophosphamide and epirubicin in wastewaters by direct injection analysis–liquid chromatography–high-resolution mass spectrometry. Environ Sci Pollut Res 19:3210–3218CrossRefGoogle Scholar
  21. Gomez-Canela C, Ventura F, Caixach J, Lacorte S (2014) Occurrence of cytostatic compounds in hospital effluents and wastewaters, determined by liquid chromatography coupled to high-resolution mass spectrometry. Anal Bioanal Chem 406:3801–3814Google Scholar
  22. Joqueviel C, Martino R, Gilard V, Malet-Martino M, Canal P, Niemeyer U (1998) Urinary Excretion of Cyclophosphamide in Humans, Determined by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy. Drug Metab Dispos 26:418–428Google Scholar
  23. Kaijser GP, Beijnen J, Bult A, Underberg WJ (1994) Ifosfamide metabolism and pharmacokinetics (review). Anticancer Res 14:517–531Google Scholar
  24. Kalhorn TF, Ren S, Howald WN, Lawrence RF, Slattery JT (1999) Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography–mass spectrometry and gas chromatography–nitrogen–phosphorus detection. J Chromatogr B Biomed Sci Appl 732:287–298CrossRefGoogle Scholar
  25. Kiffmeyer T, Götze H-J, Jursch M, Lüders U (1998) Trace enrichment, chromatographic separation and biodegradation of cytostatic compounds in surface water. Fresenius J Anal Chem 361:185–191CrossRefGoogle Scholar
  26. Kim I, Tanaka H (2009) Photodegradation characteristics of PPCPs in water with UV treatment. Environ Int 35:793–802CrossRefGoogle Scholar
  27. Kim I, Yamashita N, Tanaka H (2009) Performance of UV and UV/H2O2 processes for the removal of pharmaceuticals detected in secondary effluent of a sewage treatment plant in Japan. J Hazard Mater 166:1134–1140CrossRefGoogle Scholar
  28. Köhler C, Venditti S, Igos E, Klepiszewski K, Benetto E, Cornelissen A (2012) Elimination of pharmaceutical residues in biologically pre-treated hospital wastewater using advanced UV irradiation technology: a comparative assessment. J Hazard Mater 239–240:70–77CrossRefGoogle Scholar
  29. Kosjek T, Heath E (2011) Occurrence, fate and determination of cytostatic pharmaceuticals in the environment. TrAC Trends Anal Chem 30:1065–1087CrossRefGoogle Scholar
  30. Kosjek T, Heath E, Kompare B (2007) Removal of pharmaceutical residues in a pilot wastewater treatment plant. Anal Bioanal Chem 387:1379–1387CrossRefGoogle Scholar
  31. Kovalova L, Siegrist H, Singer H, Wittmer A, McArdell CS (2012) Hospital wastewater treatment by membrane bioreactor: performance and efficiency for organic micropollutant elimination. Environ Sci Technol 46:1536–1545CrossRefGoogle Scholar
  32. Kümmerer K, Steger-Hartmann T, Meyer M (1997) Biodegradability of the anti-tumour agent ifosfamide and its occurrence in hospital effluents and communal sewage. Water Res 31:2705–2710CrossRefGoogle Scholar
  33. Lai WW-P, Lin HH-H, Lin AY-C (2015) TiO2 photocatalytic degradation and transformation of oxazaphosphorine drugs in an aqueous environment. J Hazard Mater 287:133–141CrossRefGoogle Scholar
  34. Li F, Patterson AD, Höfer CC, Krausz KW, Gonzalez FJ, Idle JR (2010) Comparative metabolism of cyclophosphamide and ifosfamide in the mouse using UPLC–ESI-QTOFMS-based metabolomics. Biochem Pharmacol 80:1063–1074CrossRefGoogle Scholar
  35. Lin AY-C, Lin Y-C, Lee W-N (2014) Prevalence and sunlight photolysis of controlled and chemotherapeutic drugs in aqueous environments. Environ Pollut 187:170–181CrossRefGoogle Scholar
  36. Lin AY, Hsueh JH, Hong PK (2015) Removal of antineoplastic drugs cyclophosphamide, ifosfamide, and 5-fluorouracil and a vasodilator drug pentoxifylline from wastewaters by ozonation. Environ Sci Pollut Res 22:508–515CrossRefGoogle Scholar
  37. Llewellyn N, Lloyd P, Jürgens MD, Johnson AC (2011) Determination of cyclophosphamide and ifosfamide in sewage effluent by stable isotope-dilution liquid chromatography–tandem mass spectrometry. J Chromatogr A 1218:8519–8528Google Scholar
  38. Lutterbeck CA, Machado ÊL, Kümmerer K (2015) Photodegradation of the antineoplastic cyclophosphamide: a comparative study of the efficiencies of UV/H2O2, UV/Fe2+/H2O2 and UV/TiO2 processes. Chemosphere 120:538–546CrossRefGoogle Scholar
  39. Martín J, Camacho-Muñoz D, Santos JL, Aparicio I, Alonso E (2011) Simultaneous determination of a selected group of cytostatic drugs in water using high-performance liquid chromatography–triple-quadrupole mass spectrometry. J Sep Sci 34:3166–3177Google Scholar
  40. Moldovan Z (2006) Occurrences of pharmaceutical and personal care products as micropollutants in rivers from Romania. Chemosphere 64:1808–1817Google Scholar
  41. Momerency G, Van Cauwenberghe K, De Bruijn EA, Van Oosterom AT, Highley MS, Harper PG (1994) Determination of iphosphamide and seven metabolites in blood plasma, as stable trifluoroacetyl derivatives, by electron capture chemical ionization GC-MS. J High Resolut Chromatogr 17:655–661CrossRefGoogle Scholar
  42. Negreira N, de Alda ML, Barceló D (2014) Cytostatic drugs and metabolites in municipal and hospital wastewaters in Spain: Filtration, occurrence, and environmental risk. Sci Total Environ 497–498:68–77Google Scholar
  43. Nicole I, De Laat J, Dore M, Duguet JP, Bonnel C (1990) Utilisation du rayonnement ultraviolet dans le traitement des eaux: mesure du flux photonique par actinometrie chimique au peroxyde d’hydrogene. Water Res 24:157–168CrossRefGoogle Scholar
  44. Steger-Hartmann T, Kümmerer K, Schecker J (1996) Trace analysis of the antineoplastics ifosfamide and cyclophosphamide in sewage water by twostep solid-phase extraction and gas chromatography-mass spectrometry. J Chromatogr A 726:179–184CrossRefGoogle Scholar
  45. Steger-Hartmann T, Kümmerer K, Hartmann A (1997) Biological degradation of cyclophosphamide and its occurrence in sewage water. Ecotoxicol Environ Saf 36:174–179CrossRefGoogle Scholar
  46. Tasso M, Boddy A, Price L, Wyllie R, Pearson AJ, Idle J (1992) Pharmacokinetics and metabolism of cyclophosphamide in paediatric patients. Cancer Chemother Pharmacol 30:207–211CrossRefGoogle Scholar
  47. Ternes TA (1998) Occurrence of drugs in German sewage treatment plants and rivers. Water Res 32:3245–3260Google Scholar
  48. Thomas KV, Dye C, Schlabach M, Langford KH (2007) Source to sink tracking of selected human pharmaceuticals from two Oslo city hospitals and a wastewater treatment works. J Environ Monit 9:1410–1418Google Scholar
  49. Valcárcel Y, González Alonso S, Rodríguez-Gil JL, Gil A, Catalá M (2011) Detection of pharmaceutically active compounds in the rivers and tap water of the Madrid Region (Spain) and potential ecotoxicological risk. Chemosphere 84:1336–1348Google Scholar
  50. Venta MB, Castro CH, Garcia LA, Marzo AL, Lorenzo EV, Alvarez CA (2005) Effect of O3/H2O2 molar concentration ratio at different pH values on cyclophosphamide degradation. J Water Supply Res Technol AQUA 54:403–410Google Scholar
  51. Weiss RB (1991) Ifosfamide vs cyclophosphamide in cancer therapy. Oncology (Williston Park) 5(5):67–76Google Scholar
  52. Yin J, Shao B, Zhang J, Li K (2010) A preliminary study on the occurrence of cytostatic drugs in hospital effluents in Beijing, China. Bull Environ Contam Toxicol 84:39–45CrossRefGoogle Scholar
  53. Zhang J, Tian Q, Zhou S-F (2006) Clinical pharmacology of cyclophosphamide and ifosfamide. Curr Drug Ther 1:55–84CrossRefGoogle Scholar
  54. Zhang J, Chang VWC, Giannis A, Wang J-Y (2013) Removal of cytostatic drugs from aquatic environment: a review. Sci Total Environ 445–446:281–298CrossRefGoogle Scholar
  55. Zuccato E, Castiglioni S, Fanelli R (2005) Identification of the pharmaceuticals for human use contaminating the Italian aquatic environment. J Hazard Mater 122:205–209Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Marjeta Česen
    • 1
    • 2
  • Tina Kosjek
    • 1
    • 2
  • Francesco Busetti
    • 3
  • Boris Kompare
    • 4
  • Ester Heath
    • 1
    • 2
    Email author
  1. 1.Jožef Stefan InstituteLjubljanaSlovenia
  2. 2.Jožef Stefan International Postgraduate SchoolLjubljanaSlovenia
  3. 3.Curtin Water Quality Research Centre (CWQRC)Curtin UniversityPerthAustralia
  4. 4.Faculty of Civil and Geodetic EngineeringUniversity of LjubljanaLjubljanaSlovenia

Personalised recommendations