ANALYSIS OF CYTOSTATIC COMPOUNDS IN AQUATIC ENVIRONMENT Ľ ubomíra Kova ľ ová 1 under the supervision of Juliane Hollender 2 and Wolfgang Dott 1 1 RWTH Aachen University, Institute of Hygiene and Environmental Health 2 EAWAG, Swiss Federal Institute of Aquatic Science and Technology 18 January 2006 AQUAbase Workshop on ANALYTICAL METHODS
• CONTEXT • COMPOUNDS PHARMACEUTICALS IN THE ENVIRONMENT • ANALYTICAL CHALLENGE • ANALYTICAL METHOD - GENERAL AIM: • OUTLOOK - find out ENVIRONMENTAL CONCENTRATIONS - check ECOTOXICOLOGY - provide ways of ELIMINATION FROM ENVIRONMENT (if necessary)
CYTOSTATICS • CONTEXT • COMPOUNDS - COMPOUNDS USED IN CHEMOTHERAPY OF PATIENTS WITH CANCER • ANALYTICAL CHALLENGE - DESIGNED TO CAUSE MUTATION AND KILL CELLS • ANALYTICAL - PRESCRIPTION RATE: METHOD • OUTLOOK Uniklinik Aachen 2004 Denmark 2001 cytostatic kg/year cytostatic kg/year HYDROXYCARBAMID 180 5-FLUOROURACIL 2.9 5-FLUOROURACIL 8.4 CYTARABINE 1.0 IFOSFAMIDE 1.0 CYCLOPHASPHAMIDE 6.8 CYCLOPHOSPHAMIDE 0.8 METHOTREXATE 1.0 GEMCITABINE 0.7 IFOSFAMIDE 0.9 MEHOTREXATE 0.6 ETOPOSIDE 0.7
5-FLUOROURACIL CYTARABINE GEMCITABINE N N O N F N N • CONTEXT O N O N O N • COMPOUNDS F O O F O • ANALYTICAL O O CHALLENGE O O • ANALYTICAL METHOD • OUTLOOK Uniklinik Aachen 2004 HOSPITAL WASTEWATER µ g/l cytostatic kg/year unmetabolised ng/l 5-FLUOROURACIL 2.9 7.4 740 CYTARABINE 1.0 2.7 270 10% GEMCITABINE 0.7 1.8 180
5-FLUOROURACIL CYTARABINE GEMCITABINE ACIDIC BASIC BASIC N N O F N N N • CONTEXT O N O N O N • COMPOUNDS F O O O F • ANALYTICAL O O CHALLENGE O O • ANALYTICAL METHOD Metabolites: ACIDIC ACIDIC ACIDIC ACIDIC • OUTLOOK O O O O F F N N N N N O O N O N O N O F O O O O O F O O O O O O O O uracil 1- β -D- 5-fluorouridine 5-fluoro- 2’,2’-difluoro- 2 ′ -deoxyuridine arabinofuranoside deoxyuridine
• CONTEXT • COMPOUNDS ANALYTICAL CHALLENGE • ANALYTICAL CHALLENGE • ANALYTICAL METHOD - VERY POLAR COMPOUNDS • OUTLOOK Log Kow -0.8 to -2.3
ANALYTICAL METHOD • CONTEXT • COMPOUNDS • ANALYTICAL CHALLENGE CLEAN-UP / • ANALYTICAL PRECONCEN- SEPARATION DETECTION METHOD TRATION • OUTLOOK SPE HPLC MS/MS
CytR GemC mAU 5-Fu 60 50 40 ANALYTICAL METHOD 30 20 10 0 0 2 4 6 min • CONTEXT • COMPOUNDS HPLC SEPARATION I. RP columns separation retention • ANALYTICAL CHALLENGE Synergi Hydro Phenomenex • ANALYTICAL Aqua METHOD Phenomenex DETECTION • OUTLOOK CLEAN-UP Polaris C18-Ether Varian Synergi Polar Phenomenex Nucleosil C18 Machery-Nagel MZ - PAH MZ Symmetry C18 Waters RP – Reversed Phase
ANALYTICAL METHOD • CONTEXT • COMPOUNDS HPLC SEPARATION II. • ANALYTICAL HILIC columns separation retention CHALLENGE • ANALYTICAL Zorbax NH2 METHOD Agilent DETECTION • OUTLOOK CLEAN-UP TSK-Gel Amide 80 TOSOH Bioscience ZIC-HILIC SeQuant HILIC – Hydrophilic Interaction Chromatography
ANALYTICAL METHOD • CONTEXT DETECTION • COMPOUNDS • ANALYTICAL LOQ [ µ g/l] CHALLENGE • ANALYTICAL 5-Fu CytR GemC METHOD - MS/MS (electrospray ionisation) SEPARATION • OUTLOOK CLEAN-UP TSQ Quantum 50 5 10 API 4000 0.1 0.5 0.5 - UV HP 1100 300 500 500
ANALYTICAL METHOD • CONTEXT CLEAN-UP / PRECONCENTRATION • COMPOUNDS • ANALYTICAL CHALLENGE RP mechanism (pH adjusted to keep analytes neutral) • ANALYTICAL acidic basic METHOD analytes analytes SEPARATION DETECTION ISOLUTE ENV+ • OUTLOOK IST Strata-X Phenomenex Oasis HLB Waters
ANALYTICAL METHOD • CONTEXT CLEAN-UP / PRECONCENTRATION • COMPOUNDS • ANALYTICAL Cation Exchange mechanism (pH adjusted to keep analytes CHALLENGE positively charged) basic analytes • ANALYTICAL METHOD Bond Elut Certify Varian SEPARATION DETECTION • OUTLOOK Accubond Evidex Agilent Bakerbond Narc-2 JT Baker Strata-XC Phenomenex Empore MPC-SD 3M
ANALYTICAL METHOD • CONTEXT CLEAN-UP / PRECONCENTRATION • COMPOUNDS • ANALYTICAL Cation Exchange mechanism (pH adjusted to keep analytes CHALLENGE positively charged) basic analytes • ANALYTICAL METHOD SPEC MP1 Ansys SEPARATION DETECTION • OUTLOOK SPEC MP3 Ansys Speedisk H2O-philic SC-DVB JT Baker
SUMMARY • CONTEXT • COMPOUNDS - good HPLC separation and retention using HILIC - satisfactory detection • ANALYTICAL - selection of SPE retention mechanism for basic CHALLENGE compounds • ANALYTICAL METHOD OUTLOOK • OUTLOOK - to complete analytical method: choice of SPE materials optimization of SPE procedure choice of internal standards ENVIRONMENTAL CONCENTRATIONS
• CONTEXT • COMPOUNDS • ANALYTICAL thank you for your attention CHALLENGE • ANALYTICAL METHOD • OUTLOOK
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