ORIGINAL RESEARCH
Development and Validation
of SPE-HPLC-MS/MS Method for Determining
Cyclophosphamide in Surface Waters
Radosław Czernych1, Jarosław Jan Halkiewicz2, Agata Kot-Wasik3, Jacek Namieśnik3
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1Medical University of Gdansk, Department of Health Sciences, Faculty of Health Sciences with Subfaculty of Nursing
and Institute of Maritime and Tropical Medicine, Department of Environmental Toxicology,
Debowa 23, 80-204 Gdańsk, Poland
2PROLAB, Pharmaceutical Analytical Production,
Przemysłowa 3, Paterek, 89-100 Nakło n/Notecią, Poland
3Department of Analytical Chemistry, Chemical Faculty, Gdańsk University of Technology (GUT),
Narutowicza 11/12 G, 80-952 Gdańsk, Poland
Pol. J. Environ. Stud. 2014;23(5):1537-1545
KEYWORDS
ABSTRACT
A rapid and selective method for trace amounts determination of cyclophosphamide in surface water
samples has been developed. A solid phase extraction SPE method for extraction and clean-up procedure has
been optimized for determination by reversed-phase high-performance liquid chromatography with tandem
mass spectrometry. The analyses proceed in the positive ion mode by means of the electrospray ionization
method (ESI). Clean up was accomplished using a polimeric surface modified styrene – divinylobenzene SPE
column.
The final method was validated according to international chemical harmonization (ICH) standards.
High selectivity of assay procedure was obtained by choosing optimal columns (most columns contained a bed
modified with C18 groups but varied in the amount of bed: 50-500 mg, and grain size: 33-80 μm) and setting
separation conditions, such as flow rate during sorption (2 and 6 mL/min), sorbent type, pH of the sample
(samples at pH 3 and 7 were examined), solvent strength during desorption (methanol and dichloromethane),
time of evaporation (10, 20, and 30 minutes), type of solvent used for injection (HPLC) (water, methanol and
methanol-water mixture, 1:1, v/v), matrix effect (tap water and river water), and time “sample preparationanalysis”
on recovery (3, 6, and 9 days).
After optimization of sample preparation procedure and analytical conditions environmental water samples
were collected from five sampling sites situated in Gdańsk (Pomerania, Poland) and its outskirts and subjected
to validated methodology. In four samples cyclophosphamide has been quantified.