Determination of cyclophosphamide in human urine by HPLC coupled to tandem mass spectrometry

Marahatta, Anu

http://hdl.handle.net/10400.1/10745

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Authors

Marahatta, Anu

Advisor(s)

Wasik, Agata Kot

Wasik, Andrzej

Abstract(s)

Biological samples urine are much more complex due to presence of proteins, salts, acids, bases and various organic compounds with similar chemistry to analytes of interest. As a result, the extraction methods for biological samples have been difficult. The main task was to remove maximum interfering substance from urine matrix and conversion of analytes into a more suitable form for injection. Therefore, optimization of SPE method was done by changing different parameter such as different type and amount of sorbent, composition and volume of elution, washing solvent. The use of 24 well SPE plate for matrix purification significantly allows the use of small urine and solvent volumes, reduces the sample preparation time and ensures a high throughput, thus allowing the routine biological monitoring of CP as indices of human exposures. In order to investigate the effectiveness of the SPE method tests were repeated on Strata-X cartridge and method showed good recovery (above 75 %) of CP. After optimizing SPE method, CP separation conditions of the HPLC-MS/MS analysis was achieve by changing different parameter such as suitable mobile phase and its composition, flow rate, scan type, polarity. The LC separation was performed on Lichrocart® 100 RP-18 column (125 4 mm, particle size of 5 μm) with 1 mM formic acid with ammonia buffer pH 3 and methanol using gradient program at a flow rate 1 mL/min with total run time 15 min. A reversed phase HPLC system was interfaced with an ESI source coupled to tandem mass spectrometry. The triple quadrupole mass spectrometer was operated in positive ion mode and MRM was used for analysis of CP. LOD and LOQ were 0.27 ng/mL and 0.54 ng/mL. A sensitive, specific and accurate HPLC/MS/MS has been developed for monitoring CP in urine samples. The use of reversed phase HPLC coupled to tandem mass spectrometry has facilitated the analysis of CP in short retention time. Fifteen urine samples were collected from medical personnel of Medical University of Gdansk, who were exposed with CP during their work in pharmacy. SPE-HPLC-MS/MS method was applied for the analysis of health workers and all urine samples from university workers has CP below analytical limit of detection.