Sample handling for determination of free platinum in blood after cisplatin exposure

Cisplatin is a commonly used cytostatic drug that can be pharmacokinetically monitored by measurement of non-protein-bound platinum (Pt) in plasma. The present report elucidates some practical aspects on blood sample preparation with the purpose of identifying methodological error sources and simplifying sample handling. Human blood was incubated in vitro with clinically relevant doses of cisplatin and then stored at different temperatures for various periods prior to deproteinization. Pt concentrations were analyzed by inductively coupled plasma mass spectrometry, which is a very sensitive technique that is well suited for determination of free Pt in biological samples. Free Pt concentration is usually determined after ultrafiltration of plasma. An alternative approach used to study the protein-free fraction is to precipitate the proteins with ice-cold ethanol and then analyze the aliquot. We compared the Pt concentrations in protein-free plasma obtained by these two methods and the levels were very similar. Thus, ethanol deproteinization may be an attractive alternative to ultrafiltration, since it is both simple to perform and very cost-effective. Storage of nondeproteinized whole blood or plasma at 4 degrees C or 20 degrees C did not affect the free Pt fraction significantly for the first 2 h, after which a gradual decrease was noted. This suggests that saving the blood sample for approximately 1 h before processing should not have any major impact on the result. On long-term storage of frozen blood samples prior to deproteinization the freezing temperature proved to be of critical importance. At -20 degrees C there was a gradual decrease in the free Pt fraction during the study period of 14 days, whereas the free Pt concentration remained unchanged at -70 degrees C for at least 3 months. Thus, samples may be stored unprocessed at -70 degrees C for several months, whereas -20 degrees C is not sufficiently cold.