Gas chromatographic-mass spectrometric assay for N-2-chloroethylaziridine, a volatile cytotoxic metabolite of cyclophosphamide, in rat plasma.
A sensitive and specific method for the quantitative analysis of N-2-chloroethylaziridine (CEA), a volatile cytotoxic metabolite of cyclophosphamide, has been developed using gas chromatography-mass spectrometry and stable isotope dilution techniques. The high volatility problem of CEA during isolation procedure was overcome by the combined use of a deuterium-labeled analog as the internal standard and a Snyder column-concentrator assembly. The assay was found to be linear from 16.7 to 2667 ng/ml in rat plasma with a routine detection limit of 5 ng/ml. The within-run precision at 33, 333 and 1333 ng/ml (n = 6) was found to be 4.8, 4.9, and 6.1%, respectively. The between-run precision was 6.4% (n = 6). The dichloromethane extraction recoveries at 33, 333, and 1333 ng/ml were found to be 101, 98, and 91%, respectively (all at n = 6). However, the overall recovery through extraction and evaporation was only 18.3, 15.2, and 27.7% at 33, 333, and 1333 ng/ml levels, respectively. The analytical method was used to evaluate the generation of CEA from its precursors in sodium phosphate buffer, in cell culture media, and the degradation of CEA in these media. In pH 7.4, 0.067 M sodium phosphate buffer at 37 degrees C, both phosphoramide mustard (PM) and nornitrogen mustard (NNM) were degraded in an apparent first-order fashion with half-lives of 24.8 and 14.5 min, respectively. The generated CEA was rather stable in this buffer and degraded with a half-life of 20 h. It was found that 32% PM and 91% NNM were converted to CEA in pH 7.4, 0.067 M sodium phosphate buffer at 37 degrees C, respectively, and 41% PM was transformed into CEA in RPMI 1640 tissue culture media containing 10% FBS at 37 degrees C. The generated CEA was very stable in the culture media with a degradation half-life of 265 h.
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College of Pharmacy, Ohio State University, Columbus 43210, USA.
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D000818:Animals; D000970:Antineoplastic Agents; D001388:Aziridines / Q000097:blood* / Q000737:chemistry; D002021:Buffers; D003470:Culture Media; D003520:Cyclophosphamide / Q000378:metabolism*; D003903:Deuterium; D004355:Drug Stability; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods* / Q000706:statistics & numerical data; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007201:Indicator Dilution Techniques; D007700:Kinetics; D008752:Methylene Chloride; D009588:Nitrogen Mustard Compounds / Q000737:chemistry; D010752:Phosphoramide Mustards / Q000737:chemistry; D051381:Rats; D012680:Sensitivity and Specificity; D014835:Volatilization
D000970:Antineoplastic Agents; D001388:Aziridines; D002021:Buffers; D003470:Culture Media; D009588:Nitrogen Mustard Compounds; D010752:Phosphoramide Mustards; C030090:phosphoramide mustard; D008752:Methylene Chloride; C103230:N-(2-chloroethyl)aziridine; D003520:Cyclophosphamide; D003903:Deuterium; C004933:nornitrogen mustard