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Trace analysis of cyclophosphamide and its metabolites in urine by liquid chromatography-tandem mass spectrometry Troster, Charles Micah Smolkin


Cyclophosphamide (CP) is an antineoplastic drug used to treat a wide variety of cancers and immune disorders. CP is also a highly toxic alkylating agent, classified as an IARC Group 1 carcinogen. Workers in health-care environments are vulnerable to occupational exposure to CP, primarily via inhalation and dermal absorption. CP is a prodrug; both its therapeutic effectiveness and toxicity are activated through metabolism. To date, however, no study measuring occupational exposure to CP has successfully analyzed its metabolites. The main objective of this work was to develop an analytical method for CP, as well as its metabolites 4-ketocyclophosphamide (KCP) and carboxyphosphamide (CBP), in urine samples collected from health-care workers at risk of CP exposure. A liquid chromatography-tandem mass spectrometry (LC/MS-MS) method was optimized for CP, KCP and CBP on two different instruments. Post-column infusion showed that the matrix effects resulting from synthetic urine could be separated from the analyte peaks by LC. Estimated instrument limits of quantitation for CP, KCP and CBP in neat solvent were respectively 4.2, 8.2 and 57 ng/L. These parameters were sufficient to meet a quantitation target of 50 ng/L CP in urine, but suggested a need to reduce sample volume to reach a 2.5 ng/L target for KCP and CBP. Solid-phase extraction (SPE) was explored as a means to exchange the sample matrix for clean solvent and reduce sample volume. Previously developed SPE methods for CP were not designed to include the more polar metabolites, and thus required modification. The best retention of metabolites was seen on a C¹⁸ sorbent with reduced carbon loading. Retention was improved further under acidic loading conditions, but this had to be controlled carefully since CBP can decompose more rapidly at acidic pH. All three analytes were observed to elute with methanol.

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