QUANTITATION OF 4-HYDROXYCYCLOPHOSPHAMIDE ALDOPHOSPHAMIDE IN WHOLE-BLOOD

There is considerable interest in determining 4-hydroxycylcophosphamid e/aldophosphamide (4-HO-CP/AP) blood levels in patients receiving the prodrug, cyclophosphamide (CP). Phosphoramide mustard (PM), the alkyla ting metabolite of CP, is relatively impermeable to cell membranes and it is generally believed that circulating intermediary metabolites, i ncluding aldophosphamide, the immediate precursor of PM, is; transport ed by circulating blood to tumor tissue. Therefore, circulating 4-HO-C P/AP blood levels should more closely reflect the oncostatic and cytot oxic effects of CP than the parent drug. We have developed a gas chrom atographic electron-impact mass spectrometric (GC-EIMS) method suitabl e for routine monitoring of 4-HO-CP/AP levels in whole blood over the range 0.085 mu M (25 ng/ml) to 34 mu M (10 mu g/ml). The unstable meta bolites were derivatized with O-(2,3,4,5,6-pentafluorobenzyl)hydroxyla mine-HCl to form a stable aldophosphamide oxime derivative (PBOX). [H- 2(4)]PBOX was used as an internal standard. For clinical samples, tube s were prepared prior to blood drawing, which contained the derivatizi ng reagent solution and the internal standard. These solutions were st able for up to 3 months when stored at room temperature. Following add ition of blood to the reaction tubes, PBOX formation was rapid and the resulting derivative was stable under these conditions for up to 8 da ys at room temperature. Application of the method was demonstrated by quantitating 4-HO-CP/AP blood levels in patients receiving 4 g/m(2) in travenous infusion of CP over a period of 90 min.