DETERMINATION OF REACTIVE NITROGEN-MUSTARD ANTICANCER DRUGS IN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING DERIVATIZATION

A high-performance liquid chromatography (HPLC) method is described for the determination of reactive nitrogen mustard anticancer drugs in plasma after derivatization with diethyl-dithlocarbamic acid (DDTC). Three compounds were studied: two reactive species (mechlorethamine (HN2) and galactose 6-mustard (G-6-M)) and a less reactive species (melphalan (L-PAM)) included for validation experiments. Mass and NMR spectrometry confirmed that one molecule of DDTC reacts with each arm of the mustard, displacing a chlorine atom to form a stable disubstituted adduct. With the reactive mustards a 30-min incubation at 37-degrees-C is recommended for > 90% derivatization efficiency. Gradient elution was employed to analyze all three compounds using the same conditions with a mu-Bondapak C18 10-mu-m particle size column (30 cm by 3.8 mm i.d.). The retention time (t(R)) of HN2-DDTC2 was 13.1 min +/- 1.5% within day CV; t(R) of L-PAM was 7.6 min and L-PAM-DDTC2 was 14.6 min +/- 0.8% CV. G-6-M-DDTC2 yielded a double peak, t(R) = 10.7 min and 10.9 min +/- 2.9% CV. The limit of detection on column was 0.5 ng for HN2, 1 ng for L-PAM, and 5 ng for G-6-M. A solid-phase sample preparation technique using "Bond Elut" phenyl is described that extracts from plasma G-6-M-DDTC2 with > 74% efficiency and HN2-DDTC2 with > 90% efficiency. When the drugs were derivatized in plasma, recovery remained high for G-6-M (> 84%) but dropped to 50% for HN2. The chemical half-life in plasma was 9 min for G-6-M and 37 min for HN2. For pharmacokinetic studies, it is recommended that 10 mg of DDTC/mL of blood is added to specimen containers in order to trap the reactive mustard in situ prior to analysis by HPLC.