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-dithiocarbamic 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 greater than 90% derivatization efficiency. Gradient elution was employed to analyze all three compounds using the same conditions with a microBondapak C18 10-microns particle size column (30 cm by 3.8 mm i.d.). The retention time (tR) of HN2-DDTC2 was 13.1 min +/- 1.5% within day CV; tR 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, tR = 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 greater than 74% efficiency and HN2-DDTC2 with greater than 90% efficiency. When the drugs were derivatized in plasma, recovery remained high for G-6-M (greater than 84%) but dropped to 50% for HN2.