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.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of the 4-(p-nitrobenzyl) pyridine assay for measurement of melphalan and mechlorethamine in plasma.

A sensitive, reproducible spectrophotometric assay for mechlorethamine and melphalan was developed through modifications in the use of 4-(p-nitrobenzyl) pyridine as the chromogenic reagent. Key factors that led to enhanced color development and consistency of results were: (1) protein precipitation with 4 degrees C perchloric acid; (2) control of the pH of the reaction with acetate buffer; (3) precise timing of color development in alkali; and (4) sample reading at the appropriate wavelength. The modifications allowed measurement of mechlorethamine and melphalan levels in perfusate samples obtained from isolated rat liver perfusions. Half-lives calculated from perfusate decay curves after addition of mechlorethamine at 50 microgram/ml and 10 microgram/ml were 15.9 min and 15.0 min respectively. The perfusate half-life after addition of melphalan at 5 microgram/ml was 44.2 min. Plasma levels of mechlorethamine were not detectable by this assay after intrapleural administration of 15 mg to one patient but were detectable after intravenous administration of 20 mg to a second patient.

Quantitation by gas chromatography-chemical ionization mass spectrometry of cyclophosphamide, phosphoramide mustard, and nornitrogen mustard in the plasma and urine of patients receiving cyclophosphamide therapy.

Unambiguous and sensitive methods based on gas chromatography-chemical ionization mass spectrometry have been developed to quantitate cyclophosphamide and two alkylating and cytotoxic metabolites, phosphoramide mustard and nornitrogen mustard. The levels of these materials have been determined in the plasma and urine of five patients receiving cyclophosphamide, 60 or 75 mg/kg i.v. Peak plasma levels of phosphoramide mustard of 50 to 100 nmoles/ml were found at 3 hr after cyclophosphamide administration. Variable levels of nornitrogen mustard were found in the plasma. This product may be arising in part from the decomposition of other metabolites during sample storage and preparation.