High-throughput sample preparation and simultaneous column regeneration liquid chromatography-tandem mass spectrometry method for determination of nitrogen mustard metabolites in human urine.

Nitrogen mustards (NMs) are known to have DNA alkylation and strong vesicant properties. Their availability to terrorist organizations makes them a potential choice for chemical attacks on civilian populations. After an exposure, it is difficult to measure NMs directly because of their rapid metabolism in the human body. Therefore to determine an individual's level of exposure to NMs, it is necessary to analyze for NM metabolites being excreted by the body. The metabolites of NMs are generated by a hydrolysis reaction, and are easily detectable by liquid chromatography tandem mass spectrometry (LC-MS/MS). This work is focused on the development of a high-throughput assay for the quantitation of N-ethyldiethanolamine (EDEA) and N-methyldiethanolamine (MDEA) metabolites of bis (2-chloroethyl) ethylethanamine (HN1) and bis (2-chloroethyl) methylethanamine (HN2), respectively. The method uses automated 96-well plate sample preparation of human urine samples and a 2-position 10-port switching valve to allow for simultaneous regeneration of the liquid chromatography (LC) columns. Using this method, over 18 h was saved through the reduction of sample preparation and analysis time when compared to a conventional method for 96 samples. The validated method provided excellent accuracy for both EDEA (100.9%) and MDEA (100.6%) with precision better than 5.27% for each analyte.

Determination of nitrogen mustard hydrolysis products in rat urine samples using GC-MS.

A gas chromatographic-mass spectrometric method was developed, validated and demonstrated by measuring the levels of nitrogen mustard hydrolysis products in the urine collected from dosed rats. The recovery values for trimethylsilyl derivatives of EDEA and MDEA are between 82-95% and 88-112%, respectively. In vivo studies performed by using three different doses (0.5 mg/kg, 1.0 mg/kg, and 2.0 mg/kg) of HN2 base of nitrogen mustard. MDEA concentrations were between 43.1-232.2 ng/mL. The limit of detection (S/N = 3) values are 2.5 ng/mL and 1.6 ng/mL for EDEA and MDEA, respectively, and the precision of the method in terms of RSD is between 5-8%.

Quantitative determination of the hydrolysis products of nitrogen mustards in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry.

Nitrogen mustards are a public health concern because of their extreme vesicant properties and the possible exposure of workers during the destruction of chemical stockpiles. A sensitive, rapid, accurate, and precise analysis for the quantitation of ultratrace levels of N-ethyldiethanolamine (EDEA) and N-methyldiethanolamine (MDEA) in human urine as a means of assessing recent exposure to the nitrogen mustards bis(2-chloroethyl)ethylamine and bis(2-chloroethyl)methylamine, respectively, was developed. The method was based on solid-phase extraction, followed by analysis of the urine extract using isotope-dilution high-performance liquid chromatography-mass spectrometry with TurbolonSpray ionization and multiple-reaction monitoring. The method limits of detection were 0.41 ng/mL for EDEA and 0.96 ng/mL for MDEA in 1 mL of urine with coefficients of variation < 10% for both compounds.

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.

Detection of mutagenic activity in human urine using mutant strains of Salmonella typhimurium.

Histidine-requiring mutants of Salmonella typhimurium that can be reverted to prototrophy by a variety of mutagens were used mutagenic activity in the urine of patients receiving chemotherapeutic agents. Patients given cyclophosphamide and BCNU had detectable urinary mutagenic activity over a 24-hour period, with maximal levels occurring 12 to 21 hours after drug injection. Whereas native cyclophosphamide required the presence of a rat liver extract to be mutagenic in the test system, the cyclophosphamide metabolites in the urine were fully active in the absence of added liver extract. Mutagenic activity was detected in only the first voided urine specimen of patients receiving fluorouracil. Patients receiving Adriamycin, methotrexate, Mitomycin C, and low doses or oral melphalan did not have detectable mutagenic activity in their urines. One thousand and ten random urine speciments were screened for mutagenic activity. Only eight had greater than 26 revertant colonies per plate. Four of the eight had received metronidazole (Flagyl) for vaginitis while two others had received chemotherapeutic drugs. We were unable to detect increased mutagenic metabolites in the urine of 43 patients with known malignancies, using the standard assay conditions.