Ethanolysis of nitrogen mustards: A novel strategy for nitrogen mustard identification in environmental matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry.

RATIONALE: Nitrogen mustards (NMs) are blistering chemical warfare agents. The ability to detect NMs in environmental samples is very important for obtaining forensic evidence. The most common analytical techniques for NM detection are gas chromatography-mass spectrometry, which detects NMs in their intact form but is disadvantaged by high limits of detection (LODs), and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) of their hydrolysis products, which do not provide robust evidence to support NM use. METHODS: We developed a novel approach to detect and identify NMs using LC/ESI-MS/MS after chemical derivatization. The method is based on ethoxide-promoted ethanolysis prior to analysis. The effects of reaction time, temperature, ethoxide concentration and chromatography behavior were studied and optimized. In the developed procedure, 0.1% (v/v) sodium ethoxide solution is added to the NMs in ethanol and agitated for 2 h at 50°C, followed by LC/ESI-MS/MS, without any other pretreatment. RESULTS: The ethanolysis reaction efficiencies were evaluated in ethanolic extracts from soil, asphalt, and ethanol contaminated with 0.5% (v/v) diesel fortified with NMs at a five-point calibration curve. The calibration curves showed good linearity in the range of 0.05-1 ng/mL, with an R2 value of 0.99, and were similar to those of LC/MS-grade ethanol, with almost no observable matrix effects. The derivatization products were stable at room temperature, with LODs of 10 pg/mL, in all investigated extracts. CONCLUSIONS: Through this newly developed strategy, the derivatization of active NMs by ethanolysis was achieved for the first time, and these derivatization products can serve as specific indicators for the use and presence of NMs. The methodology can also verify trace levels of NM chemical warfare agents collected in war or terror scenarios in forensic investigations.

Analytical methods based on liquid chromatography for the analysis of albumin adducts involved in retrospective biomonitoring of exposure to mustard agents.

The objective of the present review is to list, describe, compare, and critically analyze the main procedures developed in the last 20 years for the analysis of digested alkylated peptides, resulting from the adduction of albumin by different mustard agents, and that can be used as biomarkers of exposure to these chemical agents. While many biomarkers of sulfur mustard, its analogues, and nitrogen mustards can easily be collected in urine such as their hydrolysis products, albumin adducts require blood or plasma collection to be analyzed. Nonetheless, albumin adducts offer a wider period of detectability in human exposed patients than urine found biomarkers with detection up to 25 days after exposure to the chemical agent. The detection of these digested alkylated peptides of adducted albumin constitutes unambiguous proof of exposure. However, their determination, especially when they are present at very low concentration levels, can be very difficult due to the complexity of the biological matrices. Therefore, numerous sample preparation procedures to extract albumin and to recover alkylated peptides after a digestion step using enzymes have been proposed prior to the analysis of the targeted peptides by liquid chromatography coupled to mass spectrometry method with or without derivatization step. This review describes and compares the numerous procedures including a number of different steps for the extraction and purification of adducted albumin and its digested peptides described in the literature to achieve detection limits for biological samples exposed to sulfur mustard, its analogues, and nitrogen mustards in the ng/mL range.

In Vitro Evaluation of DNA Damage Effect Markers toward Five Nitrogen Mustards Based on Liquid Chromatography-Tandem Mass Spectrometry.

Endogenous DNA lesions frequently occur due to internal effects such as oxidative stress, inflammation, endogenous alkylation, and epigenetic modifications. However, exposure to chemical toxicants from the environment, diet, or drugs can also induce significant endogenous DNA damage. The quantification of endogenous DNA damage effect markers might reflect the actual DNA damage level of chemical toxicants. Herein, we report a liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ MS/MS) method for simultaneous determination of eight representative endogenous DNA damage biomarkers, including five endogenous DNA damage effect markers (oxidative damage, 8-oxo-dG; lipid peroxidation, εdA and N2-Et-dG; inflammation, 5-Cl-dC; and endogenous alkylation, O6-Me-dG), and three epigenetic modifications (5-m-dC, 5-hm-dC, and N6-Me-dA). The method validation was performed, and the linear range was 0.05 pg to 2 ng (on-column), the limit of detection was 0.02 pg (on-column), and the precision, accuracy, matrix effect, and recovery were all between 85 and 115%. We then applied this method to evaluate endogenous DNA damage to human embryonic lung fibroblast cells exposed to five nitrogen mustards [NMs, i.e., HN1, HN2, HN3, chlorambucil (CB), and cyclophosphamide (CTX)], where curcumin exposure was used as a control due to its inability to induce the formation of endogenous DNA adducts. The amounts of eight DNA adducts in the low-, middle-, and high-concentration exposure groups of five NMs were almost all significantly different from those in the blank group (P < 0.05). We obtained a positive correlation between the contents of eight DNA damage biomarkers and the inhibition dose of five NMs, except for N2-Et-dG and 5-Cl-dC. Via further principal component analysis and partial least squares discriminant analysis, we clustered all NMs into three units with different cytotoxicity levels, that is, HN2 and HN1 (highly toxic), HN3 and CB (moderately toxic), and CTX (less toxic). Moreover, for the same concentration of HN1/2/3 exposure groups, as the cytotoxicity increased according to the order of HN3 < HN1 < HN2, the contents of 8-oxo-dG, 5-m-dC, 5-hm-dC, and N6-Me-dA increased, whereas the content of O6-Me-dG decreased. Therefore, the contents of these DNA damage effect markers were somewhat related to the cytotoxicity and concentration of NMs. We hope that this method will provide an alternative evaluation approach for the toxicological effects of NMs and the safety of the medication.

Extraction-spectrophotometric determination of tris(2-chloroethyl)amine using phthaleins.

Procedures for the extraction-spectrophotometric determination of tris(2-chloroethyl)amine, an alkylating agent known as a drug as well as a chemical warfare agent (nitrogen mustard HN-3), with 7 acid-base indicators of a triphenylmethane lactone type, phthaleins, were developed. Representatives of phthaleins without an oxygen bridge (thymolphthalein, o-cresolphthalein, naphtholphthalein) and with an oxygen bridge (fluorescein, 2',7'-dichlorofluorescein, eosin B and eosin Y) were used. The methods were based on the formation of ion pair complexes. Chloroform was used as a non-polar solvent for an extraction. The conditions to determine were optimized for the optimal pH of the buffer and the concentration of a phthalein as a reagent. The dependence on the reaction time in a water phase and the stoichiometry of extraction products were studied. The detection limits and the limits of the determination of separate procedures and conditional extraction constants were determined. Comparison with the spectrophotometric method of the group determination of alkyl halides and acyl halides using alkaline ethanol-water solution of thymolphthalein, the so-called T-135 agent, was conducted. While studying the selectivity, the possible interference of bis(2-chloroethyl)sulphide and 3 nitrogen mustards in the proposed procedures were verified. Copyright © 2016 John Wiley & Sons, Ltd.

Rapid screening of N-oxides of chemical warfare agents degradation products by ESI-tandem mass spectrometry.

Rapid detection and identification of chemical warfare agents and related precursors/degradation products in various environmental matrices is of paramount importance for verification of standards set by the chemical weapons convention (CWC). Nitrogen mustards, N,N-dialkylaminoethyl-2-chlorides, N,N-dialkylaminoethanols, N-alkyldiethanolamines, and triethanolamine, which are listed CWC scheduled chemicals, are prone to undergo N-oxidation in environmental matrices or during decontamination process. Thus, screening of the oxidized products of these compounds is also an important task in the verification process because the presence of these products reveals alleged use of nitrogen mustards or precursors of VX compounds. The N-oxides of aminoethanols and aminoethylchlorides easily produce [M + H](+) ions under electrospray ionization conditions, and their collision-induced dissociation spectra include a specific neutral loss of 48 u (OH + CH2OH) and 66 u (OH + CH2Cl), respectively. Based on this specific fragmentation, a rapid screening method was developed for screening of the N-oxides by applying neutral loss scan technique. The method was validated and the applicability of the method was demonstrated by analyzing positive and negative samples. The method was useful in the detection of N-oxides of aminoethanols and aminoethylchlorides in environmental matrices at trace levels (LOD, up to 500 ppb), even in the presence of complex masking agents, without the use of time-consuming sample preparation methods and chromatographic steps. This method is advantageous for the off-site verification program and also for participation in official proficiency tests conducted by the Organization for the Prohibition of Chemical Weapons (OPCW), the Netherlands. The structure of N-oxides can be confirmed by the MS/MS experiments on the detected peaks. A liquid chromatography-mass spectrometry (LC-MS) method was developed for the separation of isomeric N-oxides of aminoethanols and aminoethylchlorides using a C18 Hilic column. Critical isomeric compounds can be confirmed by LC-MS/MS experiments, after detecting the N-oxides from the neutral loss scanning method.

Stability-indicating LC method for the estimation of bendamustine hydrochloride and its related impurities.

A novel, simple, sensitive and stability-indicating high-performance liquid chromatography method was developed and validated for the quantification of impurities (process related and degradants) and the assay determination of Bendamustine hydrochloride. A chromatographic separation of Bendamustine and its impurities was achieved with an Inertsil ODS-2 analytical column, 250 × 4.6 mm, 5 µm, using gradient elution with mobile phase A consisting of a mixture of water and trifluoroacetic acid (1000:1, v/v) and mobile phase B consisting of acetonitrile. The instrumental settings included a flow rate of 1.0 mL/min, column temperature of 27°C and a detector wavelength of 233 nm, using a photodiode array detector. The tailing factor for Bendamustine was 1.10. Bendamustine hydrochloride was exposed to thermal, photolytic, hydrolytic and oxidative stress conditions and the stressed samples were analyzed by the proposed method. Peak homogeneity data of Bendamustine were obtained by using a photodiode array detector in the stressed sample chromatograms, which demonstrated the specificity of the method for estimation in the presence of degradants. The developed method was validated for parameters such as precision, accuracy, linearity, limit of detection, limit of quantification, ruggedness and robustness. The stability tests were also performed on drug substances as per International Conference on Harmonization guidelines.

Development and validation of sensitive liquid chromatography/tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue.

A liquid chromatography-tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue was developed and fully validated. Methanol was used to precipitate proteins in brain tissue. Bendamustine and internal standard (chlorambucil) were separated with reverse-phase chromatography on a C-18 column with a gradient of water and 95% methanol in 0.1% formic acid. Positive mode electrospray ionization was applied with selected reaction monitoring to achieve 5 ng/ml lower limits of quantitation in mouse brain tissue. The calibration curve for bendamustine in mouse brain was linear between 5 and 2000 ng/ml. The within- and between-batch accuracy and precision of the assay were within 15% at 10, 100 and 1000 ng/ml. The recovery and matrix effect of bendamustine in mouse brain tissue ranged from 41.1% to 51.6% and 107.4% to 110.3%, respectively. The validated method was then applied to quantitate bendamustine in an animal study. Results indicate the assay can be applied to evaluate bendamustine disposition in mouse brain tissue. This assay will be applied in the future to detect and quantify bendamustine in human brain tissue samples.

Metabolism and excretion of the novel bioreductive prodrug PR-104 in mice, rats, dogs, and humans.

PR-104 is the phosphate ester of a 3,5-dinitrobenzamide nitrogen mustard (PR-104A) that is reduced to active hydroxylamine and amine metabolites by reductases in tumors. In this study, we evaluate the excretion of [(3)H]PR-104 in mice and determine its metabolite profile in mice, rats, dogs, and humans after a single intravenous dose. Total radioactivity was rapidly and quantitatively excreted in mice, with cumulative excretion of 46% in urine and 50% in feces. The major urinary metabolites in mice were products from oxidative N-dealkylation and/or glutathione conjugation of the nitrogen mustard moiety, including subsequent mercapturic acid pathway metabolites. A similar metabolite profile was seen in mouse bile, mouse plasma, and rat urine and plasma. Dogs and humans also showed extensive thiol conjugation but little evidence of N-dealkylation. Humans, like rodents, showed appreciable reduced metabolites in plasma, but concentrations of the cytotoxic amine metabolite (PR-104M) were higher in mice than humans. The most conspicuous difference in metabolite profile was the much more extensive O-beta-glucuronidation of PR-104A in dogs and humans than in rodents. The structure of the O-beta-glucuronide (PR-104G) was confirmed by independent synthesis. Its urinary excretion was responsible for 13 +/- 2% of total dose in humans but only 0.8 +/- 0.1% in mice. Based on these metabolite profiles, biotransformation of PR-104 in rodents is markedly different from that in humans, suggesting that rodents may not be appropriate for modeling human biotransformation and toxicology of PR-104.

Single-molecule detection of nitrogen mustards by covalent reaction within a protein nanopore.

Mustards, including sulfur mustards and nitrogen mustards, form a class of cytotoxic, vesicant chemical warfare agents. Mustards have also been used to treat cancer and played a vital role in the development of chemotherapy. Additionally, because of their destructive properties, ease of synthesis, and the lack of effective antidotes, mustards are unquestionably terrorist threats. Therefore, quick and convenient detection of mustards is a critical issue. In the present study, we achieved detection of various mustards on the basis of their chemical reactivity by using engineered alpha-hemolysin (alphaHL) protein pores as sensor elements. We describe four classes of reactions for detecting mustards. These reactions occur between mustards and thiol groups contributed by cysteine side-chains within the lumen of the alphaHL pore or on an internal molecular adapter. The approach is quick and straightforward. It can confirm the existence of mustards in as little as 10 min at 50 microM or lower.

Development of a liquid chromatography-multiple reaction monitoring procedure for concurrent verification of exposure to different forms of mustard agents.

A novel liquid chromatography-multiple reaction monitoring (LC-MRM) procedure has been developed for retrospective diagnosis of exposure to different forms of mustard agents. This concise method is able to validate prior exposure to nitrogen mustards (HN-1, HN-2, and HN-3) or sulfur mustard (HD) in a single run, which significantly reduces analysis time compared to separate runs to screen for different mustards' biomarkers based on tandem mass spectrometry. Belonging to one of the more toxic classes of chemical warfare agents, these potent vesicants bind covalently to the cysteine-34 residue of human serum albumin. This results in the formation of stable adducts whose identities were confirmed by a de novo sequencing bioinformatics software package. Our developed technique tracks these albumin-derived adduct biomarkers in blood samples which persist in vitro following exposure, enabling a detection limit of 200 nM of HN-1, 100 nM of HN-2, 200 nM of HN-3, or 50 nM of HD in human blood. The CWA-adducts formed in blood samples can be conveniently and sensitively analyzed by this MRM technique to allow rapid and reliable screening.

Applying pattern recognition methods to analyze the molecular properties of a homologous series of nitrogen mustard agents.

The purpose of this research was to analyze the pharmacological properties of a homologous series of nitrogen mustard (N-mustard) agents formed after inserting 1 to 9 methylene groups (-CH2-) between 2 -N(CH2CH2Cl)2 groups. These compounds were shown to have significant correlations and associations in their properties after analysis by pattern recognition methods including hierarchical classification, cluster analysis, nonmetric multi-dimensional scaling (MDS), detrended correspondence analysis, K-means cluster analysis, discriminant analysis, and self-organizing tree algorithm (SOTA) analysis. Detrended correspondence analysis showed a linear-like association of the 9 homologs, and hierarchical classification showed that each homolog had great similarity to at least one other member of the series-as did cluster analysis using paired-group distance measure. Nonmetric multi-dimensional scaling was able to discriminate homologs 2 and 3 (by number of methylene groups) from homologs 4, 5, and 6 as a group, and from homologs 7, 8, and 9 as a group. Discriminant analysis, K-means cluster analysis, and hierarchical classification distinguished the high molecular weight homologs from low molecular weight homologs. As the number of methylene groups increased the aqueous solubility decreased, dermal permeation coefficient increased, Log P increased, molar volume increased, parachor increased, and index of refraction decreased. Application of pattern recognition methods discerned useful interrelationships within the homologous series that will determine specific and beneficial clinical applications for each homolog and methods of administration.

Applications of deactivated GC columns for analysis of nitrogen-containing chemicals related to the chemical weapons convention.

Nitrogen-containing chemicals are one of the important families of compounds relevant to the purposes of the Chemical Weapons Convention (CWC). Several applications, using various injection modes, of new deactivated columns specially designed for basic compounds are presented. These columns prove remarkably well-suited to the gas chromatographic (GC) analysis of the chemicals of interest, even to underivatized amino-alcohols, whose analysis on conventional GC columns is often difficult and hindered by poor resolution and high detection limits. Such a deactivated phase can even replace the typical GC phases used for CWC verification purposes.

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.

Identification of the nitrogen-based blister agents bis(2-chloroethyl)methylamine (HN-2) and tris(2-chloroethyl)amine (HN-3) and their hydrolysis products on soil using ion trap secondary ion mass spectrometry.

The nitrogen blister agents HN-2 (bis(2-chloroethyl)methylamine) and HN-3 (tris(2-chloroethyl)amine) were directly analyzed on the surface of soil samples using ion trap secondary ion mass spectrometry (SIMS). In the presence of water, HN-1 (bis(2-choroethyl)ethylamine), HN-2 and HN-3 undergo hydrolysis to form N-ethyldiethanolamine, N-methyldiethanolamine and triethanolamine (TEA), respectively; these compounds can be readily detected as adsorbed species on soil particles. When soil samples spiked with HN-3 in alcohol were analyzed, 2-alkoxyethylamine derivatives were observed on the sample surfaces. This result shows that nitrogen blister agents will undergo condensation reactions with nucleophilic compounds and emphasizes the need for an analytical methodology capable of detecting a range of degradation and condensation products on environmental surfaces. The ability of ion trap SIMS to isolate and accumulate ions, and then perform tandem mass spectrometric analysis improves the detection of low-abundance surface contaminants and the selectivity of the technique. Utilizing these techniques, the limits of detection for HN-3 were studied as a function of surface coverage. It was found that HN-3 could be detected at a surface coverage of 0.01 monolayer, which corresponds to 20 ppm (mass/mass) for a soil having a surface area of 2.2 m(2) g(-1). TEA, the exhaustive hydrolysis product of HN-3, was detected at a surface coverage of 0.001 monolayer, which corresponds to 0.86 ppm.

Detection of the chemical warfare agents bis-(2-chloroethyl)ethylamine (HN-1) and tris-(2-chloroethyl)amine (HN-3) in air.

This paper describes the method development and validation for detection of the chemical warfare agents HN-1 and HN-3 in air using C8 solid-phase extraction disks followed by liquid desorption and analysis by gas chromatography. The method is contrasted to the standard approach which uses solid sorbent tubes followed by thermal desorption and analysis by gas chromatography.

Oxazolidonylethyl adducts to hemoglobin and DNA following nornitrogen mustard exposure.

Formation of adducts to hemoglobin (Hb) and DNA of nornitrogen mustard (NNM) was studied with the aim of developing a method for monitoring exposure to NNM. Adducts to N-terminal valines in Hb were studied by the N-alkyl Edman method using pentafluorophenyl isothiocyanate (PFPITC) as the derivatizing reagent. In preliminary studies five major Hb adducts were shown to be formed in reaction of NNM with red cell hemolysate in vitro. Following treatment with PFPITC three of these were found to be pentafluorophenylthiohydantoins (PFPTHs) of N-alkylated valines and the fourth probably originates from NNM esters in which PFPITC had reacted with the nitrogen of N-chloroethylaminoethyl. A PFPTH was found to originate from N-2-(3-oxazolidonyl)ethylvaline, Val-OZ. Val-OZ is formed in reaction, with ring closure to oxazolidone, of CO2 with the 2-chloroethylamino group in the primary valine-N adduct. Besides a few other adducts, Val-OZ was also observed in mouse Hb following injection of NNM, and also after injection of cyclophosphamide. Following reaction in vitro of NNM with DNA, three major adducts to guanine-N-7 were observed; one of them, 7-(N'-(2-chloroethyl)-2-aminoethyl]-guanine (NNMCl), was converted by carbonate to 7-(2-3-oxazolidonyl)ethyl]guanine (Gua-OZ). In mice treated with NNM, Gua-Oz was the only DNA adduct observed. Val-Oz is a chemically stable Hb adduct, potentially useful for monitoring exposures to NNM and cyclophosphamide.

Occupational exposure to nor-nitrogen mustard: chemical and biological monitoring.

Nor-nitrogen mustard is an alkylating agent used in kilogram quantities in the production of various cytostatics. It has been shown to be mutagenic in vitro, and therefore must be regarded as a health hazard. We have studied the environment and blood of a small group of individuals working with nor-nitrogen mustard. New chemical methods for measuring surface contamination were used to make comparisons with ambient air levels of nor-nitrogen mustard. Surprisingly high levels of surface contamination were found even after decontamination, which has led to new decontamination routines and the establishment of wipe test limits of 0.5 microgram nor-nitrogen mustard/dm2. The wipe test has proved effective in following the day-to-day routine handling of nor-nitrogen mustard. Blood samples were taken before, during, and after production runs and analyzed for the frequency of chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and somatic (HPRT) mutations. No significant differences in chromosome aberrations, SCEs, or somatic mutations were found in the peripheral blood from production workers before, during, or after handling of nor-nitrogen mustard. Nor was any difference found between the production workers and a local control group. However, the local controls' chromosome and HPRT frequencies appeared to be somewhat high, demonstrating the problems involved in these types of studies, in which the group sizes are small.

Electrospray mass spectrometric analysis of new alpha-MSH analogues carrying nitrogen mustard at their amino terminus.

Two different chlorinated drugs, chlorambucil and melphalan, have been linked to the carrier alpha-melanocyte-stimulating hormone at the end of the solid-phase peptide synthesis of the hormone. The [Nle4, D-Phe7] and the [Nle4, L-Phe7] derivatives of the hormone have both been used. It was found by electrospray mass spectrometric analysis that the products undergo hydrolysis of the chlorinated moiety of the drugs, most likely due to the acidic conditions necessary for cleavage of the peptide from the resin. Only the melphalan-O(ethyl)-N(succinyl)-derivative of alpha-melanocyte-stimulating hormone [Nle4, L-Phe7] did not show a bis-hydroxylated form. It was proven by blank experiments with commercially available melphalan that this mustard occurs for some 45%-50% in the mono-hydroxylated form, which is known to be pharmacologically poorly active.

Cellular kinetics of prednimustine versus chlorambucil plus prednisolone in vitro.

Intracellular concentrations of prednimustine (PM), chlorambucil (CLB), phenylacetic acid mustard (PAAM) and prednisolone (P) were measured in different experimental tumor cell lines that had been incubated with either PM or CLB + P. For intracellular analytical determination, we modified a high-pressure liquid chromatographic method for the detection of these substances in plasma. Intact PM could be detected in the intracellular compartment of the incubated tumor cells. PM-incubated cells from PM-injected rats exhibited a higher intracellular concentration-time integral (PAAM) and longer concentration-time profiles for drugs with alkylating capacity than did cells exposed to the CLB + P mixture or to CLB. PAAM was not detectable after incubation of cells with PM, whereas in CLB-incubated cells the AUC of PAAM exceeded that of the parent drug CLB. Our in vitro results therefore favour the concept of a facilitated intracellular uptake and an increased antiproliferative effect for PM versus CLB and CLB + P.

Accumulation of estramustine and estromustine in adipose tissue of rats and humans.

The tissue distribution of estramustine and estromustine, two cytotoxic lipophilic metabolites of estramustine phosphate (Estracyt, EMP) was studied in rats and humans. A single dose of [3H]-estramustine was given i.v. to groups of rats. At 24 h after administration, the concentration of radioactivity in fat was about 20, 12, and 2 times that in muscle, plasma, and liver, respectively. Liquid chromatography verified that the radioactivity represented estramustine and estromustine. The clinical relevance of these results was investigated in pancreas cancer patients treated with a single oral dose of Estracyt at 12-16 h before surgery. As judged by gas chromatography, the concentration of estromustine, which is the main metabolite in man, was about 13 times higher in fat than in plasma and was also higher in adipose tissue than in muscle and liver. After 5 days of Estracyt treatment, the adipose uptake of estromustine was even higher, namely, about 40 times that in plasma and 8 times that in muscle and liver. Thus, our results demonstrate that estramustine and estromustine are stored in adipose tissue after the administration of EMP; this is important for the pharmacokinetics and, consequently for the therapeutic effects of Estracyt.