Identification of urinary metabolites of 2,4,6-trinitro-toluene in rats by liquid chromatography-mass spectometry

205

Toxicology Letters, 26 (1985) 205-209 Elsevier

TOXLett.

1446

IDENTIFICATION

OF URINARY

METABOLITES

OF 2,4,6-TRINITRO-

TOLUENE IN RATS BY LIQUID SPECTOMETRY*

CHROMATOGRAPHY-MASS

(TNT

aminodinitrotoluenes;

toxicity;

J. YINON

reduction

and D.-G.

processes;

diaminonitrotoluenes)

HWANG

Department of Isotope Research, The Weizmann Institute of Science, Rehovor (Israel). (Received

January

(Accepted

June 4th,

22nd,

1985)

1985)

SUMMARY Metabolites extracts cluded

of 2,4,6-trinotrotoluene

were analysed TNT

itself

2,4-diamino-6nitrotoluene,

(TNT) were found

by liquid chromatography-mass as

well

as

indicating

in the urine of rats fed with TNT. spectrometry

2-amino-4,6-dinitrotoluene, that reduction

processes

(LC-MS).

Metabolites

The urine found

4-amino-2,6-dinitrotoluene are reponsible

for the formation

inand

of these

metabolites.

INTRODUCTION

The metabolism of TNT in humans and the analysis of TNT and its metabolites in body fluids are of great importance in the biomedical and environmental fields. Munition workers are exposed to TNT in various ways, such as contact and inhalation of dust and vapor. Part of the inhaled TNT finds its way into the gastrointestinal tract [l]. The toxic action of TNT in humans may lead to toxic jaundice, aplastic anemia, cyanosis, dermatitis, nose-bleeds, constipation and giddiness [2]. Periodical analysis of the body fluids of personnel working in explosives manufacturing plants must therefore be made to check for of traces of TNT and its metabolites in blood and urine. Improper disposal of obsolete explosives may cause serious contamination pro-

*Paper

3 in the series: Metabolic

studies

Abbreviations:

4-A,4-amino-2,6-dinitrotoluene;

6-nitrotoluene;

4-HA,

of explosives. 6-A,6-amino-2.4.dinitrotoluene;

4-hydroxylamino-2,6-dinitrotoluene;

037%4274/M/$ 03.30 0 Elsevier Science Publishers

TNT,

B.V

2,4-DA,2,4-diamino-

2, 4, 6-trinitrotoluene

206

blems [3]. water and tion state Several metabolites

Trace detection of TNT and its degradation products in soil and ground in the blood and urine of animals and humans may reveal the contaminaof the area with regard to TNT. works [1,4-91 have been published on the detection and identification of of TNT in animals and humans. 4-Amino-2,6-dinitrotoluene(4-A),

6-amino-2,4-dinitrotoluene(6-A) and 4-hydroxylamino-2,6-dinitrotoluene(4-HA) were found in the urine of rabbits fed with TNT [4]. 4-A, 2,4-diamino-6-nitrotoluene(2,4-DA) and I ,3-diamino-5-nitrobenzene were found in the urine of rats fed with TNT [8]. 4-HA and 4-A were found in the urine of dogs fed with TNT [l]. 4-A [8,9] and 2-A, hydroxylaminodinitrotoluenes and diaminonitro compounds [8] were found in the urine of munition workers. Several analytical methods have been used to identify the metabolites of TNT: thin-layer chromatography (TLC) followed by UV spectroscopy or mass spectrometry [lo], gas chromatography (GC) [3,9,11], high-performance liquid chromatography (HPLC) [12], electron impact mass spectrometry (EIMS) [3,13,14] and chemical ionisation mass spectrometry (CIMS) [14]. The purpose of this research was to detect and identify urinary metabolites of TNT in rats using LC-MS as the analytical method. LC-MS incorporates good separation characteristics combined with highly specific detection, and has already proved to be adequate for the analysis of metabolites of TNT [ 151. MATERIALS

AND

METHODS

Equipment The LC-MS system consisted of an HPLC interfaced to a magnetic sector mass spectrometer by a direct liquid insertion probe interface, and is described in detail elsewhere [ 151. The HPLC column used was an RP-8 reversed-phase column, and the mobile phases were acetonitrile:water at various relative concentrations. Flow rate was 1 ml/min

and UV detector

wavelength

was 214 nm.

Standard samples and solvents Standard metabolites were synthesised and analysed by LC-MS [ 151. TNT was obtained in pure form from the Israeli Police Analytical Laboratory. Toluene was UV-grade (Aldrich Chemical Co. Inc., Milwaukee, WI), and acetone was UV-grade (Fluka, Buchs, Switzerland). The HPLC solvents used were HPLC-grade acetonitrile (Bio-Lab, Jerusalem, Israel) and triple-distilled water. The solvents were filtered through a 0.5 pm filter (Millipore, Bedford, MA).

207

Procedure Animal tests Male SPD rats weighing solved in 1 ml corn oil Each rat lected over

200-250

g were used.

Single doses of 20 mg TNT dis-

ml corn oil were fed to 10 rats by gavage. 2 Additional without TNT were used as controls. was housed in a metabolic cage during urine collection. dry ice after 12, 24 and 48 h.

rats fed with 1 Urine

was col-

Extraction Toluene extraction was chosen [9] and carried out as follows: a 20-ml sample of urine was acidified to pH 2 with a 10% HCl solution and allowed to stand for 10 min before being neutralised with an excess of sodium bicarbonate and extracted once with 10 ml toluene for 30 min. The toluene extract was drawn off and dried over anhydrous NazS04, then filtered through glass wool. The filtrate was evaporated to dryness, dissolved in 2 ml acetone, filtered through a 5-pm filter and evaporated to dryness under Nz. This residue was then diluted with acetone to 100 ~1. 1 ~1 Was injected into the LC-MS for analysis. The recovery efficiency of TNT and the aminodinitrotoluenes was found to be 90’70, while that of the diaminonitrotoluenes was only 30%.

COLUMN:RP-8 ACETONITRILE:

WATERl40:60~

UV WAVELENGTH:214007

Q i i &

loo

t

LC/MS .4ZElONITRILE:WATERl20:80

+ 2+DIAMINO-6-NITROTOLUENE $”

MH-30)’ 136

1

0

I

5

01

I

IO

15

20

130

140

I50

mln Fig. 1. HPLC-UV Fig. 2. LC-MS

160

170

m/z trace of a urine sample

mass spectrum

of 2,4-DA.

containing

metabolites

of TNT.

180

190

200

210

208

TABLE

I

METABOLITES

OF TNT FOUND Amount

Metabolite

IN THE URINE of metabolite

OF RATS

found

in urine

(fig) After

TNT

12 h

5

After

10

24 h

48 h

I-3

2-A

13 - 24

5-

4-A

11 -27

4 - 20

2,4-DA

After

0.09

0.15 - 0.24

15

2-7 1 - 10

0.16

0.02 - 0.14

Analysis Analysis was done by LC-MS. As it is difficult to find a common procedure for the total separation of all metabolites [12], we used several separate isocratic separations with acetonitrile:water at various relative concentrations [16]: 20:80, 28:72, 40:60 and 60:40. Quantitation of metabolites was obtained by plotting HPLC chromatogram peak heights vs. amount of standard metabolite injected, and thus obtaining standard curves which were used for quantitation of sample metabolites. RESULTS

AND CONCLUSIONS

Table I shows the quantities of metabolites recovered from urine. Examples of an HPLC chromatogram and an LC-MS mass spectrum are shown in Figs. 1 and 2, respectively. Fig. 1 shows an HPLC-UV trace of the 24-h urine sample of a TNTfed rat. Acetonitrile:water (40:60) was used as mobile phase. 2,4-DA and 2,6-DA, 2-A and 4-A were not separated in this run, but were separated with acetonitrile:water (2O:SO) and acetonitrile:water(28:72), respectively. Fig. 2 shows the LC-MS mass spectrum of 2,4-DA, with acetonitrile:water (20:80) which serves also as CI reagent. The results show that the main metabolic pathways of ingested TNT in rats, through excretion in the urine, are reduction processes of the nitro group to the amino group, thus forming 2-A and 4-A. This process is followed by the reduction of an additional nitro group, leading to the formation of 2,4-DA. REFERENCES R.K. Snyder, Toxicol.,

Metabolites

of 2,4,6-trinitrotoluene

(TNT) excreted

in the urine of dogs,

.I. Ind. Hyg.

28 (1946) 59-75.

C. Voegtlin,

C.W.

Hooper

and J.M. Johnson,

Trinitrotoluene

prevention, J. Ind. Hyg., 3 (1921-22) 239-254. W.E. Pereira, D.L. Short, D.B. Manigold and P.K. Roscio, and its metabolites Bull. Environm.

in groundwater Contam.

Toxicol.

by gas chromatograph-mass 21 (1979) 554-562.

poisoning Isolation

its nature,

diagnosis

and characterization

spectrometerxomputer

and

of TNT techniques.

209

4 H.J.

Channon,

G.T.

Mills and R.T.

Biochem. J., 38 (1944) 70-U. 5 R. Lemberg and J.P. Callaghan,

7

768-769. 6 R. Lemberg

and

diazotisable

amines

J.P.

Williams,

Metabolism

Callaghan,

of 2,4,6-trinitrotoluene

of symmetrical

trinitrotoluene,

of aromatic

nitro-compounds,

Metabolism

in rats’ and human

Metabolism

of aromatic

products

154 (1944)

1. Estimation Aust.

nitro-compounds,

amines in the urine after intake of TNT and of reduction

((U-TNT),

Nature,

urine after intake of 2,4,6trinitrotoluene,

Med. Sci., 23 (1945) 1-5. R. Lemberg and J.P. Caliaghan, diazotisabie

The metabolism

of

J. Exp. Biol.

2. Excretion

of

of TNT, Aust. J. Exp.

Biol. Med. Sci., 23 (1945) 6-12. 8 R. Lemberg products

and J.P. Callaghan,

~~etabolism

of 2,4,4-trinitrotoIuene

of aromatic

nitro-compounds,

3. Isolation

from the urine of rats and from human

urine,

Aust.

of reduction J. Exp. Biol.

Med. Sci., 23 (1945) 13-20. 9 J. Almog,

S. Kraus and A. Basch, Determination

of TNT metabolites

in urine, Arch. Toxicol.,

Suppl.

6 (1983) 351-353. 10 W.D.

Won,

R.J.

2,4,&trinitrotoluene, 11 D.G.

Heckly,

D.J.

Glover,

2-amino-4,6-dinitrotoluene,

Anal.

and A.M.

Kaplan,

with liquid chromatography,

Center,

No.

metabolites Yinon

and D.-G. and

D.-G.

Chim.

Structure

J.C.

Hoffsommer,

Hwang,

Metabolic

Hwang, spectrometry

76-96,

mixtures

of and

of 2,4,6-trinitrotoluene

reduction

products

via mass spectrometry. 10 September

Tetranitro

1976, Naval

azoxy- and

Surface

Weapons

MD.

studies

of explosives,

Mass Spectr. studies

of metabolites

16 J. Yinon and D.-G. Hwang, High-performance plosives, J. Chromatogr., 268 (1983) 45-53.

of

of

136 (1982) 425-428.

identification

Metabolic

Analysis

disposition

88 (1977) 381-384.

Acta,

Biom.

Metabolic

2,4-diamino-6-nitrotoluene

of mixtures

Silver Spring,

of 2,4,6-trinitrotoiuene,

chromatography-mass Appl., in press.

Acta,

NSWC/WOL/TR

White Oak Laboratory,

14 J. Yinon 15 J.

Report

Chim. Separation

Anal.

13 D.A. Kubose and D.J. Glover, azotoluenes,

and

27 (1974) 513-516. D.A. and Kubose,

4-amino-2,6_dinitrotoluene,

2,6diamino+nitrotoluene, 12 D.L. Kaplan

Clover

Appl. Microbial., J.C. Hoffsommer

of

1. El and CI mass spectrometry

explosives,

2.

of 2,4,6-trinitrototuene, liquid

of

in press.

chromatography-mass

High-performance J. Chromatogr. spectrometry

liquid Biom. of ex-