High performance liquid chromatography (HPLC) analysis of sulfur mustards and their decomposition by-products by derivatization

A method for the simultaneous detection, separation and analysis by reverse hase HPLC of sulfur mustard type compounds and their major hydrolysis and oxidation by-products utilizes a novel precolumn enhancement derivatization procedure. The method involves the reaction of these nonchromophoric sulfides with N-halogeno-N-metal arylsulfonamidates on a microscale to produce UV or visible absorbing or fluorescing arylsulfonylsulfilimine compounds. These arylsulfonylsulfilimine derivatives can then be readily separated on a low polarity C-18 column by reverse phase HPLC. The method permits quantitation of these sulfides by UV detector response in quantities as small as 20 nanograms.

BACKGROUND OF THE INVENTION 
There is a need to be able to readily detect, separate and quantify trace 
amounts of sulfur mustard agents and their decomposition by-products in 
aqueous solution in combat situations for detecting agent use as well as 
in the laboratory for analyzing intelligence samples. The decomposition 
by-products are more likely to be encountered in the environment rather 
than the agent alone. This need is also present in environmental studies 
and generally in analytical methodology for the identification of alkyl 
sulfides in an aqueous matrix. 
Sulfur mustards are currently analyzed by colorimetry using 
4-(p-nitrobenzyl)pyridine (DB-3) (Holzman, G. Jr. Swift, E. H. and 
Niemann, C. OSRD 4288, The Colorimetric Estimation of NH-3 and DB-3, 27 
Oct. 1944; Esptein, J. Rosenthal, R. W. and Ess, R. J. Uses of 
4-(p-Nitrobenzyl)pyridine As Analytical Reagent for Ethyleneimines and 
Alkylating Agents, Anal. Chem. 27, 1435-9 (1955). This is a general test 
which gives positive results for practically all alkylating agents and 
false positives for some acylating agents. However, this test is not 
effective for the decomposition by-products of sulfur mustards. 
Gas chromatography (GC) has also been employed for analyzing some mustards 
(Fisher, T. L., Jaskot, M. and Sass, S. Edgewood Arsenal Technical Report 
4321, Trace Estimation and Differentiation of Some Mustards Employing 
Gas-Liquid Chromatography). However, aqueous samples cannot be analyzed 
directly by GC but must undergo a lengthy extraction and workup procedure 
before analysis can be performed. 
High performance (pressure) liquid chromatography (HPLC) using a reverse 
phase column has achieved state-of-art analytical separation technology 
due to the development of improved columns and more sensitive detectors. 
The most commonly used sensitive detector is based on ultraviolet/visible 
absorption or fluorescence. However, sulfur mustards and their 
decomposition by-products do not absorb or fluoresce in this spectral 
region and hence as such are not amenable to HPLC analysis with a UV or 
fluorescent detector. 
SUMMARY OF THE INVENTION 
In accordance with the present invention there is provided a method for the 
simultaneous detection, separation and analysis of sulfur mustards and 
their major hydrolysis and oxidation by-products, as well as of mixtures 
of non-chromophoric aliphatic sulfides generally. The method comprises 
reacting a mixture of aliphatic sulfides of the formula R.sup.1 
--S--R.sup.2. wherein R.sup.1 and R.sup.2 are alkyl radicals which may or 
may not contain substituents and may be the same or different, with an 
alkali metal arylsulfochloramide of the formula aryl-SO.sub.2 NMe Cl, 
wherein Me is an alkali metal, to produce a mixture of corresponding 
ultraviolet fluorescent arylsulfonylsulfilimine compounds according to the 
equation: 
##STR1## 
wherein Me, R.sup.1 and R.sup.2 have the meanings defined above, 
separating the mixture into the individual arylsulfonylsulfilimine 
compounds by reverse phase high pressure liquid chromatography, and 
fluorescing the individual arylsulfonylsulfilimine compounds by 
ultraviolet radiation. The invention is particularly valuable for the 
analysis of mixtures of sulfur mustard type compounds and their 
decomposition by-products of the formula R.sup.1 --S--R.sup.2, wherein 
R.sup.1 is 2-chlorethyl, 2-hydroxyethyl, or vinyl, and R.sup.2 is 
2-chloroethyl, 2-hydroxyethyl, vinyl or an unsubstituted alkyl group of 1 
to 6 carbon atoms. 
The reaction of alkylsulfides with salts of N-chloroarylsulfonamides, such 
as chloramine-B or -T, in aqueous solution has been widely employed as a 
facile means of preparing crystalline and innocuous derivatives of alkyl 
sulfides for characterization purposes. The present invention utilizes 
this reaction on a microscale to convert alkyl sulfides, which do not 
fluoresce in the UV region, into UV absorbing arylsulfonylsulfilimines, 
which when dissolved in a suitable solvent, such as aqueous methanol or 
ethanol and aqueous acetonitrile, can then be readily separated by reverse 
phase HPLC and quantified by UV detector response. Analysis is made by 
comparison with standard solutions of known arylsulfonylsulfilimine 
derivatives of alkylsulfides. 
In HPLC the chromatographic stationary phase is relatively nonpolar while 
the solvent or mobile phase is relatively polar. This derivatization 
technique is also useful in that sample polarity is reduced, thus 
improving subsequent column separation. In this manner these sulfides can 
be quantified by photometric or fluorometric detector response in amounts 
as low as 20 nanograms.

DETAILED DESCRIPTION OF THE INVENTION 
The invention is particularly valuable for the reverse phase HPLC 
separation and analysis of mixtures of sulfur mustards and major 
hydrolysis and oxidation by-products thereof. In the following description 
a model sulfur mustard, 2-chloroethyl ethylsulfide (I), and its major 
decomposition by-products, 2-hydroxyethyl ethyl sulfide (II) and vinyl 
ethylsulfide (III) 
##STR2## 
are reacted with sodium benzenesulfochloramide (chloramine B) (IV), which 
contains a strong ultraviolet chromophore, on a microscale in aqueous 
alcoholic medium to form novel ultraviolet absorbing 
phenylsulfonylsulfilimines according to the following equation: 
##STR3## 
wherein R is ClCH.sub.2 CH.sub.2 --=I.fwdarw.V 
HOCH.sub.2 CH.sub.2 --=II.fwdarw.VI 
is CH.sub.2 .dbd.CH.sub.2 --=III.fwdarw.VII 
Compounds V, VI and VII are then separated by HPLC and quantified by UV 
detector response, as described below: 
MATERIALS 
Instrumentation 
HPLC analyses were carried out using a Waters Associates High Pressure 
Liquid Chromatograph consisting of two Model 6000A Pumps, a U6K Injector, 
a Model 440 UV Detector, a 730A Data Module, and a 720A Systems 
Controller. Separation was carried out using a Waters Associates 
Radial-PAK C18 (10.mu.) Column. 
Infrared spectra were recorded on a Perkin-Elmer 283-B Spectrophotometer. 
.sup.1 H NMR spectra were recorded using a Varian A-60-D Spectrometer. 
GC-MS analysis was carried out using a Hewlett-Packard 5985A equipped with 
a 10 m.times.0.25 mm ID glass, WCOT, SP2100 column. 
Chemicals 
Water used for HPLC was distilled and deionized (10-14 megohm-cm). 
Acetonitrile and methanol were HPLC grade (Burdick and Jackson, Muskegon, 
MI, USA). Compounds I and III were obtained from Fairfield Chemical Co., 
Inc. (Blythewood, SC, USA). Compound II was obtained from Aldrich Chemical 
Co., Inc. (Milwaukee, WI, USA). Compound IV was obtained from Eastman 
Kodak (Rochester, NY, USA) and purified. All chemicals used gave 
analytical data consistent with their chemical structure. 
METHODS 
Preparation of the V, VI and VII Standards for HPLC 
Quantities of each of the three sulfilimines were prepared for use as 
standards to determine optimum chromatographic conditions and 
effectiveness of analytical derivatization. The sulfide (1.0 m mole) and 
IV (1.1 m mole) were stirred together in 10 ml of 30% cold aqueous 
methanol for one hour. a white crystalling precipitate appeared almost 
immediately in all cases. The precipitate was filtered off, washed with a 
small quantity of water, dried, and washed with ether. The precipitate was 
then recrystallized from ethanol. Spectral data were in agreement with the 
assigned structures. 
S-Ethyl-S-chloroethyl-N-phenylsulfonylsulfilimine (V) 
The recrystallized product was obtained by the general procedure outlined 
above: yield 89%; m.p. 109.degree.-110.degree. C. (Found: C, 42.9; H, 5.1; 
Cl, 12.8; N, 5.1; S, 22.9. Calc. for C.sub.10 H.sub.14 CiNS.sub.2 O.sub.2 
: C, 42.9, H, 5.0; Cl, 12.7; N, 5.0; S, 22.9). 
S-Ethyl-S-2-hydroxyethyl-N-phenylsulfonylsulfilimine (VI) 
The recrystallized product was obtained by the general procedure outlined 
above except that water alone was used as the reaction solvent and ether: 
chloroform was used for recrystallization. The yield was 83%; m.p. 
75.degree.-76.5.degree.. (Found C, 45.8; H, 5.8; N, 5.6; S, 24.5 Calc. for 
C.sub.10 H.sub.15 NS.sub.2 O.sub.3 : C, 45.9, H, 5.8; N, 5.4; S, 24.5). 
S-Ethyl-S-vinyl-N-phenylsulfonylsulfilimine (VII) 
The recrystallization product was obtained by the general procedure 
outlined above: yield, 82%; m.P. 85.degree.-86.degree.. (Found C, 49.1; H, 
5.2; N, 6.0; S, 26.5. Calc. for C.sub.10 H.sub.13 NS.sub.2 O.sub.2 : C, 
49.3; H, 5.4; N, 5.8; S, 26.4). 
Chromatographic Procedure 
Analytical separations were performed under the following conditions: 
sample size, 20 .mu.l; flow rate, 1.5 ml/min; column temperature, ambient; 
mobile phase, 30% acetonitrile: water; UV. detector, 254 nm. 
Standard solutions of V, VI and VII were injected onto the column and their 
retention times determined. Calibration curves conforming to Beer's law 
were obtained by injecting known concentrations (1.0 .mu.g, 2.0 .mu.g, 4.0 
.mu.g, 10.0 .mu.g, and 20.0 .mu.g per ml) of the sulfides as sulfilimine 
derivatives onto the column in triplicate and measuring the resulting peak 
areas. 
Analytical Derivatization 
To one equivalent of each of the sulfides in one ml of methanol is added 
two equivalents of IV. The mixture was heated with stirring at 60.degree. 
C. for one hour. After cooling, 20 .mu.l samples were introduced into the 
column through a continuous flow loop injector. Peak areas were measured 
and computed with an on-line integrator (Data Module). 
In this way, concentrations of I, II, and III were prepared singly and in 
combined mixture at 1.0 .mu.g, 2.0 .mu.g, 4.0 .mu.g, 10.0 .mu.g, and 20.0 
.mu.g/ml for detection as the sulfilimines species. 
RESULTS AND DISCUSSION 
Arylsulfonylsulfilimines show a strong absorption peak around 230 nm (log E 
4.0-5.0) and a weak absorption peak in the area of 270 nm (log E 3.0-4.5) 
Gilchrist, T. L. and Moody, L. J. The Chemistry of Sulfilimines, Chem. 
Rev., 77 (No. 3) 409 (1977). As seen in Table I, compounds V, VI and VII 
show strong absorption peaks at 224-225 nm as well as a weak absorption at 
272 nm. Another weak peak was also observed for all three compounds at 265 
nm. Also in Table 1, the log E values at 254 nm for V, VI and VII are 
shown. 
TABLE 1 
______________________________________ 
UV Spectra of V, VI and VII in Acetonitrile 
Com- Log Log Log Log 
pound .lambda..sub.1 (nm) 
E.sub.1 
.lambda..sub.2 (nm) 
E.sub.2 
.lambda..sub.3 (nm) 
E.sub.3 
E.sub.254 
______________________________________ 
V 224 3.9 265 2.7 272 2.6 2.7 
VI 225 3.9 265 2.7 272 2.6 2.7 
VII 226 4.0 264 2.8 272 2.7 2.9 
______________________________________ 
The qualitative capability for this technique is illustrated in the 
chromatogram of FIG. 1 in which the complete separation of V,VI, and VII 
as well as IV is achieved in 15 minutes. 
The retention times for the phenylsulfonylsulfilimine derivatives and 
excess chloramine-B reagent under the stated conditions are shown in Table 
2. Excess chloramine-B reagent is unretained on the column and does not 
interfere with the analysis. 
TABLE 2 
______________________________________ 
Retention Times of the Phenylsulfonylsulfilimine 
Derivatives 
Compound Retention Time (Min) 
______________________________________ 
IV 1.05 
VI 3.32 
VII 6.72 
V 11.00 
______________________________________ 
Quantitation was also readily achieved by HPLC. This is illustrated in FIG. 
2. The reactions were reproducible and detector response was linear for I, 
II, and III in concentrations of 1.0-20.0 .mu.g/ml. The overall efficiency 
of the derivatization reaction for the three sulfides was 85-99% by 
comparison with standardized materials. The detection limits for I, II, 
and III were 10, 12 and 21 nanograms, respectively. The limits of 
detection are based on the method described by Hubaux and Vos "Decision 
and Detection, Limits for Linear Calibration Curves", Anal. Chem. 42 
(8):849 using a 95% confidence level with alpha and beta being equal to 5% 
(i.e. one out of 20 datum may fall outside 2 standard deviations of the 
fitted curve). 
The method of the present invention can be similarly employed for the 
separation and analysis of mixtures of other sulfur mustard type compounds 
and their major hydrolysis and oxidation by-products as well as other 
aliphatic sulfides generally. Suitable sulfides include 
bis(2-chloroethyl)sulfide, bis(2-hydroxyethyl)sulfide, divinylsulfide, 
dimethylsulfide, diethylsulfide, ethyl methyl sulfide, dipropyl sulfide, 
ethyl propyl sulfide and ethyl n-hexyl sulfide. Also the HPLC process of 
the present invention can be carried out using other separatory column 
materials than C18. The C.sub.18 separatory packing is made by chemically 
bonding a C.sub.18 alkyl group to micro particles of fully porous silica 
at 10% C.sub.18 alkyl by weight. The general chemical structure is as 
follows: 
##STR4## 
Reverse phase HPLC normally involves a relatively non-polar stationary 
phase (e.g. C.sub.18 mentioned above) used in conjunction with a very 
polar (e.g. aqueous) mobile phase to separate a wide variety of less polar 
solutes. 
Other separatory packing materials suitable for use in the present 
invention include micro particles of fully porous silica bonded to 
--C.sub.8 alkyl, --R--CN, --R--NH.sub.2, and 
##STR5## 
groups, wherein R is an alkylene radical. 
The nature of the separatory material is not critical to the HPLC process 
for separation and analysis of sulfur mustards and alkylsulfides.