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Screening of nitrogen mustards and their degradation products in water and decontamination solution by liquid chromatography–mass spectrometry
Journal of Chromatography A, 1102 (2006) 214–223
Screening of nitrogen mustards and their degradation products in water and decontamination solution by liquid chromatography–mass spectrometry Hoe-Chee Chua, Hoi-Sim Lee, Mui-Tiang Sng ∗ DSO National Laboratories, 20 Science Park Drive, Singapore 118230, Singapore Received 28 February 2005; received in revised form 28 October 2005; accepted 28 October 2005 Available online 28 November 2005
Abstract Analysing nitrogen mustards and their degradation products in decontamination emulsions posed a significant challenge due to the different phases present in such matrices. Extensive sample preparation may be required to isolate target analytes. Furthermore, numerous reaction products are formed in the decontamination emulsion. A fast and effective qualitative screening procedure was developed for these compounds, using liquid chromatography–mass spectrometry (LC–MS). This eliminated the need for additional sample handling and derivatisation that are required for gas chromatographic–mass spectrometric (GC–MS) analysis. A liquid chromatograph with mixed mode column and isocratic elution gave good chromatography. The feasibility of applying this technique for detecting these compounds in spiked water and decontamination emulsion was demonstrated. Detailed characterisation of the degradation products in these two matrices was carried out. The results demonstrated that N-methyldiethanolamine (MDEA), N-ethyldiethanolamine (EDEA) and triethanolamine (TEA) are not the major degradation products of their respective nitrogen mustards. Degradation profiles of nitrogen mustards in water were also established. In verification analysis, it is important not only to develop methods for the identification of the actual chemical agents; the methods must also encompass degradation products of the chemical agents as well so as to exclude false negatives. This study demonstrated the increasingly pivotal role that LC–MS play in verification analysis. © 2006 Elsevier B.V. All rights reserved. Keywords: Nitrogen mustards; Ethanolamine; GC–MS; LC–ESI–MS; Decontamination emulsion; Mixed mode chromatography
1. Introduction The nitrogen mustards belong to the same class of chemical agents with vesicant properties as lewisites and sulphur mustards. Nitrogen mustards were developed in the 1930s and stockpiled during World War II for military use. They are controlled as Schedule 1 chemicals in the Chemical Weapons Convention (CWC). The three nitrogen mustards are HN-1 (bis(2-chloroethyl)ethylamine), HN-2 (bis(2-chloroethyl) methylamine) and HN-3 (tris(2-chloroethyl))amine), and their structures are shown in Table 1. The possibility that the nitrogen mustards might pose a potential threat to water source led to an exhaustive study on the chemical reactions undergone by these agents in unbuffered water and water buffered at pH 8 [1]. The mechanism of the
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hydrolysis reaction was proposed (Fig. 1a). A cyclic ethylenimonium intermediate species was identified to be the focal point from which various hydrolysis products would subsequently form, depending on the solution pH, the nature of the nitrogen mustard and chloride concentration. The cyclic ethylenimonium ion was sufficiently stable to allow isolation and characterization as a salt of picrylsulfonic acid. Other ionic hydrolysis products were similarly isolated and characterized. Characterization and identification were mainly based on the purification and classical techniques available at that time, namely purification by crystallization, melting point determination, elemental composition analysis and titrametric techniques. The change in concentration of the degradation products were monitored by taking regular measurements of the concentrations of hydrogen ions and chloride ions and the required amount of thiosulphate titre over the course of 48–72 h. These analytical data along with the characterization of selective isolation of particular reaction species led to the elucidation of the hydrolysis mechanism of the nitrogen mustard in water shown in Fig. 1a.
H.-C. Chua et al. / J. Chromatogr. A 1102 (2006) 214–223 Table 1 Nitrogen mustards and their degraded compounds investigated Abbreviation
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Table 1 (Continued ) Abbreviation
Structure
Structure K
HN-1
A
L
B M
C TEA D N EDEA
DEA
HN-2
E
F
G
H
MDEA
HN-3
I
J
Ways to speed up the degradation of the nitrogen mustard for the purpose of decontamination have been studied, employing chlorinating bleach and decontamination emulsion with chlorinating or redox activities. Sartori [2] reported on the chemical properties of the nitrogen mustards. Chlorination resulted in the dealkylation of bis(2-chloroethyl)alkylamines. Aldehydes and bis(2-chloroethyl)amine were identified among the products of the reaction after treating the resultant solution with water, shown in Fig. 1b. Reaction of tertiary bis(2chloroethyl)amines hydrochloride with sodium hypochlorite solution buffered at pH 8 with sodium bicarbonate resulted in the formation of several products, among which N,N-[bis(2chloroethyl)]-N-chloroamine was identified. Further reaction of N,N-[bis(2-chloroethyl)]-N-chloroamine with hydrochloric acid gave N,N-bis(2-chloroethyl)amine (H), as shown in Fig. 1b. Since these studies, modern analytical techniques have been applied to the analysis of nitrogen mustards and their degradation products in a wide variety of matrices, with varying degree of efficiency and effectiveness on the sample preparation. For the analysis of intact nitrogen mustards, gas chromatographic analysis is the method of choice. Sample preparation from water using solid phase extraction (SPE) [3] and air using SPE disks [4] with subsequent gas chromatographic analysis have been reported. Analysis of hydrolysis products of nitrogen mustards in aqueous extract and determination by gas chromatography is both challenging and time consuming since concentration to dryness and derivatisation as part of sample treatment are required. The presence of extraneous materials may interfere with the derivatisation resulting in low apparent recoveries. Determination by liquid chromatography coupled to mass
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Fig. 1. (a) Hydrolysis pathways of HN-1, HN-2 and HN-3 in water. L can further undergo hydrolysis to give the product TEA via intermediate M [1]. (b) Formation of N via two chlorination pathways.
spectrometry would be suited for analysis of the hydrolysis products, since no derivatisation may be necessary and the presence of intact chemical agent could be analysed concurrently. Liquid chromatography–atmospheric chemical ionization mass spectrometry was applied as a screening method on water spiked with degradation products of chemical agents including ethanolamines. The chromatography column used for separation was either a C18 column and or a combination C8 /C18 column. Under those conditions, triethanolamine (TEA) and N-methyldiethanolamine (MDEA) were not resolved and N-ethyldiethanolamine (EDEA) gave split peak [5,6]. A liquid chromatographic–mass spectrometric (LC–MS) application using low capacity ion exchange column with electrospray ionisation (ESI) for the determination of TEA and MDEA was reported for analysis of aqueous extracts of vegetation [7]. A detection limit of 0.02 ppm was achieved, demonstrating that LC–ESI–MS and LC–ESI–MS–MS methods are well suited for direct quantitation and confirmation of TEA and MDEA with little sample cleanup. LC–MS application had also been extended successfully to more complex matrices such as plasma and urine. The use of diethyldithiocarbamic acid for derivatising HN-2 in plasma, fol-
lowed by SPE extraction of derivatised HN-2 prior to LC–MS analysis was reported [8]. Lemire and co-workers [9,10] reported a sensitive and rapid analysis for the quantitation of ultra-trace level of EDEA and MDEA in human urine. SPE cation exchange was for extraction and quantitation was by isotopic dilution with detection by multiple-reaction monitoring. In summary, analysis for intact nitrogen mustard by GC–MS and LC–MS are possible, although the former is the preferred method due to better sensitivity. However, for the analysis of the hydrolysis products, GC–MS may be difficult and time consuming since these products tend to be highly polar. As a result, sample pre-treatment must incorporate the additional steps of derivatisation and sample enrichment (for complex matrices) to promote detection sensitivity at the expense of the total analysis time. Under these circumstances, LC–MS becomes the more desirable technique. It offers the advantage of concurrent detection for the intact agent and its hydrolysis products. Few published literature is available on the applicability of LC–MS to the analysis of nitrogen mustard and its degradation products in decontamination emulsion. Wils and Hulst [11] reported the analysis of HN-2 by thermospray LC–MS as an
H.-C. Chua et al. / J. Chromatogr. A 1102 (2006) 214–223 Table 2 LC–ESI–MS and CID product ion spectra of nitrogen mustards and their degradation products Compounds
Molecular ions m/z
Fragment ions m/z
HN-1 A B C D EDEA DEA HN-2 E F G MDEA HN-3 I J K L M TEA N
170 134 152 116 108 134 106 156 120 138 102 120 204 168 186 150 168 132 150 142
142, 144, 134, 136, 106, 108, 72, 70, 63, 65 106, 108, 72, 70 134, 136, 106, 108, 72, 70 98, 88, 72, 70 80, 82, 72 116, 98, 88, 72, 70 88, 70 128, 130, 120, 122, 92, 94, 84, 63, 65, 58 92, 94, 84, 63, 65, 58 120, 122, 92, 94, 84, 63, 65, 58 58 102, 58 176, 178, 168, 170, 106, 108, 78, 80, 70, 63, 65 106, 108, 70, 63, 65 168, 170, 106, 108, 70, 63, 65 132, 134, 106, 108, 70, 63, 65 150, 152, 132, 134, 106, 108, 70, 63, 65 114, 88, 70 132, 114, 88, 70 114, 116, 106, 108, 70, 63, 65
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A commercial decontamination emulsion based on sodium dichloroisocyanurate was purchased off-the-shelf. It consists of two components. Component 1 comprises sodium dichloroisocyanurate (16.7% with approximately 10% active chlorine), talcum (16.7%), sodium chloride (33.3%) and calcium chloride dihydrate (33.3%). Component 2 contains xylene (80%) and an emulsifier. The decontamination emulsion based on a mixture of these components was prepared in accordance with the manufacturer’s instruction. The resultant mixture is a viscous white emulsion and has a pH of 8.0. Chemicals were used without further purification. Solvents used, including analytical reagent HPLC grade ACN, dichloromethane (DCM), isopropyl alcohol (IPA), and ethyl acetate were from J.T. Baker (Phillipsburg, NJ, USA). Water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). 2.1.1. Safety considerations Nitrogen mustards are known vesicants. All handling of the agents were in dilute amounts and carried out in an efficient fume hoods equipped with alkaline scrubber. Gloves and appropriate protective measures were adopted. 2.2. Instruments
acetoxy derivative. In this paper, we report a screening method by LC–MS, in a single run, that covers most of the degradation products reported for water and decontaminating solution with chlorinating activity [1,2]. The identification of degradation products was confirmed by comparison of their mass spectra to those of authentic compounds where available, and by tandem mass spectrometry (MS/MS) and nuclear magnetic spectroscopy (NMR). The LC–MS–MS data (Table 2) are presented in this paper. The developed screening method also allows the analysis of intact nitrogen mustards. This paper will discuss the differences in the compounds detected by LC–MS in the hydrolysis of nitrogen mustards in water and their chlorination products in decontamination emulsion were compared. Time profile studies were also performed to ascertain the behavior of nitrogen mustards in water. 2. Experimental 2.1. Chemicals and reagents The nitrogen mustards and their degradation products investigated, together with the abbreviations used, are shown in Table 1. MDEA (99%), EDEA (98%), TEA (98%), DEA (98%), N (98%) and spectrophotometric grade trifluoroacetic acid (TFA) (99%) were from Aldrich (Milwaukee, WI, USA). Deuterium oxide (D2 O) was from Merck (99.95%) (Darmstadt, Germany). HN-1, HN-2, HN-3, B, D, F, H, J and L were synthesized inhouse with greater than 95% purity by GC–MS. They were dissolved in acetonitrile (ACN) (1000 g ml−1 ) and stored at 4 ◦ C as stock solution. The working solution was prepared by diluting to the appropriate concentration with ACN prior to use.
2.2.1. Liquid chromatography–mass spectrometry LC–MS analyses were performed on Shimadzu HPLC (Kyoto, Japan). The system was fitted with a 150 mm × 2.1 mm i.d. Primesep 100 column (SIELC Technologies Co., USA), with ˚ pore size. The mobile phase at iso5 m particle size and 100 A cratic elution consisted of 0.1% TFA in water [mobile phase A]: 0.1% TFA in acetonitrile: water (95:5) [mobile phase B] (60:40) at a flowrate of 0.2 ml min−1 . Injections (1 l) were made using the autosampler. The column effluent was introduced into a TSQ7000 mass spectrometer (Thermo-Finnigan, San Jose, CA, USA) via an atmospheric pressure ionisation source/interface operated in ESI mode. Capillary and tube lens voltages were optimised to give maximum response to m/z 156 [M + H]+ from HN-2. This ensures that the [M + H]+ ions were prominent in the spectra. ESI conditions were as follows: capillary temperature 250 ◦ C, spray voltage 5 kV, Q0 offset −5 V, sheath gas nitrogen (Air Products, Singapore) at 60 psi and auxiliary gas nitrogen at a flow-meter reading of 20. The mass scan range was m/z 50–m/z 500. CID product ion spectra were obtained from the protonated molecules of the compound using the LC conditions described above. Argon was used as collision gas, collision offset −20 V and Q0 offset −5 V. 2.2.2. Nuclear magnetic resonance spectrometry 1 H NMR spectra were acquired on a Varian 500 MHz NMR spectrometer (Varian Inc., Palo Alto, CA, USA) and processed using the Varian VNMR software. The 1 H NMR experiments were conducted at 25 ◦ C and accumulated with a spectral width of 5000 Hz with 32,000 data points and a pulse repetition delay of 4 s. One microliter of neat agent was spiked into 1 ml of water to give a 1000 g ml−1 solution. One hundred microliters of D2 O was added to 600 l of this solution for NMR analysis.
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2.3. Nitrogen mustards study by LC–MS 2.3.1. Spiking of decontamination emulsion samples Decontamination emulsion (1 ml) was spiked individually with 5 l of neat HN-1, HN-2 and HN-3 at a concentration of 5000 g ml−1 and allowed to stand at room temperature for 1 h. 2.3.2. Spiking of water samples Deionised water (4 ml) was spiked individually with 4 l of neat HN-1, HN-2 and HN-3 at a concentration of 1000 g ml−1 and stored at room temperature for a time profile study over 72 h. The water sample was first analysed at zero hour and then every half an hour interval for 72 h. 2.4. Sample preparation ACN (1 ml) was added to the decontamination emulsion (1 ml). The mixture was vortexed for 10 s and then centrifuged at 4500 × g for 10 min. The organic and aqueous layers were transferred to separate vials for LC–MS analysis. The water samples were directly analysed. All experiments were carried out in triplicates. 3. Results and discussion 3.1. Method development for LC–MS 3.1.1. C18 versus mixed-mode column Initially a C18 column was used for chromatography. Fig. 2a showed the reconstructed ion current chromatogram of a cocktail of 14 standards prepared in ACN. To increase retention and improve the sensitivity of detection of these compounds, an acidic condition and the lowest percentage of organic mod-
ifier were used in the mobile phase. However, the column still exhibited poor retention and selectivity for the ethanolamines and the other nitrogen compounds because of their highly polar and basic characteristics. Under these circumstances, possible remedies would be to employ ion-pairing reagents in the mobile phase or a cation exchange column to enhance retention. Unfortunately, the prerequisite LC mobile phases are not conducive to ESI–MS. With these considerations, a mixed-mode column with embedded ion-pairing group was selected for this study. The column, Primesep 100, exhibits both hydrophobic and cation exchange retention mechanisms. The latter interaction would be selective towards polar basic species such as the nitrogen compounds covered in this study (Table 1). Fig. 2b showed the reconstructed ion current chromatogram of a cocktail of the standards by mixed mode column. This column provides chromatography under reversed phase conditions, which are compatible with ESI-MS. The resolution of the standards is greatly improved in comparison to the chromatogram on the C18 column. 3.1.2. Mobile phase TFA (0.1%) in water as mobile phase A and 0.1% TFA in ACN:water (95:5) as mobile phase B was used. Preliminary work with this column indicated that chromatography was best performed under isocratic rather than gradient elution. Based on isocratic runs at different composition of the mobile phase A over the range of 20–80%, an increase in the percentage of mobile phase A resulted in an increase in retention time and overall improvement in resolution, except for compounds B and N which co-eluted as one peak. With mobile phase A at isocratic condition of 60%, a good separation with almost baseline resolution for most of the
Fig. 2. Reconstructed ion current chromatogram of nitrogen mustards and their related products by (a) C18 column, and (b) mixed mode column: 1 = DEA, 2 = TEA, 3 = MDEA, 4 = EDEA, 5 = L, 6 = F, 7 = B, 8 = N, 9 = J, 10 = HN-2, 11 = HN-1, 12 = HN-3.
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compounds, except for DEA and TEA, was achieved in less than 20 min (Fig. 2b). Although these two compounds co-elute, some separation between them still exist. This is sufficient for discriminating them by performing extracted ions analysis. No degradation products were observed on running a standard mixture of the nitrogen mustards prepared in ACN, indicating the stability of these compounds under the LC conditions used. Hence, the isocratic mobile phase of 60% A:40% B was selected for general screening of water and emulsion for all the compounds proposed in Table 1. There were two problems encountered during the screening of water and emulsion sample by the developed LC conditions. The general screening LC conditions could resolve the cyclic and acylic compounds of partially hydrolysed HN3 but not the corresponding species of HN-1 and HN-2 in the water. Compounds B and F co-eluted with compounds C and G, respectively. In the context of confirmatory analysis, achieving this resolution would be essential in avoiding identification ambiguity. To resolve these pair of peaks, the mobile phase A was increased to a higher isocratic composition of 95%. However, this was at the expense of the run time, which increased from 20 to 60 min. Hence, the isocratic mobile phase of 95% A:5% B would only be used when confirmatory analysis was required. The extracts from decontamination emulsion were analysed by LC–MS using the isocratic mobile phase of 60% A:40% B. The presence of N-(2-chloroethyl)N-alkylamine species D and H, formed from the chlorination of HN-1 and HN-2, respectively, presented chromatographic problem. Compound H co-eluted with the partially hydrolysed HN-2 (F) and compound D gave a split peak. Altering to the same isocratic mobile phase of 95% A:5% B mentioned above (for resolving the acyclic/cyclic species) gave baseline resolution of H and F. For the case of compound D, re-focusing to give a single peak was achievable by increasing the TFA content from 0.1 to 0.2% in both mobile phases A and B at 60% A:40% B. 3.2. Identification The optimised capillary temperature, tube lens voltage, sheath gas nitrogen and auxiliary gas nitrogen are given in Section 2.2.1. Under these soft ionization conditions organic standards of nitrogen mustards and its degradation products in ACN gave intense protonated molecular ions [M + H]+ with no fragmentation, except for compounds D, H and N. These produced intense protonated acetonitrile adduct ions [M + ACN + H]+ . Identification of the acyclic compounds detected in the water and decontamination emulsion sample was based on matches of the obtained ESI–MS, ESI–MS–MS spectra and retention times against those of the reference standards. The shift in retention time between the reference standards and analytes was less than 0.1 min. Identification of the cyclic compounds was based on the ESI–MS spectra, ESI–MS–MS spectra (Table 2). Generally, the ESI–MS spectra of the cyclic compound exhibited intense M+ and [M + TFA]+ adduct ions. The NMR data of A and E was used to confirm the cyclic structures. The signals for A are assigned as follows: 1 H NMR (500 MHz, D2 O): ␦ 4.022 (t,
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J = 5.8 Hz, He ), 3.618 (t, J = 5.8 Hz, Hd ), 3.238 (m, Hc ), 3.337 (q, J = 7.3 Hz, Hb ), 1.365 (t, J = 7.3 Hz, Ha ).
Similarly, the signals for E are assigned as follows: 1 H NMR (500 MHz, D2 O): ␦ 4.055 (t, J = 5.8 Hz, Hd ), 3.620 (t, J = 5.5 Hz, Hc ), 3.271 (m, Hb ), 3.152 (s, Ha ).
An attempt was made to confirm the other cyclic compounds using NMR. However, due to the simultaneous existence of several compounds resulting in overlapping peaks, not all of the signals due to the compounds could be assigned. 3.3. Analysis of nitrogen mustards in water The time profile on the degradation of individual HN-1, HN2 and HN-3 in water was studied using the developed LC–MS method. Water samples were spiked separately with nitrogen mustards at 1000 g ml−1 level. For analytical convenience, since the ethanolamines had already been characterised earlier on in terms of their retention times and mass spectra, these ethanolamines were selected as internal standards for use only in the water studies to correct for instrumental variation. Prior to this, it was empirically verified that TEA could never exist as the degradation product of HN-1, DEA of HN-2 and EDEA of HN-3 (data not shown). Hence, TEA was added as an internal standard into the water spiked with HN-1, and similarly DEA and EDEA were spiked respectively in water samples separately containing HN-2 and HN-3. Subsequently, 1 l of the spiked water samples were analysed every 0.5 h over an observation period of 72 h. The 1-l aliquot was analysed directly by LC–MS without sample preparation. Figs. 3–5 show the respective time profiles, with the values for the y-axis presented in logarithmic format to fit all the data points of all the compounds on the same scale. Similar time profile in the decontamination emulsion was not performed, since no suitable internal standards were found that would not degrade in the aggressive environment of the decontamination emulsion. Fig. 3 showed the degradation of HN-1 in water. HN-1 was rapidly converted to the ethylenimonium compound A, which became the most abundant species up to 20 h. Compound A is known to be fairly stable since it had previously been isolated as a salt of picrylsulfonic acid and characterised by classical techniques [1]. The rapid conversion rate caused the concentration of HN-1 to drop below the instrument detection limit between 0.5 and 2.5 h. Compound A underwent further hydrolysis to give B, the hydroxylated ethylenimonium ion (C) and EDEA. The hydrolysis reaction released H+ , which led to the gradual pH decrease from 7 to 4 observed over 72 h.
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Fig. 3. Time profile of HN-1 in water over 72 h. Inset graph shows the first 25 h of the time profile.
From 20 h onwards, compound B became the predominant product and compound A remained present as a major species. Compound C and EDEA existed as minor products up to the end of the observation period (72 h). EDEA became detectable at 3 h onwards. The predominance of compound B could be due to the change in pH leading to the stabilization of the compound through salt formation [1]. Besides the two minor products already mentioned, HN-1 re-appeared as a minor product in sufficient amount to be detectable from 2.5 h onwards, its concentration is similar in level to compound C and EDEA. The re-appearance of HN-1 might be from the two predominating species present at that time: compound A and the chloride ions reacting with each other to reform the mustard. The appearance of the mustard at a later stage indicated a prerequisite reaction condition be present before the reversion process would occur, probably an acidic condition. Although this was not experimentally verified for HN-1, HN-2 was reported to be released when an aqueous solution of N-(2-chloroethyl)-N-methyl ethylenimonium ion was acidified with hydrochloric acid [1]. Fig. 4 showed the degradation of HN-2 in water. Due to the structural similarity to HN-1, HN-2 degraded in a similar manner. Conversion of HN-2 to the ethylenimonium ion (E) was rapid. Hydrolysis thereafter also produced compound F, hydroxylated ethylenimonium ion (G) and MDEA, with the corresponding pH change found to drop from 7 to 5 over 72 h. Initially, the concentration of HN-2 decreased to very low albeit
still detectable level in the time period between 2 and 4 h. Subsequent reversion reaction led to HN-2 increasing gradually in concentration up to the concentration level similar to that of compound G and MDEA. Unlike the degradation process of HN-1, the corresponding species of compounds F, G and E were already detectable at the very initial stage, whilst MDEA was detected only from 2.5 h onwards. But as with HN-1, these species existed as minor products. The main degradation product observed was compound F, which became the most abundant species from 15 h onwards. The reasons for the stability of compound F and the reversion to HN-2 are the same as described above in HN-1. HN-2 hydrolysis was also known to produce substantial amount of dichloro cyclic and dihydroxyl cyclic dimers [1], but they were not detected in our study. Because of its three chloroethyl groups, the degradation of HN-3 was more complicated compared to HN-1 and HN2 (Fig. 5). HN-3 was rapidly hydrolysed and became undetectable after 25 h. In contrast to HN-1 and HN-2, no reversion process occurred with HN-3, hence after 25 h it remained below the detection limit of the instrument. The hydrolysis occurred via three ethylenimonium ions (I, K and M), producing hydrolytic products that were mainly ethanolamines with either the chloroethyl (L) or dichloroethyl group (J) attached. Compound J was the predominant product up to 30 h. From 30 h onwards, compound L became the predominant product and compound J remained present as major species. Unlike HN-1
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Fig. 4. Time profile of HN-2 in water over 72 h. Inset graph shows the first 20 h of the time profile.
and HN-2, the first ethylenimonium ion (I) was not long lived and become undetectable after 2.5 h. The second and third ethylenimonium ions (K and M) were detectable in trace level throughout the observation period. The final stage of the hydrolysis process produced TEA, which was found to be present only as a minor product after 72 h. Before that, only trace amount was observed from 7 h onwards. In contrast to HN-1 and HN-2, the pH dropped appreciably from 6 to 2 over the course of the experiment. 3.4. Analysis of nitrogen mustards in decontamination emulsions Decontamination emulsions were spiked separately with HN1, HN-2 and HN-3, and left to react for 1 h before sample extraction and analysis by LC–MS. In the sample pretreatment, an organic solvent was added to breakdown the emulsion, followed by centrifugation, to separate the emulsion into three layers: organic, aqueous and sludge. Preliminary LC–MS analysis on these layers had shown that most of the analytes were found in the organic layer, with approximately 30 g ml−1 (based on a 1-point standard) or less remaining in the aqueous and sludge
layers. Since the proportion was small, the sludge was not analysed in the subsequent work. Various organic solvents ethyl acetate, ACN, DCM and IPA were evaluated for their emulsion breakdown efficiency. Both DCM and IPA caused precipitation at the interface of the organic and aqueous layers. Filtration was required prior to LC–MS analysis. No precipitate was observed when ethyl acetate and ACN were used. Hence only ethyl acetate and ACN were further considered. The organic extracts using the ACN and ethyl acetate and the extracted aqueous layer were analysed by LC–MS without prior filtration. The LC–MS analysis employed the optimized conditions determined from the water studies described earlier. Unfortunately the aqueous layer from ethyl acetate extract consistently caused blockage of the heated capillary tube, which required clearance after every three sample injections. The ACN extract, on the other hand, did not present any such problem. The active ingredient in the decontamination emulsion under study is sodium dichloroisocyanurate. It acts by producing hypochlorous acid (HClO) upon interaction with water. Generally, the LC–MS analysis on the ACN extracts of the decontamination emulsion revealed the presence of intact mustards as
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Fig. 5. Time profile of HN-3 in water over 72 h. Inset shows the first 10 h of the time profile.
well as products of chlorination and hydrolysis. In the emulsion, HN-1 degraded to give only chloroethylamine species (D and N); while HN-2 and HN-3 degraded to give partially hydrolysed nitrogen mustards (F, J and L) and chloroethylamines (H and N). The ESI–MS spectra of the compounds in the extract matched that of the reference standards. The differences in retention time as compared to the reference standard were less than 0.2 min. For HN-2 and HN-3, not all the compounds detected were identified. The identities of one unknown peak in the extract of the decontamination solution spiked with HN-2, and two unknown peaks with HN-3 are still being determined. These unidentified compounds existed in trace level. On the basis of the relative peak heights in the chromatograms, HN-3 was clearly difficult to degrade using the emulsion. Its degradation products gave noticeably smaller peaks compared to those of HN-1 and HN2. Correspondingly, the HN-3 peak was larger than the HN-2 peak, and HN-1 was completely degraded. The characterization of these compounds could prove valuable for future analyses since their identification could verify the presence of nitrogen mustards.
presence of nitrogen mustards and their degradation products in water and decontamination emulsion. There were no matrix interferences from the decontamination emulsions despite the numerous ingredients present. Acetonitrile was chosen as an extraction solvent for qualitative analysis of decontamination emulsion sample. The ethanolamines can only be detected in the aqueous layer. Both the hydrolysis and decontamination studies revealed that N-methyldiethanolamine, N-ethyldiethanolamine and triethanolamine are not the major degraded products of their respective nitrogen mustards. The partially hydrolysed nitrogen mustards are the more prominent evidence. Acknowledgements The support from the organic synthesis group of DSO National Laboratories is gratefully acknowledged. This work was funded by the Directorate of Research & Development, Defence Science & Technology Agency, Singapore. References
4. Conclusion The developed LC–MS method, based on the mixed-mode column, offers a quick and effective method of screening for the
[1] C. Golumbic, J.S. Fruton, M.A. Stahmann, M. Bergmann, J. Org. Chem. 11 (1946) 518. [2] M.F. Sartori, Chem. Rev. 48 (1951) 225. [3] N.M.A. H¨akkinen, J. High Resolut. Chromatogr. 14 (1991) 811.
H.-C. Chua et al. / J. Chromatogr. A 1102 (2006) 214–223 [4] J.R. Stuff, R.L. Cheicante, H.D. Durst, J.L. Ruth, J. Chromatogr. A 849 (1999) 529. [5] R.M. Black, R.W. Read, J. Chromatogr. A 759 (1997) 79. [6] R.W. Read, R.M. Black, J. Chromatogr. A 862 (1999) 169. [7] J.V. Headley, K.M. Peru, L.C. Dickson, Rapid Commun. Mass Spectrom. 13 (1999) 730.
223
[8] T. Karayilanoglu, E. Berenshtein, L. Kenar, N. Kitrossky, U. Kisa, M. Chevion, Turk. J. Biochem. 26 (3) (2001) 90. [9] S.W. Lemire, D.L. Ashley, A.M. Calafat, J. Anal. Toxicol. 27 (2003) 1. [10] S.W. Lemire, J.R. Barr, D.L. Ashley, C.T. Olson, T.L. Hayes, J. Anal. Toxicol. 28 (5) (2004) 320. [11] E.R.J. Wils, A.G. Hulst, Fresenius J. Anal. Chem. 342 (9) (1992) 749.
Screening of nitrogen mustards and their degradation products in water and decontamination solution by liquid chromatography–mass spectrometry Hoe-Chee Chua, Hoi-Sim Lee, Mui-Tiang Sng ∗ DSO National Laboratories, 20 Science Park Drive, Singapore 118230, Singapore Received 28 February 2005; received in revised form 28 October 2005; accepted 28 October 2005 Available online 28 November 2005
Abstract Analysing nitrogen mustards and their degradation products in decontamination emulsions posed a significant challenge due to the different phases present in such matrices. Extensive sample preparation may be required to isolate target analytes. Furthermore, numerous reaction products are formed in the decontamination emulsion. A fast and effective qualitative screening procedure was developed for these compounds, using liquid chromatography–mass spectrometry (LC–MS). This eliminated the need for additional sample handling and derivatisation that are required for gas chromatographic–mass spectrometric (GC–MS) analysis. A liquid chromatograph with mixed mode column and isocratic elution gave good chromatography. The feasibility of applying this technique for detecting these compounds in spiked water and decontamination emulsion was demonstrated. Detailed characterisation of the degradation products in these two matrices was carried out. The results demonstrated that N-methyldiethanolamine (MDEA), N-ethyldiethanolamine (EDEA) and triethanolamine (TEA) are not the major degradation products of their respective nitrogen mustards. Degradation profiles of nitrogen mustards in water were also established. In verification analysis, it is important not only to develop methods for the identification of the actual chemical agents; the methods must also encompass degradation products of the chemical agents as well so as to exclude false negatives. This study demonstrated the increasingly pivotal role that LC–MS play in verification analysis. © 2006 Elsevier B.V. All rights reserved. Keywords: Nitrogen mustards; Ethanolamine; GC–MS; LC–ESI–MS; Decontamination emulsion; Mixed mode chromatography
1. Introduction The nitrogen mustards belong to the same class of chemical agents with vesicant properties as lewisites and sulphur mustards. Nitrogen mustards were developed in the 1930s and stockpiled during World War II for military use. They are controlled as Schedule 1 chemicals in the Chemical Weapons Convention (CWC). The three nitrogen mustards are HN-1 (bis(2-chloroethyl)ethylamine), HN-2 (bis(2-chloroethyl) methylamine) and HN-3 (tris(2-chloroethyl))amine), and their structures are shown in Table 1. The possibility that the nitrogen mustards might pose a potential threat to water source led to an exhaustive study on the chemical reactions undergone by these agents in unbuffered water and water buffered at pH 8 [1]. The mechanism of the
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hydrolysis reaction was proposed (Fig. 1a). A cyclic ethylenimonium intermediate species was identified to be the focal point from which various hydrolysis products would subsequently form, depending on the solution pH, the nature of the nitrogen mustard and chloride concentration. The cyclic ethylenimonium ion was sufficiently stable to allow isolation and characterization as a salt of picrylsulfonic acid. Other ionic hydrolysis products were similarly isolated and characterized. Characterization and identification were mainly based on the purification and classical techniques available at that time, namely purification by crystallization, melting point determination, elemental composition analysis and titrametric techniques. The change in concentration of the degradation products were monitored by taking regular measurements of the concentrations of hydrogen ions and chloride ions and the required amount of thiosulphate titre over the course of 48–72 h. These analytical data along with the characterization of selective isolation of particular reaction species led to the elucidation of the hydrolysis mechanism of the nitrogen mustard in water shown in Fig. 1a.
H.-C. Chua et al. / J. Chromatogr. A 1102 (2006) 214–223 Table 1 Nitrogen mustards and their degraded compounds investigated Abbreviation
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Table 1 (Continued ) Abbreviation
Structure
Structure K
HN-1
A
L
B M
C TEA D N EDEA
DEA
HN-2
E
F
G
H
MDEA
HN-3
I
J
Ways to speed up the degradation of the nitrogen mustard for the purpose of decontamination have been studied, employing chlorinating bleach and decontamination emulsion with chlorinating or redox activities. Sartori [2] reported on the chemical properties of the nitrogen mustards. Chlorination resulted in the dealkylation of bis(2-chloroethyl)alkylamines. Aldehydes and bis(2-chloroethyl)amine were identified among the products of the reaction after treating the resultant solution with water, shown in Fig. 1b. Reaction of tertiary bis(2chloroethyl)amines hydrochloride with sodium hypochlorite solution buffered at pH 8 with sodium bicarbonate resulted in the formation of several products, among which N,N-[bis(2chloroethyl)]-N-chloroamine was identified. Further reaction of N,N-[bis(2-chloroethyl)]-N-chloroamine with hydrochloric acid gave N,N-bis(2-chloroethyl)amine (H), as shown in Fig. 1b. Since these studies, modern analytical techniques have been applied to the analysis of nitrogen mustards and their degradation products in a wide variety of matrices, with varying degree of efficiency and effectiveness on the sample preparation. For the analysis of intact nitrogen mustards, gas chromatographic analysis is the method of choice. Sample preparation from water using solid phase extraction (SPE) [3] and air using SPE disks [4] with subsequent gas chromatographic analysis have been reported. Analysis of hydrolysis products of nitrogen mustards in aqueous extract and determination by gas chromatography is both challenging and time consuming since concentration to dryness and derivatisation as part of sample treatment are required. The presence of extraneous materials may interfere with the derivatisation resulting in low apparent recoveries. Determination by liquid chromatography coupled to mass
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Fig. 1. (a) Hydrolysis pathways of HN-1, HN-2 and HN-3 in water. L can further undergo hydrolysis to give the product TEA via intermediate M [1]. (b) Formation of N via two chlorination pathways.
spectrometry would be suited for analysis of the hydrolysis products, since no derivatisation may be necessary and the presence of intact chemical agent could be analysed concurrently. Liquid chromatography–atmospheric chemical ionization mass spectrometry was applied as a screening method on water spiked with degradation products of chemical agents including ethanolamines. The chromatography column used for separation was either a C18 column and or a combination C8 /C18 column. Under those conditions, triethanolamine (TEA) and N-methyldiethanolamine (MDEA) were not resolved and N-ethyldiethanolamine (EDEA) gave split peak [5,6]. A liquid chromatographic–mass spectrometric (LC–MS) application using low capacity ion exchange column with electrospray ionisation (ESI) for the determination of TEA and MDEA was reported for analysis of aqueous extracts of vegetation [7]. A detection limit of 0.02 ppm was achieved, demonstrating that LC–ESI–MS and LC–ESI–MS–MS methods are well suited for direct quantitation and confirmation of TEA and MDEA with little sample cleanup. LC–MS application had also been extended successfully to more complex matrices such as plasma and urine. The use of diethyldithiocarbamic acid for derivatising HN-2 in plasma, fol-
lowed by SPE extraction of derivatised HN-2 prior to LC–MS analysis was reported [8]. Lemire and co-workers [9,10] reported a sensitive and rapid analysis for the quantitation of ultra-trace level of EDEA and MDEA in human urine. SPE cation exchange was for extraction and quantitation was by isotopic dilution with detection by multiple-reaction monitoring. In summary, analysis for intact nitrogen mustard by GC–MS and LC–MS are possible, although the former is the preferred method due to better sensitivity. However, for the analysis of the hydrolysis products, GC–MS may be difficult and time consuming since these products tend to be highly polar. As a result, sample pre-treatment must incorporate the additional steps of derivatisation and sample enrichment (for complex matrices) to promote detection sensitivity at the expense of the total analysis time. Under these circumstances, LC–MS becomes the more desirable technique. It offers the advantage of concurrent detection for the intact agent and its hydrolysis products. Few published literature is available on the applicability of LC–MS to the analysis of nitrogen mustard and its degradation products in decontamination emulsion. Wils and Hulst [11] reported the analysis of HN-2 by thermospray LC–MS as an
H.-C. Chua et al. / J. Chromatogr. A 1102 (2006) 214–223 Table 2 LC–ESI–MS and CID product ion spectra of nitrogen mustards and their degradation products Compounds
Molecular ions m/z
Fragment ions m/z
HN-1 A B C D EDEA DEA HN-2 E F G MDEA HN-3 I J K L M TEA N
170 134 152 116 108 134 106 156 120 138 102 120 204 168 186 150 168 132 150 142
142, 144, 134, 136, 106, 108, 72, 70, 63, 65 106, 108, 72, 70 134, 136, 106, 108, 72, 70 98, 88, 72, 70 80, 82, 72 116, 98, 88, 72, 70 88, 70 128, 130, 120, 122, 92, 94, 84, 63, 65, 58 92, 94, 84, 63, 65, 58 120, 122, 92, 94, 84, 63, 65, 58 58 102, 58 176, 178, 168, 170, 106, 108, 78, 80, 70, 63, 65 106, 108, 70, 63, 65 168, 170, 106, 108, 70, 63, 65 132, 134, 106, 108, 70, 63, 65 150, 152, 132, 134, 106, 108, 70, 63, 65 114, 88, 70 132, 114, 88, 70 114, 116, 106, 108, 70, 63, 65
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A commercial decontamination emulsion based on sodium dichloroisocyanurate was purchased off-the-shelf. It consists of two components. Component 1 comprises sodium dichloroisocyanurate (16.7% with approximately 10% active chlorine), talcum (16.7%), sodium chloride (33.3%) and calcium chloride dihydrate (33.3%). Component 2 contains xylene (80%) and an emulsifier. The decontamination emulsion based on a mixture of these components was prepared in accordance with the manufacturer’s instruction. The resultant mixture is a viscous white emulsion and has a pH of 8.0. Chemicals were used without further purification. Solvents used, including analytical reagent HPLC grade ACN, dichloromethane (DCM), isopropyl alcohol (IPA), and ethyl acetate were from J.T. Baker (Phillipsburg, NJ, USA). Water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). 2.1.1. Safety considerations Nitrogen mustards are known vesicants. All handling of the agents were in dilute amounts and carried out in an efficient fume hoods equipped with alkaline scrubber. Gloves and appropriate protective measures were adopted. 2.2. Instruments
acetoxy derivative. In this paper, we report a screening method by LC–MS, in a single run, that covers most of the degradation products reported for water and decontaminating solution with chlorinating activity [1,2]. The identification of degradation products was confirmed by comparison of their mass spectra to those of authentic compounds where available, and by tandem mass spectrometry (MS/MS) and nuclear magnetic spectroscopy (NMR). The LC–MS–MS data (Table 2) are presented in this paper. The developed screening method also allows the analysis of intact nitrogen mustards. This paper will discuss the differences in the compounds detected by LC–MS in the hydrolysis of nitrogen mustards in water and their chlorination products in decontamination emulsion were compared. Time profile studies were also performed to ascertain the behavior of nitrogen mustards in water. 2. Experimental 2.1. Chemicals and reagents The nitrogen mustards and their degradation products investigated, together with the abbreviations used, are shown in Table 1. MDEA (99%), EDEA (98%), TEA (98%), DEA (98%), N (98%) and spectrophotometric grade trifluoroacetic acid (TFA) (99%) were from Aldrich (Milwaukee, WI, USA). Deuterium oxide (D2 O) was from Merck (99.95%) (Darmstadt, Germany). HN-1, HN-2, HN-3, B, D, F, H, J and L were synthesized inhouse with greater than 95% purity by GC–MS. They were dissolved in acetonitrile (ACN) (1000 g ml−1 ) and stored at 4 ◦ C as stock solution. The working solution was prepared by diluting to the appropriate concentration with ACN prior to use.
2.2.1. Liquid chromatography–mass spectrometry LC–MS analyses were performed on Shimadzu HPLC (Kyoto, Japan). The system was fitted with a 150 mm × 2.1 mm i.d. Primesep 100 column (SIELC Technologies Co., USA), with ˚ pore size. The mobile phase at iso5 m particle size and 100 A cratic elution consisted of 0.1% TFA in water [mobile phase A]: 0.1% TFA in acetonitrile: water (95:5) [mobile phase B] (60:40) at a flowrate of 0.2 ml min−1 . Injections (1 l) were made using the autosampler. The column effluent was introduced into a TSQ7000 mass spectrometer (Thermo-Finnigan, San Jose, CA, USA) via an atmospheric pressure ionisation source/interface operated in ESI mode. Capillary and tube lens voltages were optimised to give maximum response to m/z 156 [M + H]+ from HN-2. This ensures that the [M + H]+ ions were prominent in the spectra. ESI conditions were as follows: capillary temperature 250 ◦ C, spray voltage 5 kV, Q0 offset −5 V, sheath gas nitrogen (Air Products, Singapore) at 60 psi and auxiliary gas nitrogen at a flow-meter reading of 20. The mass scan range was m/z 50–m/z 500. CID product ion spectra were obtained from the protonated molecules of the compound using the LC conditions described above. Argon was used as collision gas, collision offset −20 V and Q0 offset −5 V. 2.2.2. Nuclear magnetic resonance spectrometry 1 H NMR spectra were acquired on a Varian 500 MHz NMR spectrometer (Varian Inc., Palo Alto, CA, USA) and processed using the Varian VNMR software. The 1 H NMR experiments were conducted at 25 ◦ C and accumulated with a spectral width of 5000 Hz with 32,000 data points and a pulse repetition delay of 4 s. One microliter of neat agent was spiked into 1 ml of water to give a 1000 g ml−1 solution. One hundred microliters of D2 O was added to 600 l of this solution for NMR analysis.
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2.3. Nitrogen mustards study by LC–MS 2.3.1. Spiking of decontamination emulsion samples Decontamination emulsion (1 ml) was spiked individually with 5 l of neat HN-1, HN-2 and HN-3 at a concentration of 5000 g ml−1 and allowed to stand at room temperature for 1 h. 2.3.2. Spiking of water samples Deionised water (4 ml) was spiked individually with 4 l of neat HN-1, HN-2 and HN-3 at a concentration of 1000 g ml−1 and stored at room temperature for a time profile study over 72 h. The water sample was first analysed at zero hour and then every half an hour interval for 72 h. 2.4. Sample preparation ACN (1 ml) was added to the decontamination emulsion (1 ml). The mixture was vortexed for 10 s and then centrifuged at 4500 × g for 10 min. The organic and aqueous layers were transferred to separate vials for LC–MS analysis. The water samples were directly analysed. All experiments were carried out in triplicates. 3. Results and discussion 3.1. Method development for LC–MS 3.1.1. C18 versus mixed-mode column Initially a C18 column was used for chromatography. Fig. 2a showed the reconstructed ion current chromatogram of a cocktail of 14 standards prepared in ACN. To increase retention and improve the sensitivity of detection of these compounds, an acidic condition and the lowest percentage of organic mod-
ifier were used in the mobile phase. However, the column still exhibited poor retention and selectivity for the ethanolamines and the other nitrogen compounds because of their highly polar and basic characteristics. Under these circumstances, possible remedies would be to employ ion-pairing reagents in the mobile phase or a cation exchange column to enhance retention. Unfortunately, the prerequisite LC mobile phases are not conducive to ESI–MS. With these considerations, a mixed-mode column with embedded ion-pairing group was selected for this study. The column, Primesep 100, exhibits both hydrophobic and cation exchange retention mechanisms. The latter interaction would be selective towards polar basic species such as the nitrogen compounds covered in this study (Table 1). Fig. 2b showed the reconstructed ion current chromatogram of a cocktail of the standards by mixed mode column. This column provides chromatography under reversed phase conditions, which are compatible with ESI-MS. The resolution of the standards is greatly improved in comparison to the chromatogram on the C18 column. 3.1.2. Mobile phase TFA (0.1%) in water as mobile phase A and 0.1% TFA in ACN:water (95:5) as mobile phase B was used. Preliminary work with this column indicated that chromatography was best performed under isocratic rather than gradient elution. Based on isocratic runs at different composition of the mobile phase A over the range of 20–80%, an increase in the percentage of mobile phase A resulted in an increase in retention time and overall improvement in resolution, except for compounds B and N which co-eluted as one peak. With mobile phase A at isocratic condition of 60%, a good separation with almost baseline resolution for most of the
Fig. 2. Reconstructed ion current chromatogram of nitrogen mustards and their related products by (a) C18 column, and (b) mixed mode column: 1 = DEA, 2 = TEA, 3 = MDEA, 4 = EDEA, 5 = L, 6 = F, 7 = B, 8 = N, 9 = J, 10 = HN-2, 11 = HN-1, 12 = HN-3.
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compounds, except for DEA and TEA, was achieved in less than 20 min (Fig. 2b). Although these two compounds co-elute, some separation between them still exist. This is sufficient for discriminating them by performing extracted ions analysis. No degradation products were observed on running a standard mixture of the nitrogen mustards prepared in ACN, indicating the stability of these compounds under the LC conditions used. Hence, the isocratic mobile phase of 60% A:40% B was selected for general screening of water and emulsion for all the compounds proposed in Table 1. There were two problems encountered during the screening of water and emulsion sample by the developed LC conditions. The general screening LC conditions could resolve the cyclic and acylic compounds of partially hydrolysed HN3 but not the corresponding species of HN-1 and HN-2 in the water. Compounds B and F co-eluted with compounds C and G, respectively. In the context of confirmatory analysis, achieving this resolution would be essential in avoiding identification ambiguity. To resolve these pair of peaks, the mobile phase A was increased to a higher isocratic composition of 95%. However, this was at the expense of the run time, which increased from 20 to 60 min. Hence, the isocratic mobile phase of 95% A:5% B would only be used when confirmatory analysis was required. The extracts from decontamination emulsion were analysed by LC–MS using the isocratic mobile phase of 60% A:40% B. The presence of N-(2-chloroethyl)N-alkylamine species D and H, formed from the chlorination of HN-1 and HN-2, respectively, presented chromatographic problem. Compound H co-eluted with the partially hydrolysed HN-2 (F) and compound D gave a split peak. Altering to the same isocratic mobile phase of 95% A:5% B mentioned above (for resolving the acyclic/cyclic species) gave baseline resolution of H and F. For the case of compound D, re-focusing to give a single peak was achievable by increasing the TFA content from 0.1 to 0.2% in both mobile phases A and B at 60% A:40% B. 3.2. Identification The optimised capillary temperature, tube lens voltage, sheath gas nitrogen and auxiliary gas nitrogen are given in Section 2.2.1. Under these soft ionization conditions organic standards of nitrogen mustards and its degradation products in ACN gave intense protonated molecular ions [M + H]+ with no fragmentation, except for compounds D, H and N. These produced intense protonated acetonitrile adduct ions [M + ACN + H]+ . Identification of the acyclic compounds detected in the water and decontamination emulsion sample was based on matches of the obtained ESI–MS, ESI–MS–MS spectra and retention times against those of the reference standards. The shift in retention time between the reference standards and analytes was less than 0.1 min. Identification of the cyclic compounds was based on the ESI–MS spectra, ESI–MS–MS spectra (Table 2). Generally, the ESI–MS spectra of the cyclic compound exhibited intense M+ and [M + TFA]+ adduct ions. The NMR data of A and E was used to confirm the cyclic structures. The signals for A are assigned as follows: 1 H NMR (500 MHz, D2 O): ␦ 4.022 (t,
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J = 5.8 Hz, He ), 3.618 (t, J = 5.8 Hz, Hd ), 3.238 (m, Hc ), 3.337 (q, J = 7.3 Hz, Hb ), 1.365 (t, J = 7.3 Hz, Ha ).
Similarly, the signals for E are assigned as follows: 1 H NMR (500 MHz, D2 O): ␦ 4.055 (t, J = 5.8 Hz, Hd ), 3.620 (t, J = 5.5 Hz, Hc ), 3.271 (m, Hb ), 3.152 (s, Ha ).
An attempt was made to confirm the other cyclic compounds using NMR. However, due to the simultaneous existence of several compounds resulting in overlapping peaks, not all of the signals due to the compounds could be assigned. 3.3. Analysis of nitrogen mustards in water The time profile on the degradation of individual HN-1, HN2 and HN-3 in water was studied using the developed LC–MS method. Water samples were spiked separately with nitrogen mustards at 1000 g ml−1 level. For analytical convenience, since the ethanolamines had already been characterised earlier on in terms of their retention times and mass spectra, these ethanolamines were selected as internal standards for use only in the water studies to correct for instrumental variation. Prior to this, it was empirically verified that TEA could never exist as the degradation product of HN-1, DEA of HN-2 and EDEA of HN-3 (data not shown). Hence, TEA was added as an internal standard into the water spiked with HN-1, and similarly DEA and EDEA were spiked respectively in water samples separately containing HN-2 and HN-3. Subsequently, 1 l of the spiked water samples were analysed every 0.5 h over an observation period of 72 h. The 1-l aliquot was analysed directly by LC–MS without sample preparation. Figs. 3–5 show the respective time profiles, with the values for the y-axis presented in logarithmic format to fit all the data points of all the compounds on the same scale. Similar time profile in the decontamination emulsion was not performed, since no suitable internal standards were found that would not degrade in the aggressive environment of the decontamination emulsion. Fig. 3 showed the degradation of HN-1 in water. HN-1 was rapidly converted to the ethylenimonium compound A, which became the most abundant species up to 20 h. Compound A is known to be fairly stable since it had previously been isolated as a salt of picrylsulfonic acid and characterised by classical techniques [1]. The rapid conversion rate caused the concentration of HN-1 to drop below the instrument detection limit between 0.5 and 2.5 h. Compound A underwent further hydrolysis to give B, the hydroxylated ethylenimonium ion (C) and EDEA. The hydrolysis reaction released H+ , which led to the gradual pH decrease from 7 to 4 observed over 72 h.
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Fig. 3. Time profile of HN-1 in water over 72 h. Inset graph shows the first 25 h of the time profile.
From 20 h onwards, compound B became the predominant product and compound A remained present as a major species. Compound C and EDEA existed as minor products up to the end of the observation period (72 h). EDEA became detectable at 3 h onwards. The predominance of compound B could be due to the change in pH leading to the stabilization of the compound through salt formation [1]. Besides the two minor products already mentioned, HN-1 re-appeared as a minor product in sufficient amount to be detectable from 2.5 h onwards, its concentration is similar in level to compound C and EDEA. The re-appearance of HN-1 might be from the two predominating species present at that time: compound A and the chloride ions reacting with each other to reform the mustard. The appearance of the mustard at a later stage indicated a prerequisite reaction condition be present before the reversion process would occur, probably an acidic condition. Although this was not experimentally verified for HN-1, HN-2 was reported to be released when an aqueous solution of N-(2-chloroethyl)-N-methyl ethylenimonium ion was acidified with hydrochloric acid [1]. Fig. 4 showed the degradation of HN-2 in water. Due to the structural similarity to HN-1, HN-2 degraded in a similar manner. Conversion of HN-2 to the ethylenimonium ion (E) was rapid. Hydrolysis thereafter also produced compound F, hydroxylated ethylenimonium ion (G) and MDEA, with the corresponding pH change found to drop from 7 to 5 over 72 h. Initially, the concentration of HN-2 decreased to very low albeit
still detectable level in the time period between 2 and 4 h. Subsequent reversion reaction led to HN-2 increasing gradually in concentration up to the concentration level similar to that of compound G and MDEA. Unlike the degradation process of HN-1, the corresponding species of compounds F, G and E were already detectable at the very initial stage, whilst MDEA was detected only from 2.5 h onwards. But as with HN-1, these species existed as minor products. The main degradation product observed was compound F, which became the most abundant species from 15 h onwards. The reasons for the stability of compound F and the reversion to HN-2 are the same as described above in HN-1. HN-2 hydrolysis was also known to produce substantial amount of dichloro cyclic and dihydroxyl cyclic dimers [1], but they were not detected in our study. Because of its three chloroethyl groups, the degradation of HN-3 was more complicated compared to HN-1 and HN2 (Fig. 5). HN-3 was rapidly hydrolysed and became undetectable after 25 h. In contrast to HN-1 and HN-2, no reversion process occurred with HN-3, hence after 25 h it remained below the detection limit of the instrument. The hydrolysis occurred via three ethylenimonium ions (I, K and M), producing hydrolytic products that were mainly ethanolamines with either the chloroethyl (L) or dichloroethyl group (J) attached. Compound J was the predominant product up to 30 h. From 30 h onwards, compound L became the predominant product and compound J remained present as major species. Unlike HN-1
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Fig. 4. Time profile of HN-2 in water over 72 h. Inset graph shows the first 20 h of the time profile.
and HN-2, the first ethylenimonium ion (I) was not long lived and become undetectable after 2.5 h. The second and third ethylenimonium ions (K and M) were detectable in trace level throughout the observation period. The final stage of the hydrolysis process produced TEA, which was found to be present only as a minor product after 72 h. Before that, only trace amount was observed from 7 h onwards. In contrast to HN-1 and HN-2, the pH dropped appreciably from 6 to 2 over the course of the experiment. 3.4. Analysis of nitrogen mustards in decontamination emulsions Decontamination emulsions were spiked separately with HN1, HN-2 and HN-3, and left to react for 1 h before sample extraction and analysis by LC–MS. In the sample pretreatment, an organic solvent was added to breakdown the emulsion, followed by centrifugation, to separate the emulsion into three layers: organic, aqueous and sludge. Preliminary LC–MS analysis on these layers had shown that most of the analytes were found in the organic layer, with approximately 30 g ml−1 (based on a 1-point standard) or less remaining in the aqueous and sludge
layers. Since the proportion was small, the sludge was not analysed in the subsequent work. Various organic solvents ethyl acetate, ACN, DCM and IPA were evaluated for their emulsion breakdown efficiency. Both DCM and IPA caused precipitation at the interface of the organic and aqueous layers. Filtration was required prior to LC–MS analysis. No precipitate was observed when ethyl acetate and ACN were used. Hence only ethyl acetate and ACN were further considered. The organic extracts using the ACN and ethyl acetate and the extracted aqueous layer were analysed by LC–MS without prior filtration. The LC–MS analysis employed the optimized conditions determined from the water studies described earlier. Unfortunately the aqueous layer from ethyl acetate extract consistently caused blockage of the heated capillary tube, which required clearance after every three sample injections. The ACN extract, on the other hand, did not present any such problem. The active ingredient in the decontamination emulsion under study is sodium dichloroisocyanurate. It acts by producing hypochlorous acid (HClO) upon interaction with water. Generally, the LC–MS analysis on the ACN extracts of the decontamination emulsion revealed the presence of intact mustards as
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Fig. 5. Time profile of HN-3 in water over 72 h. Inset shows the first 10 h of the time profile.
well as products of chlorination and hydrolysis. In the emulsion, HN-1 degraded to give only chloroethylamine species (D and N); while HN-2 and HN-3 degraded to give partially hydrolysed nitrogen mustards (F, J and L) and chloroethylamines (H and N). The ESI–MS spectra of the compounds in the extract matched that of the reference standards. The differences in retention time as compared to the reference standard were less than 0.2 min. For HN-2 and HN-3, not all the compounds detected were identified. The identities of one unknown peak in the extract of the decontamination solution spiked with HN-2, and two unknown peaks with HN-3 are still being determined. These unidentified compounds existed in trace level. On the basis of the relative peak heights in the chromatograms, HN-3 was clearly difficult to degrade using the emulsion. Its degradation products gave noticeably smaller peaks compared to those of HN-1 and HN2. Correspondingly, the HN-3 peak was larger than the HN-2 peak, and HN-1 was completely degraded. The characterization of these compounds could prove valuable for future analyses since their identification could verify the presence of nitrogen mustards.
presence of nitrogen mustards and their degradation products in water and decontamination emulsion. There were no matrix interferences from the decontamination emulsions despite the numerous ingredients present. Acetonitrile was chosen as an extraction solvent for qualitative analysis of decontamination emulsion sample. The ethanolamines can only be detected in the aqueous layer. Both the hydrolysis and decontamination studies revealed that N-methyldiethanolamine, N-ethyldiethanolamine and triethanolamine are not the major degraded products of their respective nitrogen mustards. The partially hydrolysed nitrogen mustards are the more prominent evidence. Acknowledgements The support from the organic synthesis group of DSO National Laboratories is gratefully acknowledged. This work was funded by the Directorate of Research & Development, Defence Science & Technology Agency, Singapore. References
4. Conclusion The developed LC–MS method, based on the mixed-mode column, offers a quick and effective method of screening for the
[1] C. Golumbic, J.S. Fruton, M.A. Stahmann, M. Bergmann, J. Org. Chem. 11 (1946) 518. [2] M.F. Sartori, Chem. Rev. 48 (1951) 225. [3] N.M.A. H¨akkinen, J. High Resolut. Chromatogr. 14 (1991) 811.
H.-C. Chua et al. / J. Chromatogr. A 1102 (2006) 214–223 [4] J.R. Stuff, R.L. Cheicante, H.D. Durst, J.L. Ruth, J. Chromatogr. A 849 (1999) 529. [5] R.M. Black, R.W. Read, J. Chromatogr. A 759 (1997) 79. [6] R.W. Read, R.M. Black, J. Chromatogr. A 862 (1999) 169. [7] J.V. Headley, K.M. Peru, L.C. Dickson, Rapid Commun. Mass Spectrom. 13 (1999) 730.
223
[8] T. Karayilanoglu, E. Berenshtein, L. Kenar, N. Kitrossky, U. Kisa, M. Chevion, Turk. J. Biochem. 26 (3) (2001) 90. [9] S.W. Lemire, D.L. Ashley, A.M. Calafat, J. Anal. Toxicol. 27 (2003) 1. [10] S.W. Lemire, J.R. Barr, D.L. Ashley, C.T. Olson, T.L. Hayes, J. Anal. Toxicol. 28 (5) (2004) 320. [11] E.R.J. Wils, A.G. Hulst, Fresenius J. Anal. Chem. 342 (9) (1992) 749.