Anal Bioanal Chem DOI 10.1007/s00216-014-7623-0
NOTE
Rapid screening of N-oxides of chemical warfare agents degradation products by ESI-tandem mass spectrometry L. Sridhar & R. Karthikraj & V. V. S. Lakshmi & N. Prasada Raju & S. Prabhakar
Received: 31 October 2013 / Revised: 31 December 2013 / Accepted: 10 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract 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-2chlorides, 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 Published in the topical collection Analysis of Chemicals Relevant to the Chemical Weapons Convention with guest editors Marc-Michael Blum and R. V. S. Murty Mamidanna. Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-7623-0) contains supplementary material, which is available to authorized users. L. Sridhar : R. Karthikraj : V. V. S. Lakshmi : N. P. Raju : S. Prabhakar (*) National Center for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India e-mail: [email protected]
complex masking agents, without the use of timeconsuming 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. Keywords Dialkylaminoethanols . Dialkylaminoethyl-2-chlorides . N-oxides . Neutral loss scan . Rapid screening . LC-MS/MS
Introduction Detection and identification of chemical warfare agents (CWAs), their precursors/degradation products in suspected sites, or environmental samples has been an important area of research [1, 2]. It is also important for the verification process, an important component of monitoring compliance with the chemical weapons convention (CWC). Adoption of new methods for rapid screening of CWC-related chemicals is always advantageous for implementation of CWC, which an international treaty came into force in 1997 to prohibit development/production/storage/usage of chemical weapons [3, 4]. The treaty is administered by the Organization for the Prohibition of Chemical Weapons (OPCW), the Netherlands. The OPCW maintains a network of designated laboratories for
L. Sridhar et al.
off-site analysis of samples collected during inspections and other needs for the implementation of the CWC [5]. Most of the CWAs are prone to undergo hydrolysis and oxidation in environmental matrices leading to more persistent degradation products. The alkylaminoethanols and triethanolamine (S3.B.15 to S3.B.17) have their own importance since their chlorination leads to the formation of nitrogen mustards (S1.A.06). The N,N-dialkylaminoethyl-2-chlorides (S2.B.10) and N,N-dialkylaminoethanols (S2.B.11) are the precursors and the hydrolyzed products of nerve agents (VX type of compounds, S1.A.03). These compounds are prone to undergo oxidation in environmental matrices or upon decontamination process. The N-oxides of aminoethanols and aminoethylchlorides are non-scheduled chemicals, but they are relevant to the verification of CWC because the presence of these degradation products in suspected matrices such as organic, oil, soil etc. reveals alleged use of nitrogen mustards or precursors of VX compounds. Thus, screening of environmental residues for hydrolysis and oxidation products is an important part of verification process [6]. As a first attempt, we synthesized the N-oxides of CWC-related aminoethanols and aminoethylchlorides by applying know procedures, and reported characterization of all the N-oxides, including isomers, by mass spectrometry [7, 8]. The N-oxides were known to be thermally labile, thus they cannot be analyzed by GCMS. Consequently, on-site analysis of these compounds is difficult for the OPCW inspection teams as they routinely use GC-MS for verification. Therefore, a soft ionization technique, electrospray ionization (ESI), using direct flow injection or LC separation, is a facile technique for the identification of these N-oxides by mass spectrometry. The direct ESIMS analysis of target N-oxides readily formed protonated molecule ions, [M+H]+ ions. In the previous study, we reported characterization of the N-oxides based on specific/selective fragmentation of the respective [M+H]+ ions [7, 8]. In order to identify the N-oxides present in a suspected sample using this method, at first a full scan ESI mass spectrum to be recorded for the sample extract, and then perform MS/MS on each detected peak whose m/z value matches with that of the target N-oxides. The presence of an N-oxide can only be confirmed if the MS/MS spectrum matches with the standard N-oxide spectrum [7, 8]. However, in the real scenario, the target Noxide peaks may be missed or masked by the matrix constituents and background chemicals present in the environmental samples at high concentration levels. Moreover, the m/z values of the [M+H]+ ions associated with the N-oxides may match with those of other classes of CWC schedule compounds. For example, the [M+H] + ions of N-oxides of N,Ndialkylaminoethanols matches with the [M+H]+ ions of corresponding N,N-dialkylaminoethanethiols. It is possible to overcome such problems by applying specific tandem mass spectrometric (MS/MS) scan modes of analysis, such as constant neutral loss (NL) scan mode and multiple reaction
monitoring mode [6, 9]. In these lines, we have developed a sensitive, selective, and rapid screening method by applying direct ESI-MS/MS (NL scan) technique for unambiguous detection of N-oxides of CWC-related aminoethanols and aminoethylchlorides in a single step, even in the presence of a large amount of masking agents/background chemicals. The study also includes structure confirmation of critical isomers of N-oxides by LC-MS/MS analysis.
Experimental Chemicals All the N-oxides (Fig. 1) used in the present study were synthesized by the oxidation of the corresponding tertiary amine using reported methods [7, 8]. Analytical grade solvents were used for chromatographic analysis, and they were obtained from E-Merck (Mumbai, India). Ammonium acetate and triethylamine were purchased from Sigma-Aldrich (St. Louis, USA). Stock solutions (100 ppm) of individual Noxides of aminoethanols and aminoethylchlorides and mixtures of N-oxides were prepared in methanol and dichloromethane, respectively. The stock solutions were further diluted in methanol/acetonitrile for MS analysis. Water sample The stock solution of N-oxides of aminoethanol mixture (DM, ME, MP, EP, DP, MDEA, EDEA, and TEA) was spiked in a blank water sample (5 ppm) received during the 28th official OPCW PT. The blank water consists of polyethylene glycol 200 (5,000 ppm) in deionized water. Soil sample The stock solution of a mixture of N-oxides of aminoethanols (DM, ME, MP, EP, DP, and TEA) was spiked at various concentrations (500 ppb–25 ppm) in a blank soil sample received during the 30th official OPCW PT. The blank soil sample consists of diesel fuel (1,000 ppm), n-butyl phosphonic acid (10 ppm), and dibutyl phosphate (10 ppm) in sand (ISO standard). This sample was subjected to water extraction and 1 % triethylamine (TEA) in methanol extraction as per standard procedures used for proficiency test (PT) samples. Briefly, 5 g of soil sample was extracted twice with 5 ml of water by shaking for 10 min. The supernatant solutions from extractions were collected, centrifuged, combined, and concentrated to 1 ml by rotary evaporator. Similar procedure was used to extract fresh soil sample with 1 % TEA/methanol, where the combined extracts were concentrated to 100 μL with mild nitrogen flow.
Rapid screening of N-oxides of chemical warfare agents Fig. 1 Molecular structures of the studied N-oxides of aminoethanols and aminoethylchlorides
Organic sample A mixture of N-oxides of aminoethylchlorides (DM-Cl, MPCl, EP-Cl, DP-Cl, MDEA-Cl, and TEA-Cl) was spiked in a blank organic waste sample (5 ppm) received during the 28th official OPCW PT. The blank organic sample contains alkane mixture (C7–C30; 10 ppm) and petroleum ether (10 %) in nhexane.
under the control of Xcalibur software. The typical ESI source conditions were as follows: spray voltage 4.8 kV, capillary voltage 20 V, capillary temperature 300 °C, and tube lens offset voltage 10 V. Nitrogen (40 psi) was used as sheath gas and helium (1.8 mTorr) was used as damping gas and collision gas. The collision energies used for LC–MS/MS experiments were 18–30 eV. Extraction recovery
Mass spectrometry All the experiments were carried out on a Quattro LC, a triple quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with an ESI source operated in positive ion mode. All the spectra were acquired using MassLynx software (version 3.2). The typical operating conditions were capillary voltage, 3.5 kV; cone voltage, 30 V; source housing and desolvation temperatures, 200 °C. Nitrogen was used as nebulization and desolvation gas. The product ion and NL scan experiments were performed by using collision energies 18– 30 eV. Argon was used as the collision gas, and the collision cell pressure was maintained at 1.67×10−3 mbar. All the spectra were averages of 20 scans. For direct ESI-MS experiments, sample was directly infused into ESI source at 10 μL/ min using a syringe pump (Harvard). For LC-MS and LC-MS/ MS experiments, an Alliance 2695 HPLC system (Waters) with an autoinjector and a Zorbax Hilic Plus C18 (4.6 mm× 100 mm, 3.5 μm) column (Agilent Technologies, CA) was used. The injection volume of the sample was 10 μL. The mobile phase consisted of 5 mM ammonium acetate solution (A) adjusted to pH 3 with acetic acid and acetonitrile (B) in 15:85 ratio. LC-MS and LC-MS/MS experiments were also performed using LCQ ion trap mass spectrometer (Thermo Fisher, San Jose, CA, USA) coupled with Surveyor HPLC system, using the same column. The data acquisition was
Extraction recoveries for all the N-oxides were determined by spiking the N-oxides at four levels of concentration (1, 10, 25, 50 ppm) before and after extraction. In spiking experiment before extraction, the N-oxides were spiked in 2 g of the soil sample and extracted with water and TEA/methanol (independently). In spiking experiment after extraction, 2 g of soil sample was extracted with water and TEA/methanol (separately), and then the N-oxides were spiked to each blank extract. The extracts were analyzed by direct ESI-MS/MS (NL method) in triplicate, and the peak areas of the N-oxide peak (s) in the NL mass spectra were used to calculate the extraction recovery values. Validation The validation of the NL scan screening method was performed according to Eurachem guidelines for qualitative validation [10, 11]. The main purpose of qualitative screening methods is to distinguish between negative samples and positive samples at a determined level; the method is considered as validated when positive findings are reported in all samples. The limit of detection (LOD) was obtained by spiking the Noxides in blank samples at five different concentration levels (100 ppb–1 ppm). The specificity of the NL method was tested by analyzing with or without spiking N-oxides in
L. Sridhar et al.
solvent blank samples, blank water/soil sample extracts, and a test sample mixture containing several other classes of CWCrelated chemicals, i.e., VX, sulfur mustards, nitrogen mustards, aminoethanethiols, aminoethanols, thiodiglycol, bis-(2-hydroxyethyl)-disulfide, and phosphonic acids.
Results and discussions NL method for screening N-oxides Direct ESI-MS analysis of all the N-oxides (Fig. 1) show abundant [M+H]+ ions. Previously, we have studied the collision-induced dissociation (CID) spectra of the [M+H]+ ions from N-oxides of aminoethanols and aminoethylchlorides, and found that they show a specific product ion due to a constant neutral loss, i.e., CH4O2 (48 u) and CH3ClO (66 u), respectively (see Electronic Supplementary Material Fig. S1). This specific fragmentation process can be used for rapid screening of these compounds in a complex mixture by applying NL scan mode of analysis. A NL scan method was developed for the N-oxides of aminoethanols and aminoethylchlorides by optimizing typical instrument conditions including collision energy (18–20 eV). A NL of 48 u is set for N-oxides of aminoethanols, and 66 u for Noxides aminoethylchlorides. When a mixture of N-oxides of aminoethanol was subjected for NL scan of 48 u by injecting the sample directly into ESI source, the spectrum exclusively showed the peaks due to the [M+H]+ ions of the corresponding N-oxides of aminoethanols. Similarly, when a mixture of Noxides of aminoethylchlorides was subjected for NL scan of 66 u, the spectrum specifically showed the [M+H]+ ions of the corresponding N-oxides of aminoethylchlorides. We have also recorded NL scan spectra for a mixture of some N-oxides of aminoethanols and aminoethylchlorides by selecting two NL channels (channel 1, 48 u and channel 2, 66 u) in a single experiment. The spectra clearly show the [M+H]+ ions of all the N-oxide compounds in the mixture (see Electronic Supplementary Material Fig. S2). Thus, the presence/absence of N-oxides of aminoethanols and aminoethylchlorides in a complex environmental sample can be rapidly ascertained based on the NL scan method without involving any chromatography separation techniques.
peaks. However, when the same samples subjected for NL scan mode (48 and 66 u), the spectra exclusively showed the [M+H]+ ions of all the N-oxides that were spiked (Fig. 2 and see Electronic Supplementary Material Fig. S3). Thus, it is possible to detect the target N-oxides in a complex mixture unambiguously by applying NL scan experiment. We have also applied the NL scan method to a soil sample, where the analysis of oxidation products is a challenging task because this matrix involves sample preparation step. To demonstrate the viability of this method to soil samples, a mixture of N-oxides of aminoethanols was spiked in a blank soil sample of the 30th official OPCW PT. The soil sample was subjected to water and TEA/methanol extractions. The extracts were analyzed by direct ESI-MS method and NL scan method. The full scan ESI mass spectra of these samples appeared very complex; however, the NL scan spectra clearly showed the [M+H]+ ions of all the spiked aminoethanol Noxides (Fig. 3 and see Electronic Supplementary Material Fig. S4). The extraction recoveries were found to be in the range from 65 to 82 % in water extraction and 52 to 64 % in TEA/methanol extraction. Validation of NL scan method The NL scan method used in the current study is a qualitative method, i.e., a method of analysis whose response is either the presence or absence of the analyte, detected directly in a certain amount of sample; this method is validated as per the guidelines of EURACHEM [10]. LOD of the N-oxides in NL scan method was tested by injecting the samples of N-oxides
Application of the NL method for test samples A mixture of N-oxides of aminoethanols was spiked in a blank water sample received during the 28th official OPCW PT. A mixture of N-oxides of aminoethylchlorides was spiked in a blank organic waste sample received during the 28th official OPCW PT. These samples were directly analyzed by full scan ESI-MS and by NL scan mode. The ESI mass spectra of these samples showed a complex spectrum where the matrix/ background chemicals peaks obscured the target compounds
Fig. 2 a Full scan mode b neutral loss (66 u) scan mode mass spectra of organic sample spiked with a mixture of N-oxides of aminoethylchlorides under ESI conditions
Rapid screening of N-oxides of chemical warfare agents
Isomeric N-oxides The direct NL scan method can be applied for rapid screening of N-oxides in environmental samples, which reveals presence or absence of the target N-oxides. However, MS/MS technique needs to apply to obtain full structure of N-oxides. In our previous reports, we have discussed the structure indicative fragment ions during CID of [M+H]+ ions from the N-oxides of aminoethanols and aminoethylchlorides [7, 8]. Thus, it is possible to confirm the structure of an N-oxide by direct ESI-MS/MS method. However, it is problematic when a mixture of isomeric N-oxides is present in the same sample. Under such conditions, direct ESI-MS/MS can only be useful where each isomer results in a specific/selective fragment ion pertinent to its structure. For example, the base peak in the MS/MS spectrum of MP is the ion at m/z 86 (loss of 48 u), while it is m/z 92 in the case of MIP (loss of 42 u). In the MS/ MS spectrum of a mixture of MP and MIP, both the ions were Fig. 3 a Full scan mode b neutral loss (48 u) scan mode mass spectra of water extract of soil sample spiked with a mixture of N-oxides of aminoethanols under ESI conditions
100
a
DM 16.0
MP 9.7 EP 8.3
of aminoethanols and aminoethylchlorides at different concentration levels (100 ppb to 5 ppm), and LOD of the method was estimated for each analyte at a signal-to-noise ratio of 3:1 and the LOD was found to be 500 ppb. The specificity of the NL method, i.e., the ability to measure an analyte in the presence of other compounds and matrix components, was tested by analyzing different samples with or without spiking N-oxides. When the solvent blank samples and blank water/ soil sample extracts, and a test sample mixture containing several other classes of CWC-related chemicals without Noxides, were analyzed by the NL scan method (NL of 48 and 66 u), no peaks were detected in the spectra (see Electronic Supplementary Material Fig. S5). Absence of peaks in the NL scan mass spectra of the above-negative samples reveals the specificity of the method. All the samples spiked with Noxides showed the expected peaks in the NL scan. No interference was evidenced in detection of N-oxides in the spiked samples by NL method (Fig. 2 and see Electronic Supplementary Material Fig. S3). At LOD level, neither false-positive nor false-negative samples were detected; the sensitivity, defined as the percentage of true-positive identified as NL scan method, is 100 %, and the specificity, defined as the percentage of samples that were not true positives and were identified as negative by NL scan method, is 100 %. The false-positive and false-negative rates are 0 %, and they witness the precision of the method. The extract was found to be stable over a period of 1 month in refrigerated conditions. After this period, all the analytes were successfully detected by NL method.
%
MDEA EDEA ME 12.9 10.6 12.4
1.5 2.3
DP 6.5 5.2
TEA 9.3
0 5.00
100
10.00
15.00
20.00 Time
DM 16.0
b
EP 8.2
MP 9.7 MDEA EDEA
%
10.5 12.3
DP 6.5
ME 12.8
TEA 9.2
0 5.00
10.00
15.00
20.00 Time
Fig. 4 a Full scan mode b neutral loss (48 u) scan mode chromatograms of a mixture of N-oxides of aminoethanols under LC-MS conditions
L. Sridhar et al.
dominant (see Electronic Supplementary Material Fig. S6). Similar is the case with the isomeric pair, EP and EIP, in which the ion at m/z 100 (loss of 48 u) is dominant in EP and the ion at m/z 106 (loss of 42 u) is dominant in EIP; the mixture of EP and EIP shows both the fragment ions in high abundance (see Electronic Supplementary Material Fig. S7). On the other hand, when the isomeric compounds present in a mixture result in closely similar MS/MS spectra, it may be better to separate such isomeric compounds by chromatography techniques prior to MS/MS.
LC-MS analysis of N-oxides of aminoethanols LC-MS analysis of the mixture of N-oxides of aminoethanols was performed using Hilic Plus C18 column with the mobile phase consisting of 5 mM ammonium acetate solution adjusted to pH 3, with acetic acid (A) and acetonitrile (B) at different gradient conditions indicating that the chromatography was best performed under isocratic conditions rather than a gradient elution. Better chromatograms were obtained at the isocratic condition of 80 % B at the flow rate of 0.8 mL/min. The LC-ESI-MS chromatogram obtained for a mixture of eight N-oxides was presented in Fig. 4. The chromatogram of NL scan (48 u) for the same sample exclusively showed the N-oxides peaks (Fig. 4). The same LC-MS method was also tested for a few N-oxides of aminoethyl chlorides (MP-Cl and EP-Cl), and they were separated from corresponding N-oxides of aminoethanols. In the case of isomeric N-oxides of aminoethanols, three sets of isomers were possible (molecular weight 133, 147, and 161) on the basis of CWC scheduled list. All the three sets were analyzed by LC-MS/MS using the above-discussed LC method. Among the first set (molecular weight 133), the isomeric compound DE (RT=8.1 min) is separated from MP/MIP compounds (RT=7.4; see Electronic Supplementary Material Fig. S8). The isomers MP and MIP appear as a single peak, but the extracted ion chromatogram of the ion at m/z 92 (loss of propene) showed a marginal separation of MP and MIP (RT=7.4 and 7.8, respectively; see Electronic Supplementary Material Fig. S8). The LC-MS/MS chromatogram of the second set of isomers (EP and EIP) shows that the EP and EIP were separated marginally (RT=6.6 and 7.2, respectively) (see Electronic Supplementary Material Fig. S8). At their respective peak top, the MS/MS spectra of the two isomers were different. In the LC-MS/MS chromatogram of the third set of isomers (three compounds of molecular weight 161), two peaks were visible, in which the first one is of DP (RT= 5.2 min) and DIP and PIP are co-eluting at RT 5.9 min (see Electronic Supplementary Material Fig. S8). Under the used experimental conditions, it is difficult to separate the coeluting DIP and PIP.
Conclusions A sensitive, specific, and rapid qualitative screening method was developed and validated for the detection of N-oxides of aminoethanol and aminoethylchlorides, which are the hydrolysis and oxidation products of chemical warfare agents. In this method, the samples were directly analyzed by NL (48 and 66 u) scan mode, without involving chromatography steps. The method was successfully used for unambiguous detection of the N-oxides of aminoethanols and aminoethylchlorides in complex environmental samples in the presence of matrix/background chemicals. This rapid screening method is useful for off-site OPCW verification program as well as for participating in official OPCW PTs. The structure of N-oxides can be confirmed by the MS/MS experiments on the detected peaks in the NL scan method. An LC-MS method was also developed for the separation of mixture of the N-oxides using a C18 Hilic column. The NL scanning after LC separation was advantageous for the analysis of isomeric N-oxides. Acknowledgments The authors thank the Director, CSIR-IICT, Hyderabad, India for facilities and encouragement. The authors LS and RK thank the University Grants Commission, New Delhi and Council of Scientific and Industrial Research, New Delhi, respectively, for the award of senior research fellowships. The authors acknowledge financial support to the CSC-0406 project by the Council of Scientific and Industrial Research, New Delhi.
References 1. Black RM, Read RW (1997) Application of liquid chromatographyatmospheric pressure chemical ionization mass spectrometry, and tandem mass spectrometry, to the analysis and identification of degradation products of chemical warfare agents. J Chromatogr A 759(1–2):79–92 2. Tak V, Kanaujia PK, Pardasani D, Gupta AK, Palit M, Srivastava RK, Dubey DK (2006) Electrospray ionization tandem mass spectral analysis of oxidation products of precursors of sulfur mustards. Rapid Commun Mass Spec 20:2387–2394 3. Convention on the prohibition of the development, production, stockpiling and use of chemical weapons and their destruction (1997) Technical Secretariat of the Organization for Prohibition of Chemical Weapons, The Hague. http://www.opcw.org 4. Krutzsch W, Trapp R (1994) A Commentary of CWC. Nijhoff M, Netherlands 5. Mesilaakso M (2005) Chemical weapons convention chemical analysis. John Wiley, Chichester 6. Lakshmi VVS, Murty MRVS, Reddy TJ, Ravikumar M, Prabhakar S, Vairamani M (2006) Electrospray ionization mass spectral studies on hydrolysed products of sulfur mustards. Rapid Commun Mass Spec 20:981–986 7. Sridhar L, Karthikraj R, Murty MRVS, Raju NP, Vairamani M, Prabhakar S (2011) Mass spectral analysis of N-oxides of chemical weapons convention related aminoethanols under electrospray ionization conditions. Rapid Commun Mass Spec 25:533–542 8. Sridhar L, Karthikraj R, Murty MRVS, Raju NP, Vairamani M, Prabhakar S (2013) Mass spectral analysis of N-oxides of nitrogen
Rapid screening of N-oxides of chemical warfare agents mustards and N, N-dialkylaminoethyl-2-chlorides under electrospray ionization conditions. Int J Mass Spectrom 333:15–20 9. Compagnone D, Curini R, Ascenzo GD, Carlo MD, Montesano C, Napoletano S, Sergi M (2011) Neutral loss and precursor ion scan tandem mass spectrometry for study of activated benzopyrene-DNA adducts. Anal Bioanal Chem 401(6):1983–1991
10. Eurachem Working Group (1998) A laboratory guide to method validation and related topics. Eurachem, Teddington 11. Susanna V, Donata F, Giampietro F, Sergio M, Guido V, Roberta S, Pietro T, Santo DF (2010) Validation of a fast screening method for the detection of cocaine in hair by MALDI-MS. Anal Bioanal Chem 396:2435–2440
NOTE
Rapid screening of N-oxides of chemical warfare agents degradation products by ESI-tandem mass spectrometry L. Sridhar & R. Karthikraj & V. V. S. Lakshmi & N. Prasada Raju & S. Prabhakar
Received: 31 October 2013 / Revised: 31 December 2013 / Accepted: 10 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract 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-2chlorides, 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 Published in the topical collection Analysis of Chemicals Relevant to the Chemical Weapons Convention with guest editors Marc-Michael Blum and R. V. S. Murty Mamidanna. Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-7623-0) contains supplementary material, which is available to authorized users. L. Sridhar : R. Karthikraj : V. V. S. Lakshmi : N. P. Raju : S. Prabhakar (*) National Center for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India e-mail: [email protected]
complex masking agents, without the use of timeconsuming 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. Keywords Dialkylaminoethanols . Dialkylaminoethyl-2-chlorides . N-oxides . Neutral loss scan . Rapid screening . LC-MS/MS
Introduction Detection and identification of chemical warfare agents (CWAs), their precursors/degradation products in suspected sites, or environmental samples has been an important area of research [1, 2]. It is also important for the verification process, an important component of monitoring compliance with the chemical weapons convention (CWC). Adoption of new methods for rapid screening of CWC-related chemicals is always advantageous for implementation of CWC, which an international treaty came into force in 1997 to prohibit development/production/storage/usage of chemical weapons [3, 4]. The treaty is administered by the Organization for the Prohibition of Chemical Weapons (OPCW), the Netherlands. The OPCW maintains a network of designated laboratories for
L. Sridhar et al.
off-site analysis of samples collected during inspections and other needs for the implementation of the CWC [5]. Most of the CWAs are prone to undergo hydrolysis and oxidation in environmental matrices leading to more persistent degradation products. The alkylaminoethanols and triethanolamine (S3.B.15 to S3.B.17) have their own importance since their chlorination leads to the formation of nitrogen mustards (S1.A.06). The N,N-dialkylaminoethyl-2-chlorides (S2.B.10) and N,N-dialkylaminoethanols (S2.B.11) are the precursors and the hydrolyzed products of nerve agents (VX type of compounds, S1.A.03). These compounds are prone to undergo oxidation in environmental matrices or upon decontamination process. The N-oxides of aminoethanols and aminoethylchlorides are non-scheduled chemicals, but they are relevant to the verification of CWC because the presence of these degradation products in suspected matrices such as organic, oil, soil etc. reveals alleged use of nitrogen mustards or precursors of VX compounds. Thus, screening of environmental residues for hydrolysis and oxidation products is an important part of verification process [6]. As a first attempt, we synthesized the N-oxides of CWC-related aminoethanols and aminoethylchlorides by applying know procedures, and reported characterization of all the N-oxides, including isomers, by mass spectrometry [7, 8]. The N-oxides were known to be thermally labile, thus they cannot be analyzed by GCMS. Consequently, on-site analysis of these compounds is difficult for the OPCW inspection teams as they routinely use GC-MS for verification. Therefore, a soft ionization technique, electrospray ionization (ESI), using direct flow injection or LC separation, is a facile technique for the identification of these N-oxides by mass spectrometry. The direct ESIMS analysis of target N-oxides readily formed protonated molecule ions, [M+H]+ ions. In the previous study, we reported characterization of the N-oxides based on specific/selective fragmentation of the respective [M+H]+ ions [7, 8]. In order to identify the N-oxides present in a suspected sample using this method, at first a full scan ESI mass spectrum to be recorded for the sample extract, and then perform MS/MS on each detected peak whose m/z value matches with that of the target N-oxides. The presence of an N-oxide can only be confirmed if the MS/MS spectrum matches with the standard N-oxide spectrum [7, 8]. However, in the real scenario, the target Noxide peaks may be missed or masked by the matrix constituents and background chemicals present in the environmental samples at high concentration levels. Moreover, the m/z values of the [M+H]+ ions associated with the N-oxides may match with those of other classes of CWC schedule compounds. For example, the [M+H] + ions of N-oxides of N,Ndialkylaminoethanols matches with the [M+H]+ ions of corresponding N,N-dialkylaminoethanethiols. It is possible to overcome such problems by applying specific tandem mass spectrometric (MS/MS) scan modes of analysis, such as constant neutral loss (NL) scan mode and multiple reaction
monitoring mode [6, 9]. In these lines, we have developed a sensitive, selective, and rapid screening method by applying direct ESI-MS/MS (NL scan) technique for unambiguous detection of N-oxides of CWC-related aminoethanols and aminoethylchlorides in a single step, even in the presence of a large amount of masking agents/background chemicals. The study also includes structure confirmation of critical isomers of N-oxides by LC-MS/MS analysis.
Experimental Chemicals All the N-oxides (Fig. 1) used in the present study were synthesized by the oxidation of the corresponding tertiary amine using reported methods [7, 8]. Analytical grade solvents were used for chromatographic analysis, and they were obtained from E-Merck (Mumbai, India). Ammonium acetate and triethylamine were purchased from Sigma-Aldrich (St. Louis, USA). Stock solutions (100 ppm) of individual Noxides of aminoethanols and aminoethylchlorides and mixtures of N-oxides were prepared in methanol and dichloromethane, respectively. The stock solutions were further diluted in methanol/acetonitrile for MS analysis. Water sample The stock solution of N-oxides of aminoethanol mixture (DM, ME, MP, EP, DP, MDEA, EDEA, and TEA) was spiked in a blank water sample (5 ppm) received during the 28th official OPCW PT. The blank water consists of polyethylene glycol 200 (5,000 ppm) in deionized water. Soil sample The stock solution of a mixture of N-oxides of aminoethanols (DM, ME, MP, EP, DP, and TEA) was spiked at various concentrations (500 ppb–25 ppm) in a blank soil sample received during the 30th official OPCW PT. The blank soil sample consists of diesel fuel (1,000 ppm), n-butyl phosphonic acid (10 ppm), and dibutyl phosphate (10 ppm) in sand (ISO standard). This sample was subjected to water extraction and 1 % triethylamine (TEA) in methanol extraction as per standard procedures used for proficiency test (PT) samples. Briefly, 5 g of soil sample was extracted twice with 5 ml of water by shaking for 10 min. The supernatant solutions from extractions were collected, centrifuged, combined, and concentrated to 1 ml by rotary evaporator. Similar procedure was used to extract fresh soil sample with 1 % TEA/methanol, where the combined extracts were concentrated to 100 μL with mild nitrogen flow.
Rapid screening of N-oxides of chemical warfare agents Fig. 1 Molecular structures of the studied N-oxides of aminoethanols and aminoethylchlorides
Organic sample A mixture of N-oxides of aminoethylchlorides (DM-Cl, MPCl, EP-Cl, DP-Cl, MDEA-Cl, and TEA-Cl) was spiked in a blank organic waste sample (5 ppm) received during the 28th official OPCW PT. The blank organic sample contains alkane mixture (C7–C30; 10 ppm) and petroleum ether (10 %) in nhexane.
under the control of Xcalibur software. The typical ESI source conditions were as follows: spray voltage 4.8 kV, capillary voltage 20 V, capillary temperature 300 °C, and tube lens offset voltage 10 V. Nitrogen (40 psi) was used as sheath gas and helium (1.8 mTorr) was used as damping gas and collision gas. The collision energies used for LC–MS/MS experiments were 18–30 eV. Extraction recovery
Mass spectrometry All the experiments were carried out on a Quattro LC, a triple quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with an ESI source operated in positive ion mode. All the spectra were acquired using MassLynx software (version 3.2). The typical operating conditions were capillary voltage, 3.5 kV; cone voltage, 30 V; source housing and desolvation temperatures, 200 °C. Nitrogen was used as nebulization and desolvation gas. The product ion and NL scan experiments were performed by using collision energies 18– 30 eV. Argon was used as the collision gas, and the collision cell pressure was maintained at 1.67×10−3 mbar. All the spectra were averages of 20 scans. For direct ESI-MS experiments, sample was directly infused into ESI source at 10 μL/ min using a syringe pump (Harvard). For LC-MS and LC-MS/ MS experiments, an Alliance 2695 HPLC system (Waters) with an autoinjector and a Zorbax Hilic Plus C18 (4.6 mm× 100 mm, 3.5 μm) column (Agilent Technologies, CA) was used. The injection volume of the sample was 10 μL. The mobile phase consisted of 5 mM ammonium acetate solution (A) adjusted to pH 3 with acetic acid and acetonitrile (B) in 15:85 ratio. LC-MS and LC-MS/MS experiments were also performed using LCQ ion trap mass spectrometer (Thermo Fisher, San Jose, CA, USA) coupled with Surveyor HPLC system, using the same column. The data acquisition was
Extraction recoveries for all the N-oxides were determined by spiking the N-oxides at four levels of concentration (1, 10, 25, 50 ppm) before and after extraction. In spiking experiment before extraction, the N-oxides were spiked in 2 g of the soil sample and extracted with water and TEA/methanol (independently). In spiking experiment after extraction, 2 g of soil sample was extracted with water and TEA/methanol (separately), and then the N-oxides were spiked to each blank extract. The extracts were analyzed by direct ESI-MS/MS (NL method) in triplicate, and the peak areas of the N-oxide peak (s) in the NL mass spectra were used to calculate the extraction recovery values. Validation The validation of the NL scan screening method was performed according to Eurachem guidelines for qualitative validation [10, 11]. The main purpose of qualitative screening methods is to distinguish between negative samples and positive samples at a determined level; the method is considered as validated when positive findings are reported in all samples. The limit of detection (LOD) was obtained by spiking the Noxides in blank samples at five different concentration levels (100 ppb–1 ppm). The specificity of the NL method was tested by analyzing with or without spiking N-oxides in
L. Sridhar et al.
solvent blank samples, blank water/soil sample extracts, and a test sample mixture containing several other classes of CWCrelated chemicals, i.e., VX, sulfur mustards, nitrogen mustards, aminoethanethiols, aminoethanols, thiodiglycol, bis-(2-hydroxyethyl)-disulfide, and phosphonic acids.
Results and discussions NL method for screening N-oxides Direct ESI-MS analysis of all the N-oxides (Fig. 1) show abundant [M+H]+ ions. Previously, we have studied the collision-induced dissociation (CID) spectra of the [M+H]+ ions from N-oxides of aminoethanols and aminoethylchlorides, and found that they show a specific product ion due to a constant neutral loss, i.e., CH4O2 (48 u) and CH3ClO (66 u), respectively (see Electronic Supplementary Material Fig. S1). This specific fragmentation process can be used for rapid screening of these compounds in a complex mixture by applying NL scan mode of analysis. A NL scan method was developed for the N-oxides of aminoethanols and aminoethylchlorides by optimizing typical instrument conditions including collision energy (18–20 eV). A NL of 48 u is set for N-oxides of aminoethanols, and 66 u for Noxides aminoethylchlorides. When a mixture of N-oxides of aminoethanol was subjected for NL scan of 48 u by injecting the sample directly into ESI source, the spectrum exclusively showed the peaks due to the [M+H]+ ions of the corresponding N-oxides of aminoethanols. Similarly, when a mixture of Noxides of aminoethylchlorides was subjected for NL scan of 66 u, the spectrum specifically showed the [M+H]+ ions of the corresponding N-oxides of aminoethylchlorides. We have also recorded NL scan spectra for a mixture of some N-oxides of aminoethanols and aminoethylchlorides by selecting two NL channels (channel 1, 48 u and channel 2, 66 u) in a single experiment. The spectra clearly show the [M+H]+ ions of all the N-oxide compounds in the mixture (see Electronic Supplementary Material Fig. S2). Thus, the presence/absence of N-oxides of aminoethanols and aminoethylchlorides in a complex environmental sample can be rapidly ascertained based on the NL scan method without involving any chromatography separation techniques.
peaks. However, when the same samples subjected for NL scan mode (48 and 66 u), the spectra exclusively showed the [M+H]+ ions of all the N-oxides that were spiked (Fig. 2 and see Electronic Supplementary Material Fig. S3). Thus, it is possible to detect the target N-oxides in a complex mixture unambiguously by applying NL scan experiment. We have also applied the NL scan method to a soil sample, where the analysis of oxidation products is a challenging task because this matrix involves sample preparation step. To demonstrate the viability of this method to soil samples, a mixture of N-oxides of aminoethanols was spiked in a blank soil sample of the 30th official OPCW PT. The soil sample was subjected to water and TEA/methanol extractions. The extracts were analyzed by direct ESI-MS method and NL scan method. The full scan ESI mass spectra of these samples appeared very complex; however, the NL scan spectra clearly showed the [M+H]+ ions of all the spiked aminoethanol Noxides (Fig. 3 and see Electronic Supplementary Material Fig. S4). The extraction recoveries were found to be in the range from 65 to 82 % in water extraction and 52 to 64 % in TEA/methanol extraction. Validation of NL scan method The NL scan method used in the current study is a qualitative method, i.e., a method of analysis whose response is either the presence or absence of the analyte, detected directly in a certain amount of sample; this method is validated as per the guidelines of EURACHEM [10]. LOD of the N-oxides in NL scan method was tested by injecting the samples of N-oxides
Application of the NL method for test samples A mixture of N-oxides of aminoethanols was spiked in a blank water sample received during the 28th official OPCW PT. A mixture of N-oxides of aminoethylchlorides was spiked in a blank organic waste sample received during the 28th official OPCW PT. These samples were directly analyzed by full scan ESI-MS and by NL scan mode. The ESI mass spectra of these samples showed a complex spectrum where the matrix/ background chemicals peaks obscured the target compounds
Fig. 2 a Full scan mode b neutral loss (66 u) scan mode mass spectra of organic sample spiked with a mixture of N-oxides of aminoethylchlorides under ESI conditions
Rapid screening of N-oxides of chemical warfare agents
Isomeric N-oxides The direct NL scan method can be applied for rapid screening of N-oxides in environmental samples, which reveals presence or absence of the target N-oxides. However, MS/MS technique needs to apply to obtain full structure of N-oxides. In our previous reports, we have discussed the structure indicative fragment ions during CID of [M+H]+ ions from the N-oxides of aminoethanols and aminoethylchlorides [7, 8]. Thus, it is possible to confirm the structure of an N-oxide by direct ESI-MS/MS method. However, it is problematic when a mixture of isomeric N-oxides is present in the same sample. Under such conditions, direct ESI-MS/MS can only be useful where each isomer results in a specific/selective fragment ion pertinent to its structure. For example, the base peak in the MS/MS spectrum of MP is the ion at m/z 86 (loss of 48 u), while it is m/z 92 in the case of MIP (loss of 42 u). In the MS/ MS spectrum of a mixture of MP and MIP, both the ions were Fig. 3 a Full scan mode b neutral loss (48 u) scan mode mass spectra of water extract of soil sample spiked with a mixture of N-oxides of aminoethanols under ESI conditions
100
a
DM 16.0
MP 9.7 EP 8.3
of aminoethanols and aminoethylchlorides at different concentration levels (100 ppb to 5 ppm), and LOD of the method was estimated for each analyte at a signal-to-noise ratio of 3:1 and the LOD was found to be 500 ppb. The specificity of the NL method, i.e., the ability to measure an analyte in the presence of other compounds and matrix components, was tested by analyzing different samples with or without spiking N-oxides. When the solvent blank samples and blank water/ soil sample extracts, and a test sample mixture containing several other classes of CWC-related chemicals without Noxides, were analyzed by the NL scan method (NL of 48 and 66 u), no peaks were detected in the spectra (see Electronic Supplementary Material Fig. S5). Absence of peaks in the NL scan mass spectra of the above-negative samples reveals the specificity of the method. All the samples spiked with Noxides showed the expected peaks in the NL scan. No interference was evidenced in detection of N-oxides in the spiked samples by NL method (Fig. 2 and see Electronic Supplementary Material Fig. S3). At LOD level, neither false-positive nor false-negative samples were detected; the sensitivity, defined as the percentage of true-positive identified as NL scan method, is 100 %, and the specificity, defined as the percentage of samples that were not true positives and were identified as negative by NL scan method, is 100 %. The false-positive and false-negative rates are 0 %, and they witness the precision of the method. The extract was found to be stable over a period of 1 month in refrigerated conditions. After this period, all the analytes were successfully detected by NL method.
%
MDEA EDEA ME 12.9 10.6 12.4
1.5 2.3
DP 6.5 5.2
TEA 9.3
0 5.00
100
10.00
15.00
20.00 Time
DM 16.0
b
EP 8.2
MP 9.7 MDEA EDEA
%
10.5 12.3
DP 6.5
ME 12.8
TEA 9.2
0 5.00
10.00
15.00
20.00 Time
Fig. 4 a Full scan mode b neutral loss (48 u) scan mode chromatograms of a mixture of N-oxides of aminoethanols under LC-MS conditions
L. Sridhar et al.
dominant (see Electronic Supplementary Material Fig. S6). Similar is the case with the isomeric pair, EP and EIP, in which the ion at m/z 100 (loss of 48 u) is dominant in EP and the ion at m/z 106 (loss of 42 u) is dominant in EIP; the mixture of EP and EIP shows both the fragment ions in high abundance (see Electronic Supplementary Material Fig. S7). On the other hand, when the isomeric compounds present in a mixture result in closely similar MS/MS spectra, it may be better to separate such isomeric compounds by chromatography techniques prior to MS/MS.
LC-MS analysis of N-oxides of aminoethanols LC-MS analysis of the mixture of N-oxides of aminoethanols was performed using Hilic Plus C18 column with the mobile phase consisting of 5 mM ammonium acetate solution adjusted to pH 3, with acetic acid (A) and acetonitrile (B) at different gradient conditions indicating that the chromatography was best performed under isocratic conditions rather than a gradient elution. Better chromatograms were obtained at the isocratic condition of 80 % B at the flow rate of 0.8 mL/min. The LC-ESI-MS chromatogram obtained for a mixture of eight N-oxides was presented in Fig. 4. The chromatogram of NL scan (48 u) for the same sample exclusively showed the N-oxides peaks (Fig. 4). The same LC-MS method was also tested for a few N-oxides of aminoethyl chlorides (MP-Cl and EP-Cl), and they were separated from corresponding N-oxides of aminoethanols. In the case of isomeric N-oxides of aminoethanols, three sets of isomers were possible (molecular weight 133, 147, and 161) on the basis of CWC scheduled list. All the three sets were analyzed by LC-MS/MS using the above-discussed LC method. Among the first set (molecular weight 133), the isomeric compound DE (RT=8.1 min) is separated from MP/MIP compounds (RT=7.4; see Electronic Supplementary Material Fig. S8). The isomers MP and MIP appear as a single peak, but the extracted ion chromatogram of the ion at m/z 92 (loss of propene) showed a marginal separation of MP and MIP (RT=7.4 and 7.8, respectively; see Electronic Supplementary Material Fig. S8). The LC-MS/MS chromatogram of the second set of isomers (EP and EIP) shows that the EP and EIP were separated marginally (RT=6.6 and 7.2, respectively) (see Electronic Supplementary Material Fig. S8). At their respective peak top, the MS/MS spectra of the two isomers were different. In the LC-MS/MS chromatogram of the third set of isomers (three compounds of molecular weight 161), two peaks were visible, in which the first one is of DP (RT= 5.2 min) and DIP and PIP are co-eluting at RT 5.9 min (see Electronic Supplementary Material Fig. S8). Under the used experimental conditions, it is difficult to separate the coeluting DIP and PIP.
Conclusions A sensitive, specific, and rapid qualitative screening method was developed and validated for the detection of N-oxides of aminoethanol and aminoethylchlorides, which are the hydrolysis and oxidation products of chemical warfare agents. In this method, the samples were directly analyzed by NL (48 and 66 u) scan mode, without involving chromatography steps. The method was successfully used for unambiguous detection of the N-oxides of aminoethanols and aminoethylchlorides in complex environmental samples in the presence of matrix/background chemicals. This rapid screening method is useful for off-site OPCW verification program as well as for participating in official OPCW PTs. The structure of N-oxides can be confirmed by the MS/MS experiments on the detected peaks in the NL scan method. An LC-MS method was also developed for the separation of mixture of the N-oxides using a C18 Hilic column. The NL scanning after LC separation was advantageous for the analysis of isomeric N-oxides. Acknowledgments The authors thank the Director, CSIR-IICT, Hyderabad, India for facilities and encouragement. The authors LS and RK thank the University Grants Commission, New Delhi and Council of Scientific and Industrial Research, New Delhi, respectively, for the award of senior research fellowships. The authors acknowledge financial support to the CSC-0406 project by the Council of Scientific and Industrial Research, New Delhi.
References 1. Black RM, Read RW (1997) Application of liquid chromatographyatmospheric pressure chemical ionization mass spectrometry, and tandem mass spectrometry, to the analysis and identification of degradation products of chemical warfare agents. J Chromatogr A 759(1–2):79–92 2. Tak V, Kanaujia PK, Pardasani D, Gupta AK, Palit M, Srivastava RK, Dubey DK (2006) Electrospray ionization tandem mass spectral analysis of oxidation products of precursors of sulfur mustards. Rapid Commun Mass Spec 20:2387–2394 3. Convention on the prohibition of the development, production, stockpiling and use of chemical weapons and their destruction (1997) Technical Secretariat of the Organization for Prohibition of Chemical Weapons, The Hague. http://www.opcw.org 4. Krutzsch W, Trapp R (1994) A Commentary of CWC. Nijhoff M, Netherlands 5. Mesilaakso M (2005) Chemical weapons convention chemical analysis. John Wiley, Chichester 6. Lakshmi VVS, Murty MRVS, Reddy TJ, Ravikumar M, Prabhakar S, Vairamani M (2006) Electrospray ionization mass spectral studies on hydrolysed products of sulfur mustards. Rapid Commun Mass Spec 20:981–986 7. Sridhar L, Karthikraj R, Murty MRVS, Raju NP, Vairamani M, Prabhakar S (2011) Mass spectral analysis of N-oxides of chemical weapons convention related aminoethanols under electrospray ionization conditions. Rapid Commun Mass Spec 25:533–542 8. Sridhar L, Karthikraj R, Murty MRVS, Raju NP, Vairamani M, Prabhakar S (2013) Mass spectral analysis of N-oxides of nitrogen
Rapid screening of N-oxides of chemical warfare agents mustards and N, N-dialkylaminoethyl-2-chlorides under electrospray ionization conditions. Int J Mass Spectrom 333:15–20 9. Compagnone D, Curini R, Ascenzo GD, Carlo MD, Montesano C, Napoletano S, Sergi M (2011) Neutral loss and precursor ion scan tandem mass spectrometry for study of activated benzopyrene-DNA adducts. Anal Bioanal Chem 401(6):1983–1991
10. Eurachem Working Group (1998) A laboratory guide to method validation and related topics. Eurachem, Teddington 11. Susanna V, Donata F, Giampietro F, Sergio M, Guido V, Roberta S, Pietro T, Santo DF (2010) Validation of a fast screening method for the detection of cocaine in hair by MALDI-MS. Anal Bioanal Chem 396:2435–2440
