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 Table of Contents  
Year : 2016  |  Volume : 2  |  Issue : 4  |  Page : 190-194

Application of Spectra Accuracy for Analysis of Organic Explosive: 2,4,6-trinitrotoluene by AccuTOF-DART

1 Institute of Forensic Science, Ministry of Public Security, Beijing, China
2 Lumiere Tech. Ltd., Beijing, China

Date of Web Publication9-Jan-2017

Correspondence Address:
Zhanfang Liu
Institute of Forensic Science, Ministry of Public Security, 17# Building, Muxidi Road, Beijing 100038
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2349-5014.197927

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This paper, through mass spectrometric (MS) analysis for nitro compound explosives on a direct analysis in a real-time time-of-flight MS, indicates that even on a high-resolution MS with accurate mass measurement capabilities, there is no guarantee to obtain the unique molecular formula of a compound. By calculating spectra accuracy, highly accurate isotope pattern matching can be conducted to significantly improve performance of compound confirmation or identification.

Keywords: 2,4,6-trinitrotoluene, direct analysis in real-time time-of-flight mass spectrometer, isotope pattern, mass spectrometric analysis, spectral accuracy

How to cite this article:
Liu Z, Sun Z, Zhang G, Zhu J, Mei H, Li H, Li B, Xu J, Zhou H. Application of Spectra Accuracy for Analysis of Organic Explosive: 2,4,6-trinitrotoluene by AccuTOF-DART. J Forensic Sci Med 2016;2:190-4

How to cite this URL:
Liu Z, Sun Z, Zhang G, Zhu J, Mei H, Li H, Li B, Xu J, Zhou H. Application of Spectra Accuracy for Analysis of Organic Explosive: 2,4,6-trinitrotoluene by AccuTOF-DART. J Forensic Sci Med [serial online] 2016 [cited 2023 Feb 5];2:190-4. Available from: https://www.jfsmonline.com/text.asp?2016/2/4/190/197927

  Introduction Top

2, 4, 6-trinitrotoluene, also known as TNT, is the most commonly-used explosive. Due to high energy density, great instantaneous power, and other characteristics, it is extensively used in military, industry, agriculture, and construction. In recent years, bomb attacks and emergencies based on TNT happened from time to time, which threatened people's life and property security. Accurate analysis for nitro compounds such as TNT, therefore, is important for fighting against terrorism and securing public safety.

High-resolution mass spectrometric (MS) systems (such as time-of-flight [TOF]) are capable of quickly and sensitively measuring the accurate molecular weight and determining the elemental composition of a compound. They have become the most crucial identification means for analysis of TNT and other trace explosives in a complex matrix. Although high mass accuracy plays an important role in determining unknown compounds, mass accuracy at 1 ppm or better can result in many possible chemical formulas. On the other hand, isotope distribution information on high-resolution MS is equally important in qualitative MS analysis of unknown compounds as it helps exclude those chemical formulas with poor isotope pattern matching.[1],[2],[3],[4] Recently, a new MS software product, MassWorks, based on mass spectral peak shape calibration technology, allows highly effective isotope pattern matching between calibrated and theoretically calculated spectra to significantly improve the performance of elemental composition determination for unknown compound identification.[5],[6] Through data processing with MassWorks on MS data of TNT acquired on a direct analysis in real time (DART) MS system, we demonstrate that a more accurate and desirable result can be obtained by utilizing not only high mass accuracy but also, more importantly, high spectral accuracy.[7]

  Materials and Methods Top

Instrument and reagent

All data acquisitions were performed on an AccuTOF-MS high-resolution TOFMS (Electron JEOL Company of Japan, JMS-T100 LP) with a DART ion source (U.S Ion Sense Company). Elemental composition determination with spectral accuracy was done by MS Software MassWorks (Cerno Bioscience, USA). Acetone (Sinopharm Chemical Reagent Co., Ltd., China) was used for TNT sample preparation.

Test conditions

  • DART parameters: distance between DART and MS taperhole – 25 mm; Voltage of spray point: 4000V; Outlet deflecting voltage: -100V; DART ionization: negative ion mode
  • AccuTOF-MS parameters: MS resolving power – above 6000; mass scanning range – 50–500 Da.

Sample preparation

TNT was the standard sample provided by Xi'an Modern Chemistry Research Institute.

Organic explosive of TNT was prepared with acetone to have a concentration of 0.001 mg/ml.

Data processing

Mass calibration was performed on DART-TOFMS spectra to achieve high mass accuracy. To determine the elemental composition of compounds, mass tolerance of 5 mD was used including possible elements C (0–10), H (0–20), O (0–20), and N (0–20). With MassWorks, peak shape calibration was conducted on DART-TOFMS spectra using sCLIPS function available from MassWorks; then, elemental composition determination was done by utilization of the same search condition completely consistent with AccuTOF.

  Results and Discussion Top

2, 4, 6-trinitrotoluene fragmentation by direct analysis in real time

With the negative ion mode of DART, [M] and [M-H] of compounds were often observed. This is also true with the TNT compounds [Figure 1]. It is also reported [8],[9],[10],[11] that during the MS decomposition process of TNT, [M-OH] with m/z at 210, [M-NO] with m/z at 197, and [M-NO2] with m/z at 181 are also produced. In the DART-TOFMS spectra of relevant standard samples of TNT, these ions can be observed. Therefore, it can be roughly confirmed that under the DART negative ion mode, three fragmentation pathways can be found: [M-OH] produced by loss of OH ; [M-NO] from loss of NO ; [M-NO2] with loss of nitro from the side chain of TNT.
Figure 1: 2,4,6-trinitrotoluene standard substance mass spectra obtained from direct analysis in real-time time-of-flight mass spectrometer

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Mass spectrometric analysis for 2, 4, 6-trinitrotoluene compositions

Elemental composition determination by mass accuracy only

Elemental composition determination based on mass accuracy only with AccuTOF is mainly through the combination of the possible elements with certain mass tolerance. This method is, currently, the most common way to determine the elemental composition of unknown compounds by high-resolution MS. In [Figure 1], the accurate mass after calibration of [M-H] produced by TNT is 226.0108, which has mass errors <5 mDa. The proposed chemical formulas composed of C, H, O, and N are shown in [Table 1].
Table 1: Proposed formulas of [M-H]: In order of mass accuracy

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Elemental composition determination with MassWorks

In MassWorks, the monoisotopic peak of [M-H] was used to perform peak shape calibration, which then applied to the entire isotope clusters to allow calculating spectral accuracy. In addition to the search conditions according to mass accuracy used previously, the spectral accuracy provides effectively isotope pattern matching to further improve the formula search performance. Unlike the general high-resolution MS analytical results, the formulas proposed by MassWorks are sorted according to spectral accuracy as shown in [Table 2].
Table 2: Proposed formulas of [M-H] ions according to spectra accuracy

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Comparison of mass accuracy and spectra accuracy

Although the mass accuracy is of vital importance for MS analysis of compounds, it should not be taken as the only basis for MS analysis of unknown compounds. Within the mass error of 5 mDa, all eight proposed [M-H] formulas of TNT are possibly the correct one. In the case of determining elemental composition based on mass accuracy only, C8N7O2, with the minimum mass errors, appears to be most likely correct formula. However, the true and correct one is C7H4N3O6 which ranks as the third.

Fortunately, high-resolution MS has also provided us with abundant isotope distribution information of compounds which significantly improved elemental composition determination of unknown compounds. Through peak shape calibration on original mass spectrum, a nearly perfect isotope pattern matching (spectral error of <2%) between the experimental spectrum and the theoretical one is achieved as shown in [Figure 2] and [Figure 3]. The correct elemental composition of C7H4N3O6 was identified as number one choice with spectral accuracy of 98.37%. [Figure 2] and [Figure 3] show that the matching results of calibration spectrum and theoretical spectrum of C7H4N3O6 and C9H6O7 are demonstrated, i.e., the C7H4N3O6 isotope peak type matches perfectly while minor difference exists for C9H6O7 with the theoretical spectrum on 228 for m/z.
Figure 2: Overlay of theoretical spectrum (red) and calibration spectrum (black) of 2,4,6-trinitrotoluene (the first formula with spectra accuracy of 98.36%)

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Figure 3: Overlay of theoretical spectrum (red) of the second formula (C9H6O7) and calibration spectrum (black) of 2,4,6-trinitrotoluene (with spectra accuracy of 98.28%)

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Isotopic ratio distortion due to ion interference

The isotope distribution information of irons can facilitate elemental composition determination of unknown compounds, but cautions still have to be given when isotope pattern is distorted due to ion interference. In TNT DART-TOF spectra, molecular ion peaks [M] at m/z 226 and quasi-molecular ion peaks [M-H] at m/z 227 simultaneously appear, leading to the overlying of “A + 1” isotopic peaks of [M-H] at m/z 227 peaks. Therefore, it is not the real isotopic distribution pattern of either [M] or [M-H] within the mass range from 226 to 228 in [Figure 1], but the superposition of isotope abundances of the two ions. The theoretical isotope ratio 226/227 of C7H4N3O6 is approximately 100:9 while the observed ratio appears to be 100:70 as shown in [Figure 1]. In this case, the wrong qualitative results might be obtained based on only simple observation for isotope abundances. MassWorks, on the other hand, can perform mixture analysis for the both components of [M] and [M-H] and effectively rule out the isotopic abundance interference of the known ions, thus leading to more accurate qualitative results. [Figure 4] shows the differences between the actual calibration spectrum of TNT and the theoretical spectrum of [M] and [M-H] at m/z 227 and 228.
Figure 4: Comparison between the calibration spectrum of 2,4,6-trinitrotoluene (blue) and theoretical spectrum of 2,4,6-trinitrotoluene [M] (green) and [M-H] (red)

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Compound composition rules

Under specified ionization pattern, the molecular ions of compounds have different electron states, carrying on either odd or even number of electrons. For example, the molecular ions generated from electron impact ionization are odd-electron ions while the quasi-molecular ions generated from atmosphere pressure ionization are even-electron ions. [M-H] ion generated by DART is an even-electron ion, but no considerations have been given to the electron states of the ions during the elemental composition determination process with AccuTOF so that the determination is found to have many “false positives” with odd-electron ions, thus increasing the probability of misidentification. With MassWorks, all the odd-electron ions during the determination of elemental composition are filtered and the number of proposed [M-H] chemical formulas are decreased from 8 to 4 [Table 3], significantly decreasing the qualitative difficulty. The results also exclude unreasonably composed chemical formulas. In a combination of the selection of electron states with “Empirical Rule,”[12] MassWorks can further filter out formulas inconsistent with the rules such that the proposed formula C7H4N3O6 is the only finally determined. Therefore, by correctly selecting electron states under fixed ionization mode, the application of MassWorks can rule out unreasonable chemical formulas and significantly decrease the number of “false positive” chemical formulas, lower down the qualitative difficulty, and ensure qualitative accuracy.
Table 3: Even-electron [M-H] elemental composition determination results

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Case analysis

[Figure 5] shows the DART-TOFMS spectrum of TNT explosion in the real case, from which we can clearly observe the existence of [M], [M-H], and [M-NO2] which is consistent with the fragmentation patterns of TNT by DART. As shown in [Table 4], high mass accuracy of 2.02 mD and high spectral accuracy of 98.15% were achieved for [M-H] ion of C7H4N3O6. Mass accuracy ranks as the fifth among all the ten proposed chemical formulas. According to spectrum accuracy, the correct formula of TNT in this analysis ranks as the third among all proposed chemical formulas. After ruling out all the “false positive” results, the correct formula C7H4N3O6 becomes the only choice through MassWorks analysis with nearly perfect matching between the actually calibrated isotope pattern and the theoretically calculated one [Figure 6]. The analytical results of [M-NO2] [Table 5] also confirms that the compound is, truly, TNT.
Figure 5: Direct analysis in real-time time-of-flight spectrum for case

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Table 4: Proposed m/z 226 ion chemical formula results involved in explosives

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Figure 6: Theoretical spectrum and actual calibrated spectrum of formula C7H4N3O6 with spectra accuracy at 98.13% in Case 1

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Table 5: Proposed m/z 181 ion chemical formula involved in explosives

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  Conclusions Top

High-resolution TOF spectrum provides high mass accuracy which, however, is not adequate for high confident compound confirmation or identification, but the isotope patterns of compounds provides additional information to significantly facilitate elemental composition determination. With the help of spectra accuracy, MassWorks allows us to accurately determine elemental composition from numerous proposed chemical formulas with certain mass tolerance. Therefore, the combination of mass accuracy and spectra accuracy is, currently, the most effective way identifying unknown compounds.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Kind T, Fiehn O. Metabolomic database annotations via query of elemental compositions: Mass accuracy is insufficient even at less than 1 ppm. BMC Bioinformatics 2006;7:234-7.  Back to cited text no. 1
Stoll N, Schmidt E, Thurow K. Isotope pattern evaluation for the reduction of elemental compositions assigned to high-resolution mass spectral data from electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. J Am Soc Mass Spectrom 2006;17:1692-9.  Back to cited text no. 2
Zhang J, Gao W, Cai J, He S, Zeng R, Chen R. Predicting molecular formulas of fragment ions with isotope patterns in tandem mass spectra. IEEE/ACM Trans Comput Biol Bioinform 2005;2:217-30.  Back to cited text no. 3
Kuehl D, Wang Y. Peak shape calibration method improves the mass accuracy of mass spectrometers. J BioPharm Int 2006;19:32-8.  Back to cited text no. 4
Erve JC, Gu M, Wang Y, DeMaio W, Talaat RE. Spectral accuracy of molecular ions in an LTQ/Orbitrap mass spectrometer and implications for elemental composition determination. J Am Soc Mass Spectrom 2009;20:2058-69.  Back to cited text no. 5
Zhou W, Zhang Y, Xu H, Gu M. Determination of elemental composition of volatile organic compounds from Chinese rose oil by spectral accuracy and mass accuracy. Rapid Commun Mass Spectrom 2011;25:3097-102.  Back to cited text no. 6
Wang Y, Gu M. The concept of spectral accuracy for MS. Anal Chem 2010;82:7055-62.  Back to cited text no. 7
Yinon J, Zitrin S. Modern Methods and Applications in Analysis of Explosives. New York: John Wiley and Sons;1996.  Back to cited text no. 8
Zhou Y, Wang W, Lai J. Measurement of trace TNT on surface by desorption electrospray ionization mass spectrometry determination method. J Mass Spectrom 2006;27 Suppl:129-31(in Chinese).  Back to cited text no. 9
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Kind T, Fiehn O. Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. BMC Bioinformatics 2007;8:105.  Back to cited text no. 12


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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