Forensic Chemistry 10 (2018) 5–14
Contents lists available at ScienceDirect
Forensic Chemistry
journal homepage: www.elsevier.com/locate/forc
Development and validation of fast liquid chromatography
high-resolution mass spectrometric (LC-APCI-QToF-MS) methods
for the analysis of hexamethylene triperoxide diamine (HMTD)
and triacetone triperoxide (TATP)
Lisa Dunn ⇑, Hamda Sultan Ali Al Obaidly, Saif Eldin Khalil
General Department of Forensic Science and Criminology, Dubai Police General Headquarters, Dubai, United Arab Emirates
a r t i c l e
i n f o
Article history:
Received 7 September 2017
Received in revised form 26 June 2018
Accepted 26 June 2018
Available online 30 June 2018
a b s t r a c t
Two semi-quantitative, fast liquid chromatography-mass spectrometry methods have been developed
using an atmospheric pressure chemical ionisation source in conjunction with an accurate mass detector
(LC-APCI-QToF-MS) for the analysis of traces of the peroxide explosives hexamethylene triperoxide diamine (HMTD) and triacetone triperoxide (TATP). A comprehensive validation was conducted on each
method and is presented herein. The limits of detection (LOD) for HMTD and TATP using these methods
were determined to be 0.5 ng and 10 ng on column, respectively. The high mass accuracy and narrow
mass detection window offer high selectivity with <2 ppm mass difference between measured and calculated values for HMTD.
This paper supports recent work performed using ESI-TOF and Orbitrap that demonstrates that the
207.0614 ion observed during the analysis of HMTD is in fact [TMDDD + H]+, and not [M-1]+.
Ó 2018 Elsevier B.V. All rights reserved.
1. Introduction
Hexamethylene triperoxide diamine (HMTD) and triacetone
triperoxide (TATP) are peroxide explosives, Fig. 1. Both HMTD
and TATP can be made from readily available precursor materials;
the principal ingredients are usually hydrogen peroxide and acetone (for TATP), or hexamine (for HMTD). Preparation is done in
the presence of an acid catalyst, typically: citric, sulphuric or
hydrochloric acid. Detailed instructions and videos are available
on the internet. Although these explosives are relatively easy to
make, the process is hazardous since these primary high explosives
are very sensitive to initiation and are relatively powerful; HMTD
and TATP have TNT equivalences of around 60% and 88%, respectively [1–3]. TATP was first encountered in the 1980s in Israel,
and in the early 1990s in the UK [4,5]. Terrorist use of peroxide
explosives has not waned in recent years; in fact, the use of TATP
has continued to be used across much of the world [6–8]. HMTD
and TATP are not used commercially or by the military due to their
high sensitivity and poor stability; this means that traces of these
explosives would not be expected to be encountered in the general
environment. In any case, both explosives exhibit low persistence
in the environment and in the case of TATP, persistence is very
⇑ Corresponding author.
E-mail address: ldunn@dubaipolice.gov.ae (L. Dunn).
https://doi.org/10.1016/j.forc.2018.06.003
2468-1709/Ó 2018 Elsevier B.V. All rights reserved.
low because it readily sublimes, so any microscopic residue
remaining after an explosion would be rapidly lost. This means
that the presence of nanogram quantities of HMTD and TATP in
both pre-blast and post-blast trace samples can be highly significant; its detection can aid in establishing links between a suspect
and an explosives incident [9]. Thus, there is a need for reliable
and sensitive analytical methods that can unambiguously detect
these peroxide explosives.
Analysis of HMTD and TATP is relatively straightforward for visible quantities of sample. Methods are well-documented, and predominantly focus on techniques such as presumptive colour tests,
thin layer chromatography (TLC), FTIR and Raman spectroscopy.
However, there are limitations associated with these methods, particularly when analysis of trace amounts of sample is required.
Commercially available presumptive colour tests designed to
detect peroxide explosives generally lack specificity and can give
false indications if interferents are present in the sample; hydrogen
peroxide itself can give positive results for these tests. The LODs
tend to be in the region of 100 ng/mL for HMTD and TATP; this is
insufficient for most trace analysis casework. Published TLC methods mainly focus on using visualisation reagents comprising either
1% diphenylamine in sulphuric acid or 1% thymol in sulphuric acid.
The methods are time consuming and the LODs achievable are in
the region of 30–50 ng for both analytes; although an improvement on colour tests, TLC is not adequately sensitive for most trace
6
L. Dunn et al. / Forensic Chemistry 10 (2018) 5–14
Fig. 1. Structure of (a) TATP, (b) HMTD and (c) TMDDD.
analyses. Additionally, TLC results need to be confirmed via
another technique due to its poor specificity. Since HMTD and
TATP lack chromophores, direct analysis by HPLC-UV is not possible so UV absorption cannot be used as another identification criterion. HMTD and TATP have characteristic FTIR and Raman
spectra. However, interpretation of spectra can become complex
when samples also contain other FTIR and Raman active components; this is the case for post-blast samples which typically comprise complex matrices. For such samples, analytical methods must
exhibit a high degree of selectivity to enable the analyte of interest
to be distinguished from other substances present in the sample.
For analysis of some analytes, gas chromatographic (GC) separation
prior to FTIR analysis could simplify interpretation of results,
whilst simultaneously providing additional analyte confirmation
in the form of retention time. However, analysis of peroxide explosives by GC methods is difficult to achieve because of thermal
decomposition and activation of the column stationary phase; in
the case of TATP, this can happen rapidly [10–20].
On the other hand, LC/MS is well suited for analysing peroxide
explosives since thermal decomposition of the analytes is less
likely to occur. In general, the greater the number of characteristic
ions seen, the greater the level of confidence in the analyte being
correctly identified. Analysis of these analytes using LC/MS and
LC/MS/MS with APCI+ ionisation is well-documented [20–23]. Of
the two techniques, LC/MS/MS analysis is preferred due to
increased sensitivity and the ability to monitor selected reactions.
LC/MS analysis for HMTD results in the formation of many characteristic ions that typically include m/z 209.0768 and 207.0975,
corresponding to [M + H]+ and [C7H15O5N2]+, respectively [10,22–
26]. In contrast, TATP does not form stable ions under APCI conditions, thus a chemical such as ammonium acetate or formate is
often added to the mobile phase to facilitate the formation of
stable adduct ions [20,21]. TATP has few characteristic ions, typically m/z 240.1441 and 89.0597, corresponding to [TATP + NH4]+
and [C4H9O2]+ [10,20,21,24,26]. To achieve a high degree of confidence for TATP identifications, it is common for forensic laboratories to analyse samples in full scan and MS/MS mode for the
positive identification of TATP [21,24]. The downside to performing
LC/MS and followed by LC/MS/MS analysis is the increased analysis
time required.
Advancements in LC/MS have led to liquid chromatography
time-of-flight mass spectrometry (LC-QToF-MS) instruments. High
resolution analysis of organic and peroxide explosives has been
reported using quadrupole time-of-flight mass spectrometers and
using Orbitrap instruments [24–26]. LC-QToF-MS analysis allows
analysts to gain more information about analytes due to the high
resolution that offers greater ability to separate the target mass
from interfering masses. Since mass information is accurate to
within 3 ppm, the number of potential compounds is limited and
therefore greater confidence in analyte identification is achieved
as the possible number of candidate compounds is considerably
lower than with conventional mass spectrometry systems [27].
Additionally, the feature of all-scan, all-the-time TOF data also
allows retrospective data analysis to search for analytes that may
be present, but which were not originally sought. This could be
useful for historical searching for other analytes that are not routinely screened for at this laboratory, such as diacetone diperoxide
(DADP) and methyl ethyl ketone peroxide (MEKP), in cases where
it is later suspected that they might have been manufactured or
used. This is a significant advantage over traditional LC/MS/MS,
which only acquires data for specific ion transitions.
The aim of this work was to develop fast and accurate methods
for the analysis of HMTD and TATP using an ultra-performance liquid chromatography (UPLC) column in conjunction with an Agilent
6540 Ultra High Definition Accurate-Mass QToF Mass Spectrometer. This paper presents two separate methods developed and comprehensively validated for the semi-quantitative analysis of HMTD
and TATP. This paper supports recent work performed using ESITOF and Orbitrap that demonstrates that the 207.0611 ion
observed during the analysis of HMTD is in fact [TMDDD + H]+,
and not [M-1]+, as widely reported [28,29]. The authors believe
that this paper is the first to confirm the presence of this TMDDD
ion using accurate mass with APCI-TOF.
2. Materials and equipment
2.1. Materials
Explosive standards of hexamethylene triperoxide diamine
(HMTD) and triacetone triperoxide (TATP) were obtained from
AccuStandard Inc., (New Haven, USA). Both standards (M-8330ADD-25 and M-8330-ADD-24) were supplied as 100 ng/mL solutions in acetonitrile. HMTD and TATP standards were diluted using
LC-MS CHROMOSOLVÒ acetonitrile (Fluka Analytical, 34967-2.5L)
to obtain the desired concentration of analyte in solution.
The mobile phases were prepared using LC-MS CHROMOSOLVÒ
methanol (Fluka Analytical, 34966-2.5L) and deionised water (>18.
2 MXcm), purified using a Millipore Milli-Q Gradient A10 system.
Ammonium acetate with a purity >99% (Fluka Analytical, 73594100G-F) was used as a buffer for the TATP method mobile phase.
A Waters Acquity UPLC HSS C18, 1.8 mm, 2.1 x 50 mm column
(www.waters.com, product code: 186003532) was used for both
methods.
The internal reference mass solution was prepared using an
API-TOF reference mass solution kit (Agilent G1969-85001) and
solvents as follows: LC-MS CHROMOSOLVÒ acetonitrile (95 mL),
deionised water (5 mL), HP-121 (200 mL) and HP-921 (80 mL).
Real samples were collected using cotton swabs obtained from
the Forensic Explosives Laboratory (UK), previously analysed and
proven to be free from explosives traces. A solution comprising
analytical grade ethanol and deionised water in the ratio 50:50
was used for swab sampling and extraction. The sample extracts
were then cleaned-up using Biotage ISOLUTEÒ ENV + 200 mg/6 m
L solid phase extraction cartridges (www.biotage.com, product
code: 915-0020-C).
2.2. Instrumental
An Agilent 1260 series liquid chromatograph was used in conjunction with an Agilent 6540 Ultra High Definition AccurateMass Quadrupole Time of Flight Mass Spectrometer. Agilent Mass
Hunter Software, version B.06.01, enabled data processing. The
MS operated in 4 GHz, high resolution, low mass mode. Full scan
L. Dunn et al. / Forensic Chemistry 10 (2018) 5–14
mode was used with a mass range of m/z 60–1000 and an acquisition time of 1000 ms/spectrum.
3. Method development
A series of experiments were carried out to obtain the optimum
method parameters for the detection of HMTD and TATP. Each
parameter was independently varied, while the remaining parameters remained constant. Responses were compared as a function
of peak area (m/z 209.0766 for HMTD and m/z 240.1443 for TATP).
The results of these experiments are discussed in detail below.
3.1. HMTD analysis
The proportion of methanol in the mobile phase was varied
from 10% to 30% (v/v), in increments of 5%. The optimum HMTD
response was obtained when the mobile phase comprised 80:20
water/methanol; at this ratio the HMTD response was 16% better
compared with a mobile phase comprising 30% methanol. Decreasing the methanol content further did not have any significant effect
on the HMTD response, though the retention times were markedly
increased, as would be expected. With regards to the MS conditions, several key parameters were observed to affect the HMTD
response, including: the vaporiser and gas temperatures, the nebuliser and drying gas flows and the skimmer voltage, Fig. 2. From
the figure, it can be seen that the HMTD response increased significantly as the nebuliser flow was increased from 20 psig to 45 psig.
The strongest HMTD response (45 psig) was 147% greater than the
weakest response (20 psig). The strongest HMTD response was
obtained at a vaporiser temperature of 300 °C; a 50% increase in
response was observed at the optimum vaporiser temperature
(300 °C) compared with the weakest response (350 °C). The optimum drying gas setting was determined to be 5 L/min; the
response was improved by 47% at the optimum value compared
with the weakest response (7 L/min). The skimmer voltage was
varied between 40 V and 65 V, with the strongest response for
HMTD obtained with a skimmer voltage of 50 V; this response
was 39% greater than the weakest response (60 V). The gas temperature was varied between 180 °C and 320 °C. Comparison between
7
the strongest response (240 °C) and weakest response (300 °C)
showed that a 19% increase in response was observed at the optimum gas temperature. Fine tuning was then performed and the
greatest HMTD response was found to occur with a gas temperature of 250 °C. The fragmentor voltage was varied between 50 V
and 110 V; the HMTD response was a little lower at 40 V compared
with the other values, but was relatively consistent between 60 V
and 110 V, with the relative standard deviation (RSD) for the
response being 3.5% across this voltage range. Similar experiments
were carried out with the capillary and octopole RF voltages
(1500 V to 2700 V, and 240 V to 260 V); it was found that varying
these parameters resulted in relatively insignificant improvements
in HMTD response.
3.2. TATP analysis
The optimum mobile phase composition was determined to be
60:40 methanol/water. As the methanol content was decreased
from 70% to 60%, the TATP response increased by approximately
6%. The TATP response increased as the ammonium acetate concentration in the mobile phase was increased from 4 mmol to
10 mmol, until the ammonium acetate concentration reached
10 mmol; at this concentration the TATP response decreased by
22%. The column temperature was observed to have an effect on
the TATP response; temperatures ranging 15 °C to 25 °C were evaluated and it was observed that the TATP response was 11% greater
when the column temperature was 15 °C compared to 25 °C. With
regards to the MS conditions, several parameters were observed to
significantly affected the TATP response, including: the vaporiser
temperature, the nebuliser and drying gas flows, and the capillary
and fragmentor voltages, Fig. 3. It was observed that the capillary
voltage had the most significant effect on the TATP response. The
capillary voltage was varied between 900 V and 2500 V; an
increase of 1259% was observed at a voltage of 1000 V compared
with 900 V. At voltages >1000 V, the TATP response gradually
decreased with increasing voltage; there was a reduction in
response of 39% between the optimum response (1000 V) and
the response at the highest voltage (2500 V). The vaporiser temperature also significantly affected TATP response; as the vaporiser
Fig. 2. Optimisation of HMTD method parameters. (Duplicate measurements performed for each parameter setting and the average value plotted).
8
L. Dunn et al. / Forensic Chemistry 10 (2018) 5–14
Fig. 3. Optimisation of TATP method parameters. (Duplicate measurements performed for each parameter setting and the average value plotted).
temperature was increased from 100 °C to 340 °C, there was little
change in TATP response until 220 °C, above which the response
increased markedly with increasing temperature. The maximum
response (300 °C) was 430% greater than the weakest response
(100 °C). The drying gas was varied between 2 L/min and
7 L/min, with the strongest TATP response (5 L/min) being 257%
greater than the weakest response (2 L/min). The strongest TATP
response was obtained with a nebuliser flow of 25 psig; at this
setting, the response was 117% greater than the weakest response
(15 psig). Improvements in response of 21% and 9% were achieved
by optimising the fragmentor and skimmer voltages, respectively.
Varying the gas temperature and octopole RF made little difference
to TATP response, with the RSD of TATP response across the ranges
tested being <3% and 5%, respectively.
It was observed that the degree of contamination of the corona
pin and APCI source significantly affected the TATP response. To
examine this in further detail, a sequence comprising 14 consecutive TATP standards was analysed, Fig. 4. As can be seen in the figure, the TATP response decreases rapidly for the first few analyses,
and then decreases more slowly as the run progresses. The TATP
response drops by 47% over the run. This effect was not observed
for the HMTD response. The TATP response was also observed to
be substantially affected when the internal reference mass solution
flowed continuously into the APCI source. Reference mass solutions comprising various proportions of HP-121 and HP-921 were
tested and all were found to reduce the TATP response to approximately one third of the response obtained without the reference
mass solution. Given the effect of the reference mass solution on
Fig. 4. Monitoring response for consecutive TATP standards (50 ng/mL).
L. Dunn et al. / Forensic Chemistry 10 (2018) 5–14
Table 1
HMTD and TATP method parameters.
Parameter
HMTD method
TATP method
Solvent
20:80 MeOH:H2O
Run time/min
Gas temperature/°C
Vaporiser temperature/°C
Drying gas/L/min
Nebuliser/psig
Fragmentor voltage/V
Skimmer voltage/V
OCT1RF/V
Capillary voltage/V
Corona/mA
Reference mass(es)
4
250
310
5
40
85
50
250
1700
12
121.0509, 922.0098
60:40 MeOH:H2O,
with 8 mM ammonium
acetate
8
100
300
5
25
155
60
260
1000
12
149.0233
the TATP response, it was decided that a stable background ion
should be used instead. An ion with mass of 149.0233 was identified as being suitable reference mass for this method; this ion is
due to phthalic anhydride contamination in the system.
3.3. Optimised methods
Both optimised methods for HMTD and TATP utilised isocratic
elution at a flow rate of 200 mL/min, with autosampler and column
chamber temperatures set to 15 °C. A 1 mL injection volume was
injected from an insert vial. Sample ionisation was achieved using
Atmospheric Pressure Chemical Ionisation (APCI) in positive ion
mode. Method specific parameters are given in Table 1. For the
HMTD method, the continuous flow of the internal reference mass
solution to the APCI source enabled the monitoring of two reference masses (121.0508 and 922.0098). As discussed in section
3.2, no reference solution was used for the TATP method. On completion of TATP analysis, the column was flushed with nonbuffered mobile phase for a minimum of 30 min.
4. Validation
4.1. Validation methodology
The validation assessed the selectivity, limits of detection and
quantification, linearity, repeatability and reproducibility of the
methods. The limit of detection (LOD) and limit of quantitation
(LOQ) were determined (for 10 replicates) at a signal-to-noise ratio
(S/N) of 3 and 10, respectively. The characteristic ions evaluated
were m/z 209.0766 and 207.0973 for HMTD, and m/z 240.1443
for TATP. Standards of concentrations ranging from the LOD to
100 ng/mL were analysed in triplicate to determine the linearity
of the methods. The standards were analysed in a random order
to help eliminate any bias effects. Retention time, peak area and
accurate mass values were used for determining the repeatability,
reproducibility and robustness of the methods. Peak area was used
for quantitation. All standards and samples were interspersed with
blanks comprising acetonitrile to assess whether any HMTD or
Table 2
Identity of real samples collected for validation study.
Sample ID
Description
1
2
3
4
Post explosion debris
Desk, monitor and keyboard
Laboratory floor
Laboratory bench
9
TATP carryover occurred. The repeatability was assessed using 10
replicates analysed within 1 day for each of three concentrations
(HMTD: 5 ng/mL, 40 ng/mL and 100 ng/mL, TATP: 30 ng/mL,
50 ng/mL and 100 ng/mL). The reproducibility and ruggedness data
was acquired by different analysts conducting analyses over an
extended time period (HMTD 11 weeks, TATP 14 weeks). The RSD
was calculated to provide some measure of the reproducibility of
the method, factoring in any effects of inadvertent changes, such
as, cleanliness of the source, exact composition of the mobile
phase, and contamination of the LC column and capillary.
4.2. Preparation and analysis of real samples
The selectivity of the method was assessed using real samples
spiked with explosives. Cotton swabs moistened with ethanol/
water were used to swab a variety indoor and outdoor surfaces
to obtain samples expected to contain a matrix similar to what
might be encountered in real casework samples, Table 2. Swabs
were extracted twice with 5 mL ethanol/water, and the combined
extract of 10 mL then cleaned-up by elution through an Isolute
ENV+ cartridge. Adsorbents were removed from the cartridge using
acetonitrile (1 mL) [5,26,30]. Sample extracts were then passed
through a 0.2 mm filter. Each acetonitrile sample extract was spilt
into two portions; one aliquot was left unchanged, whilst the
remaining aliquot was spiked with standards to give a concentration of approximately 10 ng/mL HMTD and 40 ng/mL TATP. The acetonitrile sample extracts were then analysed in triplicate. A
calibration curve comprising 6 standards was analysed immediately prior to sample analysis. Additionally, a standard was
injected after every three sample injections (10 ng/mL HMTD, 50
ng/mL TATP).
Since the purpose of the spiked samples was to monitor the
effect of the sample matrices on signal suppression, mass accuracy
error and retention time shift, and given that the authors have not
yet fully validated the clean-up method, the explosives were added
post-clean-up so as not to distort the results. Validation of the
clean-up method will be performed in due course.
The recovery of analytes in casework samples varies according
to the sampling, extraction and clean-up methods used in processing the evidence. This is in addition to any losses that may occur at
the scene prior to, or during, evidence packaging. Many forensic
laboratories only report that explosives were found on evidence,
not the actual quantity. Quantitation in explosives trace analysis
is generally used as an indication as to whether evidence is likely,
or unlikely, to have been in recent direct contact with an
unwrapped quantity of explosive, or materials contaminated with
explosive. The authors wanted to determine whether semiquantitation using a 1-point calibration would be sufficient for this
purpose. Certainly, performing a 1-point calibration is markedly
quicker than performing a full calibration prior to each set of casework samples. Thus, quantitation was performed using two methods; the concentration of analyte in the sample was calculated
from the calibration curve and it was also determined using a 1point calibration using the nearest preceding standard.
5. Results and discussions
5.1. HMTD
HMTD was resolved from the solvent front (0.9 min) and eluted
at around 1.8 min. HMTD was detected using seven ions, Table 3.
The ions with m/z 209.0766 and 207.0973 were considered to
be characteristic ions for HMTD; these were required to be present
to confirm the presence of HMTD in a sample. It is widely reported
in the literature that the identity of the heavier ion is [M + 1]+. The
10
L. Dunn et al. / Forensic Chemistry 10 (2018) 5–14
Table 3
Accurate mass values for characteristic ions.
Analyte
HMTD
TATP
Calculated m/z
209.0768
207.0975
179.0662
145.0608
118.0499
90.0550
88.0393
240.1441
89.0597
Identity
+
[HMTD + H]
[C7H15O5N2]+
[C5H11O5N2]+
[C5H9O3N2]+
[C4H8O3N]+
[C3H8O2N]+
[C3H6O2N]+
[TATP + NH4]+
[C4H9O2]+
lighter ion is considered to be a reaction product between HMTD
and methanol, [C7H15O5N2]+ [10,22–26].
An ion with m/z 207.0614 was also observed, Figs. 5 and 6. It has
been previously reported that the identity of this ion is [M-1]+
[21,25,31]. The need to be able to unequivocally identify analyte
ions is important in forensic work as the reported results can have
significant implications. Additionally, if significance is reported
with regards to the quantity of that analyte present, analysts need
to ensure they are using appropriate ions for quantification calculations. If the response of the 207 ions is used for quantification,
then an erroneous result could be obtained.
Accurate mass experiments performed using ESI-TOF and Orbitrap APCI-MS have indicated that the true identity of this ion is
more likely to be [TMDDD + H]+ [28,29]. TMDDD (tetramethylene
diperoxide diamine dialdehyde) is itself considered to be an energetic peroxide similar in structure to HMTD, though two aldehyde
functional groups replace one of the peroxo groups, Fig. 1 [32].
TMDDD also appears to be a degradation product of HMTD. The
calculated m/z for [TMDDD + H]+ is 207.0611; the mass error associated with this ion on this instrument was determined to be
1.45 ppm, thus providing good corroborating evidence that this
is the true identity of this ion and that it is not [HMTD-1]+.
Measured m/z
Mass error/ppm
209.0766
207.0973
179.0661
145.0605
118.0499
90.0548
88.0393
240.1443
89.0597
1.07
1.10
0.83
1.85
0.00
1.71
0.05
4.84
0.06
5.2. TATP
TATP eluted at around 6.0 min, being well resolved from the
solvent front (0.8 min). TATP was detected using ions 240.1443
and 89.0597, Table 2. The observed ions correspond well with literature values [10,20,21,24,26]. A second peak was present in both
of these extracted ion chromatograms; it eluted almost 1 min after
the main TATP peak. The peak was present when the TATP concentration exceeded 12 ng/mL. The peak shape and signal to noise ratio
was poor, and the peak area was not reproducible. The presence of
a second peak in LC chromatograms has been previously observed
and it has been reported that it is due to the existence of an alternative structural conformer [20,33]. The presence of this peak in
the 240.1443 extracted ion chromatogram (EIC) can be used to provide additional confirmation that TATP is present in a sample.
5.3. Method validation
The LOD and LOQ for were determined to be 0.50 ng and 2 ng on
column for HMTD, and 10 ng and 25 ng on column for TATP. These
values are comparable with other published LC/MS methods
[20,21,23,25]. Both methods were observed to exhibit good linear-
Fig. 5. HMTD APCI-TOF mass spectrum.
11
L. Dunn et al. / Forensic Chemistry 10 (2018) 5–14
Fig. 6. Zoomed in region of HMTD mass spectrum.
Table 4
RSD of mass accuracy, repeatability n = 10, reproducibility n = 64 (HMTD) and n = 70 (TATP).
Analyte
Measured mass
Theoretical mass
HMTD
209.0766
207.0973
240.1430
89.0597
209.0768
207.0975
240.1441
89.0597
TATP
ity between the LOD and 100 ng/mL (the most concentrated standard commercially available), with R2 > 0.99 (HMTD: 209.0766,
207.0973, TATP: 240.1443).
The measured accurate mass values showed good agreement
with the theoretical accurate mass values; both identifying ions
for HMTD showed around 1 ppm mass error, Table 3. The TATP
240 ion was within 5 ppm of the theoretical value. The TATP conformers exhibited identical accurate mass values.
The response repeatability exhibited low RSD values of 7.3% for
HMTD and 6.6% for TATP. Other studies have obtained similar
repeatability values [24]. The reproducibility was observed to be
significantly higher; the RSD values were 32% for HMTD and 31%
for TATP. It is the opinion of the authors that the low reproducibility is due to differing degrees of source contamination on the different days that the analyses were performed. It is recommended
that samples are always analysed in the same run as appropriate
standards.
The retention time repeatability was shown to be highly
repeatable within one day; the RSD was <1.1% and <0.4% for
HMTD and TATP, respectively. The reproducibility was good
for both analytes, with a RSD of <4.0% for HMTD and <2.4%
for TATP.
Accurate mass determination was performed by averaging
across the full peak. The results exhibited good precision and
appeared stable over a short-timescale, Table 4.
Difference/ppm
1 day
7 days
0.06
0.69
4.26
4.88
1.07
1.10
4.84
0.06
5.3.1. Selectivity
No HMTD or TATP was detected in any of the unspiked samples,
i.e. there were no false positive results. Each mobile phase was
shown to be free from interfering background ions with masses
within 2 ppm of the exact masses of the analyte of interest. No
detectable HMTD or TATP carryover was detected during the validation study.
HMTD and TATP were detected in all of the spiked samples. All
7 HMTD ions were observed in all of the samples, with a signal to
noise ratio greater than 3. Frequently during TATP analyses m/z 89
channel was extremely noisy, and the peak often exhibited poor
peak shape during this experiment. The calibration curves used
for determining the concentration of analyte in the spiked samples
are given in Figs. 7 and 8. Example chromatograms for the spiked
samples are given in Figs. 9 and 10.
It was observed that the retention time and accurate mass values for the real samples were stable throughout the analyses,
regardless of the presence of common contaminants. There was
good agreement between the measured and actual concentration
of HMTD for spiked samples 1–3, Table 5 and Fig. 11. The concentration of HMTD in sample 4 was a lower than expected; it is possible that contaminants in the sample either degraded the HMTD
or affected the formation of characteristic ions. Both methods for
determining the concentration of HMTD in the samples produced
similar results.
12
L. Dunn et al. / Forensic Chemistry 10 (2018) 5–14
Fig. 7. HMTD calibration curve (m/z 209.0766) used for quantitation of spiked samples. (The error bars represent ± one standard deviation, n = 3).
Fig. 8. TATP calibration curve (m/z 240.1430) used for quantitation of spiked samples. (The error bars represent ± one standard deviation, n = 3).
Fig. 9. Mass spectrum for spiked sample 4 (HMTD), background subtracted.
L. Dunn et al. / Forensic Chemistry 10 (2018) 5–14
13
Fig. 10. EIC (240.1200-240.1700) for spiked sample 2 (TATP).
Table 5
Concentration (mean ± one standard deviation, n = 3) of explosives in spiked samples
1–4: A calculated from calibration curve, B determined from 1-point calibration.
Sample
HMTD
Conc
1
2
3
4
TATP
(A)
/ppm
9.3 ± 0.8
10.3 ± 0.8
9.9 ± 1.0
8.7 ± 0.7
Conc
(B)
/ppm
9.1 ± 0.2
10.9 ± 0.2
10.1 ± 0.4
8.2 ± 0.1
Conc
(A)
/ppm
33.4 ± 2.9
31.9 ± 1.4
30.4 ± 1.9
29.3 ± 2.2
Conc
(B)
/ppm
37.0 ± 1.3
38.0 ± 3.0
37.7 ± 2.5
42.5 ± 2.5
The concentration of TATP in the spiked samples, as determined
by the calibration curve, was observed to be significantly lower
than the actual concentration of 40 ppm. From the table it is evident that when the concentration was determined form the calibration curve, the TATP concentration decreased with increasing
sample number, i.e. the response for TATP reduced as the run progressed. This was an expected outcome given the observations
recorded during the method development phase. In contrast, the
TATP concentrations derived using a 1-point calibration based on
the peak are of the nearest preceding standard yielded significantly
more accurate results.
From the results obtained, it was concluded that semiquantitation using a 1-point calibration would be sufficient for
determining the concentration of analytes in casework samples
to give an indication as to whether evidence is likely to have been
in recent direct contact with explosives, or materials contaminated
with explosives.
Fig. 11. Concentration of explosives in spiked samples 1–4: (A) calculated from
calibration curve, (B) determined from 1-point calibration. (The error bars represent
± one standard deviation, n = 3).
6. Conclusions
In this paper, the development and validation of fast and robust
LC-QToF methods for the trace analysis of HMTD and TATP have
been presented. The methods exhibit good chromatography, with
14
L. Dunn et al. / Forensic Chemistry 10 (2018) 5–14
the peroxide analytes being sufficiently retained on the column to
prevent solvent peak overlap. Sufficient mass accuracy is achieved
to unambiguously confirm the presence of HMTD and TATP.
Thorough method validations were performed using real samples to demonstrate good method repeatability and robustness,
with complex matrices. As shown from the analysis of spiked samples, the presence of common contaminants had no significant
effect on the retention time and accurate mass values of the real
samples. The LODs for HMTD and TATP are considered to be practical for forensic applications. The authors determined that semiquantification for TATP achieved via 1-point calibration using the
peak area of the nearest preceding standard provides sufficient
accuracy for estimating the quantity of explosive in a sample.
This paper supports recent work performed using ESI-TOF and
Orbitrap that demonstrates that the 207.0614 ion observed during
the analysis of HMTD is in fact [TMDDD + H]+, and not [M-1]+. The
authors believe that this paper is the first to confirm the presence
of this TMDDD ion using accurate mass with APCI-TOF.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Acknowledgements
We would like to acknowledge the advice provided during the
method development and validation stages of this work by Dr
Andrew Crowson (Forensic Explosives Laboratory, UK). The authors
wish to acknowledge the strong support of this project from colleagues in the General Department of Forensic Science and Criminology, Dubai Police.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.forc.2018.06.003.
References
[1] Encyclopedia of explosives and related items, U.S. Army Research and
Development Command, 1960, vol 1–10.
[2] J. Oxley, J. Smith, Peroxide explosives, in: H. Schubert, A. Kuznetsov (Eds.),
Detection and disposal of improvised explosives, Springer, Dordrecht, 2006,
pp. 113–121.
[3] T. Urbanski, Chemistry and Technology of Explosives volume 3, Pergamon
Press, UK, 1964.
[4] S. Zitrin, S. Kraus, B. Glattstein, Identification of two rare explosives, Proc. 1st
Int. Symp. Anal. Detectioof Explos. (1983) 137–141.
[5] S. Doyle, Quality and the trace detection and identification of organic high
explosives, in: A. Beveridge (Ed.), Forensic Investigation of Explosions, CRC
Press, 2012, p. 541.
[6] R. Callimachi, A. Rubin, L. Fourquet, A view of ISIS’s evolution in new details of
Paris attacks. The New York Times, https://www.nytimes.com/2016/03/
20/world/europe/a-view-of-isiss-evolution-in-new-details-of-paris-attacks.
html?_r=0, 2016 (accessed 07.03.17).
[7] K. Connolly, Syrian man seized in Germany ’was planning Isis bomb attack’,
The Guardian, 2016, https://www.theguardian.com/world/2016/oct/10/
german-police-capture-syrian-man-suspected-of-planning-bomb-attack
(accessed 07.03.17).
[8] T. Gibbons-Neff, Brussels terrorists probably used explosive nicknamed ‘the
Mother
of
Satan’,
The
Washington
Post,
2016,
https://www.
washingtonpost.com/news/checkpoint/wp/2016/03/23/the-type-of-bombsused-in-brussels-have-been-seen-before/ (accessed 07.03.17).
[9] J. Oxley, J. Smith, H. Chen, E. Cioffi, Determination of the vapour density of
triacetone triperoxide (TATP) using a gas chromatography headspace
technique, Propellants Explos. Pyrotech. 30 (2) (2005) 127–130.
[10] A. Peña-Quevedo, J. Laramee, H. Durst, S. Hernández-Rivera, Cyclic organic
peroxides characterization by mass spectrometry and raman microscopy, IEEE
Sens. J. 11 (4) (2011) 1053–1060.
[11] A. Peña-Quevedo, N. Mina-Calmide, N. Rodríguez, D. Nieves, R. Cody, S.
Hernández-Rivera, Synthesis, characterization and differentiation of high
energy amine peroxides by MS and vibrational microscopy, Proc. SPIE 6201,
Sensors, and Command, Control, Communications, and Intelligence (C3I)
Technologies for Homeland Security and Homeland Defense V, 62012E (2006);
doi:10.1117/12.666202.
[12] J. Chladek, in: Advances in analysis and detection of explosives, Kluwer
Academic, Publishers, 1993, pp. 73–76.
[13] S. Zitrin, T. Tamari, Analysis of Explosives by Infrared Spectroscopy, in: A.
Beveridge (Ed.), Forensic Investigation of Explosions, CRC Press, 2012, p. 680.
[14] Dubai Police, General Department of Forensic Science and Criminology,
standard method FSD 75-EXP-SM-01 ‘Colour spot tests for explosives and
related materials’.
[15] Dubai Police, General Department of Forensic Science and Criminology, FSD
75-EXP-SM-01-V, internal report, unpublished work.
[16] Dubai Police, General Department of Forensic Science and Criminology,
standard method FSD 75-EXP-SM-03 ‘TLC analysis for peroxide explosives’.
[17] Dubai Police, General Department of Forensic Science and Criminology, FSD
75-EXP-SM-03-V, internal report, unpublished work.
[18] A. Peña-Quevedo, R. Cody, N. Mina-Camilde, M. Ramos, S. Hernández-Rivera,
Characterization and differentiation of high energy amine peroxides by direct
analysis in real time TOF/MS, Proc. SPIE 6538 (2007) 653828.
[19] A. Peña-Quevedo, S. Hernández-Rivera, Mass spectrometry analysis of
hexamethylene triperoxide diamine by its decomposition products, Proc.
SPIE 7303, Detection and Sensing of Mines, Explosive Objects, and Obscured
Targets XIV, 730303 (2009); doi:10.1117/12.819080.
[20] L. Widmer, S. Watson, L. Schlatter, A. Crowson, Development of an LC/MS
method for the trace analysis of triacetone triperoxide (TATP), Analyst 127
(2002) 1627–1632.
[21] X. Xu, A.M. van de Craats, E. Kok, P.C.A.M. de Bruyn, Trace analysis of peroxide
explosives by high performance liquid chromatography-atmospheric pressure
chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) for
forensic applications, J. Forensic Sci. 49 (2004) 1230–1236.
[22] C. Marsh, R. Mothershead, M. Miller, Post-blast analysis of hexamethylene
triperoxide diamine using liquid chromatography-atmospheric pressure
chemical ionization-mass spectrometry, Sci. Justice 55 (2015) 299–306.
[23] A. Crowson, M. Beardah, Development of an LC/MS method for the trace
analysis of hexamethylenetriperoxidediamine (HMTD), Analyst 126 (2001)
1689–1693.
[24] X. Xu, M. Koeberg, C. Kuijpers, E. Kok, Development and validation of highly
selective screening and confirmatory methods for the qualitative forensic
analysis of organic explosive compounds with high performance liquid
chromatography coupled with (photodiode array and) LTQ ion trap/Orbitrap
mass spectrometric detections (HPLC-(PDA)-LTQ Orbitrap), Sci. Justice 54
(2014) 3–21.
[25] D. DeTata, P. Collins, A. McKinley, A fast liquid chromatography quadrupole
time-of-flight mass spectrometry (LC-QToF-MS) method for the identification
of organic explosives and propellants, Forensic Sci. Int. 233 (2013) 61–74.
[26] H. Rapp-Wright, G. McEneff, B. Murphy, S. Gamble, R. Morgan, M. Beardah, L.
Barron, Suspect screening and quantification of trace organic explosives in
wastewater using solid phase extraction and liquid chromatography-high
resolution accurate mass spectrometry, J. Hazard. Mater. 329 (2017) 11–21.
[27] R. Kinghorn, C. Milner, J. Zweigenbaum, Analysis of Trace Residues of Explosive
Materials by Time-of-Flight LC/MS, Agilent Technologies, Wilmington, DE,
2005.
[28] T. Krawczyk, Enhanced electrospray ionization mass spectrometric detection
of hexamethylene triperoxide diamine (HMTD) after oxidation to
tetramethylene diperoxide diamine dialdehyde (TMDDD), Rapid Commun.
Mass Spectrom. 29 (2015) 2257–2262.
[29] J. Oxley, J. Smith, M. Porter, L. McLennan, K. Colizza, Y. Zeiri, R. Kosloff, F.
Dubnikova, Synthesis and degredation of hexamethylene triperoxide diamine
(HMTD), Propellants Explos. Pyrotech. 41 (2016) 334–350.
[30] A. Crowson, R. Cawthorne, Quality assurance testing of an explosives trace
analysis laboratory –Further improvements to include peroxide explosives,
Sci. Justice 52 (2012) 217–225.
[31] G. Asher Newsome, L. Ackerman, K. Johnson, Humidity affects relative ion
abundance in direct analysis in real time mass spectrometry of hexamethylene
triperoxide diamine, Anal. Chem. 86 (2014) 11977–11980.
[32] T. Klapötke, T. Wloka, in: The Chemistry of Peroxides Volume 3 Part 1, John
Wiley & Sons Inc, 2014, p. 520.
[33] N. Haroune, A. Crowson, B. Campbell, Characterisation of triacetone
triperoxide (TATP) conformers using LC-NMR, Sci. Justice 51 (2) (2011) 50–56.