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Original Article

Determinationof Diphenylamine in Gunshot Residue by

HPLCMS/MS
Hongcheng Mei, Yangke Quan, Wenhao Wang1, Hong Zhou, Zhanfang Liu, Huixia Shi, Peng Wang2
Institute of Forensic Science, Ministry of Public Security, 1College of Forensic Science, Peoples Public Security University of China, Beijing,
2
Xianyang Public Security Bureau, Shanxi, China

Abstract
A high performance liquid chromatography tandem mass spectrometry/mass spectrometry (HPLC-MS/MS) protocol was developed for the
determination of diphenylamine(DPA). Four productions of DPA were selected for qualitative assay and the peak area of the main product ion for
quantitation. By means of separation using an Agilent Extend-C18 column (CA, USA) (150mm4.6mm, 5 m) with methanolwater(90:10)
as the mobile phase, DPA was detected by electrospray ionization(ESI) tandem mass spectrometry in positive mode. The linearity of the peak
area versus concentration ranged 5500 ng/mL, r2=0.9978. The limit of detection (S/N =3) of this method was 0.3ng/mL. This method is
applicable for the determination of DPA in gunshot residue.
Key words: Diphenylamine, gunshot residue, high performance liquid chromatography-mass spectrometry/mass spectrometry

Introduction
Diphenylamine(DPA) is an important component of a
gun propellant, where it is used as a stabilizer that can
bond with the degradation products of explosives, such as
nitrocellulose and nitroglycerine, and slow down the rate of
their decomposition.[13] Because DPA is commonly present
in smokeless gun powder, it may remain on the hands of
firearm users. Thus, the determination of DPA can provide
forensic evidence for the identification of suspects in
gunrelated crimes. DPA determination is currently performed
by a variety of methods, such as the electrochemical
method,[4] single sweep squarewave polarography,[5] gas
chromatographynitrogen phosphorus detector(GCNPD),[6]
high performance liquid chromatography (HPLC), [7]
capillary electrophoresis(CE),[8] gas chromatographymass
spectrometry(GCMS), [2] desorption electrospray
ionizationmass spectrometry(DESIMS),[9] ion mobility
spectrometry(IMS),[1012] and so on. Most of these methods
are suitable for the determination of DPA in gun propellants.
However, only trace levels of DPA remain on the hands of
firearm users;[13] thus, it is hard to identify DPA if the detection
method is not sufficiently sensitive. In order to meet the
requirements of forensictype assay of DPA, a method based
on HPLC and electrospray ionization(ESI) tandem mass
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DOI:
10.4103/2349-5014.162808

18

spectrometry was established. Four product ions of DPA

were selected for precise qualitative assay and the peak area
of the main product ion was used for quantitation. With this
method, DPA in gunshot residues can be identified.

Experimental
Reagents and apparatus

DPA was purchased from Sigma-Aldrich (St Louis, USA).

Methanol(HPLC) and acetone were obtained from Beijing
Chemical Plant(Beijing, China). The deionized water used
herein was purified using a Milli-Q system (Millipore,
Massachusetts, USA).
An Agilent 1,200 high performance liquid chromatograph
(CA, USA) fitted with an autoinjection system and an Agilent
Extend-C18 column (CA, USA)(150mm4.6mm, 5 m)
along with API 2000 triple quadrupole mass spectrometer
(Wisconsin, USA)fitted with an ESI interface were
utilized; a METTLER AE 240 electronic balance (Zrich,
Switzerland)was used for weighing the sample.

Instrumental conditions

HPLC
The HPLC analysis was performed by using the autoinjection
system and the Agilent Extend-C18 column (CA, USA)
(150mm4.6mm, 5 m). The mobile phase comprised
methanol and water, and the optimal elution ratio was 90:10,
which was optimized for the experiment. The flow rate,
Address for correspondence: Dr.Hongcheng Mei,
Institute of Forensic Science, Ministry of Public Security, MuxidiStreet,
South Lane 17, DistrictXicheng, Beijing100038, China.
EMail:meihongcheng@163.com

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Mei, etal.: Determination of Diphenylamine in GSR

injection volume, and column temperature were 800 L/min,

10 L, and 20C, respectively.
MS/MS
An ESI ion source was used for MS/MS in positive ionization
mode, with multiple reaction monitoring(MRM). In order
to improve the sensitivity of detection, all of the parameters
mentioned in Table1 were optimized for the experiment.

Solution preparation

Different concentrations of DPA standard solutions were

prepared with methanol. Astock standard solution (100 g/mL)
was prepared by dissolving 0.025g DPA in 250mL of methanol;
serial dilutions of 500ng/mL, 250ng/mL, 100ng/mL,
50ng/mL, 10ng/mL, and 5ng/mL of DPA were then prepared
by appropriate dilution of the stock solution.

Extraction of DPA in gunshot residue

Acetone was confirmed to be the most effective solvent for

DPA extraction in many studies;[10,14] thus, acetone was selected
as the solvent for DPA extraction. After manually firing a gun,
the gunshot residue in the bullet shell was extracted by soaking
the shell in 3mL of acetone for 2min; the acetone solution
was then sucked into a new tube and evaporated to dryness
and dissolved by the addition of 0.1mL methanol. Gunshot
residue on the shooters hand was extracted carefully with
a cotton swab soaked with acetone. The acetone solution in
the cotton swab was squeezed out and filtered prior to being
placed in a beaker, then evaporated to dryness, and dissolved
with 0.1mL methanol. Ablank was prepared by similar
treatment of the hand of a person who never fired a gun. The
final methanol solution was analyzed by HPLCMS/MS using
the established method.

declustering potential(DP), focus potential(FP), and

entrance potential(EP), the more abundant product ions m/z
152.0, m/z 93.0, m/z 77.0, and m/z 65.0 were selected as the
qualification ions. In order to improve the sensitivity for each
of the product ions, these parameters were again optimized in
MRM mode, and the parameters, collision energy (CE), and
collision cell exit potential (CXP) for each product ion were
also optimized, the MRM mass spectrum of DPA is shown
in Figure 1. All of the optimized parameters for MS/MS are
listed in Table1, and the ensuing experiments were carried
out under these conditions.

Optimization of HPLC conditions

Under the optimal conditions presented in Table 1,

we investigated the effect of the composition of the mobile
phase on the separation and MS/MS determination of DPA.
Three types of mobile phases were selected(acetonitrilewater,
methanolwater, and methanolwater with 0.1% trifluoroacetic
acid[TFA]) as candidates. The results demonstrated that
methanolwater was better than acetonitrilewater as a mobile
phase under the optimal conditions; the addition of 0.1% TFA
lowered the detection intensities significantly. To determine
the optimal separation time and MS/MS intensities, different
ratios of methanol to water(90:10, 80:20, 70:30, and 50:50)
were investigated. The results showed that lower methanol
content led to increased intensity of the baseline, and the
intensity of the DPA signal decreased with an increase of
the peak width. Thus, methanolwater(90:10) was selected
as the mobile phase where the retention time of DPA under

Results and Discussion

Optimization of MS/MS conditions

One of the advantages of tandem mass spectrometry is that

multiple product ions of a molecular ion can be selected
for qualitative assessment, leading to greater accuracy. In
positive electronic spray ion mode, a 1.0 mg/mL methanolic
solution of DPA was used for the molecular ion scan. The
molecular ion[M+H] + of DPA, m/z 170.2 was easily
selected from the full scan mass spectrum. By adjusting
the MS/MS parameters, including the ion sprayvoltage
(IS), curtain gas(CUR), temperature(TEM), ion source
gas1(GS1), ion source gas2(GS2), collision gas(CAD),

Figure1: MRM mass spectrum of DPA

Table1: Optimized parameters for MS/MS

Product ion

CE

CXP

CUR

IS

TEM

GS1

GS2

CAD

DP

FP

EP

152.0
37.2 V
24.85 V
25
4000 V
450C
80
85
8
30.59 V
392.01 V
9.17 V
93.0
49.98 V
12.01 V
77.0
53.36 V
9.11 V
65.0
38.79 V
7.93 V
CE: Collision energy, CXP: Collision cell exit potential, CUR: Curtain gas, IS: Ionspray voltage, TEM: Temperature, GS1: Ion source gas1,
GS2: Ion source gas2, CAD: Collision gas, DP: Declustering potential, FP: Focusing potential, EP: Entrance potential

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Mei, etal.: Determination of Diphenylamine in GSR

Figure2: HPLC chromatogram of 250ng/mL DPA

Figure3: Calibration curve of DPA

these conditions was 3.4308min. The HPLC chromatogram

of 250ng/mL DPA using this mobile phase composition
followed by tandem mass spectrometry detection is shown
in Figure2.

sensitive, easy to operate, and provides rapid measurement.

The MS/MS response of DPA versus its concentration is
linear in the range of 5500ng/mL, and the detection limit
concentration(S/N=3) is 0.3ng/mL. DPA in gunshot residue
could be detected not only in the bullet shell, but also on the
shooters hand. This method may be applicable for sample
analysis in casework.

Quantitative analysis

A series of DPA calibration standard solutions,

5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, and
500 ng/mL, were used to investigate the linearity of the MS/
MS peakarea(PA) versus the concentration(cDPA) curve under
the optimal MS/MS and HPLC conditions described above.
The results showed that the linearity range from 5ng/mL to
500ng/mL, PA=10617cDPA+228094, r2=0.9978[Figure3]. All
of the DPA calibration standard solutions were determined with
five replicate injections, and the relative standard deviations
were less than 5%. The detection limit concentration(S/N =3)
was 0.3ng/mL.

Detection of DPA in gunshot residue

In order to test the practicability of the method described

above, a Chinese 54 pistol (China Ordnance Equipment Group
Corporation)that is widely used by Chinese police was used
for the gunfire experiment. DPA in the gunshot residue in
the remaining bullet shell and on the shooters hand were
extracted 1 h after shooting by using the methods described
in Section Quantitative analysis, and DPA was determined
using HPLCMS/MS. The concentration of DPA in the
remaining bullet shell was 892.48.9ng/mL according to the
calibration curve for DPA. The response value from tandem
mass spectrometric analysis of DPA on the shooters hand was
not within the range of the calibration curve, and the signal
to noise ratio was 8.2. The sample extracted from the hand of
a person who never fired a gun was taken as a blank sample,
and no interference was observed in the spectrum.

Conclusion
A method for the determination of DPA by HPLCMS/
MS was developed in this study. This method is highly

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Acknowledgment
This study was supported by the Basal Research Fund Program
of Institute of Forensic Science, Ministry of Public Security,
China(2014JB006).

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How to cite this article: Mei H, Quan Y, Wang W, Zhou H, Liu Z, Shi H,
et al. Determination of Diphenylamine in Gunshot Residue by HPLC-MS/MS.
J Forensic Sci Med 2016;2:18-21.
Source of Support: Nil. Conflict of Interest: None declared.

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