JForensicSciMed2118-1377315_034933

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Journal of Forensic Science and Medicine ¦ January 2016 ¦ Volume 2 ¦ Issue 118

Original Article

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.[1-3] Because DPA is commonly present

in smokeless gun powder, it may remain on the hands of

rearm users. Thus, the determination of DPA can provide

forensic evidence for the identification of suspects in

gun-related crimes. DPA determination is currently performed

by a variety of methods, such as the electrochemical

method,[4] single sweep square-wave polarography,[5] gas

chromatography-nitrogen phosphorus detector (GC-NPD),[6]

high performance liquid chromatography (HPLC), [7]

capillary electrophoresis (CE),[8] gas chromatography-mass

spectrometry (GC-MS), [2 ] desorption electrospray

ionization-mass spectrometry (DESI-MS),[9] ion mobility

spectrometry (IMS),[10-12] 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

rearm users;[13] thus, it is hard to identify DPA if the detection

method is not sufciently sensitive. In order to meet the

requirements of forensic-type assay of DPA, a method basedon HPLC and electrospray ionization (ESI) tandem mass

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 identied.

EXPERIMENTAL

Reagents and apparatusDPA was purchased from Sigma-Aldrich (St Louis, USA).

Methanol (HPLC) and acetone were obtained from Beijing

Chemical Plant (Beijing, China). The deionized water usedherein was purified using a Milli-Q system (Millipore,

Massachusetts, USA).

An Agilent 1,200 high performance liquid chromatograph

(CA, USA) tted with an auto‑injection system and an Agilent

Extend-C18 column (CA, USA) (150 mm × 4.6 mm, 5 µm)

along with API 2000 triple quadrupole mass spectrometer

(Wisconsin, USA) fitted with an ESI interface were

utilized; a METTLER AE 240 electronic balance (Zürich,

Switzerland) was used for weighing the sample.

Instrumental conditionsHPLC

The HPLC analysis was performed by using the auto-injectionsystem and the Agilent Extend-C18 column (CA, USA)

(150 mm × 4.6 mm, 5 µm). The mobile phase comprised

methanol and water, and the optimal elution ratio was 90:10,

which was optimized for the experiment. The ow rate,

Determination of Diphenylamine in Gunshot Residue byHPLC-MS/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, People’s 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) (150 mm × 4.6 mm, 5 µm) with methanol-water (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 5-500 ng/mL, r 2 = 0.9978. The limit of detection (S/N =3) of this method was 0.3 ng/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

Address for correspondence: Dr. Hongcheng Mei,Institute of Forensic Science, Ministry of Public Security, Muxidi Street,

South Lane 17, District - Xicheng, Beijing - 100038, China.E-Mail: [email protected]

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Mei, et al .: Determination of Diphenylamine in GSR

Journal of Forensic Science and Medicine ¦ January 2016 ¦ Volume 2 ¦ Issue 1 19

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

10 µL, and 20°C, 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 Table 1 were optimized for the experiment.

Solution preparationDifferent concentrations of DPA standard solutions were

prepared with methanol. A stock standard solution (100µg/mL)

was prepared by dissolving 0.025 g DPA in 250 mL of methanol;

serial dilutions of 500 ng/mL, 250 ng/mL, 100 ng/mL,

50 ng/mL, 10 ng/mL, and 5 ng/mL of DPA were then prepared

by appropriate dilution of the stock solution.

Extraction of DPA in gunshot residueAcetone was conrmed 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 ring a gun,

the gunshot residue in the bullet shell was extracted by soakingthe shell in 3 mL of acetone for 2 min; the acetone solution

was then sucked into a new tube and evaporated to dryness

and dissolved by the addition of 0.1 mL methanol. Gunshot

residue on the shooter’s hand was extracted carefully with

a cotton swab soaked with acetone. The acetone solution in

the cotton swab was squeezed out and ltered prior to being

placed in a beaker, then evaporated to dryness, and dissolved

with 0.1 mL methanol. A blank was prepared by similar

treatment of the hand of a person who never red a gun. The

nal methanol solution was analyzed by HPLC‑MS/MS using

the established method.

RESULTS AND DISCUSSION

Optimization of MS/MS conditionsOne 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 µg/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 spray voltage

(IS), curtain gas (CUR), temperature (TEM), ion source

gas1 (GS1), ion source gas2 (GS2), collision gas (CAD),

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

qualication 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 shownin Figure 1. All of the optimized parameters for MS/MS are

listed in Table 1, and the ensuing experiments were carried

out under these conditions.

Optimization of HPLC conditionsUnder 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 (acetonitrile-water,

methanol‑water, and methanol‑water with 0.1% triuoroacetic

acid [TFA]) as candidates. The results demonstrated that

methanol-water was better than acetonitrile-water as a mobile

phase under the optimal conditions; the addition of 0.1% TFA

lowered the detection intensities signicantly. 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, methanol-water (90:10) was selected

as the mobile phase where the retention time of DPA under

Table 1: 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 450°C 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: Ion-spray voltage, TEM: Temperature, GS1: Ion source gas1,

GS2: Ion source gas2, CAD: Collision gas, DP: Declustering potential, FP: Focusing potential, EP: Entrance potential

Figure 1: MRM mass spectrum of DPA



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Journal of Forensic Science and Medicine ¦ January 2016 ¦ Volume 2 ¦ Issue 120

these conditions was 3.4308 min. The HPLC chromatogram

of 250 ng/mL DPA using this mobile phase composition

followed by tandem mass spectrometry detection is shown

in Figure 2.

Quantitative analysisA series of DPA calibration standard solutions,

5 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, 250 ng/mL, and

500 ng/mL, were used to investigate the linearity of the MS/

MS peak-area (PA) versus the concentration (cDPA

) curve under

the optimal MS/MS and HPLC conditions described above.

The results showed that the linearity range from 5 ng/mL to

500 ng/mL, PA = 10617cDPA

+ 228094, r 2 = 0.9978 [Figure 3]. All

of the DPA calibration standard solutions were determined with

ve replicate injections, and the relative standard deviations

were less than 5%. The detection limit concentration (S/N =3)

was 0.3 ng/mL.

Detection of DPA in gunshot residueIn 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 gunre experiment. DPA in the gunshot residue in

the remaining bullet shell and on the shooter’s hand were

extracted 1 h after shooting by using the methods described

in Section Quantitative analysis, and DPA was determined

using HPLC-MS/MS. The concentration of DPA in the

remaining bullet shell was 892.4 ± 8.9 ng/mL according to the

calibration curve for DPA. The response value from tandem

mass spectrometric analysis of DPA on the shooter’s hand wasnot 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 red 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 HPLC-MS/

MS was developed in this study. This method is highly

sensitive, easy to operate, and provides rapid measurement.

The MS/MS response of DPA versus its concentration is

linear in the range of 5-500 ng/mL, and the detection limit

concentration (S/N = 3) is 0.3 ng/mL. DPA in gunshot residuecould be detected not only in the bullet shell, but also on the

shooter’s hand. This method may be applicable for sample

analysis in casework.

ACKNOWLEDGMENTThis study was supported by the Basal Research Fund Program

of Institute of Forensic Science, Ministry of Public Security,

China(2014JB006).

REFERENCES1. Dalby O, Butler D, Birkett JW. Analysis of gunshot residue and

associated materials-a review. J Forensic Sci 2010;55:924-43.

2. Dalby O, Birkett JW. The evaluation of solid phase micro-extraction

bre types for the analysis of organic components in unburned propellant

powders. J Chromatogr A 2010;1217:7183-8.

3. Tong Y, Wu Z, Yang C, Yu J, Zhang X, Yang S, et al . Determination

of diphenylamine stabilizer and its nitrated derivatives in smokeless

gunpowder using a tandem MS method. Analyst 2001;126:480-4.

4. Vuki M, Shiu KK, Galik M, O’Mahony AM, Wang J. Simultaneous

electrochemical measurement of metal and organic propellant

constituents of gunshot residues. Analyst 2012;137:3265-70.

5. Zhang Cui-mei. Chinese Journal of Explosives & Propellants

2007;1:30-2.

6. Burleson GL, Gonzalez B, Simons K, Yu JC. Forensic analysis

of a single particle of partially burnt gunpowder by solid phase

micro-extraction-gas chromatography-nitrogen phosphorus detector.

J Chromatogr A 2009;1216: 4679-83.7. Xu T, Li S, Wang X, Luan Z, Wei X. Phys Test Chem Anal Part B:Chem

Anal 2001;37:227.

8. Northrop DM. Gunshot residue analysis by micellar electrokinetic

capillary electrophoresis: Assessment for application to casework.

Part I. J Forensic Sci 2001;46:549-59.

9. Morelato M, Beavis A, Ogle A, Doble P, Kirkbride P, Roux C. Screening

of gunshot residues using desorption electrospray ionisation-mass

spectrometry (DESI-MS). Forensic Sci Int 2012;217:101-6.

10. Arndt J, Bell S, Crookshanks L, Lovejoy M, Oleska C, Tulley T, et al .

Preliminary evaluation of the persistence of organic gunshot residue.

Forensic Sci Int 2012;222:137-45.

Figure 2: HPLC chromatogram of 250 ng/mL DPA Figure 3: Calibration curve of DPA



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Journal of Forensic Science and Medicine ¦ January 2016 ¦ Volume 2 ¦ Issue 1 21

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. Conict of Interest: None declared.

11. West C, Baron G, Minet JJ. Detection of gunpowder stabilizers with ion

mobility spectrometry. Forensic Sci Int 2007;166:91-101.

12. Jordan M,Suzanne B. International Journal for Ion Mobility

Spectrometry 2013;16:247-58.

13. Laza D, Nys B, Kinder JD, Kirsch-De Mesmaeker A, Moucheron C.

Development of a quantitative LC-MS/MS method for the analysis of

common propellant powder stabilizers in gunshot residue. J Forensic Sci

2007;52:842-50.

14. Haynes WM. CRC Handbook of Chemistry and Physics. 92nd ed. Boca

Raton, Florida: Taylor and Francis/CRC Press; 2011.


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JForensicSciMed2118-1377315_034933