Journal of Forensic Science and Medicine

: 2016  |  Volume : 2  |  Issue : 1  |  Page : 18--21

Determination of Diphenylamine in Gunshot Residue by HPLC-MS/MS

Hongcheng Mei1, Yangke Quan1, Wenhao Wang2, Hong Zhou1, Zhanfang Liu1, Huixia Shi1, Peng Wang3,  
1 Institute of Forensic Science, Ministry of Public Security, Beijing, China
2 College of Forensic Science, People's Public Security University of China, Beijing, China
3 Xianyang Public Security Bureau, Shanxi, China

Correspondence Address:
Hongcheng Mei
Institute of Forensic Science, Ministry of Public Security, Muxidi Street, South Lane 17, District - Xicheng, Beijing - 100038


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.

How to cite this article:
Mei H, Quan Y, Wang W, Zhou H, Liu Z, Shi H, Wang P. Determination of Diphenylamine in Gunshot Residue by HPLC-MS/MS.J Forensic Sci Med 2016;2:18-21

How to cite this URL:
Mei H, Quan Y, Wang W, Zhou H, Liu Z, Shi H, Wang P. Determination of Diphenylamine in Gunshot Residue by HPLC-MS/MS. J Forensic Sci Med [serial online] 2016 [cited 2020 Feb 23 ];2:18-21
Available from:

Full Text


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],[2],[3] 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 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],[11],[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 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 forensic-type assay of DPA, a method based on 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 identified.


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 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 (Zόrich, Switzerland) was used for weighing the sample.

Instrumental conditions


The HPLC analysis was performed by using the auto-injection system 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 flow rate, injection volume, and column temperature were 800 μL/min, 10 μL, and 20°C, respectively.


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.{Table 1}

Solution preparation

Different concentrations of DPA standard solutions were prepared with methanol. A stock standard solution (100 μg/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 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 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 filtered 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 fired a gun. The final methanol solution was analyzed by HPLC-MS/MS using the established method.

 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 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 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 [Table 1], and the ensuing experiments were carried out under these conditions.{Figure 1}

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 (acetonitrile-water, methanol-water, and methanol-water with 0.1% trifluoroacetic 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 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, methanol-water (90:10) was selected as the mobile phase where the retention time of DPA under 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].{Figure 2}

Quantitative analysis

A 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 five replicate injections, and the relative standard deviations were less than 5%. The detection limit concentration (S/N =3) was 0.3 ng/mL.{Figure 3}

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 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 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.


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 residue could 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.


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


1Dalby O, Butler D, Birkett JW. Analysis of gunshot residue and associated materials-a review. J Forensic Sci 2010;55:924-43.
2Dalby O, Birkett JW. The evaluation of solid phase micro-extraction fibre types for the analysis of organic components in unburned propellant powders. J Chromatogr A 2010;1217:7183-8.
3Tong 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.
4Vuki 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.
5Zhang Cui-mei. Chinese Journal of Explosives & Propellants 2007;1:30-2.
6Burleson 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.
7Xu T, Li S, Wang X, Luan Z, Wei X. Phys Test Chem Anal Part B:Chem Anal 2001;37:227.
8Northrop DM. Gunshot residue analysis by micellar electrokinetic capillary electrophoresis: Assessment for application to casework. Part I. J Forensic Sci 2001;46:549-59.
9Morelato 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.
10Arndt 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.
11West C, Baron G, Minet JJ. Detection of gunpowder stabilizers with ion mobility spectrometry. Forensic Sci Int 2007;166:91-101.
12Jordan M,Suzanne B. International Journal for Ion Mobility Spectrometry 2013;16:247-58.
13Laza 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.
14Haynes WM. CRC Handbook of Chemistry and Physics. 92 nd ed. Boca Raton, Florida: Taylor and Francis/CRC Press; 2011.