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

Emily C. Lennert





gunshot residue, diphenylamine, high performance liquid chromatography, tandem, mass spectrometry, HPLC-MS/MS

Article Reviewed

Mei, H.; Quan, Y.; Wang, W.; Zhou, H.; Liu, Z.; Shi, H., Wang, P. Determination of diphenylamine in gunshot residue by HPLC-MS/MS. Journal of Forensic Science and Medicine. 2016, 2(1), 18-21.


The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.


Diphenylamine (DPA) is a common component of smokeless powders, i.e. gun powder. DPA is a stabilizer that is added to smokeless powder to bind the degradation products of other components of the smokeless powder, i.e. nitrocellulose and nitroglycerin, which degrade over time. DPA serves to stabilize the smokeless powder by slowing the decomposition of the product. The identification of DPA from gunshot residue may be useful in establishing whether an individual has fired a weapon, and can aid in the identification of suspects. After a weapon is fired, trace levels of DPA and other gunshot residues will remain on the shooter’s hands. To detect these trace levels, a sufficiently sensitive method is required, such as high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). In this study, HPLC-MS/MS is used for the identification and quantification of diphenylamine. Qualitative identification was based on the presence of four product ions of DPA, and quantification was performed using the peak area of the main product ion.

A HPLC-MS/MS system was utilized in this experiment. The HPLC portion utilized a mobile phase of methanol and water in a 90:10 ratio. The mobile phase is the liquid in which analytes are separated within the HPLC column. An electrospray ionization (ESI) source was utilized to ionize the samples. The MS/MS portion consisted of a triple quadrupole mass spectrometer. For more information on triple quadrupole mass spectrometers, see Review: Rapid Analysis of Drugs of Abuse and their Metabolites in Human Urine Using Dilute and Shoot Liquid Chromatography – Tandem Mass Spectrometry. Optimized parameters are given in Table 1 within the study.

A DPA stock solution of 100 μg/mL DPA in methanol was prepared and diluted to prepare solutions of 500 ng/mL, 250 ng/mL, 100 ng/mL, 50 ng/mL, 10 ng/mL, and 5 ng/mL. Gunshot residue samples were prepared from fired ammunition samples 1 h after shooting. The residue within the cartridge casing was extracted in acetone by placing the casing in 3 mL acetone for 2 min. The acetone was then transferred to a new tube, evaporated until dry, and reconstituted in 0.1 mL of methanol. Gunshot residue was also collected from shooter’s hand using a cotton swab soaked in acetone. The hand was swabbed, then the swab was squeezed out and filtered into a beaker. After filtration, the extract was evaporated to dryness and reconstituted in 0.1 mL of methanol. A blank was prepared by swabbing the hand of a non-shooter. Extracts were then analyzed by HPLC-MS/MS.

Using MS/MS, the analyst may monitor selected ions. The molecular ion m/z 170.2, [M + H]+, was selected for monitoring, as well as four product ions: m/z 152.0, 93.0, 77.0, and 65.0. Parameters were optimized in multiple reaction monitoring (MRM) mode to improve sensitivity for each product ion. The optimized parameters are given in Table 1 within the study. HPLC conditions were also optimized. Of three potential mobile phases, methanol:water provided the best detection of DPA. The ratio of methanol and water was optimized to 90:10, which gave the best signal for DPA.

To allow for quantitative analysis, a calibration curve was prepared using the series of DPA dilutions. Results indicated that the curve was linear between 5 ng/mL and 500 ng/mL, with a R2 value of 0.9978. This means that the instrument is accurately recording the abundance of DPA in the sample over a series of concentrations. This will also allow the analyst to calculate the concentration of DPA in an unknown smokeless powder sample. The limit of detection, i.e. signal to noise ratio of 3, was determined to be 0.3 ng/mL.

Analysis of the sample extracted from the cartridge casing yielded a concentration of 892.4 ± 8.9 ng/mL DPA based on the calibration curve. The response of the extract from the shooter’s hand was below the range of the calibration curve, and thus a concentration of DPA was not determined. However, the signal to noise ratio was 8.2, and was therefore higher than the limit of detection.

Scientific Highlights

  • LOD was determined to be 0.3 ng/mL for DPA by the presented HPLC-MS/MS method.
  • DPA in the cartridge casing was quantifiable based on the calibration curve that was created from the standard solutions.
  • DPA in the residue from the shooter’s hand was detectable by the given method, but was not quantifiable.


The detection of DPA may be useful in the identification of shooting suspects.

Potential Conclusions

The given HPLC-MS/MS method may be applicable to the detection of DPA in casework samples.