Human Factors and Mechanical Engineering for Defense and Safety (2019) 3: 8

https://doi.org/10.1007/s41314-019-0024-6

ORIGINAL PAPER

Evaluation of Natural Capsaicin (N.Cap) in Pepper Spray

by GC-MS/FID, NMR and HPLC as an Alternative to the Use
of Oleoresin Capsicum (OC)
N. Oliveira Junior 1 & V. Padulla 1 & I. Flores 2 & D. Annunciação 2 & M. Neiva 3 & V. Brant 3

Received: 22 February 2019 / Revised: 29 April 2019 / Accepted: 17 May 2019 / Published online: 31 July 2019
# Springer Nature Singapore Pte Ltd. 2019

Abstract
GC-FID/MS, NMR and HPLC were used to study natural capsaicin (N.Cap) in pepper sprays in order to show the capsaicinoids in
raw material as well as a solution in a spray canister. Natural capsaicin was used instead of Oleoresin Capsicum (OC) while
maintaining the usual effect with the advantage of better chemical control. In addition, the non-flammability safety characteristics
have been increased since N.Cap does not contain the flammable substances present in OC. GC-FID/MS, NMR and HPLC results
showed 2:1 ratio of capsaicin and dihydrocapsaicin, respectively, agreeing with a typical ratio expected. Other capsaicinoids were
detected in lower concentrations. Substances such as oils and resins, which are normally found in OC, were not detected in
significant concentrations. The HPLC alternative method was evaluated regarding resolution and capsaicinoids ratio measurement.
The dilution of raw materials (N.Cap) in dichloromethane avoids the intermediary processes of extraction or concentration of an
analyte. It was possible to carry out an analysis with a solution directly collected from a spray by only one dilution step.
Contaminants such as PAVA and others could be observed in the same analysis by GC-MS/FID without changes in the method-
ology. A review of the literature was carried out comparing the usual aspects regarding capsaicinoids contents in OC spray solutions,
where findings reported between 1 and 40% of capsaicinoids in the solutions showing a large range of active substance concen-
tration in commercial products. The method allows controlling the production and safety evaluation of the product by the user.

Keywords Pepper . Capsaicin . Dihydrocapsaicin . Capsaicinoids . Non-lethal . Riot control

Introduction
* N. Oliveira Junior
[email protected]
Since 1980, pepper spray has been used as a non-lethal (or less
V. Padulla than lethal) resource by law enforcement forces around the
[email protected] world [1]. The pulverised solution’s irritating effect allows
I. Flores dissuading the opponent by providing some advantage in
[email protected] crowd controlling and disturbances. There are two expected
D. Annunciação
main effects: involuntary closure of the eyelids and lacrima-
[email protected] tion, both perceived immediately. Side effects that take a few
seconds more to be noticed include burning eyes, cough, nasal
M. Neiva
[email protected] discharge, difficulty breathing and burning in the mouth and
other affected regions. Rarer effects may still occur, such as
V. Brant
disorientation and mild loss of motor action. These effects,
[email protected]
added to the psychological effects, render the individual tem-
1
Condor Non-Lethal Technology S/A, 160 Armando Dias Pereira, porarily incapacitated by allowing law enforcement to take
Nova Iguaçu, Rio de Janeiro, Brazil restraining measures, leading to the dispersal of clusters of
2
Instituto Federal de Goiás, São Bartolomeu-Vila Esperança, individuals and diminishing the ability to counteract or resist.
Luiziânia, Goiás, Brazil This state of temporary incapacitation lasts for about 10 to
3
Instituto deTecnologia Ambiental Firjan SENAI, 53 Moraes e Silva, 15 min and after a few hours, the individual regains full ca-
Tijuca, Rio de Janeiro, Brazil pacity without any permanent effect.
8 Page 2 of 10 Hum Factors Mech Eng Def Saf (2019) 3: 8

There are reports of injuries involving the use of this type the region of the planet where it is cultivated, as well as a
of resource and reported cases of death are rare. Rohini et al. number of other process and production factors. Typically,
[2] reported that in 7156, documented cases of pepper spray OC has a concentration of 0.1 to 1.0% capsaicinoids on a dry
injuries 6.0% presented serious lesions and there were no re- basis [13].
ports of death. Kearney et al. [3] reported that in 3671 cases, The biological action mechanism of capsaicinoids involves
6.8% were severe, but without any reports of death [4–6]. the activation of peripheral nerve receptors in the mucous
Epidemiological studies show a range of 2.7 to 15.0% of se- membranes. Contact with capsaicinoids initiates the release
vere cases involving pepper spray, even without any records of inflammatory substances (substance-P, cytosines and
of deaths. In general, the rarely reported cases of death always neuropeptides) triggering a series of physiological processes
involve adverse circumstances, either due to the environment such as altered potassium flow in the cells, blood vessel dila-
or the individual, which lead to death for reasons other than tation, nasal mucus production and lacrimation along with
the effect of the irritant, such as asphyxia by use in closed sensory responses causing a burning sensation and pain [14].
places or extreme and prolonged exposure [1]. On the other Prolonged contact may cause nerve endings to be desensitised
hand, there are reports of cases of product inefficiency, placing due to a reduced availability of neuropeptides for release;
law enforcement agents at risk due to non-perceived disabling however, it does not lead to a permanent desensitisation state
effects by the affected individual [7, 8]. Both extreme cases and can be reversed by discontinuing contact.
must be contemplated in the development of this type of prod- The presence of capsaicin and dihydrocapsaicin is the most
uct by determining an optimum concentration range so that important factor for its markedly superior irritant properties
there is efficacy in the disabling effect without causing perma- among capsaicinoids [13]. Therefore, the greater the content
nent damage, intoxication or even death. of capsaicin and dihydrocapsaicin in the spray solution, the
The most widely used irritant agent in the production of greater the disabling effect of the product. Thus, the best way
pepper sprays is the Oleoresin Capsicum (OC) [1–9]; OC is a to evaluate the effectiveness of capsaicinoid concentration in a
viscous liquid extracted from the pepper fruit (Capsicum) pepper spray solution is to measure the capsaicin together with
which contains more than a hundred different types of sub- dihydrocapsaicin present in the packaged solution. Obviously,
stances and does not have a simple chemical formula. some correlations between the concentration and the irritant
The most important substances present in the OC are organoleptic effect must be taken into account.
capsaicinoids since they produce the desired irritant effect. The Scoville test, whose unit of measure is SHU, is another
Other substances such as phenols, acids, alcohols, aldehydes, known form of evaluating the effect of “pungency” or blazing
carotenes, esters, oils and resins are also present in the OC [10] peppers. This method was developed in 1912 by Wilbur
but cause little or no irritant effect compared with capsaicinoids. Scoville [15] giving a five-level scale for pungency: non-
It should be noted that oils and resins are potentially flammable pungent (0–700 SHU), low pungency (700–3000 SHU), mod-
and they become undesirable by increasing the flammability of erate pungency (3000–25000 SHU), high pungency (25000−
the spray solution. 70000 SHU) and very high pungency (> 80000 SHU).
Capsaicinoids are amides formed by the binding of a chem- However, the method is based on the dilution of the pepper
ical group called vanillamide with fatty acids having nine to extract and subsequent organoleptic test, which makes it sub-
11carbon atoms in the chain. The biochemical synthesis of jective and difficult to reproduce. Modern methods have many
these substances occurs in the fruit of the Capsicum genus, advantages including the amplification of qualitative and
and the concentration in the fruit varies with the plant species, quantitative information by instrumental chemical methods,
cultivation conditions, soil, seasonality and so on [11]. especially chromatographic methods [11].
Among capsaicinoids, capsaicin is the main substance from This work presents an improvement in the production of
the point of view of irritant effect. Capsaicinoids include cap- sprays using a raw material different from OC that has a more
saicin and dihydrocapsaicin, which both correspond to 90% of homogeneous chemical composition that can be more effort-
the total capsaicinoids present in the fruit. The typical distri- lessly controlled than OC. This raw material is another type of
bution is 60–70% capsaicin, 20–30% dihydrocapsaicin, 3–7% pepper extract named natural capsaicin (N.Cap) that generates
nordihydrocapsaicin, ≈ 1% homocapsaicin and ≈ 1% better quality sprays. The absence of oils and resins contrib-
monodihydrocapsaicin [1]. Many species have 0.5–1.0% utes to eliminate the risk of flammability of the solution, for
nonivamide that has the same chemical structure as PAVA example. In practice, the same effects are known in OC
[12]. Thus, the molecular formula C18H27NO3 is not the OC sprays, but with a better defined chemical composition and
formula but only the capsaicin molecular formula, which is other advantages like absence of red dyes and seasoning
contained in OC. smell, besides generating totally non-flammable products
The chemical composition of OC used in the manufacture of when in an adequate solution.
pepper spray depends on the type of plant, the extraction meth- The characterisation of the N.Cap presented was performed
od, the season of the year in which the crop was harvested and by GC-MS, NMR and HPLC, in a qualitative and quantitative
Hum Factors Mech Eng Def Saf (2019) 3: 8 Page 3 of 10 8

way. Also studied were spray solutions produced with this raw comparative purposes, the reported concentration values were
material and a procedure was established to analyse this type recalculated to a base in g/m3. The work of Reilly et al. [14]
of product so that the concentration of capsaicin and (2001) carried out on five different types of commercial pepper
dihydrocapsaicin present in the spray composition could be sprays shows a variation from 700 to 40500 g/m3. However, it
readily verified. is not clear in the literature what would be the ideal concentra-
Another substance that some manufacturers have been tion to guarantee effectiveness in the incapacitation of the indi-
using is nonivamide, also called pelargonic acid vanillylamide vidual, ensuring that there is no permanent injury.
or PAVA. This type of irritant can be used as a single active The reported reviews do not indicate the inhalation of cap-
ingredient or, on the other way, combined with OC to produce saicin being critically dangerous and do not report non-
a variety of pepper sprays. Two major points differ PAVA from reversible injures when capsaicin was applied directly to face.
capsaicin: PAVA is much less irritating than capsaicin and can Prolonged contact presented attention points but not regarding
be obtained synthetically on an industrial scale, generating a permanent injuries or death. On the other hand and curiously,
low-cost raw material. On the other hand, PAVA is also natu- capsaicin can have a number of health benefits such anticancer
rally present in pepper extracts like N.Cap or OC but appears activity, anti-inflammatory activity, anti-obesity activity and
in very less concentration than capsaicin. analgesia. Therefore, capsaicin can be considered a secure
An important point is the action of this natural capsaicin in irritant agent to be used as a less lethal alternative for crowd
the body compared with OC. In this sense, it is important to control.
report that in both the use of OC and the use of natural cap-
saicin, the disabling effect is always due to the content of Natural Capsaicin Raw Material
capsaicinoids (capsaicin and dihydrocapsaicin) present in the
solution. A bibliographical review of specific inhalation stud- The natural capsaicin used in this study was imported from
ies of pure capsaicin [4, 16–18] was carried out verifying the India from the manufacturer Chillies Export House Limited
use of this chemical agent in inhaled solutions and their con- that supplies pepper extracts to the world market since 1963.
sequences. George et al. [19] report the capsaicin nasal re- The product is in the form of white crystalline powder with
sponse effects by spraying a solution in the nose. Capsaicin some agglomerates in the form of thin blades or flat surfaces.
sprayed into the nose caused burning, lacrimation and It has a strongly pungent and very penetrating odour. Its spec-
rhinorrhoea and there are no permanent injuries mentioned. ification meets a minimum of 95% total capsaicin. The re-
Marabini et al. [20] reported benefit intranasal use of capsaicin maining 5% consists of the extraction residues present at phar-
in patients with vasomotor rhinitis where nasal disease was maceutical control levels and by other lower concentration
markedly reduced by capsaicin treatment. Saito et al. [21] capsaicinoids. Other control parameters are maximum drying
reported oral LD50 values were 118.8 mg/kg for male and loss of 1%, maximum ignition residue of 1% and residual
97.4 mg/kg for female mice, and 161.2 mg/kg for male and solvent (methanol) max 3000 ppm (reference USP-467). It
148.1 mg/kg for female rats. Major toxic symptoms in mice has a melting range of 57–66 °C, metal content < 10.0 ppm
were salivation, skin erythema, staggering gait, bradypnea and and arsenic < 2.0 ppm.
cyanosis. Some animals showed tremor, clonic convulsion,
dyspnea and lateral or prone position and then died 4 to Pepper Spray Solution
26 min after dosing.
Table 1 reports the concentrations used in a pure capsaicin The pepper spray solution presented in this study refers to
inhalation study and a study of commercial OC sprays. For N.Cap dissolved in ethyldiglycol and water. In this solution

Table 1 The concentrations used in pure Capsaicin inhalation studies and commercial OC sprays for comparative purposes

Reported references Reported value Recalculated value Work focus

Spray studies This work 2.64 mg/mL 2640 g/m3 Concentration of Cap.N in solution before
filling in the spray canister.
Reilly et al. [14] 0.7 ~ 40.5 mg/mL 700 ~ 40,500 g/m3 Capsaicinoid spray concentration study for
self-defence. Five different types of spray.
Inhalation studies J.G. Collier et al. [24] 2 ~ 65 μmol/L 0.6 ~ 19.8 g/m3 Inhalation of Capsaicin by nebulization of the
solution and use of mask for application with
duration of 1 min.
P. Blanc, et al. [25] 0.3 ~ 3.0 μmol/L 0.092 ~ 0.92 g/m3 Study of ingestion tolerance of Capsaicin in
workers in the pepper product industry.
G. Philip et al. [20] 6.6 mg/L 6.6 g/m3 Study of nasal response to Capsaicin
8 Page 4 of 10 Hum Factors Mech Eng Def Saf (2019) 3: 8

type, the presence of water contributes to a non-inflammable type and do not contain unexpected substances [25]. The sub-
behaviour of the spray when dispersed against flame and stances shown in Table 2 are about 95% of the chromatogram
ethyldiglycol is used as vehicle. The exact formulation was total area. The last 5% was due side substances or method
purposely omitted to protect the manufacturer. issues, such as no use of split and other detection and peak
integration details.
The method was improved to avoid analytical issues and to
Experiment and Results get quantitative results. Figure 2 shows the chromatogram of
spray solution made with N.Cap previously dissolved on
GC-MS Analyses ethyldiglycol and water, in the same way that the solution
spray is made in production processes. An aliquot of 500 μL
The first step was to run a qualitative study by GC-MS on was diluted to 10.0 mL in dichloromethane and then, 2 mL
N.Cap directly dissolved in dichloromethane (HPLC/UV was transferred to the vial. At this time, the split was set to
grade) to identify substances present in the raw material and 50:1 and the injection volume was 1 μL.
verify the column wise separation of each molecule. 0.03 g of The peak at r.t. 1.8 min in Fig. 2 refers to dichloromethane
the N.Cap was weighed and quantitatively transferred to a used as a solvent and the peak at r.t. 6.7 min refers to the
10-mL volumetric flask with dichloromethane. After the prep- ethyldiglycol vehicle. The capsaicin and dihydrocapsaicin
aration, the solution was sent for analysis by GC-MS. The peaks can be seen respectively around r.t. 23 min and the %
analysis of capsaicin was conducted in a split less mode in area ration of the peaks was about 2:1, respectively.
order to increase the detection sensitivity of the impurities With the purpose of quantifying, these two peaks containing
present. capsaicin and dihydrocapsaicin are integrate together and the
The experiments were performed using an Agilent calibration curve was made by this result against standard so-
Technologies 7890B GC System (USA), coupled with a flame lutions made which Sigma-Aldrich Capsaicin (purity capsaicin
ionisation detector (FID) and hyphened with mass spectrom- 61.1%, dihydrocapsaicin 31.2%; LOT no.: LRAA9221 09
eter model 5977A (Agilent). HP-5MS low bleeding capillary September 2015). A standard solution of 0.33 g/L was prepared
column (Agilent) (30 m × 0.25 mm × 0.25 μm film) was used dissolving the standard in dichloromethane and subsequent di-
for separation, using helium as carrier gas, with a flow of lutions were made to obtain the concentration range for the
1.2 mL/min. Operation conditions are the following: temper- capsaicin standard 0.066, 0.132, 0.198, 0.264 and 0.330 g/L.
ature of injector and MS were set at 300 and 260 °C, respec- The regression coefficient R2 was 0.999 and uncertainties were
tively. MS was operated in the electron ionisation mode and less than 0.9%. The subsequent dilution was satisfactory by the
mass spectra was collected within m/z from 50 to 300. For statistical linear fit results, but some issues can be improved by
analysis by GC FID, the detector temperature was set at preparation of solution standards separately. In this way, it is
350 °C. The flow rate for hydrogen was 35 mL/min and for possible to quantify de capsaicinoids, that is, capsaicin and
air 350 mL/min, and the makeup flow of nitrogen was 30 mL/ dihydrocapsaicin together, on the pepper spray solution.
min.
Figure 1 shows the chromatogram resultant of injection of NMR Analyses
the N.Cap solution in a chromatograph. This step was carried
out to guide the procedure evolution and to have just informa- Nuclear magnetic resonance (NMR) emerges as an informa-
tion to pre-scale the analysis. Table 2 shows the retention times tive analytical tool for identification and quantification of dif-
(r.t.), substance name, % area and structure of the molecules. ferent chemical species present in complex mixtures. Its abil-
The first two groups in Fig. 1, at r.t. 9–15 min, are fat and ity to analyse samples in different physical states, with mini-
aromatic-like molecules that show a good separation between mum pre-treatment, allows a safe analysis that preserves the
the two principal peaks: capsaicin at r.t. 24.2 min and original characteristics of the same. In the specific case of
dihydrocapsaicin at r.t. 24.4 min. The nonivamide (PAVA) at organic molecules, their sensitivity is increased by the detec-
r.t. 23.3 min is a natural capsaicinoide present in pepper ex- tion of the isotope 1H, which is the one of greater natural
tracts and can be seen separately of both capsaicin and abundance of the element hydrogen and found in the molec-
dihydrocapsaicin peak. ular chemical groups.
Capsaicin and dihydrocapsaicin signals’ separation and Thus, the ability of NMR to evaluate the quality control of the
identification were satisfactory and agree with previously pub- non-lethal arms industry becomes clear, where it can act in the
lished papers [22–24]. There were deformations of the chro- characterisation and choice of the raw material, in the process
matographic peaks, markedly on the capsaicin and control and safety of the final product. It is also observed that the
dihydrocapsaicin peaks, due to the overload of the analytes information allows identifying problems such as adulterations,
in the chromatographic split less injection. The substances taking into account that the results are able to detect compounds
presented were the same expected ones for this pepper extract of interest concomitant to the other non-target present.
Hum Factors Mech Eng Def Saf (2019) 3: 8 Page 5 of 10 8

For verification of the main components present in the performed at 25 °C using glass capillary for the quantification
N.Cap, a 26.4 mg sample was weighed and solubilized in of the maleic acid present in the solution with 100 mmol of
600 μL of CDCl3 in a 5-mm NMR tube. The analysis was D2O. The Broad Band Inverse (BBI) probe, inserted into a

Abundance TIC: Qualificacao Capsaicina_20180413_R1.D\data.ms

6.8e+07

6.6e+07

6.4e+07

6.2e+07

6e+07

5.8e+07

5.6e+07

5.4e+07

5.2e+07

5e+07

4.8e+07

4.6e+07

4.4e+07

4.2e+07

4e+07

3.8e+07

3.6e+07

3.4e+07

3.2e+07

3e+07

2.8e+07

2.6e+07

2.4e+07

2.2e+07

2e+07

1.8e+07

1.6e+07

1.4e+07

1.2e+07

1e+07

8000000

6000000

4000000

2000000

Time--> 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

Fig. 1 Total iron chromatogram of N.Cap analysis by GC-MS

8 Page 6 of 10 Hum Factors Mech Eng Def Saf (2019) 3: 8

Table 2 Results of analysis of the raw material capsaicin, by GC-MS

Bruker Avance III 11.75 T equipment, was used. The simple was possible to identify all the signals of the chemical groups
pulse sequence used was zg. The spectra were processed with of the main components present in the OC sample. Groups in
0.3 Hz line-broadening and zero-filling. bold are those that are clear and can be used for quantification.
Figure 3 shows the graphical results and the signal assign- Doublet signals were used, with numberings 10
ments (chemical shift, integration relative to the amount of (dihydrocapsaicin) and 11 (capsaicin), for relative integration
hydrogens, signal multiplicity and coupling constant) of cap- of the signals. In addition to being unobstructed, they had a
saicin and dihydrocapsaicin. Through the NMR analyses, it higher signal-to-noise ratio due to having six hydrogens

Response_ Signal: 7862-3.D\FID2B.ch

Fig. 2 Chromatogram of pepper
spray solution by GC-FID. The
peak at r.t. 1.8 min refers to 900000
dichloromethane used as a solvent
and the peak at r.t. 6.7 min refers 800000
to ethyldiglycol vehicle. The
capsaicin and dihydrocapsaicin 700000
peaks can be seen respectively
around r.t. 23 min
600000

500000

400000

300000
Capsaicina_Dihidrocapsaicina

200000

100000

0
Time 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Hum Factors Mech Eng Def Saf (2019) 3: 8 Page 7 of 10 8

Fig. 3 1H NMR spectrum results

and the capsaicin and
dihydrocapsaicin signal
assignments

contributing to the integration of each signal. Thus, it was capsaicin (majority), the latter being twice the concentration
possible to verify the proportion of these compounds among of the former.
themselves present in the mixture (Fig. 4). This led to the The NMR technique proved efficient in the identification
proportion of 33% dihydrocapsaicin (minority) and 66% and relative quantification of the components present in the
mixture of pepper derivatives. The spectra were acquired
quickly and with a simple sample preparation step. In this
way, it was possible to maintain the characteristics of the ma-
trix avoiding unwanted transformations.
The analyses allowed a broad view of the absence of other
compounds, even if minority, proving the purity of the mate-
rial and absence of degradation products. On the other hand,
the presence of other components could be identified concom-
itantly in a mixture of higher complexity. Thus, this work
suggests that 1H NMR can be used as a quality control tool
in the non-lethal arms industry.

HPLC Analyses

The pepper solution (N.Cap 0.3% w/w) was collected and

dissolved in acetonitrile/H 2 O for further injection into
the chromatograph. The chromatograms are presented
in Fig. 5. It was possible to verify that capsaicin and
dihydrocapsaicin present similar retention times for the three
samples analysed.
Taking into account only the peaks of capsaicin and
dihydrocapsaicin, seen respectively at r.t. 4.87 min and
6.62 min, 69% and 26% peak areas, respectively, were found,
following the contributions published in Molecules 2011, 16,
8919–8929 which analyses them by HPLC. The run was per-
formed under the same conditions, the eluent being acetoni-
Fig. 4 Amplification of the 1H NMR spectrum with the numerical
trile/H2O, on a PHENOMENEX C18 × 4.6 ID × 5 μm col-
indication of the chemical groups used in the relative integration of the umn, at a temperature of 25 °C, with a flow rate of 1 mL/
compounds capsaicin and dihydrocapsaicin min and an injection of 10 μL.
8 Page 8 of 10 Hum Factors Mech Eng Def Saf (2019) 3: 8

Datafile Name:Capsaicina_AM18c.lcd
Fig. 5 HPLC chromatogram of Sample Name:Capsaicina_AM18c
pepper spray solution Sample ID:Policia Federal
mAU

4,870/385671/0,000
222nm,4nm

40,0

37,5

35,0

32,5

30,0

27,5

25,0

22,5

20,0

6,621/144510/0,000
17,5

15,0

12,5

10,0
4,559/23613/0,000

7,5

5,0

2,5

0,0

0,0 2,5 5,0 7,5 10,0 12,5 15,0 min

Discussion structure as pelargonic acid vanillylamide, known as PAVA,

and made artificially. Thus, PAVA is not a natural extract. The
The raw material namely natural capsaicin (N.Cap) was eval- PAVA irritant effects are less than the capsaicin effect and it
uated satisfactorily showing purity beyond that found in oily takes longer to be perceived by the opponent. A much larger
extracts of pepper, known as Oleoresin Capsicum (OC). amount of PAVA is required to have the effect of capsaicin in
N.Cap has the same effect found in the pepper spray solution pepper spray solutions. The nonivamide can be naturally
made with OC and has analytical advantages due the much found in pepper extracts, but when the pepper spray solution
smaller component number of substances in the raw material. is made by PAVA additions, the amount is much higher than
This fact improves the chemical analyses because there are no the natural occurrences. Thus, if the manufacturer uses PAVA,
side natural substances interfering in the analytical results it can be detected by the method presented here.
when the N.Cap is used. NMR technique proved to be an excellent option in identi-
GC-MS was used to identify capsaicinoids in the raw ma- fication and quantification. These results encourage the appli-
terial and GC-FID to quantify in the spray solution. No con- cation of this technique for process control and a work in this
centration steps were used and the solution can be analysed direction is being carried out by the authors.
directly without filtrations, solvent extracts, chemical changes HPLC was important as it showed agreement with re-
or others to avoid manipulations and their interferences. The sults of the other techniques presented. However, the use of
integration of the capsaicin and dihydrocapsaicin peaks to- this technique has lower range power than GC-MS and
gether can be used to quantify the major capsaicinoids present NMR. In addition, in this case, no pre-treatment of samples
in the spray solution and report a unique key value to distin- was required. The quantification can be carried out with
guish different types of pepper spray solutions. relative success and HPLC is an efficient auxiliary tech-
The nonivamide can be separated by the GC-MS and this is nique. In this study, it was not possible to separate the
important because the nonivamide has the same molecular nonivamide signal from the capsaicin signal by HPLC.
Hum Factors Mech Eng Def Saf (2019) 3: 8 Page 9 of 10 8

This makes it difficult to identify PAVA additions in the 5. Brown L, Takeuchi D, Challoner K (2000) Corneal abrasions asso-
ciated with pepper spray exposures. Am J Emerg Med 18(3):271–
pepper solutions with HPLC in this study. The HPLC re-
272. https://doi.org/10.1016/S0735-6757(00)90120-7
sults showed different ratios between capsaicin and 6. Watson WA, Stremel KR, Westdorp EJ (1996) Oleoresin capsicum
dihydrocapsaicin because there is no good separation peak (cap-stun) toxicity form aerosol exposure. Ann Pharmacother 30(7–
between nonivamide and capsaicin. 8):733–735. https://doi.org/10.1177/106002809603000704
7. Broadstock M (2002) What is the safety of “pepper spray” use by
The capsaicin and dihydrocapsaicin 2:1 ratio was very close
law enforcement or mental health service staff? NZHTA Tech Brief
in both GC-FID and NMR results. The concentrations found Series 1(2) ISBN 1-877235-39-3
are close to the N.Cap manufacturer certificate report, which 8. Canadian Police Research Center (1993) A Toxicological Review
indicates 68.7% of capsaicin and 31.3% of dihydrocapsaicin. of Capsaicinoid (Oleoresin of Capsaicums) TR-02-93
9. Eugene J, Harry S (2001) Riot control agents: pharmacology, tox-
In addition, the ratio reported by the Sigma-Aldrich capsaicin
icology, biochemistry and chemistry. J Appl Toxicol 21:355–391
standard also agrees with the results reported here. Review Article. https://doi.org/10.1002/jat.767
10. Keller U, Flath RA, Mon RA, Teranishi R Volatiles from red pepper
(Capsicum spp.). In Quality of selected fruits and vegetables in
North America (symposium series). Am Chem Soc 12:137–146.
Conclusions https://doi.org/10.1021/bk-1981-0170.ch012
11. Zeid AAO, Yacine BHA, Mohamed AH, Ayman AG (2011)
The use of N.Cap was a satisfactory improvement in pep- Determination of capsaicin and dihydrocapsaicin in Capsicum
per spray production. The purity of the N.Cap is very high fruit samples using high performance liquid chromatography.
Molecules 16(10):8919–8929. https://doi.org/10.3390/
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molecules16108919
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