Rapid Colorimetric Analysis of para-Phenylenediamine in Henna-based, Non-permanent Tattoo Color Mixtures

Tattoos have been used throughout history as body decorations and they comprise an integral element of various cultures. However, many individuals are uncomfortable with the fact that tattooing is virtually irreversible since dyes are injected under the skin into the dermis. An alternative that has grown in popularity is temporary tattoos based on henna. This treatment is normally safe and does not injure the skin surface; it involves exposing the skin externally to a henna tattoo mixture that contains ground henna leaves mixed with water or oil.

The natural dye in henna leaves is 2-hydroxy-1,4-naphtoquinone (Lawsone), which reacts on the skin surface with skin proteins, leading to a temporary orange-brown coloring of the skin. Natural henna tattoo mixture—i.e., without para-phenylenediamine (PPD), is applied using semi-conical containers to make it easier to achieve elaborate designs and is left on the skin for three to six hours. The longer the exposure time, the darker the final color, which is never black as is often assumed, but a dark orange-brown. By adding PPD to the henna tattoo mixture, the application time can be significantly reduced and the results appear more like the desired black color. However, the autoxidation products of PPD including Bandrowski’s base, a trimer of PPD, are very strong potential skin sensitizers, thus the addition of PPD is responsible for most reported skin complications arising from the use of black henna, such as allergic contact dermatitis, post-inflammatory hyperpigmentation, or even worse, an allergic sensitization to PPD and the many substances that cross-react with it, including para-aminophenol, para-toluenediamine or many azoic dyes, such as disperse orange 3.

Previously, studies using high-performance liquid chromatography (HPLC)-based methods for the identification and quantification of PPD in black henna have been published. For instance, using HPLC with a refractive index detector, Al-Suwaidi and Hamed12 investigated 25 henna samples with 11 black henna samples among them, which were purchased from 15 different henna salons in the United Arab Emirates. PPD was found in all black henna samples in concentrations ranging from 0.4% to 29.5% w/w. Brancaccio et al.13 also detected PPD at 15.7% w/w by investigating the black henna tattoo mix to which their patient developed contact allergy using HPLC with a diode array detector and an electrospray mass-spectrometric detector.

Such reported high concentrations of PPD in black henna tattoo mixtures give cause for concern, as even a 10% concentration was shown to sensitize all 24 subjects in a human maximization study.14 And due to PPD’s allergenicity, the European Union has legislated against allowing its direct application to skin or for dyeing eyebrows and eyelashes. Further, the US Food and Drug Administration has prohibited the application of PPD directly on the skin.13

Due to the current consumer relevance of henna tattooing, an assay for detecting PPD in henna mixtures rapidly and during on-site controls would be an advantage, as suspected samples could be immediately confiscated by control authorities. However, literature on such assays has not previously been published. Therefore, the aim of the present study was to develop such an assay without the need for complex sample preparation and using the fewest chemicals and simplest equipment as possible. Formulators are also expected to benefit from such an assay, as it could be applied for testing henna supplies for use in finished formulations.

Materials and Methods

The following materials were first obtained: resorcinol (meta-dihydroxybenzene)a; PPD, copper(II) sulfate pentahydrate, hydrochloric acid (1 M) and methanolb; hydrogen peroxide (30%)c; and screw cap vials (10 mL, 30 mL) and single-use plastic syringes from 1 mL to 10 mLd. Only purified water was used. The following solutions were then prepared: a resorcinol solution, 100 mg/mL in water/methanol (1:1) v/v; a copper(II) sulfate solution, 25 mg/mL in water; and a PPD solution, 2.7 mg in 5 mL methanol (~ 5 mM).

The henna sample (1 g + 0.01g) was weighed in a 30-mL screw cap vial and a volume of 10 mL methanol was added. This suspension was shaken for 2 min by hand. After removing the cap and allowing the vial to stand for 5 min so the particles can sediment, the supernatant formed is directly used for colorimetric detection by visual analysis or for spectrophotometry.

Colorimetric Detection Procedure

The rapid visual colorimetric assay developed is based specifically on the chemical property of PPD to form dyes by coupling reactions. The underlying mechanism is the same as for coupling components in oxidizing hair dyes. These contain two sorts of dye precursors; firstly, developers, which are aromatic systems containing at least two electron-donating groups in para or ortho-position. Secondly, couplers, which are aromatic systems as well, that contain two electron-donating groups in meta-position. After being oxidized, e.g. with hydrogen peroxide, to the corresponding quinoid systems, the developers can react with the couplers by an addition reaction and reconstitution of the aromatic system. Further coupling and oxidation reactions induce a dark-brown dye. PPD belongs to the group of developers. A very simple and prominent representative for a coupler is resorcinol (meta-dihydroxybenzene). The possible mechanism of the dye formation is shown in Figure 1.15

To conduct the colorimetric assay, the following solutions were added consecutively in a 10-mL screw cap vial and marked as the “reaction solution”: 1 mL resorcinol solution, 1 drop hydrochloric acid (1 M), 3 drops copper(II) sulfate solution and 5 drops hydrogen peroxide (30%). The resulting solution is colorless. Separately, another solution was prepared in a 10-mL screw cap vial and marked as the “blank solution.” For this, 1 drop of hydrochloric acid (1 M) was added to water until the vial had the same volume as the reaction solution. The blank solution serves as an optical reference point for the test.

After the two solutions were prepared, 1–5 drops of the methanolic henna extract were added into both solutions. The number of drops required depends upon the color intensity of the extract; if the extract is colored intensely black or brown, just 1–2 drops is sufficient. It should be noted that more drops would color the solutions so intensely that the formation of the dye would not be detected. However, five drops could be used if the extract were colored green. It is important to add an equal amount of drops in both solutions. If no dye formation occurs after 5 min, 1 drop of the PPD solution would be added to the reaction solution to ensure the test system works properly.


All spectrophotometric measurements were performed on a dual beam spectrometere equipped with an automatic cell changer and softwaref. The spectra were acquired in a range between 400 nm and 700 nm with a data interval of 1.0 nm. All measurements were made against a blank; i.e., all reagents besides PPD.

Results and Discussion

Method development and optimization: For this rapid assay, the optimal conditions were investigated to assure that an intense dye was formed in the presence of PPD within a reaction time of only 5 min. Further, if PPD was not present in a sample, no dye formation should occur. In order to analyze the henna tattoo mixtures, the test sample is subjected to methanolic extraction. The extract is added to the prepared reaction solution containing resorcinol and hydrogen peroxide. If a dark brown dye forms in the solution, this indicates the presence of PPD in the henna tattoo mixture.

The methanolic henna extract shows a variable color (green to brown) of its own even if it is PPD-free. During sample preparation the extract is diluted so that this effect is negligible. Nevertheless, it is important to have an optical reference point—i.e., a blank solution, which demonstrates the original color conditions before the dye forms. After the henna extract is added to both solutions, dye formation can easily be detected by comparing the color of the solutions.

During the method development, it was elucidated that hydrochloric acid must be added to both solutions because without the addition of hydrochloric acid to the reaction solution, a brown dye formed without any PPD present; this is likely due to the oxidation and further reaction of the resorcinol. An acidic pH milieu suppresses this mechanism without negatively influencing the reaction of PPD and resorcinol. Since the presence of hydrochloric acid influences the color of the henna extract, it also must be added to the blank solution so that both solutions show an equal color condition at the beginning of the colorimetric detection. Using this procedure, the authors did not observe any brown dye forming in the blank solution. This also confirms the selectivity of the assay—i.e., only certain aromatic compounds could potentially interfere; not common cosmetic ingredients such as aromatic amino acids.

During the initial method development steps, it was further determined that for the necessary oxidation of PPD, hydrogen peroxide alone was not sufficient to oxidize PPD, so to improve the kinetics of the oxidation reaction, copper(II) sulfate was added, which significantly reduced the oxidation process time. Under these conditions, the authors’ aim to facilitate the reaction at room temperature in only 5 min was achieved and the procedure was used for all further steps. Using spectrophotometric measurements (see Figure 2), the authors finally proved there is a clear linear dependency between PPD concentration and absorption (R2 = 0.99). Photometry may also be applied to obtain a semi-quantitative estimation of the concentration against an external calibration curve; this approach warrants further investigation as the primary focus was to provide a rapid screening assay while for quantitative measurements, more specific methods such as HPLC-based methods are already available.

Sensitivity of the visual assay: The detection level of the assay was investigated by preparing two different commercial PPD-free henna powders spiked with 0.05%, 0.1%, 0.5% and 1% w/w PPD. Then, 1 g of henna sample was extracted with 10 mL methanol; 5 drops of this extract was used for the dye forming reaction and the reaction time was 5 min. The henna powder spiked with 0.05% PPD did not show a brown dye formation—i.e., the color of the reaction solution color was not significantly different from the blank solution. However, the sample spiked with 0.1% PPD showed a visibly detectable brown dye formation—i.e., the color of the reaction solution color was significantly different from the blank solution.

As far as the samples with 0.5% and 1% PPD were concerned, the brown dye formed was intensive and could easily be detected. For the evaluation of the detection limit, the subjective visual detection of the brown dye formation in the reaction solution in comparison with the blank solution was assessed. Figure 3 shows the reaction solution marked with “+” and the blank solution marked with “-” of a henna powder spiked with 0.1% w/w PPD after 5 min reaction time. According to the unanimous judgment by the authors’ research team, the detection limit was determined to be 0.1% w/w PPD.


The main advantage of the described assay is its ability to be quickly and easily performed as a screening analysis in the lab as well as during on-site controls. Within less than 15 min, including sample preparation and reaction time, a result is obtained and positive samples can be subjected to further analyses such as HPLC-based analysis, if necessary. During on-site controls, suspicious samples can be directly confiscated by the competent authorities. For a rapid on-site assay, the authors suggest for the extraction procedure that instead of weighing 1 g of sample and pipetting 10 mL of methanol, a vial with two markings be used—the first marking indicating the approximate volume of a 1 g sample and the second marking indicating the approximate amount of 10 mL of methanol.

The determined detection level of 0.1% w/w PPD in henna tattoo mixtures is suitable to detect even very low concentrations of PPD. In real black henna samples, the levels of PPD are much higher than 0.1%;12, 13 moreover, it would also make no sense for an adulterator to use such low levels as they would not have the desired effect—i.e., of improving the henna tattoo coloring significantly. Therefore, the sensitivity of this assay is judged to be sufficient for control purposes.

For this method development, it should be noted that the authors did not succeed in purchasing a real black henna tattoo sample; all black henna tattoo samples purchased over the Internet turned out to be natural henna. However, two authentic black henna hair dyes were available for investigation and the assay worked as desired for these authentic samples as both were PPD-positive. This result was also confirmed by HPLC analysis.

As a limitation of the assay, its low specificity must be pointed out, which is inherent to these types of simple visual colorimetric tests. In this case, other developers such as para-toluenediamine (PTD), which are able to react with couplers such as resorcinol, are also detected. As an example, the authors tested a red henna hair dye containing the prohibited developer 2-nitro-para-phenylenediamine, as found through an HPLC analysis. After having added two drops of the red henna extract into the reaction solution, a brown dye formed that was similar to the PPD reaction, leading to a false-positive result. However, in the authors’ opinion, this is not a significant problem for the applicability of the method. On one hand, there is no evidence that developers other than PPD are added to henna tattoo mixtures.

On the other hand, such other developers are certainly as undesirable as PPD, as they also may lead to adverse health outcomes16—especially if used without labeling. Thus, the authors conclude that it is neither possible nor necessary to increase the specificity of the assay. More specific approaches such as HPLC-based methods or thin layer chromatography are available, but are clearly not suitable for rapid screening or particularly on-site controls. For product developers and formulators in the lab, the assay could be used for raw material checks or importation controls of henna plant extracts.

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