Determination of PTCA concentrations in hair by UHPLC-MS/MS and fluorescence techniques: a study on a wide multi-ethnic population with forensic applications

Aim Nowadays forensic hair analysis plays an important role in assessing abstinence from drugs or ethanol abuse. Over the years, several studies have shown that many factors are likely to affect the content of xenobiotics in hair and thus complicate the interpretation of analytical results. These factors include melanin content, physicochemical and structural properties of drugs, external contamination, but also cosmetic hair treatment. Indeed, one of the major drawbacks in hair analysis is the affectability of deposited xenobiotics by cosmetic treatments which could be eventually used to adulterate the sample. It is well known that some cosmetic treatments containing hydrogen peroxide (H₂O₂), such as permanent coloring or bleaching, lead to the formation of 1-H‐pyrrole‐2,3,5‐tricarboxylic acid (PTCA), a melanin degradation metabolite. Considering that PTCA is an endogenous compound, spontaneously formed by natural oxidation of melanin, its only detection in hair is not enough to confirm a cosmetic treatment. For this reason, a study on PTCA endogenous level in a wide multi-ethnic population was performed. Methods In the present study, an ultra-high-performance liquid chromatographic–tandem mass spectrometry (UHPLC–MS/MS) procedure for the assay of PTCA was developed and validated in order to study its level in different hair samples and treatment conditions. First, PTCA was determined in natural (not treated) hair samples (n = 166) belonging to different ethnic groups: Caucasians, Indians, Blacks, Half-castes, Arabians and Asians. After that, a typical cosmetic treatment (Wella, Darmstadt, Germany) containing 12% H₂O₂ was applied to different natural hair samples for 30 minutes, according to official recommendations. Finally, since it is demonstrated that melanin oxidation leads to an increase in fluorescence emission(1), natural and treated hair samples from the previously mentioned 6 ethnic groups were inspected by fluorescence microscopy. Results PTCA baseline level were detected as follows: Caucasians (2.26 ± 1.01 ng/mg, mean ± SD, n = 26); Indians (6.70 ± 3.00 ng/mg, n = 25); Blacks (6.85 ± 3.39 ng/mg, n = 24); Half-castes (3.87 ± 1.63 ng/mg, n = 29); Arabians (4.79 ± 2.81 ng/mg, n = 31); Asians (4.69 ± 2.61 ng/mg, n = 31). After 30 minutes incubation of hair samples in the dark with hair bleaching products containing 12% H₂O₂, the PTCA content increased by 500%. Cosmetic treatment did not provide any changing in PTCA content in light-blond as well as white hair. Discussion Lower PTCA baseline level was found in Caucasians, while higher one in Indians and Blacks. Moreover, the levels detected in Caucasians were found to be significantly different compared to each other considered ethnic groups. All treated hair samples have shown a comparable percentage increase in PTCA concentration, except for naturally low melanin content sample such as light‐blond or white hair. The increase of the PTCA content in comparison to the untreated hair was statistically significant (p < 0.001). The changing in PTCA concentration, previously determined by UHPLC-MS/MS, were found to be directly correlated with the observed autofluorescence intensity. Conclusion In order to obtain a clear evidence of an applied cosmetic hair treatment, we aim to analyze in the next future a greater number of non-treated hair samples to establish a PTCA cut-off level as diagnostic marker in forensic field.