What do we know about the positional isomers of 2C-B? Study through real hair cases: The role of high-resolution mass spectrometry (HRMS)

Abstract

The objectives, besides the identification of 4-Bromo-2,5-Dimethoxyphenylethylamine (2C-B) in at least 8 cases of hair, is to evaluate the additional presence of positional isomers obtained by chemical positional derivations, such as 2-Br-4,5-dimethoxyphenylethylamines (2-Br-DMPEA). A target analysis for hallucinogenic substances confirmation in hair samples by high resolution mass spectrometry (Q-Orbitrap) was performed in the National Institute of Toxicology and Forensic Sciences (INTCF). The method consists in an analysis by LC-HRMS/MS (Q-Orbitrap). It is based on the acquisition of two independent signals: MS-1 and MS-2, as follow, the first one obtained in MS-1 in a full scan mode (FS) analysis, and the second signal was obtained through a MS-2 mode in Parallel Reaction Monitoring (PRM). Only one injection was accomplished. The parameters included in method validation for 2C-B, were: Selectivity ( n = 10 in ten different hair blanks), matrix effect (ME%) n = 10 for each concentration, 20 pg/mg (ME = 85% RSD% = 15.9%, and 200 pg/mg (ME = 73.6%, RSD% = 11%). Limit of detection (LOD = 2 pg/mg) and limit of quantification (LOQ = 5 pg/mg), and carryover. Five calibration curves were performed, from 5 to 1000 pg/mg range and a lineal mode excluded origin and 1/x factor were applied. Precision (RSD%) and accuracy (E%) were assessed for low, medium and high concentrations, in inter-day conditions ( n = 15). The values of accuracy and precision were noteworthy according to the acceptance criteria (<15%). Quantitative results obtained by MS-1 mode for 2C-B in real hair analysis were: from 5.4 to 452.4 pg/mg. In five of eight cases only 2C-B was detected. Surprisingly in the other three cases others additional peaks were observed for this precursor. In the scientific literature, at least five positional isomers of 2C-B have been described: 6-Br-2,3-dimethoxyphenylethylamine (DMPEA), 2-Br 3,5-DMPEA, 2-Br 4,5-DMPEA, 5-Br 2,4-DMPEA and 3-Br 2,6-DMPEA. Unfortunately, it is very difficult to obtain these substances as material references certificated (MRC) for being systematically included in our laboratory. At the present moment only 2-Br 4,5-DMPEA was available in the laboratory coming from proficiency test material in matrix of blood. For this reason, only in one of two different positional isomers of 2C-B was detected and it has been able to be compared through the retention time of 2-Br 4,5-DMPEA MRC. Interestingly, the same retention time in real hair case and MRC of 2-Br 4,5-DMPEA were obtained they were coincident. Its retention time was very different in comparison to the retention time of 2C-B eluting-0.7 min in advance. Regarding the characteristics of cluster isotopic of the molecule of 2C-B [C10H14BrNO2], the atom of bromine and its isotopes abundance [Br 79 ] and [Br 81 ] is a very specific isotopic pattern. This specific information and fragmentation in source, of primary amine of 2C-B observed in optimization of voltage of s-lens were used for detecting the neutral loss of amine (NH 3 = 17.0265), in both precursor isomers for Bromine. The identification of 2Br 4,5-DMPEA was compared with MRC from a proficiency test performed last year. At this moment, there is scarce literature reporting 2C-B consumption through hair analysis. As a consequence the present work shows the highest number of positives in hair samples to the date. The presence of positional isomers of 2C-B that do not coelute (different retention time) with 2C-B can be evaluated in hair analysis of real cases. Interestingly, the hair is an accumulative matrix for a long period and several consumptions can be studied in one aliquot analysis.