Phenethylamines are a broad family of chemical variants of the core compounds, i.e., amphetamine, methamphetamine and MDMA [41]. 2 C-x is a general name of substituted phenethylamines which contain methoxy groups on the 2 and 5 positions of the benzene ring of phenethylamine and a variety of lipophilic substituents at position 4. The substances of this category show a prevalent hallucinogenic and psychedelic effect. The N-benzyl substituted phenethylamines (NBOMEs) are highly potent hallucinogens even in doses of micrograms, as the N-benzyl substitution of phenethylamines dramatically increases their affinity with the serotonin 2A (5-HT_2A) receptors [42].

Piperazine-like compounds fall within the category of NPS and have already been found in the illegal drug market as substitute or constituent of MDMA in suspected MDMA seized pills. Piperazine-like compounds may act as stimulants and can produce euphoria, whereas some compounds of this class (e.g. TFMPP and mCPP) have small hallucinogenic potential [33].

Six studies described methods for the detection and quantification of piperazines in hair. In their previously mentioned multi-analyte LC–ESI-MS/MS assay after SPE, Imbert et al. included the determination of mCPP [25]. Using their UHPLC–MS/MS method after extraction in 0.1% formic acid Strano-Rossi et al. determined mCPP and benzylpiperazine [24]. Piperazines were not detected in none of the latter studies, when applied to real cases.

Another research group developed and validated a sensitive and simple gas chromatography-mass spectrometry (GC-MS) method for the identification and quantification of mCPP, TFMPP and MeOPP in hair [33]. A 20 mg decontaminated and cut hair sample was cleaned up by mixed mode solid-phase extraction after short hair incubation (40 min) with 1M NaOH at 50^oC. Extracts were derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide with 5% trimethylchlorosilane and p-tolylpiperazine (pTP) was used as IS. A gas chromatograph equipped with a mass selective detector and capillary column (30m×0.25mm I.D., 0.25μm film thickness) with 5% phenylmethylsiloxane was used for the analysis.

The method was applied to both post- and ante-mortem (subjects under antidepressant treatment with trazodone for the detection of mCPP and individuals undergoing psychiatric evaluation) real samples. Although results from the authentic hair samples were not presented, authors stated that the method could find application in forensic toxicology routine analysis.

Lendoiro et al. determined mCPP and TFMPP using the previously reported LC-MS/MS method which employed SPE for analytes extraction [26]. When the method was applied to 16 authentic hair specimens, ten positive results for mCPP were obtained in the range of 341.7-4000 pg/mg; however, in all cases trazodone identification (2085.3–4000 pg/mg) indicated a licit origin of mCPP as a metabolite of trazodone.

In their UHPLC–MS/MS multi-analyte assay Salomone et al. included hair testing of mCPP [27] and Rust and co-workers determined mCPP, benzylpiperazine and TFMPP using their method which employs 2 step extraction and LC-MS/MS analysis [28]. After the application of the latter method to 325 real cases, mCPP was found positive in 34 cases and TFMPP in one case. Five mCPP cases were also positive for trazodone.

Synthetic cathinones are chemical analogs of cathinone, a psychoactive monoamine alkaloid present in the shrub of Khat plant (Catha edulis). Cathinone derivatives are the β-keto (βk) analogues of the corresponding phenethylamine. Psychoactive stimulant substances belonging to the class of synthetic cathinones have been linked to varying degrees of agitation, confusional states, hyper arousal, and various types of psychotic behavior, up to and including a syndrome indistinguishable from excited delirium syndrome [43]. Moreover, synthetic cathinones can produce neurological and cardiovascular side-effects consistent with sympathomimetic toxicity [44].

Nine studies investigated the presence of synthetic cathinones in hair of possible consumers.

Namera et al. [35] were the first in detecting MDPV in hair sample coming from a deceased subject, whose cardiac blood contained 1200 ng/ml MDPV and 200 ng/ml α-PBP. Although the death was attributed to MDPV poisoning, hair test was performed to investigate chronic use. Hair samples were extensively washed with both aqueous and organic solutions and cut into 10 mm segments up to ten cm. Each segment was then treated with alkaline solution, in the presence of IS, and then extracted, evaporated and re-dissolved in methanol aqueous solution, before injected into the LC-MS system. Separation was achieved using a phenyl-hexyl column (150 mm x 2.1 mm i.d.,3 μm) with a mobile phase of methanol/ammonium formate solution (10 mM ammonia aqueous solution containing 0.1% formic acid, pH 3.3) (35:65, v/v).

Segments analysed were positive for MDPV and α-PVP, with the highest MDPV concentration being 22 ng/10-mm hair, confirming previous intake of MDPV and α-PVP. Traces of α-PBP were also detected in the first segment (the one closet to the scalp). However, the concentrations provided per 10 mm and not per mg of hair did not allow any comparison with results from other studies. The authors reported that their findings suggest chronic MDPV and α-PVP use for at least 5 months, since both drugs were detected in the first 5 segments of 10 mm each.

In their multi-analyte method, Salomone et al. [27] determined α-PVP, buphedrone, amfepramone, 4-MEC, ethcathinone, mephedrone, methylone, pentedrone and MDPV after an extraction in methanol and determination in UHPLC–MS/MS. When they applied the method to 23 cases coming from MDMA and ketamine users, 3 were found positive to the latter drugs. In detail, in one case 4-MEC and mephedrone were detected at the concentration of 330 and 50 pg/mg, respectively; in another case, mephedrone was found at 59 pg/mg. In the last case of this group, α-PVP was detected together with methylone, MDPV and other NPS at the concentration of 1040 pg/mg, <LOQ and 120 pg/mg, respectively. In one out of 54 authentic samples from driver’s license regranting which had been found negative, when tested for common drugs of abuse, methylone was quantified in a sample at 28 pg/mg.

In their above mentioned UHPLC–MS/MS method for NPS hair testing, Strano-Rossi et al. [24] included 3,4-DMMC, 4-MEC, ethcathinone, ethylone, mephedrone, methedrone, methylone, naphyrone, pentedrone, pentylone, 4-FMC and MDPV after hair treatment with formic acid 0.1%.

Three samples out of 50, coming from forensic cases, were positive for cathinones: MDPV at a concentration of 50 pg/mg in one case, 4-MEC at a concentration below LOQ in another and at 26 pg/mg in the last one.

In their LC-MS/MS method for the evaluation of the prevalence of NPS by hair testing, Rust et al. included the investigation of ethylone, mephedrone, methcathinone, methylone and MDPV in hair, after two step sample extraction with methanol and acidic methanol [28]. Methylone was detected in one out of 325 samples tested and mephedrone in 3% of the cases.

Pubic hair drug testing for the detection of 3-MMC and its metabolites, by means of liquid chromatography–high resolution/ high accuracy Orbitrap mass spectrometry (LC-HRMS), was used to document their potential intake by a drug dealer [7]. Pubic hair samples were decontaminated, pulverized using a ball mill, extracted in the presence of IS by overnight incubation with methanol:trifluoroacetic acid 9:1 and analysed.

The LC-HRMS system was equipped with a TFS Hypersil Gold PFP analytical column (2.1 x 100 mm, 1.9 μm particle size) and coupled to a TFS single-stage Exactive high energy collisional dissociation (HCD) MS system, interfaced with a TFS heated electrospray ionization HESI-II Ion Max source. MP A was water with 0.05% formic acid and 10 mM ammonium formate and MP B was acetonitrile with 0.05% formic acid.

Pubic hair specimen was positive for 3-methylmeth-cathinone (3-MMC) which was quantified at 25.8 ng/mg. 3-methylephedrine and 3-methylnorephedrine, metabolites of 3-MMC, were also detected in the same sample, suggesting 3-MMC intake by the drug dealer.

In their above reported multi-analyte LC-MS/MS assay, Lendoiro et al. [26] included hair testing of mephedrone, methedrone, methylone, 4-FMC and MDPV, after SPE sample treatment. However, none of those substances was detected in real cases tested.

After developing and validating a method for the determination of mephedrone in hair by GC-MS, Martin et al. reported the first series of mephedrone positive findings in hair samples [45]. Hair samples were decontaminated and incubated overnight in Soerensen buffer pH 7.0 in the presence of IS. Liquid-liquid extraction (LLE) was performed after alkalinisation of the incubation medium. The dry extract was derivatized, evaporated and reconstituted in ethyl acetate. Gas chromatographic separation was achieved with a 5-MS capillary column and detection by single ion monitoring in electron impact mode.

Thirteen out of 67 hair tested samples, were found positive for mephedrone with concentrations ranging from 0.2 to 313.2 ng/mg with a mean concentration of 26.8 ng/mg, showing enough method sensitivity to demonstrate both occasional and regular mephedrone intake.

Shah et al. were the first authors in developing and validating an UHPLC-MS/MS method for the quantification of not only mephedrone but also its two metabolites, 4-methylnorephedrine and 4-methylephedrine in hair [30]. Hair samples were enzymatically digested and extracted using LLE technique. The UHPLC-MS/MS system was operated in selective reaction monitoring (SRM) mode. The method was then applied to 154 real samples, 5 of which were confirmed as positive just for mephedrone (only one quantified at the concentration of 21.11 pg/mg).

Pichini et al. presented for the first time a case of a neonatal withdrawal syndrome in a baby born to a woman who was a chronic consumer of 4-methylethcathinone [19]. 4-MEC was identified and quantified by LC-MS/MS in neonatal meconium and in 4 subsequent 3 cm segments of maternal hair accounting for maternal consumption during the whole pregnancy (4.3, 4.0, 4.0 and 3.9 ng/mg hair starting from most proximal segment) demonstrating that maternal hair testing could provide useful information on chronic gestational use of 4-MEC in relation to neonatal outcome.

Synthetic cannabinoids are among the most commonly used NPS, which all act as agonists at the cannabinoid CB1 receptor. They are usually purchased as cannabis-like drugs, marketed as herbal blends and perceived as risk-free by inexperienced users. Synthetic cannabinoids may lead to severe health consequences, including tachycardia, anxiety, violent behavior, hallucinations and psychosis [46, 47].

Salomone et al. in their multi-analyte method included also MXE, 4-MeO-PCP and diphenidine after an extraction in methanol and determination by UHPLC–MS/MS [27]. Analyzing hair samples from MDMA or ketamine users, MXE was detected in three samples with a range of concentration between 7.7 and 28 pg/mg and diphenidine in one sample at the concentration of 4400 pg/mg.