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Article

Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry and High-Sensitivity Gas Chromatography-Mass Spectrometry Screening of Classic Drugs and New Psychoactive Substances and Metabolites in Urine of Consumers

1
National Centre on Addiction and Doping, Istituto Superiore di Sanità, V.Le Regina Elena 299, 00161 Rome, Italy
2
Drug Addiction Program, Institut de Neuropsiquiatria i Addicions, Institut Hopsital del Mar d’Investigacions Mèdiques (INAD-IMIM), Parc de Salut Mar, 08003 Barcelona, Spain
3
Department of Psychiatry and Pharmacology, Therapeutics and Toxicology, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, Spain
4
Clinical Pharmacology Unit, Hospital Universitari Germans Trias i Pujol and Institut de Recerca GermansTrias i Pujol (HUGTiP-IGTP), 08916 Badalona, Spain
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(8), 4000; https://doi.org/10.3390/ijms22084000
Received: 1 April 2021 / Revised: 12 April 2021 / Accepted: 12 April 2021 / Published: 13 April 2021
(This article belongs to the Special Issue Molecular Insights of New Psychoactive Substances (NPS))

Abstract

:
The use of the new psychoactive substances is continuously growing and the implementation of accurate and sensible analysis in biological matrices of users is relevant and fundamental for clinical and forensic purposes. Two different analytical technologies, high-sensitivity gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) were used for a screening analysis of classic drugs and new psychoactive substances and their metabolites in urine of formed heroin addicts under methadone maintenance therapy. Sample preparation involved a liquid-liquid extraction. The UHPLC-HRMS method included Accucore™ phenyl Hexyl (100 × 2.1 mm, 2.6 μm, Thermo, USA) column with a gradient mobile phase consisting of mobile phase A (ammonium formate 2 mM in water, 0.1% formic acid) and mobile phase B (ammonium formate 2 mM in methanol/acetonitrile 50:50 (v/v), 0.1% formic acid) and a full-scan data-dependent MS2 (ddMS2) mode for substances identification (mass range 100–1000 m/z). The GC-MS method employed an ultra-Inert Intuvo GC column (HP-5MS UI, 30 m, 250 µm i.d, film thickness 0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40–550 m/z). Urine samples from 296 patients with a history of opioid use disorder were examined. Around 80 different psychoactive substances and/or metabolites were identified, being methadone and metabolites the most prevalent ones. The possibility to screen for a huge number of psychotropic substances can be useful in suspected drug related fatalities or acute intoxication/exposure occurring in emergency departments and drug addiction services.

1. Introduction

A new psychoactive substance (NPS) is defined as “a new narcotic or psychotropic drug, in pure form or in preparation, that is not controlled by the United Nations drug conventions, but which may pose a public health threat comparable to that posed by substances listed in these conventions” [1].
In Europe, seizures of NPS mainly concern synthetic cannabinoids which together with synthetic cathinones account for more than 70% of NPS seizures [2]. Nevertheless, the more recent and most toxic NPS showed to be the novel synthetic opioids (NSOs). Since 2009, 57 new NSOs have been detected on Europe’s drug market [2]. Several NSOs were originally synthesized by pharmaceutical companies in their research for analgesic drugs as compounds with a similar chemical structure to natural opiates without addictive properties, but their toxicity or abuse potential posed a very high risk of poisoning to consumers. Whereas some of them were then marketed as prescription drugs, some others were eliminated from the licit market and some others were chemically modified to exclusively enter illicit market [3,4,5].
The chemical variety of NSOs, ranging from several illicit analogs of fentanyl and derivatives to newly synthesized molecules, make their identification extremely difficult and need the investigation of qualified analysts/toxicologists [6].
Since NSOs and particularly fentanyl-related compounds are active in very low doses, due to their potency and many users are unknowingly consuming these as adulterants in products sold as heroin, or as pain killers [7,8], parent drugs and metabolites are present in biological material at extremely low concentrations. One consequence of this is that they may escape detection because routine testing of these drugs is rarely performed and requires dedicated analytical methods with sufficiently high sensitivity and specificity [9].
The 2020 COVID-19 pandemic has transformed daily life and the different intensity of the lockdown across countries showed important consequences on drug users. The legal restrictions modified their ability to access classic illicit drugs (e.g., heroin, cocaine, cannabinoids) and shifted consumptions towards prescription psychoactive drugs, frequently available at home or from the use of psychoactive recreational NPS (e.g., synthetic cathinones, synthetic cannabinoids, phenethylamines to narcotic analgesics such as NSOs or to anxiolytics such as new benzodiazepines [10,11]. Nine new uncontrolled NSOs have been reported during 2020 [12]and the global shortage of heroin due to pandemic may have forced regular users to take other substances with similar effects, such as fentanyl analogs and NSOs [13].
In 2018 the JUSTSO project (analysis, dissemination of knowledge, implementation of Justice and special tests of new synthetic opioids), funded by European Commission, intended to evaluate, test profile and feedback into education and prevention, knowledge related to the NSO currently used in Europe, their nature, effects and associated harm [14].
Our main involvement in the project was to develop and validate analytical methodologies for the screening analysis of NSO and their metabolites, together with all other possible psychoactive drugs in urine samples of drug users collected in different settings (detoxification units, methadone maintenance clinics, drug addiction services, etc.).
Targeted/untargeted screening workflows based on gas or liquid chromatography coupled with mass spectrometry or tandem mass spectrometry (GC-MS, LC-MS and LC-MS/MS, respectively) play a central role in the daily activities of analytical laboratories operating in clinical and forensic toxicology. Specifically, urinalysis with multiple analytical technologies can increase the number of licit and illicit drugs band metabolites with different physicochemical properties that can be determined [15,16,17,18,19,20,21,22,23]. New pharmacologically active substances, both licit and illicit, are constantly being introduced and this occurrence has increased demand for new MS solutions that go beyond conventional GC-MS and LC-MS/MS. High-resolution mass spectrometry (HRMS) enables determination of the exact molecular formula (<5 ppm mass error) that can be useful for presumptive assignment of unknowns in general toxicology screenings [18].
Few previous studies performed in this field used one or more than one analytical tool for identification of a high number of unreported psychotropic substances in biological matrices of users.
Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methodology has been applied not only to detect, but also to quantify 87 NPS and 32 classic illicit drugs and their metabolites in hair and nails [16] and 77 among the most abused NPS in blood, urine and oral fluid [17]. These two assays used only one type of instrument, but required the availability of all the pure standards of analytes under investigation for their quantification. Others screening methods coupled LC or GC with detection methods as time-of-flight mass spectrometry for analytical determination of NPS in seized samples [19] or in serum of consumers [20]. Moreover, to solve a complex toxicological fatal case due to NPS, several different analytical methodologies, including 1H nuclear magnetic resonance (NMR), GC–MS and UPLC–MS/MS to examine unambiguously seized material and biological fluids [21].
Finally, a combination of last generation GC-MS and UHPLC-HRMS has been recently employed by our investigation group to determine a selection of synthetic cannabinoids in oral fluid of consumers. Specifically, GC-MS has proven useful to identify and quantify parent compounds whereas UHPLC-HRMS also confirmed the presence of their metabolites in oral fluid [22].
Using the same combination of analytical methodologies, we hereby propose a screening method for urinalysis of principal NSOs, classical drugs of abuse and other NPS with main metabolites using a fast sample extraction.

2. Results

2.1. GC-MS and UHPLC-HRMS Methods

A simple and selective screening analysis with simultaneous use of high-sensitivity GC-MS and UHPLC-HRMS was applied for the identification of classic drugs of abuse, new psychoactive substances and metabolites in urine of drug addicts. The extraction procedure was tested with above reported fortified urine samples using different solvents. The mixture of chloroform and isopropanol has been found as the best compromise for the extraction of drugs and with acceptable signal-to noise ratio in an analytical screening, optimizing the extraction times and costs. Furthermore, even if the total analysis time was not short (each chromatographic run was completed in 32 min in GC/MS and 15 min for UHPLC-HRMS) the combined use of two instruments allowed to screen with a high percentage of compounds matched several different substances.
The characteristic retention times and monitored m/z ions used for the identification of mostly found substances monitored in urine samples are reported in Table 1.
The results obtained by screening proficiency urine testing samples from UNODC International Quality Assurance Program and those from in “NPS-LABVEQ” project showed an excellent agreement (98% agreement as screened substances) between substances declared and those found in the samples. Since these latter substances were analyzed at a concentration of 1 ng/mL urine with a signal to noise ratio, calculated at the baseline, always higher than 10, we could assume that our methodologies could screen substances present in concentrations equal or above 1 ng/mL. Moreover, from the analysis of blank urine no additional peaks due to endogenous substances, which could have interfered with the detection, were observed.

2.2. Methods Application

Drug screening applied to 296 former heroin addicts under methadone maintenance therapy urine disclosed the presence of different psychoactive prescription drugs, classical drugs of abuse, NSO, NPS and their metabolites. The presence of a certain drug and/or metabolites was confirmed only if both methodologies identified the molecules, which occurred in 95% cases.
Pharmaceuticals like benzodiazepines, antidepressants, antipsychotics, anticonvulsants and opioids were detected. Drugs of abuse (opioids, amphetamines, cocaine and cannabinoids), NPS (synthetic cannabinoids, synthetic cathinones), fentanyls, NSO and other drug classes were also found. The frequency of different drug classes found in urine samples using the developed GC-MS and LC–HRMS screening methods is reported in Figure 1.
The most frequent found substances (about 90%) were methadone and its metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP). Urine samples resulted positive also to benzodiazepines (mainly Clonazepam, Diazepam and their metabolites), antipsychotics (principally Risperidone, Quetiapine and their metabolites), antidepressants (Citalopram, Mirtazapine and their metabolites, Trazadone and its psychoactive metabolite meta-Chlorophenylpiperazine) and Gabapentin. Additional findings included samples positives for cocaine and its metabolites BZE and EME, cannabinoids, amphetamine and synthetic cathinone methylenedioxypyrovalerone and synthetic cannabinoids from JWH family.
In urine samples in which methadone was not found, screening analysis revealed the presence of the opiates (buprenorphine, 6-MAM, morphine, codeine, dextromethorphan), cocaine, cannabinoids and fentanyl and analogs.

2.3. Fentanyl, Fentanyl Analogs and Novel Synthetic Opioids

Toxicological screening analysis revealed the presence of fentanyl and analogs and/or metabolites in 23 (7.8%) out of 296 screened urine samples. No other NSOs were found.
In 4 out of 23 samples, the substances matched while in other cases, parent drug was identified by one method and metabolite by the other, or similar compounds were determined.
Chromatogram sin GC-MS and UHPLC-HRMS of 2 positive fentanyl samples are shown in Figure 2 and Figure 3, respectively, and screening results on fentanyl positive samples were reported in Table 2.

2.4. Other NPS

The NPS, other than NSOs, detected in the 296 analyzed samples by both methodologies belonged to the class of synthetic cathinones (4.4%) and to that of synthetic cannabinoids (1.3%) (Table 3).
The analysis by UHPLC-HRMS method of real sample obtained from the subject that results positive to UR-144 showed two peaks with different retention time but similar mass spectrum (Figure 4).

3. Discussion

Methadone is frequently prescribed for the maintenance therapy of opioid addiction detoxification. Patients needing treatment with this and other medications often have co-occurring medical and mental illnesses that require medication treatment [24].
Untargeted mass spectrometry techniques have become essential tools for toxicological analysis [25].
The poor availability of reference standards for many NPS and metabolites presents a large challenge to forensic toxicology laboratories when trying to detect and identify both known and unknown NPS and other xenobiotics. What toxicologists expect both in clinical and forensic analysis from a general unknown screening procedure is the unequivocal identification of the xenobiotics involved in intoxication cases, even when they have no evidences to guide the search.
In general, the combination of different complementary methods (immunoassays, liquid chromatography and gas chromatography) was shown to be a good approach for screening samples in forensic and clinical toxicology [26]. Currently, the most competent approach for compound identification involves mass spectral library search [27].
We here presented two complementary analytical methods for screening of classic drugs of abuse and new psychoactive substances and metabolites in urine samples. Low resolution GC-MS and high-resolution instruments (UHPLC-HRMS) can both be used to develop efficient screening workflows. It was possible to obtain an identification, based on the obtained mass spectrometry information, of different xenobiotics.
The main purpose of this initial screening technique has been to identify samples positive to classical drugs of abuse, NPS and NSOs while simultaneously eliminating negative specimens from any subsequent analytical examination. Once a NPS or a NSO are detected, quantification could be further performed to provide information regarding concentrations found in urine of users and in cases of fatal and non-fatal intoxications.
The principal limitation of the presented methodology was the difficulty associated with data processing to get the information from single sample analysis that required qualified expertise. Moreover, in some cases it can be extremely difficult to chromatographically separate certain NPS to facilitate identification via mass spectrometry, such as in the case of isomers and isobaric compounds which display the same or significantly related chemical formulae [22,28,29,30,31,32].
In the current method and in agreement with a previous study [29] the isomers JWH-019 and JWH-122 as well the two metabolites of JWH-122 (JWH 122 N-4-Hydroxypentyl and N-5-Hydroxypentyl) and JWH-210 (JWH 210 N-4-Hydroxypentyl and N-5-Hydroxypentyl) were not distinguishable, since their masses and retention times matched. Otherwise, the isomers UR-144 and UR-144-pyr could be distinguished (Figure 4). Moreover, the opiate family contains a number of isobaric couples that can complicate the correct identification of e.g., morphine versus hydromorphone, or codeine versus hydrocodone. Other potential isobaric/isomeric interfering compounds that we found in our run were amitriptyline versus EDDP, and Tramadol versus O-desmethylvenlafaxine. Nevertheless, in our developed methodology, the above reported substances exhibited different retention times.
Isomeric and isobaric substances require gas or liquid chromatographic conditions that enable adequate separation of the compounds prior to MS analysis or include other mass spectrometry data such as m/z, isotope pattern, retention time and fragmentation information [22,30,31,32].
However, even if the total analysis time was not short, this method could screen several psychoactive substances of different chemical structures in epidemiological studies aimed to disclose the use of compounds with a high risk of toxicity, leading to severe acute intoxications and overdoses. Moreover, High resolution full scan data also provides retrospective analysis for identifying previously unknown drugs of abuse [31].
Indeed, for this particular study, no reference standards were used, but only mass spectrometric libraries and the coupling of both methodologies. As above reported, positivity to a certain substance was only provided when both methodologies, independently run by different operators, matched with the identification of a specific molecule.
In agreement with previous studies [15,19,21], the HRMS procedure was shown to be superior to screening by GC-MS, the costs still limit the widespread distribution in routine laboratories.
On the other hand, a last generation GC-MS assay highlighted the similar specificity of UHPLC-HRMS and therefore the simultaneous use of the two instruments allowed to demonstrate that a simple and traditional methodology can be used to screen unknown samples this also due to the presence of the latest generation of libraries present in support to toxicologist whose experience allows to identify unknown substances or to exclude false positives.
In this concern, analytical methodologies used for the identification of NPS continuously emerging in illicit markets should be developed, validated, updated and analytical data should always be shared across different communication platforms to help health professionals involved in clinical and forensic toxicology issue [6,33].
In addition, once substances identification has been accomplished, it can be of interest to confirm and quantify identified substances to expand information on concentration found in biological fluids of consumers and eventually associate obtained data with clinical evidence. In this concern, pure standards of parent compounds and/or metabolites are needed an extensive method validation whatever is the applied methodology (e.g., LC-MS/MS, GC-MS, GC-MS/MS or HRMS) considering the maximum cost-benefit ration for a high throughput laboratory facing with this kind of analyses.

4. Materials and Methods

4.1. Chemicals and Reagents

Water, methanol (MeOH) and acetonitrile (ACN) MS grade, chloroform, isopropanol and formic acid analytical grade were purchased by Carlo Erba (Milan, Italy). Ammonium formate, phosphate buffer and N,O-bis-trimethylsilyl-trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was obtained from Sigma–Aldrich (Milan, Italy).

4.2. Study Design

Urine samples collection took place at Consorcio Mar Parc De Salut De Barcelona, Spain and Hospital Universitari Germans Trias i Pujol from March, 2019 through October, 2020. Here, 296 patients with a history of opioid use disorder were enrolled in this study. All individuals were under methadone maintenance therapy (MTT). In this case, 109 patients provided identified urine samples after obtaining a signed informed consent, while 187 accepted to provide an anonymous sample, but no personal information was collected.
In order to secure the participants’ privacy, the survey data and collected urine were coded and the local Human Research Ethics Committee of both centers (ref. 2018/2138/I and PI-18-126) approved the study protocol. Prior to analysis aliquots of urine were stored at −20 °C.

4.3. Sample Preparation for Screening Analysis by GC-MS and UHPLC-HRMS

A liquid-liquid extraction was performed after diluting 0.5 mL of urine in 1 mL 0.1 M phosphate buffer pH 3.0 and 0.5 mL of the same sample in 0.1 M phosphate buffer pH 10 (the desired pH was eventually adjusted using drops of 1 N HCl or 1N KOH, respectively). The samples were vortex mixed and then the solutions were extracted twice with 1.5 mL chloroform/isopropanol (9:1, v:v). After centrifugation, the organic layer from each buffered sample was divided into two 1.5 mL aliquots and evaporated to dryness at 40 °C under a nitrogen stream.
The first dry aliquot was derivatized with a mixture of 25 μL of acetonitrile and 25 μL of N,O-bis-trimethylsilyl-trifluoroacetamide (BSTFA) with 1%trimethylchlorosilane (TMCS) at 70 °C for 30 min. The second dry aliquot was dissolved in 50 μL ethyl acetate. A 1 μL amount of underivatized and derivatized acid and alkaline extracts were injected into the GC-MS system.
After the analysis in GC-MS, the underivatized samples were evaporated to dryness under a nitrogen stream and then dissolved in 150 μL of a mixture of mobile phase A (Ammonium formate 2 mM, 0.1% HCOOH) and B (Ammonium formate 2 mM in MeOH/ACN 50/50, 0.1% HCOOH, 1% H2O) (50:50, v/v). 5 μL were injected into UHPLC-HRMS.

4.4. Gas Chromatography-Mass Spectrometry (GC-MS) Instrumentation

The GC-MS instrument consisted of an Agilent 7890 A gas chromatograph coupled with 5975 C mass spectrometry detector (Agilent Technologies, PaloAlto, CA, USA). Ultra-Inert GC column Zebron (ZB-Drug-1, 15m × 250 µm i.d, film thickness 0.25 µm; Phenomenex, Milan, Italy) was installed.
The GC-MS condition for the screening procedure was as follows: splitless injection mode; helium (purity 99%) carrier gas flow 1.2 mL/min; the injection port, ion source, quadrupole and transfer line temperatures were 260, 230, 150 and 320 °C, respectively; column temperature was programmed at 70 °C for 2 min and increased to 190 °C at 30 °C/min and then increased to 290 °C at 5 °C/min for 10 min. Subsequently the programed temperature was increased to 340 °C at 40 °C/min to eliminate impurities from the column.
The electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40–550 m/z).
The full scan data files were processed by an Agilent Workstation (Agilent Technologies). The mass spectra international libraries used for peaks identification were NIST Research Library (National Institute of Standards and Technology)

4.5. Ultra-High-Performance Liquid Chromatography-High-Resolution Accurate Masses Spectrometry (UHPLC-HRMS) Instrumentation

The UHPLC/ESI Q-Orbitrap system consisted of an Ultimate3000 LC pump and an Ultimate 3000 autosampler coupled with a QExactive Focus mass spectrometer equipped with a heated electrospray ionization (HESI) probe operating in positive ionization mode and the system was controlled by Trace finder 4.0 software (Thermo Fisher Scientific, Bremen, Germany).
Separation was performed on an Accucore™ phenyl Hexyl (100 × 2.1 mm, 2.6 μm, Thermo, USA). They were maintained at 40 °C. The flow rate was set at 500 μL/min. Elution was achieved as follow: 99% A for 1 min, linear gradient to 99% B in 10 min, held for 1.5 min. The column re-equilibration was performed with a linear gradient to 99% A in 0.01 min, held for 4.0 min. A heated electrospray ionization (HESI) source in positive/negative ion mode was used for the ionization of compounds.
The mass parameters were as follows: ionization voltage was 3.0 kV; sheath gas and auxiliary gas were 35 and 15 arbitrary units, respectively; S-lens RF level 60; vaporizer temperature and capillary temperature were setting both at 320 °C. Nitrogen was used for spray stabilization, for collision induced dissociation experiments in the HCD cell and as the damping gas in the C-trap. The instrument was calibrated in the positive and negative modes every week.
Data were acquired in full-scan in data-dependent MS2 (ddMS2) mode. In this mode, both positive and negative high-resolution, full-scan data at resolution of 70 k were collected with a scan range of 100–1000 m/z, then MS2 spectra at a resolution of 17.5 k with an isolation window of 2 m/z were triggered for compounds entered in the inclusion list and expected retention times of the target analytes, with a 1 min time window.
The MS and fragmentation data acquired in full scan is processed by Thermo Scientific TraceFinder™ software. This specific software performs a thorough interrogation of the database by making use of the built-in database and mass spectral library of over 1400 compounds, retention times, isotope pattern matching, elemental composition determinations to identify and confirm drugs and metabolites in the analyzed samples. Moreover, mzCloud Mass Spectral Library was used as mass spectra international library for unknown peak identification (Advanced Mass Spectral Database; www.mzcloud.org, accessed on 1 April 2021).

4.6. Analytical Performance

To check the robustness and the reliability of the developed analytical methods, 10 different proficiency urine testing samples from UNODC International Quality Assurance Program (some with no analytes, some with one and some with more substances), whose previous qualitative and quantitative GC–MS results were available, were re-analyzed using the present methods.
Moreover, we also tested 10 urine samples fortified with 1 ng/mL 40 different most popular NPS and main metabolites prepared within the framework of an Italian Project (“NPS-LABVEQ” project) founded by Italian antidrug policy department aimed to allow pharmacotoxicological laboratories along the Italian peninsula to identify these substances in biological and non-biological matrices with different NPS [34]. Finally, 20 blank urine samples from laboratory personnel were also tested to check for false positives during the different batches.

5. Conclusions

This study presents a comprehensive gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (UHPLC)–high-resolution mass spectrometry (HRMS) general screening procedure for classic drugs and new psychoactive substances in urine of consumers involving an easy, quick and low-cost sample preparation. This screening method based on two different chromatographic and mass spectrometry methodologies can be applied to disclose suspected drugs of abuse related fatalities or acute intoxications occurring in emergency departments and drug addiction services.

Author Contributions

“Conceptualization, S.P., M.F., M.T. and R.P.; methodology, E.M., M.P. and M.A.F.; validation, E.M., M.P. and M.A.F.; investigation, S.P., E.M. and M.P. writing—original draft preparation, E.M., M.P.; writing—review and editing, all the authors. All authors have read and agreed to the published version of the manuscript.

Funding

Project funded from the European Commission under the Call JUST-2017-AG-DRUG SUPPORTING INITIATIVES IN THE FIELD OF DRUGS POLICY) Topic: JUST-2017-AG-DRUG; Type of action: JUSTDRUGS-AG (GRANT AGREEMENT NUMBER: 806996-JUSTSO).Other funding from Instituto de Salud Carlos III (ISCIII, Fondo de Investigación en Salud (FIS)-Fondo Europeo de Desarrollo Regional (FEDER), Grant Numbers: FIS PI14/00715 and FIS PI17/01962, ISCIII-Red de Trastornos Adictivos RTA Grant Number: RD16/0017/0003 and RD16/0017/0010, AGAUR Gencat Suport Grups de Recerca, Grant Number: 2017 SGR 316 and 2017 SGR 530. The Presidency of the Ministers Council, Department of Antidrug Policies, in Italy.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Human Research Ethics Committees of Parc de Salut MAR (protocol code 2018/8138/I and date of approval 7 February 2019) and the Hospital Universitary Germans Trias i Pujol (protocol code PI-18-126 and date of approval 20 June 2018).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Acknowledgments

The authors thank Laura Martucci, Simonetta Di Carlo, Michele Sciotti and Antonella Bacosi for administrative and technical support.

Conflicts of Interest

The authors declare no conflict of interest

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Figure 1. Percentage plot of classic drugs and new psychoactive substances found in 296 urine samples from former heroin users at methadone maintenance clinics and drug addiction services.
Figure 1. Percentage plot of classic drugs and new psychoactive substances found in 296 urine samples from former heroin users at methadone maintenance clinics and drug addiction services.
Ijms 22 04000 g001
Figure 2. Representative selected ion monitoring GC-MS chromatograms of: urine samples positive to Fentanyl (A,B) and mass spectrometry or tandem mass spectrometry (MS/MS) full scan mass spectrum used for substance identification (C).
Figure 2. Representative selected ion monitoring GC-MS chromatograms of: urine samples positive to Fentanyl (A,B) and mass spectrometry or tandem mass spectrometry (MS/MS) full scan mass spectrum used for substance identification (C).
Ijms 22 04000 g002
Figure 3. Representative extracted-ion UHPLC-HRMS chromatograms of: (A) urine sample positive to Fentanyl and Norfentanyl (B) urine sample positive to Fentanyl, Norfentanyl and β-hydroxyfentanyl and MS/MS full scan mass spectrum used for substances identification (C).
Figure 3. Representative extracted-ion UHPLC-HRMS chromatograms of: (A) urine sample positive to Fentanyl and Norfentanyl (B) urine sample positive to Fentanyl, Norfentanyl and β-hydroxyfentanyl and MS/MS full scan mass spectrum used for substances identification (C).
Ijms 22 04000 g003
Figure 4. Full mass spectra of UR-144-pyr and UR-144.
Figure 4. Full mass spectra of UR-144-pyr and UR-144.
Ijms 22 04000 g004
Table 1. List of different target compounds, retention times (Rt) and monitored ions (m/z) using for the screening gas chromatography-mass spectrometry (GC/MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis.
Table 1. List of different target compounds, retention times (Rt) and monitored ions (m/z) using for the screening gas chromatography-mass spectrometry (GC/MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis.
CompoundFormulaGC/MSUHPLC-HRMS
Rt
(min)
Target
m/z ion
(Q)
Fragment
m/z ions
(Q/q) a
Rt
(min)
Target
m/z ion [M+H]+
(Δ-error, ppm) b
Fragment
m/z ions
Anticonvulsants
CarbamazepineC15H12N2O15.9236193(0.21)
165(1.4)
5.83237.1022(−2.53)194.0963
192.0805
GabapentinC9H17NO27.74171153(0.06)
110(0.13)
81(0.05)
2.79172.1332(−3.48)154.1227
137.0961
95.0860
LevetiracetamC8H14N2O27.75170126(0.05)
98 (0.33)
69(0.13)
2.85171.1128(−3.51)154.0863
126.0914
PregabalinC8H17NO26.54159141(0.05)
103(0.03)
84(0.04)
2.61160.1332(−3.75)142.1227
97.1016
83.0861
TopiramateC12H21NO8S5.12324206(2.61)
189 (2.61)
127(1.62)
5.19357.1326 * (2.20)264.0532
184.0970
127.0391
Antidepressants
AmitriptylineC20H23N9.59277215 (0.33)
202 (0.17)
58 (0.02)
5.98278.19033 (−1.94)233.1332
191.0861
105.0700
BupropionC13H18ClNO17.53239139 (0.14)
100 (0.02)
44 (0.01)
4.52240.1150 (−2.08)184.0521
166.0419
131.0731
CitalopramC20H21FN2O17.27324238 (0.33)
208 (0.37)
58 (0.02)
5.44325.1711(−1.59)262.1026
234.0712
109.0452
ClomipramineC19H23ClN217.22314268 (0.34)
85 (0.23)
58 (0.11)
6.23315.1623(−1.59)270.1044
86.0964
58.0651
DesmethylcitalopramC19H19FN2O17.42310238 (0.23)
138 (0.56)
44 (0.05)
5.40311.1554(−1.60)293.1446
262.1025
109.0451
DesmethylmirtazapineC16H17N319.71251208 (2.50)
195 (0.08)
4.08252.1495(−2.38)235.1229
209.1073
195.0918
MirtazapineC17H19N319.51265208(0.39)
195(0.05)
167(0.5)
4.24266.1652(−1.88)209.1076
195.0917
72.0816
TrazodoneC19H22ClN5O30.50371278(0.26)
205(0.05)
176(0.16)
4.95372.1586(−1.34)176.0819
148.0505
96.0446
Antipsychotics
LevomepromazineC19H24N2OS19.32328282(6.34)
100(6.01)
58(0.79)
6.12329.1682(−1.82)242.0633
100.1126
58.0660
NorquetiapineC17H17N3S20.01295239(0.46)
227(0.09)
210(0.16)
5.26296.1216(−1.69)221.1080
210.0373
139.2405
OlanzapineC17H20N4S19.01312242(0.20)
229(0.25)
213(0.33)
3.09313.1481(−1.92)256.0901
213.0480
84.0814
QuetiapineC21H25N3O2S19.38383239(0.09)
210(0.04)
144(0.06)
5.61384.1740(−1.56)279.0949
253.0792
221.1071
RisperidoneC23H27FN4O28.1410233(0.09)
191(2.04)
177(1.30)
4.78411.2191(−1.22)191.1179
110.0600
69.0334
Amphetamines
AmphetamineC9H13N5.4013591(0.04)
44(0.005)
2.84136.1121(−3.67)119.0857
91.0547
EthylamphetamineC11H17N6.98163148 (0.11)
91 (0.02)
72 (0.005)
3.38164.1434 (−3.05)119.0858
91.0547
MDAC10H13NO26.67179136(0.03)
77(0.08)
44(0.02)
3.24180.1019(−3.33)163.0753
135.0439
105.0699
MDMAC11H15NO26.88193135(0.10)
77(0.83)
58(0.01)
3.31194.1176(−2.58)163.0753
135.04393
105.06986
MethamphetamineC10H15N5.80149134(0.25)
91(0.04)
58(0.01)
3.20150.1277(−3.99)119.0855
91.0541
Benzodiazepines
7-AminoclonazepamC15H12ClN3O12.60285256(1.15)
222(6.82)
194(6.82)
4.06286.0742(−1.75)250.0974
222.1025
194.0831
7-AminoflunitrazepamC16H14FN3O11.33283264(5.00)
255(1.53)
240(5.55)
4.65284.1194(−1.76)227.0978
256.1243
148.0631
7-AminonitrazepamC15H13N3O15.12251222(1.64)
195(5.55)
110 (5.55)
3.20252.1131(−2.38)224.1182
146.0714
121.0762
AlprazolamC17H13ClN413.54308279(0.64)
245(2.29)
204(0.83)
6.38309.0902(−1.62)274.1208
241.0528
205.0747
ClonazepamC15H10ClN3O312.34315288(1.14)
280(0.73)
234(1.14)
6.18316.0484(−1.58)302.0448
241.0521
214.0415
ClonazolamC17H12ClN5O217.99353324 (0.60)
249 (1.00)
203 (0.82)
5.65354.0752 (−1.69)326.0563
319.1064
DiazepamC16H13ClN2O17.66284283(0.77)
256(0.59)
221(1.43)
6.83285.0789(−2.10)222.1150
193.0885
154.0417
EtizolamC17H15ClN4S18.01342313 (2.64)
266 (3.22)
137 (4.83)
6.54343.0779 (−1.46)314.0388
259.0216
FlubromazolamC17H12BrFN4 370341 (0.60)
222 (0.45)
195 (2.25)
6.22371.0302 (−1.62)343.0096
292.1105
237.0951
FlunitrazepamC16H12FN3O322.31313312(0.71)
285(0.65)
266(0.95)
6.25314.0936(−1.59)300.0902
268.1003
239.0976
FlualprazolamC17H12ClFN4 326297 (0.55)
257 (2.75)
222 (0.61)
327.0806 (−2.14)299.0625
292.1124
223.0662
NitrazepamC15H11N3O324.08281280 (0.44)
253 (0.64)
206(0.78)
5.96282.0873(−2.12)268.0842
236.0944
207.0918
NordiazepamC15H11ClN2O18.66270242(1.04)
235(3.61)
207(4.87)
6.41271.0633(−1.84)208.0994
165.0214
140.0261
OxazepamC15H11N2O2Cl16.70286268(0.06)
239(0.07)
205(0.06)
6.11287.0581(−2.09)241.0525
269.0475
104.0498
TemazepamC16H13ClN2O219.93300273(0.35)
271(0.12)
256(0.86)
6.51301.0738(−1.99)283.0630
256.0715
255.0681
Cocaine
BenzoylecgonineC16H19NO415.15289168(0.27)
124(0.07)
105(0.22)
3.84290.1387(−1.72)168.1019
105.0335
82.0650
CocaethyleneC18H23NO415.03317196(0.23)
82(0.11)
105(0.35)
4.72318.1704(−0.31)196.1330
82.0657
105.0341
CocaineC17H21NO414.27303272(2.00)
182(0.24)
82(0.17)
4.25304.1543(−1.97)182.1175
82.0657
105.0337
Ecgonine methyl esterC10H17NO37.12199182(1.63)
94(0.39)
82(0.31)
0.6200.1281(−2.99)182.1177
150.0911
82.0658
Cannabinoids
11-OH-THCC21H30O315.91330300(0.74)
299(0.16)
41(1.86)
8.15331.2267(−1.81)313.2161
193.1224
105.0703
CannabidiolC21H30O216.42314246(0.53)
231(0.06)
193(0.75)
8.64315.2319(−1.59)193.1225
135.1169
93.0704
CannabinolC21H26O217.30310295(0.11)
238(0.79)
165(2.36)
8.88311.2006(−1.61)293.1901
241.1224
223.1118
Delta-9-tetrahydrocannabinolC21H30O216.90314299(0.79)
271(1.66)
231(1.01)
9.02315.2319(−1.59)193.1223
123.0441
93.0701
THC-COOHC21H28O417.20344329 (0.70)
299(0.41)
41(0.40)
8.26345.2060(−1.74)327.1953
299.2004
193.1223
Fentanyls and NSOs
4-ANPPC19H24N218.16280189(0.08)
146(0.07)
91(0.24)
5.04281.2012(−2.13)188.1435
134.0965
105.0703
Acetyl fentanylC21H26N2O18.01322231(0.03)
188(0.08)
146(0.05)
4.89323.2118 (−1.54)188.1434
105.0703
132.0809
AH-7921C16H22Cl2N2O11.22329172(0.20)
144(0.20)
126(0.05)
3.73329.1182(−1.52)284.0610
189.9555
172.0610
AlfentanilC21H32N6O318.47416289(0.01)
268(0.03)
140(0.04)
5.35417.2609(−1.20)268.17651
197.1284
165.10223
Alpha-methylfentanylC23H30N2O18.30350259(0.05)
146(0.20)
91(0.25)
5.50351.2431(−1.42)202.1588
119.0856
91.0546
Beta-HydroxyfenatnylC22H28N2O217.52352245 (0.02)
189 (0.05)
146 (0.03)
4.90353.2224 (−1.42)204.1384
186.1276
132.0809
CarfentanilC24H30N2O318.72394303(0.01)
187(0.05)
105(0.08)
5.60395.2329(−1.52)134.0965
105.0702
113.0600
Despropionyl para-fluorofentanylC19H23FN218.45298207(0.08)
164(0.08)
136 (0.40)
5.33299.1918(−2.01)188.1435
134.0966
105.0703
FentanylC22H28N2O18.89245189(2.77)
146(1.57)
105(4.27)
5.38337.2279 (−0.30)188.1436
105.0703
132.08010
FluorofentanylC22H27FN2O17.05354263(0.01)
207(0.04)
164(0.02)
3.55355.2180(−1.69)234.1289
188.1433
105.0699
IsotonitazeneC23H30N4O317.76410236 (0.40)
107 (0.12)
86 (0.01)
7.02411.2391(−1.21)250.1077
100.1109
72.0809
MT-45C24H32N212.01348257(0.01)
165(0.17)
91(0.05)
4.03349.2638(−1.72)181.1011
169.1699
87.0916
N-methyl NorfentanylC15H22N2O17.32246189(0.12)
96(0.08)
82(0.22)
4.20247.1805(−2.02)150.0915
98.0969
69.0707
NorfentanylC14H20N2O17.90232175(0.09)
159(0.12)
83(0.05)
3.77233.1649 (−2.14)204.1038
150.0914
84.0814
OcfentanilC22H27FN2O217.34370279(0.01)
176(0.05)
105(0.05)
4.83371.2129(−1.62)188.1434
134.0966
105.0702
RemifentanilC20H28N2O516.81376227(0.02)
212(0.02)
168(0.01)
4.48377.2071(−1.33)228.1230
146.0964
113.0600
SufentanilC22H30N2O2S18.50386289(0.01)
140(0.03)
93(0.03)
5.97387.2101(−1.29)355.1838
238.1257
111.0266
Thienyl fentanylC19H24N2OS17.99328179(0.20)
97(0.03)
82(0.04)
4.87329.1682(−1.82)97.0111
82.0657
U-47700C16H22Cl2N2O10.80329172(0.05)
125(0.02)
84(0.01)
3.52329.1182(−1.52)284.0596
172.9579
81.0699
Opioids and SOs
6-MonoacetylmorphineC19H21NO418.83327268(0.92)
214(2.44)
162(4.40)
3.37328.1543(−1.82)268.1327
211.0753
165.0698
BuprenorphineC29H41NO432.0467434 (0.33)
410(0.17)
378 (0.04)
5.72468.3108(−1.28)396.2165
84.0808
55.0544
CodeineC18H21NO316.94299229(3.33)
214(5.00)
162(3.00)
2.88300.1594(−1.28)243.1012
215.1065
58.0659
EDDPC20H23N11.96277262(2.17)
220(3.09)
165(3.82)
5.61278.1903(−1.43)249.1509
234.1275
186.1275
EMDPC19H21N11.60263208(0.08)
130(0.17)
115(0.20)
5.95264.1747(−1.89)235.1355
234.1275
220.1121
HydrocodoneC18H21NO316.01299284(7.80)
242(1.50)
185(2.44)
3.35300.1594(−1.99)283.175
133.0860
89.0602
HydromorphoneC17H19NO316.35285229(3.12)
214(4.08)
200(5.30)
2.49286.1438(−1.75)185.0597
227.0699
199.0753
MethadoneC21H27NO13.41309178(0.33)
165(0.25)
72(0.03)
6.15310.2165(−1.93)105.0338
265.1584
223.1116
MorphineC17H19NO317.18285268(6.67)
215(2.50)
162(2.13)
1.91286.1438(−1.75)201.0912
229.0857
183.0807
NorcodeineC17H19NO316.84285242(6.67)
215 (2.00)
148 (2.50)
2.91286.1438(−1.74)268.13263
215.10689
225.09088
NormorphineC16H17NO316.12271201(0.02)
150(1.05)
148(1.33)
1.23272.1281(−2.20)254.1173
201.0916
121.0649
NoroxycodoneC17H19NO415.32301216(1.76)
201(4.14)
188(3.63)
3.17302.1387(−1.65)284.1281
227.0941
187.0754
NoroxymorphoneC16H17NO415.30287253(5.93)
202(1.63)
174(4.15)
1.78288.1230 (−2.08)270.1122
213.0783
173.0597
OxycodoneC18H21NO415.83315258(4.42)
230(1.91)
187(7.64)
3.21316.1543(−1.90)298.1438
256.1330
241.1093
OxymorphoneC17H19NO416.25301244(9.07)
216(2.62)
203(6.18)
2.24302.1387(−1.65)284.1278
242.1173
227.0934
TramadolC16H25NO214.41263188 (2.00)
135 (2.00)
58(0.13)
4.13264.1958(−2.27)58.0659
Synthetic Cannabinoids
JWH 018C24H23NO8.55341284(1.50)
214(1.31)
127(0.82)
8.74342.1852(−1.75)214.1224
155.0605
144.0444
JWH 073C23H21NO6.98327284(1.62)
200(0.98)
127(0.84)
8.58328.1696(−1.52)230.1172
155.0489
125.0962
JWH 073 N-4-HydroxybutylC23H21NO211.10343270(0.95)
144(1.11)
127(0.77)
7.32344.1645(−1.74)155.0490
127.1062
JWH 081C25H25NO211.57371314(2.00)
214(1.43)
185(1.43)
8.92372.1958(−1.61)214.1223
185.0596
144.0443
JWH 081 4-HydroxynaphtylC24H23NO212.44357300(1.32)
214(1.31)
171(1.48)
8.36358.1802(−1.39)214.1222
171.0438
144.0443
JWH 081 N-5-HydroxypentylC25H25NO319.93387314(1.45)
230(1.50)
185(0.90)
7.70388.1907(−1.55)230.1172
185.0596
144.0443
JWH 122C25H25NO9.33355338(1.82)
298(1.38)
214(1.45)
8.91356.2009(−1.40)214.1223
169.0646
141.0697
JWH 122 N-4-HydroxypentylC25H25NO213.26371284(0.66)
169(0.92)
144(0.96)
7.81372.1958(−1.61)169.0647
141.0698
JWH 122 N-5-HydroxypentylC25H25NO215.87371284(1.57)
141(1.29)
115(1.56)
7.80372.1958(−1.61)169.0646
141.0697
JWH 210C26H27NO10.77369352(1.71)
312(1.64)
214(0.90)
9.21370.2165(−1.62)214.1223
183.0804
144.0443
JWH 210 N-4-HydroxypentylC26H27NO214.56385298(0.64)
183(0.86)
144(0.90)
8.08386.2115(−1.29)183.0804
155.0854
144.0443
JWH 210 N-5-HydroxypentylC26H27NO217.79385368(2.75)
230(3.24)
144(2.20)
8.06386.2115(−1.29)230.1172
183.0803
155.0853
UR 144C21H29NO9.94311296(0.98)
214(0.13)
144(0.40)
9.07312.2322(−1.60)214.1223
125.0962
97.1016
UR 144 N-5-HydroxypentylC21H29NO210.70327231 (0.33)
230(0.001)
144(0.10)
7.85328.2271(−1.83)230.1172
125.0962
97.1016
XLR 11C21H28FNO10.73329314(0.90)
232 (0.09)
144(0.36)
8.64330.2228(−1.51)232.1129
125.0962
97.1016
XLR 11 N-4-HydroxypentylC21H28FNO211.73345330(0.83)
248(0.11)
144(0.29)
7.57346.2177(−1.44)248.1077
144.0443
67.0550
AM-2201C24H22FNO10.35359342 (0.20)
284 (1.25)
232 (1.30)
360.1764
Synthetic Cathinones
MDPVC16H21NO38.23275149(0.25)
126(0.01)
119(0.50)
4.35276.1594 (−2.17)126.1278
149.0232
4-MECC12H17NO6.43191119(0.33)
91(0.17)
72(0.03)
3.66192.1383 (−2.60)174.1277
159.1040
119.0857
ButyloneC12H15NO38.72221149(0.10)
121(0.20)
72(0.02)
3.52222.1125(−2.25)204.1018
174.0913
72.0815
MephedroneC11H15NO6.45177119(0.20)
91(0.10)
58(0.02)
3.37178.1226(−3.36)160.1121
145.0886
119.0857
MethcathinoneC10H13NO5.98163105(0.20)
77(0.07)
58(0.02)
2.67164.107(−1.83)146.0965
131.0731
105.0703
PentyloneC13H17NO38.13235149(0.17)
121(0.25)
86(0.01)
4.16236.1281(−2.54)218.1174
188.1069
86.0969
Miscellaneous
4-FAC9H12FN4.81153109(0.06)
83(0.10)
44(0.01)
2.95154.1027(−3.24)109.0451
137.0761
114.0917
4-MA or PMAC10H15NO9.50165122(0.03)
78(0.08)
44(0.01)
3.27166.1226(−3.61)150.0499
137.0419
117.0701
PMMAC11H17NO10.33179121(0.10)
78(0.13)
58(0.01)
3.43180.1383(−2.78)149.0961
121.0649
m-CPPC10H13ClN27.39196154 (0.25)
138 (2.00)
3.97197.0840(−3.04)154.0416
119.0730
KetamineC13H16ClNO8.28237209(0.07)
179(0.02)
125(0.08)
3.77238.0993(−2.51)207.0574
179.0622
125.0154
a, Q/q ion abundance ratio; b, delta error (ppm); * Sodium adduct; MDA: 3,4-Methylenedioxyamphetamine; MDMA: 3,4-Methylenedioxymethylamphetamine;11-OH-THC: 11-Hydroxy-delta-9- tetrahydrocannabinol; THC-COOH: 11-nor-9-carboxy-delta-9- tetrahydrocannabinol carboxilc acid; NSOs: novel Synthetic Opioids;4-ANPP: 4-Aminophenyl-1-phenethylpiperidine or Despropionyl fentanyl; AH 7921: 3,4-dichloro-N-[[1-(dimethylamino)cyclohexyl]methyl]-benzamide;U-47700: trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzamide; MT-45: 1-cyclohexyl-4-(1,2-diphenylethyl)-piperazine, dihydrochloride; SO: Synthetic Opioids; EDDP: 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EMDP: 2-ethyl-5-methyl-3,3-diphenylpyrroline; MDPV: 3,4-Methylendioxy Pyrovalerone; 4-MEC: 4-Methylethcathinone;4-FA: 4-Fluoroamphetamine; 4-MA or PMA: 4-Methoxyamphetamine or para-methoxymethylamphetamine; PMMA: para-methoxymethamphetamine; m-CPP: 1-(3-Chlorophenyl)piperazine, SO: synthetic opioids.
Table 2. Comparison of GC/MS and UHPLC-HRMS fentanyl and/or its metabolites and analogs urine sample screening and confirmation results.
Table 2. Comparison of GC/MS and UHPLC-HRMS fentanyl and/or its metabolites and analogs urine sample screening and confirmation results.
Sample CodeDetected Compound
(GC/MS)
Detected Compound
UHPLC-HRMS
MI-1029NDFentanyl
Norfentanyl
MI-1077FluorfentanylND
MI-1078N-(3-ethylindole) NorfentanylND
MI-1079FluorfentanylFentanyl
Beta-Hydroxyfentanyl
Norfentanyl
BS-2003FentanylFentanyl
MI-3009Fluoro acetyl FentanylND
MI-5016Fluoro Valeryl fentanylND
US-010FentanylFentanyl
Norfentanyl
US-017FentanylNorfentanyl
US-039FentanylBeta-hydroxyfentanyl
Fentanyl
Norfentanyl
US-059FluorfentanylND
US-060NDNorfentanyl
US-065FluorfentanylND
US-077Fluoro Valeryl fentanylND
US-083Fluoro Valeryl fentanylND
US-095Fluoro Valeryl fentanylND
US-109Fluoro Valeryl fentanylND
US-139Acetyl-methylfentanyl
2’-fluoro ortho-Fluorofentanyl
ND
US-142ThiofentanylND
US-144FentanylFentanyl
Norfentanyl
Beta-Hydroxyfentanyl
US-145FluorfentanylND
US-148FentanylNorfentanyl
US-155ThiofentanylNorfentanyl
ND: not detected.
Table 3. New psychoactive substances(NPS) found in urine samples under investigation.
Table 3. New psychoactive substances(NPS) found in urine samples under investigation.
NPS ClassesSubstances (n)
synthetic cathinonesMDPV (2)
4-cloro N butylcathinone (1)
4-Methyl-PV8 (6)
Fenethylline (4)
synthetic cannabinoidsJWH-122 (1)
JWH-032 (1)
JWH-200 (1)
UR-144 (1)
n = number of positive samples; MDPV: 3,4-Methylendioxy Pyrovalerone; 4-Methyl-PV8: 2-(pyrrolidin-1-yl)-1-(p-tolyl)heptan-1-one.
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Marchei, E.; Ferri, M.A.; Torrens, M.; Farré, M.; Pacifici, R.; Pichini, S.; Pellegrini, M. Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry and High-Sensitivity Gas Chromatography-Mass Spectrometry Screening of Classic Drugs and New Psychoactive Substances and Metabolites in Urine of Consumers. Int. J. Mol. Sci. 2021, 22, 4000. https://doi.org/10.3390/ijms22084000

AMA Style

Marchei E, Ferri MA, Torrens M, Farré M, Pacifici R, Pichini S, Pellegrini M. Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry and High-Sensitivity Gas Chromatography-Mass Spectrometry Screening of Classic Drugs and New Psychoactive Substances and Metabolites in Urine of Consumers. International Journal of Molecular Sciences. 2021; 22(8):4000. https://doi.org/10.3390/ijms22084000

Chicago/Turabian Style

Marchei, Emilia, Maria Alias Ferri, Marta Torrens, Magí Farré, Roberta Pacifici, Simona Pichini, and Manuela Pellegrini. 2021. "Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry and High-Sensitivity Gas Chromatography-Mass Spectrometry Screening of Classic Drugs and New Psychoactive Substances and Metabolites in Urine of Consumers" International Journal of Molecular Sciences 22, no. 8: 4000. https://doi.org/10.3390/ijms22084000

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