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Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry and Gas Chromatography-Mass Spectrometry Screening of Classical Psychoactive Substances, Novel Psychoactive Substances, and Metabolites in Postmortem Urine Samples

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01 September 2026

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02 September 2026

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Abstract
The use of new psychoactive substances (NPS), whether pure or in mixtures, has grown steadily, increasing the need for accurate confirmatory analysis in biological matrices given their chemical diversity, structural variability, and potential postmortem redistribution and degradation. Forensic interventions require comprehensive, broad-coverage screening strategies. Two complementary platforms, ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-ESI(+/-)-Orbitrap-HRMS) and gas chromatography-mass spectrometry (GC-MS), were used to screen classical psychoactive substances (CPS), NPS, and their metabolites in 25 postmortem urine samples with suspected consumption of NPS, using Platform-specific sample preparation and identification workflows. Forty-four drugs/metabolites were identified. Ketamine was the most prevalent drug (92% LC-MS, 84% GC-MS), along with its phase I metabolites norketamine, hydroxyketamine, and hydroxynorketamine. Cocaine (68% LC-MS, 12% GC-MS) and its main metabolite, benzoylecgonine (88% LC-MS, 52% GC-MS), were also frequent on both platforms, while amphetamine-type drugs, benzodiazepines, and opioids showed marked technique-dependent detection (e.g., 3,4-methylenedioxyamphetamine: 32% LC-MS, 4% GC-MS). UHPLC-ESI-Orbitrap-HRMS provided broader polarity coverage of parent drugs and metabolites; GC-MS reliably detected parent drugs and derivatizable xenobiotics. Combined use of both platforms provided complementary, high-confidence coverage for forensic toxicology urine screening; quantitative validation and larger cohorts are needed for diagnostic, impairment, and epidemiological extrapolation.
Keywords: 
new psychoactive substances
;  
mass spectrometry
;  
urine screening
;  
forensic drug screening
;  
UHPLC-Orbitrap-HRMS
;  
GC/MS
;  
forensic toxicology
Subject: 
Biology and Life Sciences  -   Other

1. Introduction

The increase in the abuse of classical psychoactive substances (CPS) and novel psychoactive substances (NPS), especially anesthetics and synthetic opioids, has created a public health crisis. In several countries, the rates of deaths and intoxications related to this kind of drug use have risen, as well as phenomena related to criminality, adverse events, and mental health problems, which are remarkably present in school-age teenagers and young adults [1,2,3].
Despite the opioid crisis in North America, it is essential to consider that there is not a unique NPS consumption global trend, and that these phenomena could shift according to social determinants, the type of drug, consumer, region, supply, and demand. There are distinct effects for different substances, each associated with specific compounds that trigger imbalances and dysregulation in pathways across several systems (e.g., dopaminergic, serotonergic, GABAergic). Each altered pathway could produce a distinctive pharmacological profile, which is why there is an imperative need for the forensic toxicology system to determine the best methodologies for the reliable identification of any kind of drugs and their metabolites [4,5,6,7], for contributing to elucidate the mechanism of drug-impairment, intoxication, or death in the forensic toxicology system, and the generation of adverse event treatment at the clinical toxicology and emergency medicine practice [8,9,10,11,12,13].
Several methods for extracting NPS and their metabolites have been reported in the literature, including solid-phase extraction (SPE) using various sorbents. The traditional liquid extraction (LLE), miniaturized and eco-friendly techniques such as hollow fiber solvent bar microextraction, liquid-liquid microextraction, membrane-protected molecularly imprinted polymers, and QuEChERS or dispersive extraction [14,15,16,17,18,19]. Urine is the preferred matrix for forensic and clinical analysis due to its easy, non-invasive collection, a broader diagnostic window (compared to blood), relatively high concentrations of drugs and metabolites, and ease of extraction, often requiring hydrolysis and chemical derivatization before chromatographic analysis [20,21,22]. Regarding instrumental analysis, publications report the use of universal detectors for targeted screening, low mass-resolution GC and LC in single quadrupole in full-scan mode, as well as triple quadrupole systems (QqQ-MS) for targeted analysis and quantification, and high-resolution LC-MS/MS for both targeted and non-targeted analysis, focusing on the simultaneous confirmation and quantification of compounds using data-dependent and independent acquisition modes [23,24,25,26]. The selection of the system to use must account for the physicochemical differences between the lipophilicity of Phase I metabolites and the hydrophilicity of Phase II metabolites; steps or modifications should be applied to increase extraction efficiency. Recently, several approaches have aimed to elucidate drug-drug interactions and potential metabolites using in silico ADMETox and quantitative structure-activity relationship models, as well as in vitro analyses using cell and animal models [27,28,29].
This study aimed to determine a selection of NPS, CPS, and their metabolites in urine using GC/MS and UHPLC-ESI (+/-)-Orbitrap-HRMS as analytical methods for the screening in samples provided by the Forensic Toxicology Laboratory at the National Institute of Legal Medicine and Forensic Sciences, with suspected consumption of tusibi, a cocktail of drugs with high use growth and popularity mainly in Latin America, Caribbean and Spain. No detailed report on this subject has been previously published. While GC/MS helped identify parent compounds, LC/MS provided a more comprehensive profile of urinary excretion for both parent drugs and metabolites. The complementarity of the two techniques for rapid and reliable screening of CPS and NPS in urine was demonstrated. This qualitative approach helps expand the forensic metabolic chemical space and links forensic analytics with clinical and epidemiological practice by describing toxicological findings in relation to the recorded manner of death. These findings may inform future studies on drug-drug interaction-related impairment and support harm-reduction strategies.

2. Results

Analysis of Forensic Samples by GC/MS and UHPLC-ESI (+/-)-Orbitrap-HRMS

A simple, sensitive, and selective screening method was successfully applied to screen CPS, NPS, and metabolites in forensic urine. Compound identification was assigned two confidence levels: level 1 (confirmed structure), where the proposed identification was confirmed by comparison with reference standards and spectral libraries. Level 2 corresponds to a probable or tentative structure proposed using evidence from repositories and spectral libraries. [34] Selectivity assessment showed the absence of coeluting interferents from endogenous or exogenous compounds at the retention time and with the same mass spectra of drugs of interest. Carryover was negligible, as solvent and sample blanks and negative controls showed no signals for CPS, NPS, or metabolites. Limits of detection ranged from 21 to 438 ng/mL and 8.7 to 30.8 ng/mL for GC/MS and LC/MS, respectively (see Table 1). Samples underwent comprehensive preparation for GC and LC analysis. Although the total analysis time was not short (chromatographic runs were 38 and 20 min for GC/MS and LC/MS, respectively), using two complementary instruments enabled high-confidence identification.
Table 2 details the xenobiotics of forensic interest identified in 25 screened samples and relates the findings to the cause of death. Identification data were anonymized to meet ethical research requirements.
Dissociative anesthetic ketamine (bay food, bump, cat killer, k, Special k…), known on the streets of Colombia as “tusi”, was found in 92% and 84% of the samples by LC/MS and GC/MS, respectively. Norketamine, the main active metabolite, was detected in 80% and 28% of the samples by LC/MS and GC/MS, respectively. This compound is the product of N-dealkylation catalyzed by Cytochrome P450 in the liver, acts as a non-competitive NMDA receptor antagonist, and is 5 times less potent than the parent drug. The hydroxylated forms, hydroxynorketamine and hydroxyketamine, were confirmed only by LC/MS in 64% and 56% of samples, respectively. Both are products of the hydroxylation of the cyclohexanone ring of ketamine and norketamine, respectively. This analysis found no conjugation to glucuronic acid (phase II metabolism) for ketamine (Figure S1) [35]. Cocaine, one of the most widely abused substances, a CNS stimulant and a CPS that can exert multisystemic cytotoxicity, was identified in 88% and 12% of the samples for LC/MS and GC/MS, respectively. It is extensively biotransformed via both enzymatic and non-enzymatic pathways to the pharmacologically inactive benzoylecgonine (BE), detected in 88% and 52% of the samples by LC/MS and GC/MS, respectively, a product of cocaine hydrolysis catalyzed by carboxylesterases. Ecgonine methyl ester (EME), an inactive metabolite formed by enzymatic hydrolysis of cocaine, was detected in 64% and 36% of the samples by LC/MS and GC/MS, respectively. Ecgonine, also an inactive metabolite produced by hydrolysis of BE and EME, was present in 64% of the samples analyzed by LC/MS. When cocaine and alcohol are used together, some cocaine undergoes transesterification, forming in the body at least two new substances. Cocaethylene (present in 32% of the samples for LC/MS) is pharmacologically similar to cocaine, with a slight difference in terms of longer elimination half-life, slower renal clearance, and larger volume of distribution. Ecgonine ethyl ester (EEE), an inactive metabolite formed during the hydrolysis of cocaethylene, which is catalyzed by carboxylesterase, was detected in 24% of the samples analyzed by LC/MS (see Figure S2) [36,37,38,39,40].
The hallucinogen Δ9-tetrahydrocannabinol, the main active constituent of Cannabis sativa, undergoes various phase I transformations until the formation of the main urinary inactive metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), along with its phase II metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol-glucuronide, which were detected only by LC/MS in 60% and 8% of the samples, respectively [41,42].
Central nervous system stimulants, entactogens, and hallucinogens, 3,4-methylenedioxymethamphetamine (MDMA) or ecstasy, were present in 12% of the samples, both in GC/MS and LC/MS. 3,4-methylenedioxyamphetamine (MDA) was found in 32% and 4% of the samples for LC/MS and GC/MS analysis. Both NPS were also confirmed indirectly through their hydroxylated forms, HHMA (32%), HHA (16%), HMA (12%), and HMMA (8%). Methamphetamine (MA) was confirmed in 32% of the LC/MS samples, and its main hepatic metabolites, 4-HA and 4-HMA, were confirmed in 8% of the samples analyzed by LC/MS. Figure S3 and Figure S4 show the putative urinary metabolic products of amphetamine-type drugs. Amphetamine was found in 12% of the samples by LC/MS [43,44].
Central nervous system depressants consisted mainly of the benzodiazepine oxazepam, an active metabolite of diazepam, nordazepam, and temazepam, which was present in 52% of the samples; midazolam, and its metabolite α-hydroxymidazolam, found in 12% and 4% of the samples for LC/MS and GC/MS, respectively. The synthetic opioid fentanyl and analog norfentanyl were present in 24% and 4% of the samples for LC/MS and GC/MS, respectively; the metabolites hydroxyfentanyl and hydroxynorfentanyl were found in one sample analyzed by LC/MS. Morphine-3-glucuronide, a phase II metabolite of morphine, was detected in 1 sample. [45,46,47,48,49].
Two active substances known as adulterants, or cuttings, were found. First, the local anesthetic lidocaine was detected in 64% and 8% of the samples by LC/MS and GC/MS, respectively. Its main metabolite, hydroxylidocaine, was detected in 28% of the samples analyzed by LC/MS. Caffeine, a mild stimulant, and its metabolites were present in 84% and 76% of the samples by LC/MS and GC/MS, respectively; theobromine and 1,3,7-trimethyluric acid were confirmed in the LC/MS analysis for 96% and 56% of the samples. Further discussion is needed on caffeine’s role in drug-drug interactions and its origin, since forensic laboratories report its use as an adulterant; however, its origin could also be linked to the widespread practice of coffee consumption in the Colombian population [50].
The implemented GC/MS setup, using deconvolution software with an in-house AMDIS library and commercial NIST 2023 library, proved useful for identifying parent compounds and some phase I metabolites. LC/MS analysis exhibited a more complete urinary excretion profile of parent drugs and metabolites. No hydroxylated or dehydrated adducts, conjugated to sulfate, or glucuronic metabolites were found by GC/MS analysis. In this methodology, cleaning steps are preceded by cleavage of conjugate ester glucuronides of carboxy derivatives by acid, alkaline, or the preferred (time-consuming, yet analyte-gentle) enzymatic hydrolysis. Derivatization is necessary for polar metabolites containing amino, hydroxy, and carboxylic groups. Common procedures include acylation, trifluoroacetylation, pentafluoropropionylation, heptafluorobutyration, trimethylsilylation, and alkylation. Each methodology has its own pros and cons. In the present work, enzymatic hydrolysis and trimethylsilylation were selected as a compromise between simplicity, sensitivity, chemical coverage, reaction time, experimental conditions, low sample degradation, chromatographic properties, and high library coverage for TMS adducts [51].
NPS, on average, have a half-life of approximately 2 h; the bioavailable dose is excreted in urine as the unchanged parent compound or as a metabolite. Differences in the time between ingestion and sampling, as well as in ADME profiles, could affect detection capability [52,53,54,55].
Both methodologies, used complementarily, were suitable for determining several CPS, NPS, and their metabolites. GC/MS showed limitations in the analysis of some phase I and II oxidized, hydroxylated, and conjugated forms, despite the positive impact on sensitivity associated with TMS-derivatization and glucuronide-conjugated cleavage during enzymatic hydrolysis. Sample preparation for LC/MS was more straightforward and faster. Data analysis in both methodologies requires a certain level of expertise, given the possibility of coelutions, mass spectra with several endogenous matrix compounds, derivatization, or ESI adducts. Identification confidence levels in both methodologies relied on confirmation with a reference standard, retention time, and identity match. For LC/MS runs, identification confidence was higher than for GC/MS due to mass accuracy and error, as recommended in the literature [56]. It is worth noting that the AIF (all-ion-fragmentation) spectra did not suit the MS/MS tree spectra structure for the library, usually calling for a specific isolation width for filtering candidates and better matching (mzCloud); however, they exhibited fragments used for identification that were in good agreement with the low error calculated against the mass lists used and compared with the in silico libraries integrated search. The identification phase was easier with GC/MS due to the high coverage of commercial, in-house, and free-downloadable low-resolution GC spectral libraries containing EI and silylated compounds. The combination of bioinformatic tools and commercial and homemade libraries provided us with a faster way to analyze the spectral data [57].
Phase II metabolites, 11-Hydroxy-Δ9-THC-glucuronide, nordazepam-glucuronide, and morphine-3-glucuronide, were detected in the LC/MS analysis associated with the respective parent drugs. No phase II metabolites (e.g., glucuronidation conjugates) were detected for any CPS, NPS, or metabolite by GC/MS. Conjugate metabolites often contain polar functional groups, such as -OH and -COOH, as part of sugars and amino acids; they form strong intermolecular interactions that could affect chromatography and ionize poorly under standard ESI conditions, resulting in weak or missing signals. Glucuronic conjugates were detected in GC/MS as the parent drug after enzymatic hydrolysis, extraction, and derivatization. This allowed the proposal of phase I and phase II biotransformation mechanisms for the xenobiotics under study, in good agreement with studies reporting increased molecular polarity following sequential oxidation and hydroxylation reactions [58,59]. Forensic laboratories must keep pace with the ever-changing dynamics of consumption and update their analytical methodologies to meet these requirements. Once the substance, or group of substances, has been identified, quantification must be carried out to establish levels of impairment and toxic or lethal doses, requiring the acquisition of certified reference materials of parent compounds and metabolites, and the extensive validation of a quantitative method in forensic and clinical interest matrices [60,61].

3. Discussion

The implemented GC/MS setup, using deconvolution software with an in-house AMDIS library and commercial NIST 2023 library, proved useful for identifying parent compounds and some phase I metabolites. LC/MS analysis exhibited a more complete urinary excretion profile of parent drugs and metabolites. No hydroxylated or dehydrated adducts, conjugated to sulfate, or glucuronic metabolites were found by GC/MS analysis. In this methodology, cleaning steps are preceded by cleavage of conjugate ester glucuronides of carboxy derivatives by acid, alkaline, or the preferred (time-consuming, yet analyte-gentle) enzymatic hydrolysis. Derivatization is necessary for polar metabolites containing amino, hydroxy, and carboxylic groups. In the present work, enzymatic hydrolysis and trimethylsilylation were selected as a compromise between simplicity, sensitivity, chemical coverage, reaction time, experimental conditions, low sample degradation, chromatographic properties, and high library coverage for TMS adducts [51].
NPS, on average, have a half-life of approximately 2 h; the bioavailable dose is excreted in urine as the unchanged parent compound or as a metabolite. Differences in the time between ingestion and sampling, as well as in ADME profiles, could affect detection capability [52,53,54,55].
Both methodologies, used complementarily, were suitable for determining several CPS, NPS, and their metabolites. GC/MS showed limitations in the analysis of some phase I and II oxidized, hydroxylated, and conjugated forms, despite the positive impact on sensitivity associated with TMS-derivatization and glucuronide-conjugated cleavage during enzymatic hydrolysis. Sample preparation for LC/MS was more straightforward and faster. Data analysis in both methodologies requires a certain level of expertise, given the possibility of coelutions, mass spectra with several endogenous matrix compounds, derivatization, or ESI adducts. Identification confidence levels in both methodologies relied on confirmation with a reference standard, retention time, and identity match. For LC/MS runs, identification confidence was higher than for GC/MS due to mass accuracy and error, as recommended in the literature [56]. It is worth noting that the AIF (all-ion-fragmentation) spectra did not suit the MS/MS tree spectra structure for the library, usually calling for a specific isolation width for filtering candidates and better matching (mzCloud); however, they exhibited fragments used for identification that were in good agreement with the low error calculated against the mass lists used and compared with the in silico libraries integrated search. Generally speaking, the identification phase was easier with GC/MS due to the high coverage of commercial, in-house, and free-downloadable low-resolution GC spectral libraries containing EI and silylated compounds. The combination of bioinformatic tools and commercial and homemade libraries provided us with a faster way to analyze the spectral data [57].
Phase II metabolites, 11-Hydroxy-delta-9-THC-glucuronide, nordazepam-glucuronide, and morphine-3-glucuronide, were detected in the LC/MS analysis associated with the respective parent drugs. No phase II metabolites (e.g., glucuronidation conjugates) were detected for any CPS, NPS, or metabolite by GC/MS. Conjugate metabolites often contain polar functional groups, such as -OH and -COOH, as part of sugars and amino acids; they form strong intermolecular interactions that could affect chromatography, and ionize poorly under standard ESI conditions, resulting in weak or missing signals. Glucuronic conjugates in GC/MS were detected as the parent drug analysis after enzymatic hydrolysis, extraction, and derivatization. It was possible to propose phase I and phase II biotransformation mechanisms for the xenobiotics under study, in good agreement with studies reporting increased molecular polarity following sequential oxidation and hydroxylation reactions [58,59]. Forensic laboratories must keep pace with the ever-changing dynamics of consumption and update their analytical methodologies to meet these requirements. Once the substance, or group of substances, has been identified, quantification must be carried out to establish levels of impairment and toxic or lethal doses, requiring the acquisition of certified reference materials of parent compounds and metabolites, and the extensive validation of a quantitative method in forensic and clinical interest matrices [60,61].
Further research should address the need for quantitative analysis, using a larger sample size for prevalence and causality interpretation, and include a broad panel of biological matrices (blood, bile, vitreous humor) to assess matrix effects in recoveries.

4. Materials and Methods

Reagents and Chemicals

Acetonitrile, methanol, and ethanol of analytical grade were purchased from Mallinckrodt (Dublin, Ireland); dichloromethane was purchased from Merck (Darmstadt, Germany), and ultrapure helium 99.995% (Messer—Bucaramanga, Colombia) was used as the mobile phase in chromatographic analysis.
All standard solutions for quality control were prepared at a concentration of 10 mg mL-1 (stock solution) and then diluted to a working solution of 1 mg mL-1. Table S1 lists potential target drugs or metabolites to monitor during analysis, including their molecular formula, retention time (tR), and experimental exact and average masses.

Sample Preparation by GC/MS and UHPLC-ESI (+/-)-Orbitrap-HRMS

Figure 1 reports the workflow for urine analysis in both methodologies. The urine samples of forensic origin were provided by the National Institute of Legal Medicine and Forensic Sciences. Each sample was previously analyzed by immunoassay using EMIT®, looking for drugs of abuse and their metabolites (cocaine, opioids, cannabinoids, benzodiazepines, amphetamines, methadone, ketamine, and barbiturates). A total of 25 urine samples tested positive for ketamine or amphetamine-type drugs. Aliquots of 6 mL were taken and stored at -20 °C until analysis. Two extraction methodologies were applied. Extraction methods were based on the forensic laboratory’s protocols, slightly modified to enable the identification of new molecules and their metabolites [30,31,32]. For confirmatory GC/MS analysis, 100 μL of flurazepam (ISTD) was added to 2 mL of the forensic sample, followed by 300 μL of sodium acetate buffer pH 4.5, then 50 μL of β-glucuronidase (Helix pomatia), and incubation at 56 °C for 2 h. The pH was adjusted to 10 by adding 500 μL of sodium tetraborate buffer, followed by 6 mL of dichloromethane. The mixture was vortexed for 1 min, then centrifuged at 2490 g for 10 min. The organic phase was dried under a gentle nitrogen flow, and 100 μL of BSTFA+TMCS mixture (99:1) and ethyl acetate were added to the dry extract, followed by incubation at 80 °C for 20 min. 1 μL was injected into the GC/MS in full scan mode. For UHPLC-ESI (+/-)-Orbitrap-HRMS analysis, 20 μL of oxazepam-d5 (ISTD) was added to 200 μL of urine, followed by 600 μL of ice-cold acetonitrile (ACN), vortex agitation for 1 min, and centrifugation for 10 min at 2490 g. The supernatant was dried under a gentle stream of nitrogen, then reconstituted with mobile phase, and 2 μL was injected into the liquid chromatograph.
To check the reliability of the two methodologies, sample runs included: solvent blank, used between sample injection to clean the system and avoid carryover; matrix blank, to determine the presence of endogenous compounds that may co-elute with the analytes of interest, and isobars that could produce false positive; negative control in a real matrix, to discard solvent, reactants and system contamination, assess drifts in retention time (due to internal standard), and a positive control in both aqueous and real matrix samples enriched with reference materials at final concentrations of 100 ng/mL for LC/MS and 1000 ng/mL for GC/MS, to estimate shifts in retention times, the efficiency of the extraction, the detection capacity of the libraries and establish a contrast medium. Pooled Quality Control Samples (Pool QCs) were prepared by mixing 20 µL aliquots of each sample to assess the repeatability of retention times and analytical response. Controls were treated as samples and analyzed within the same analytical sequence as the study samples, using univariate statistics.

Gas Chromatography–Mass Spectrometry Analysis

GC/MS analysis was conducted on a gas chromatograph (GC 7890A System Plus, Agilent Technologies, Palo Alto, CA, USA) coupled with a selective mass detector (Agilent Technologies, MSD 5975C) using electron ionization at 70 eV. NPS and their metabolites were separated on a capillary column with 5%-phenyl-95% poly (dimethylsiloxane) (DB-5MS 30 m × 250 μm I.D., 0.25 μm film thickness, J&W Scientific, Folsom, CA, USA). The ionization source and interface temperatures were 230 °C and 300 °C, respectively. Injection was performed via pulsed splitless mode at 270 °C in the injection port. Full-scan data was acquired over a mass range of 40–450 m/z at a rate of 3.58 scans per second. The oven was initially set to 80 °C for 1 min, then heated at 15 °C min-1 to 200 °C for 5.3 min, followed by heating at 10 °C min-1 to 300 °C for 12 min. The total run time was 38.3 min. Standard solutions of the analytes were prepared in an authentic matrix at 0.5 μg/mL. Data analysis involved an automated workflow using Agilent MSD ChemStation Software (Ver. F.01.03.2357, Agilent Technologies, Santa Clara, CA, USA) for mass spectral deconvolution, peak detection, base peak integration, and compound identification via comparison with NIST 2023 library and an in-house library containing tR and m/z of confirmed reference standards.

Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry Analysis

The urine extracts were analyzed in a VanquishTM ultra-high-performance liquid chromatograph (UHPLC, Thermo Scientific, Waltham, MA, USA), equipped with a degassing unit, a gradient binary pump, and an autosampler maintained at 10 °C. A Zorbax Eclipse XDB C18 chromatographic column (Sigma Aldrich, St. Louis, MO, USA) of 50 mm length × 2.1 mm I.D. and 1.8 μm particle diameter was used. The flow rate of the mobile phases containing Type I water (A) and methanol (B), both containing ammonium formate (5 mM) and formic acid (0.1%), was 300 μL min-1. The elution gradient program was as follows: 100% A for 8 min, then 100% B for 5 min, followed by 100% A for 7 min. The total run was 20 min, and the injection volume was 2 μL. The UHPLC was connected to a Q-Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) with a heated electrospray ionization source (HESI-II) and polarity switching for periods < 500 ms, with HCD fragmentation. The capillary voltage was 3.5 kV. The nebulizer and capillary temperatures were 350 °C and 320 °C, respectively. The mass resolution was set to 70,000 (full width at half maximum, FWHM) at m/z 200, with an automatic gain control target of 3 × 106, a maximum C-trap injection time of 200 ms, and a mass range of m/z 80–1000. The ions injected into the HCD via C-trap were fragmented with normalized collision energies from 10 eV to 40 eV. Mass spectra were collected in all-ion fragmentation mode for each collision energy, employing a mass resolution of 35,000. The instrument was fully calibrated using the Pierce LTQ Velos ESI positive ion calibration solution and the Pierce ESI negative ion calibration solution (Thermo Scientific, Rockford, IL, USA). Data were analyzed using Thermo Xcalibur 3.1 software (Ver. 4.6.67.17 Thermo Scientific, San Jose, CA, USA) and Compound Discoverer (Ver. 3.4 Thermo Scientific). The compounds were identified by comparing the tR and accurate mass (Δ ppm < 1) with standard substances prepared in an authentic matrix at 0.1 μg mL-1, analyzed in the same batch as the samples, and those reported in the local PCDL and NIST 2020 library. Additional putative metabolite identifications were performed by comparing the observed exact masses of each molecule with those reported in NIST 2023 and in silico platforms, including mzCloud, CFM-ID, ChemSpider, DrugBank, and MassBank of North America [30,31,32].

Analytical Performance

Method performance for both methodologies was evaluated according to ANSI/ASB 036-2019 Standard Practices for Method Validation in Forensic Toxicology. Carryover was addressed continuously as part of the run’s quality assurance. Solvent blanks were injected after every sample, especially positive controls. Selectivity was assessed against solvent, matrix blanks, and negative controls; retention time and mass-accuracy drift were monitored via positive controls in authentic matrix and pooled QC samples across the analytical sequence (acceptance criterion: for GC/MS, tR drift ± 0.1 min, base peak ion present; for LC/MS, tR drift ± 0.1 min, and Δppm < 1). Linearity and limits of detection were estimated using a linear calibration model based on the standard deviation of the intercept and the slope. Table 2 shows the analytical performance results for all the CPS, NPS, and metabolites. Quality assurance for the analytical run assessed repeatability in retention times and base-peak areas after response normalization (Figure S5 and Figure S6).

5. Conclusions

This study presents a comprehensive ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-ESI(+/-)-Orbitrap-HRMS) and gas chromatography-mass spectrometry (GC/MS) screening method for the analysis of CPS, NPS, and metabolites. The methods were successfully applied to 25 authentic postmortem urine samples, revealing that they could be used alone or in combination for the screening of such substances in postmortem and ante-mortem urine samples in drug-related chronic or acute intoxications, sexual assaults, and fatalities, in emergency medicine departments, and clinical and forensic toxicology services.
Urinary drug profiles reveal a distribution of CPS, NPS, and metabolites in a wide lipophilic distribution range, and indicate concurrent psychoactive drug use, which could explain drug-drug interactions associated with impairment (and the registered cause of death), alteration of enzymatic activity responsible for absorption and metabolism, sympathetic system activation, or depression, cardiac and vascular effects.
In summary, this study not only establishes a reliable screening method but also elucidates the metabolic characteristics of CPS and NPS that could impact several fields related to forensic work.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org, Table S1. List of potential drugs or metabolites to monitor. Retention times (tR) and monitored ions (m/z) used for the screening gas chromatography-mass spectrometry (GC/MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry analysis of forensic samples; Figure S1. Putative ketamine phase I metabolic products; Figure S2. Putative Cocaine metabolic products; Figure S3. Putative MDMA and MDA metabolic products; Figure S4. Putative Methamphetamine metabolic products; Figure S5. Boxplot of the normalized area of ketamine obtained by LC/MS analysis; Figure S6. Boxplot of the normalized area of ketamine obtained by GC/MS analysis.

Author Contributions

Conceptualization, Eleazar Vargas-Mena, Milton Rosero-Moreano, Gonzalo Taborda-Ocampo and Elena E. Stashenko; methodology, Eleazar Vargas-Mena, Milton Rosero-Moreano, Gonzalo Taborda-Ocampo and Elena E. Stashenko; validation, Eleazar Vargas-Mena; software, Eleazar Vargas-Mena, Julio César España-Amortegui, and Andrés F. González Suárez; formal analysis, Eleazar Vargas-Mena, Julio César España-Amortegui, and Andrés F. González Suárez; data curation, Eleazar Vargas-Mena, and Julio César España-Amortegui; writing—original draft preparation, Eleazar Vargas-Mena, and Julio César España-Amortegui; writing—review and editing, Eleazar Vargas-Mena, Milton Rosero-Moreano, Gonzalo Taborda-Ocampo, Julio César España-Amortegui, Andrés F. González Suárez and Elena E. Stashenko. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Instituto Nacional de Medicina Legal y Ciencias Forenses in Pereira, Colombia, and the Ministry of Science and Technology through the project “formación del capital humano de alto nivel corte 2”. Universidad de Caldas, code BPIN 2021000100028.

Institutional Review Board Statement

This investigation was approved by the Institutional Scientific Investigation Research Department of the Colombian National Institute of Legal Medicine and Forensic Sciences (INMLCF). Bioethical approval was granted by the Comité de Ética en Investigación Científica de la Universidad Industrial de Santander (CEINCIUIS), with approval code 4110 of 4 July 2021. This Ethics Committee operates within the framework of cooperative inter-institutional agreements with the INMLCF. The biological samples derived from the study were identified and collected by professionals from the INMLCF in their routine work as Forensic pathologists and were sent to the Forensic Toxicology Laboratory for analysis. The collection of the samples was carried out in strict compliance with Colombian national laws, particularly governed by the provisions of Resolution 008430 of 1993 of the Colombian Ministry of Health, “which establishes the scientific, technical, and administrative standards for health research”, and Resolution 382 of 2015 of the INMLCF Directorate, “which regulates the registration of entities for the obtaining of cadavers, anatomical components and tissues for transplant, teaching and research purposes and dictates other provisions”.

Data Availability Statement

All data will be made available on request.

Acknowledgments

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Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
2C-B: 4-Bromo-2,5-dimethoxyphenetylamine
4-HA: 4-Hydroxyamphetamine
4-HMA: 4-Hydroxymethamphetamine
BSTFA: N,O-Bis(trimethylsilyl)trifluoroacetamide
ESI: Electrospray ionization
eV—electron volt
GC/MS: Gas chromatography coupled to mass spectrometry
GC-MS/MS: Gas chromatography coupled to tandem mass spectrometry
HHA: 3,4-Dihydroxiamphetamine
HHMA: 3,4-Dihydroximethamphetamine
HMA: 4-Hydroxy-3-methoxyamphetamine
HMMA: 4-Hydroxy-3-methoxymethamphetamine
HNK: Hydroxynorketamine
HK: Hydroxyketamine
HRMS: High-resolution mass spectrometry
ISTD: internal standard
LC-MS/MS: Liquid chromatography coupled to tandem mass spectrometry
MDA: 3,4-Methylenedioxyamphetamine
MDMA: 3,4-Methylenedioxymethamphetamine
NK: Norketamine
ppm: parts per million
QqQ: Triple quadrupole mass spectrometry
SNC: Central nervous system
THC-COOH: 11-nor-9-carboxy-Δ9-tetrahydrocannabinol
TMCS: Trimethylchlorosilane
TOF: Time-of-flight
UHPLC- ESI(+/-)-Orbitrap-HRMS, abbreviated as LC-MS, stands for:
UHPLC: Ultra-high performance liquid chromatography

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Preprints 231114 g001
Figure 1. Workflow for urine screening analysis. Oxazepam-d5 was used as an internal standard for LC/MS, and flurazepam for GC/MS.
Figure 1. Workflow for urine screening analysis. Oxazepam-d5 was used as an internal standard for LC/MS, and flurazepam for GC/MS.
Preprints 231114 g001
Table 1. Analytical performance results for CPS, NPS, and metabolites.
Table 1. Analytical performance results for CPS, NPS, and metabolites.
Compound GC/MS UHPLC-ESI (+/-)-Orbitrap-HRMS
R2 LOD ng/mL R2 LOD ng/mL
MDA 0.839 183.74 0.985 28.8
MDMA 0.732 253.72 0.983 30.81
6-Monoacetylmorphine 0.954 114.47 0.991 16
7-aminoclonazepam 0.931 143.06 0.985 31.5
7-aminoflunitrazepam 0.961 105.42 0.991 17.5
Alprazolam 0.921 153.98 0.986 29.6
Amitriptyline 0.985 62.83 0.987 27.8
Amphetamine 0.962 83.43 0.986 30.8
Benzocaine 0.953 116.48 0.987 29.7
Benzoylecgonine 0.963 103.78 0.992 0.72
Bromazepam 0.908 166.48 0.989 35.7
Caffeine 0.841 227.91 0.908 41.3
Carbamazepine 0.914 160.82 0.987 19.1
Chlorpromazine 0.949 121.15 0.991 25.2
Clonazepam 0.794 266.94 0.968 35.3
Clozapine 0.947 123.61 0.991 24.6
Cocaethylene 0.931 143.58 0.981 18.1
Cocaine 0.988 46.26 0.990 10.2
Codeine 0.996 25.72 0.992 22.6
Diazepam 0.9830 68.939 0.982 26.7
Diltiazem 0.972 89.90 0.992 13.3
Dimethyltryptamine 0.918 156.65 0.997 18.1
Ecgonine 0.824 242.07 0.941 35.9
Ecgonine methyl ester 0.925 148.51 0.985 17.7
Ethylmorphine 0.997 21.14 0.991 15.7
Fentanyl 0.986 48.53 0.991 23.7
Flurazepam 0.991 50.752 0.993 12.3
Hydrocodone 0.974 67.93 0.993 12.1
Hydromorphone 0.955 90.84 0.991 24.1
Imipramine 0.930 143.17 0.952 23.2
Ketamine 0.977 78.99 0.987 10.2
Levomepromazine 0.987 59.97 0.995 20.2
Lidocaine 0.983 69.02 0.994 8.7
Lorazepam 0.947 123.46 0.981 27.3
Meperidine 0.996 24.44 0.991 11.9
Methadone 0.990 41.51 0.991 25.3
Methamphetamine 0.478 438.31 0.986 28.7
Midazolam 0.946 124.50 0.991 17.2
Morphine 0.994 32.32 0.991 14.8
Nitrazepam 0.662 373.97 0.975 38.7
Nordazepam 0.930 143.49 0.967 24.4
Norketamine 0.967 96.52 0.991 17.5
Oxazepam 0.939 132.85 0.984 17.9
Oxycodone 0.968 76.27 0.991 13.8
Papaverine 0.963 81.54 0.987 13.8
Phencyclidine 0.983 67.94 0.996 19.1
Temazepam 0.974 85.80 0.987 14.6
Thioridazine 0.792 269.26 0.987 16.2
Tramadol 0.981 59.00 0.997 10.8
Triazolam 0.923 151.62 0.985 17.7
Zolpidem 0.896 178.95 0.993 13.2
Table 2. Detailed GC/MS and UHPLC-ESI (+/-)-Orbitrap-HRMS urine screening findings.
Table 2. Detailed GC/MS and UHPLC-ESI (+/-)-Orbitrap-HRMS urine screening findings.
Code Cause of death GC/MS a LC/MS
04SUP23 Suicide BE-2TMS, caffeine, cocaine, EME-TMS 4-HA c, caffeine b, HNK c, HK c, ketamine b, NK b, theobromine c
665PEI23 Homicide caffeine, ketamine, NK 1,3,7-trimethyluric acid c, BE b, caffeine b, cocaine b, EME b, HNK c, HK c, HMA c, HMMA c, ketamine b, MDMA b, NK b, oxazepam b, theobromine c, THC-COOH b
03PEI23 Homicide caffeine, BE-2TMS, cocaethylene, EME-TMS, ketamine, MDA, NK 4-HA c, 4-HMA c, BE b, caffeine b, cocaine b, HNK c, HK c, ketamine b, lidocaine b, MA b, NK b, oxazepam b, theobromine c, THC-COOH b
187MZ23 Suicide BE-2TMS, caffeine, cocaine, EME-TMS, ketamine, MDMA 1,3,7-trimethyluric acid c, amphetamine b, BE b, caffeine b, cocaethylene b, ecgonine b, EEE b, EME b, HMA c, HMMA c, HHMA c, MDA, theobromine c
265AR23 Homicide ketamine BE b, cocaethylene b, ketamine b, NK b
142MZ23 Suicide caffeine, ketamine 1,3,7-trimethyluric acid c, BE b, caffeine, cocaine b, HK c, ketamine b, NK b, oxazepam b, theobromine c
705PEI22 Traffic accident α-hydroxymidazolam, caffeine, EME-TMS, fentanyl, ketamine, lidocaine, midazolam α-hydroxymidazolam b, BE b, caffeine b, cocaine b, cocaethylene b, ecgonine b, EME b, fentanyl b, HNK c, HK c, hydroxyfentanyl c, hydroxynorfentanyl c, hydroxylidocaine c, ketamine b, lidocaine b, midazolam b, nordiazepam-glucuronide c, NK b, norfentanyl b, oxazepam b, theobromine c, THC-COOH b
04PEI23 Suicide BE-2TMS, caffeine, EME-TMS, ketamine, lidocaine, NK 1,3,7-trimethyluric acid c, α-hydroxymidazolam b, BE b, caffeine b, cocaine b, cocaethylene b, ecgonine b, EEE b, EME b, HNK c, HK c, hydroxylidocaine c, ketamine b, lidocaine b, midazolam b, nordiazepam-glucuronide c, NK b, norfentanyl b, oxazepam b, theobromine c, THC-COOH b
757PEI22 Homicide BE-2TMS, caffeine, ketamine, lidocaine 1,3,7-trimethyluric acid c, BE b, caffeine b, HNK c, HK c, ketamine b, MDA b, NK b, oxazepam b, theobromine c, THC-COOH b, THC-glu c
244PEI23 Homicide caffeine, ketamine BE b, cocaine b, caffeine b, HK c, HNK c, ketamine b, nordiazepam-glucuronide c, NK c, oxazepam b, theobromine c
129ARM23 Homicide caffeine, ketamine 1,3,7-trimethyluric acid c, BE b, caffeine b, ecgonine b, hydroxylidocaine c, ketamine b, lidocaine b, NK b, oxazepam b, theobromine c, THC-COOH b
020SUP23 Homicide BE-2TMS, caffeine, cocaine, cocaethylene, EME-TMS, ketamine, MDMA BE b, caffeine b, cocaethylene b, cocaine b, ecgonine b, EEE b, EME b, HNK c, HK c, HHMA c, hydroxylidocaine c, ketamine b, lidocaine b, MA b, MDA b, NK b, oxazepam b, theobromine c, THC-COOH b
697PEI23 Homicide BE-2TMS, caffeine 1,3,7-trimethyluric acid c, BE b, caffeine b, cocaethylene b, cocaine b, EEE b, EME b, HHMA c, theobromine c, THC-COOH b
045SUP22 Traffic accident caffeine, ketamine 1,3,7-trimethyluric acid c, caffeine b, BE b, cocaine b, hydroxylidocaine c, HHMA c, lidocaine b, oxazepam b, theobromine c, THC-COOH b
185PEI22 Undefined BE-2TMS, caffeine, cocaine, EME-TMS, hydromorphone, ketamine, methaqualone, morphine-2TMS 1,3,7-trimethyluric acid c, 4-HA c, BE, caffeine b, cocaine b, codeine b, HHMA c, HNK c, HK c, hydromorphone c, ketamine b, methaqualone c, morphine b, morphine-3-glucuronide c, NK b, norhydromorphone c, oxazepam b, theobromine c
709PEI23 Traffic accident caffeine, ketamine, lidocaine, NK 1,3,7-trimethyluric acid c, caffeine, cocaine b, fentanyl b, HHMA c, HK c, HNK c, hydroxylidocaine c, ketamine b, NK b, lidocaine b, MDA b, nordiazepam-glucuronide c, norfentanyl b, oxazepam b, theobromine c, THC-COOH b
370PEI23 Homicide BE-2TMS, caffeine, EME-TMS, ketamine, lidocaine 1,3,7-trimethyluric acid c, BE b, caffeine b, cocaine b, fentanyl b, HNK c, HK c, HMA c, ketamine b, levetiracetam c, lidocaine b, MDA b, MDMA b, NK b, norfentanyl b, theobromine c, THC-COOH b
03CTG23 Homicide BE-2TMS, caffeine 1,3,7-trimethyluric acid c, BE b, caffeine b, cocaine b, cocaethylene b, ecgonine b, EEE b, EME b, HHMA c, HNK c, HK c, hydroxylidocaine c, hydroxynorfentanyl c, ketamine b, lidocaine b, MDA b, NK b, norfentanyl b, oxazepam b, theobromine c, THC-COOH b
666PEI23 Traffic accident caffeine BE b, caffeine b, cocaine b, hydroxylidocaine c, HHA c, ketamine b, lidocaine b, nordiazepam-glucuronide c, norfentanyl b, oxazepam b, theobromine c, THC-COOH b
153PEI23 Homicide caffeine, codeine-TMS, ketamine, MDA, MDMA, NK
1,3,7-trimethyluric acid c, amphetamine b, BE b, cocaine b, cocaethylene b, EME b, HK b, HNK c, ketamine b, lidocaine b, MDA b, MDMA b, NK b, theobromine c, THC-COOH b
275PEI23 Suicide ketamine BE b, caffeine b, cocaine b, ketamine b, lidocaine b, MDA b, MDMA b, midazolam b, oxymorphone c, theobromine c
014PEI24 Homicide BE-2TMS, caffeine, ketamine 4-HA c, HHA c, BE b, caffeine b, ecgonine b, EEE b, EME b, ketamine b, lidocaine b, MDA b, NK b, oxymorphone c, theobromine c
379ARM23 Undefined Ketamine 4-HA c, amphetamine b, BE b, caffeine b, ecgonine b, EEE b, ketamine b, lidocaine b, MDA b, MDMA b, midazolam b, oxymorphone c, theobromine c
102DOR23 Homicide BE-2TMS, ketamine BE b, caffeine b, cocaine b, ecgonine b, EEE b, HHA c, HHMA c, HK c, HNK c, ketamine b, lidocaine b, MDA b, MDMA b, NK b, oxycodone c, oxymorphone c, theobromine c
396ARM23 Homicide BE-2TMS, ketamine BE b, caffeine b, HHA c, HK c, HNK c, ketamine b, lidocaine b, midazolam b, MDA b, MDMA b, NK b, theobromine c
a All GC/MS identifications correspond to level 1. b Confirmatory identification (level 1). c Tentative identification (level 2). 11-nor-9-carboxy-Δ9-tetrahydrocannabinol THC-COOH, 11-Hydroxy-Δ9-THC-glucuronide THC-glu, 4-hydroxyamphetamine 4-HA, 4-hydroxymethamphetamine 4-HMA, 3,4-dihydroxyamphetamine HHA, 3,4-methylenedioxymethamphetamine MDMA, 3,4-methylenedioxyamphetamine MDA, 4-hydroxy-3-methoxyamphetamine HMA, 4-hydroxy-3-methoxymethamphetamine HMMA, 3,4-dihydroximethamphetamine HHMA, benzoylecgonine BE, ecgonine ethyl ester EEE, ecgonine methyl ester EME, Hydroxyketamine HK, hydroxynorketamine HNK, Norketamine NK, methamphetamine MA, trimethylsilyl adduct -TMS.
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