Novel psychoactive substances, such as synthetic cathinones, are constantly evolving. Examples of recently reported substances within this class include eutylone, N,N-dimethylpentylone, N-propyl butylone, and pentylone. Given their occurrence in forensic casework, methods for toxicological analysis of these compounds are needed. The application of dried blood spots (DBS) to the analysis of traditional and novel stimulants provides multiple benefits such as low volume needed for sampling and facilitated transportation and storage. This chapter describes a fast and simple procedure for the simultaneous screening of traditional stimulants (amphetamine, methamphetamine, MDMA, MDA and cocaine) and synthetic cathinones (eutylone, N,N-dimethylpentylone, N-propyl butylone and pentylone) in DBS using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Several analytical methods have been developed for identifying and quantifying piperazines, mainly in forensic toxicology. These compounds are commonly detected in cases of drug-facilitated crimes and illicit drug use. This study presents distinct methods for determining piperazines, employing solid-phase extraction (SPE) for whole blood and solid-phase microextraction (SPME) for urine samples, followed by gas chromatography-mass spectrometry (GC-MS). The developed methods ensure high selectivity, sensitivity, and reproducibility, making them suitable for forensic applications.

Tryptamines constitute a class of organic compounds structurally derived from tryptamine, a natural alkaloid found in plants, fungi, and the human body. This group encompasses biologically significant molecules such as serotonin and melatonin, as well as psychoactive compounds like DMT and psilocybin. Tryptamines are typically classified into simple tryptamines and ergolines, with further subclassifications based on structural modifications on the indole ring system. Their primary mechanism of action involves modulation of serotonergic receptors, especially the 5-HT2A, often resulting in psychedelic effects. While some tryptamines are naturally occurring, others are synthetically produced and increasingly encountered as new psychoactive substances. The study presents a rapid and robust LC-MS/MS method for the simultaneous detection and quantification of 37 tryptamines in human urine and plasma. The method employs a ThermoFisher UHPLC-LC-MS/MS system using a Hypersil GOLD C18 column and gradient elution, with MS^n acquisition in positive ESI mode. Analytical validation demonstrated high selectivity, sensitivity, and reproducibility. Sample preparation was performed through a combination of liquid-liquid and protein precipitation extraction procedures. The method successfully identified tryptamines using product ion spectra compared to a reference spectral library, with detection limits, recovery, and matrix effects systematically evaluated. This analytical approach provides a reliable tool for forensic and clinical toxicology, particularly in the context of the increasing interest in tryptamines for both recreational and therapeutic purposes.

Novel synthetic opioids include various analogs of fentanyl and emerging nonfentanyl compounds with different chemical structures, such as AH-7921, MT-45, and U-47700. In recent years, these drugs have rapidly emerged on the drug market, and their abuse has been increasing worldwide. Furthermore, common drugs like heroin have been replaced or cut with fentanyl or new designer opioids, such as nitazenes. In this scenario, the detection of synthetic opioids in hair can provide insight into their current diffusion among the population and social characteristics of these synthetic drug users. Detection of new psychoactive substances and synthetic opioids is generally performed by means of targeted methods in mass spectrometry, as they generally provide adequate sensitivity and specificity. Additionally, new opportunities are offered by the nontargeted approaches allowed by modern UHPLC-HRMS instrumentation. In this chapter, we describe a simple, fast, specific, and sensitive UHPLC-MS/MS method able to detect 13 synthetic opioids (including fentanyl analogs) and metabolites in hair samples. Furthermore, the method includes the detection of 4-anilino-N-phenethyl-piperidine (4-ANPP), which is considered both a precursor and a metabolite of several fentanyl analogs. Also, we present a comprehensive and validated workflow that combines the use of UHPLC-QTOF-HRMS instrumentation with a simple hair sample extraction procedure for the detection of a variety of fentanyl analogs and metabolites.

The rapid emergence of Novel Psychoactive Substances (NPS) poses challenges for forensic toxicology laboratories. Hair offers a unique matrix for retrospective drug exposure monitoring due to its long detection window, noninvasive collection, and high stability. Here, we describe a fast, high-throughput method for the detection and quantitation of over 30 emerging NPS from multiple drug classes, including synthetic opioids, cathinones, cannabinoids, dissociative drugs, designer benzodiazepines, and hallucinogens, in human hair using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This procedure uses bead mill pulverization, methanolic extraction, and LC-MS/MS analysis with a multianalyte calibration curve.

The Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method is the established approach for pesticide residue analysis in Europe and possesses the qualities required for routine application across diverse analytical fields. In forensic toxicology, the analysis often involves limited sample amounts while demanding high sensitivity to support legal proceedings. Therefore, miniaturizing a versatile and rapid method such as QuEChERS can offer a greener and more efficient alternative for routine forensic laboratories. This chapter presents two µ-QuEChERS methodologies as clean-up procedures for the determination of ketamine and norketamine, and cocaine and its metabolites, in hair samples, followed by analysis using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Both methods achieved the cut-off values recommended by the Society of Hair Testing (SoHT).

This chapter presents a sensitive and efficient method for the determination of ketamine, its primary metabolites (norketamine and 6-hydroxynorketamine), and its analogs 2-fluorodeschloroketamine (2F-DCK) and deschloroketamine (DCK) in urine samples. The procedure is based on dispersive liquid-liquid microextraction (DLLME) followed by LC-MS/MS detection, offering several advantages aligned with Green Analytical Toxicology principles, such as reduced solvent consumption, low sample volume, and short processing time. The method demonstrated good linearity and sensitivity and was successfully applied to authentic urine samples as a proof of concept, confirming its suitability for forensic and clinical toxicological investigations.

Opioid consumption has remained a public health problem, contributing to a significant number of fatal overdoses. Both opioids and metabolites can be detected in bile specimens due to hepatic metabolism and enterohepatic circulation, offering higher concentrations and a wide detection window compared to blood samples. Thus, bile is a suitable specimen that can provide supplementary data for routine forensic toxicology cases. This chapter presents a sensitive and specific method for the qualitative confirmation of morphine, codeine, and 6-monoacetylmorphine, a heroin consumption marker, in bile samples. The proposed methodology involves a solid-phase extraction procedure and subsequent analysis by gas chromatography coupled with mass spectrometry.

The identification and quantification of drugs of abuse in biological specimens remain a key objective in forensic toxicology. Oral fluid has become a valuable alternative matrix due to its simple, noninvasive collection, minimal risk of adulteration, and good correlation with recent drug use. This chapter presents a validated gas chromatography-mass spectrometry (GC-MS) method for the detection and quantification of multiple drugs of abuse and metabolites in oral fluid samples using the dried saliva spot (DSS) technique. The target analytes include amphetamine (ANF), methamphetamine (MANF), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), cocaine (COC), benzoylecgonine (BE), Δ9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-Δ9-tetrahydr ocannabinol (THCCOOH), morphine (MOP), codeine (COD), 6-monoacetylmorphine (6-MAM), and phencyclidine (PCP). The described GC-MS protocol is a practical and robust approach for routine forensic analysis of oral fluid. DSS offers a convenient and cost-effective sampling and extraction strategy, requiring only small sample volumes and allowing for simplified storage and transport. The analytical protocol provides high sensitivity and specificity for multiple analytes using minimal sample preparation. Its applicability in forensic toxicology makes it a useful tool for routine drug testing.

Oral fluid is an increasingly valuable biological matrix in Forensic and Clinical Toxicology due to its advantages over blood and urine, such as non-invasive and directly supervised sample collection, minimal risk of adulteration, and its ability to reflect recent drug intake. Consequently, oral fluid is now widely employed in various applications such as anti-doping control, therapeutic drug monitoring, and, most notably, workplace drug monitoring and roadside drug testing. This chapter describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the confirmatory analysis of 21 illicit and licit drugs in oral fluid. The protocol includes detailed procedures for sample conditioning, clean-up by solid-phase extraction (SPE) and LC-MS/MS analysis. The method enables the simultaneous quantification of major drug classes, including cannabis, cocaine, opioids, amphetamines, benzodiazepines, and other psychoactive substances, with a limit of quantification (LOQ) of 1 ng/mL for all analytes.

Synthetic cannabinoids (SCs) remain a prominent and dynamic class of new psychoactive substances (NPS), frequently encountered in forensic toxicology casework. Their extensive metabolism, low concentrations in biological matrices, and continual emergence of new analogues demand analytical strategies that are both sensitive and adaptable. The present work describes streamlined and sensitive workflows for the identification of SCs and their metabolites in urine and hair using LC-MS-based screening and confirmation approaches. Enzymatic hydrolysis, clean-up using supported liquid extraction or solid-phase extraction, and hair pulverization enable efficient recovery, while data-dependent acquisition and defined identification criteria ensure reliable qualitative findings. The sample preparation procedures, analytical parameters, and identification criteria are fully presented.

Cannabinoids are the most frequently detected psychoactive substances in forensic toxicology. Their presence in blood is indicative of recent cannabis consumption, which is critical in contexts such as driving under the influence of drugs, workplace drug testing, drug-facilitated crimes, and postmortem investigations. In this chapter, we describe a method for the determination of several cannabinoids and their metabolites in 100 μL of human whole blood. The procedure features an optimized clean-up step that combines protein precipitation-using a chilled acetonitrile/methanol mixture containing deuterated internal standards-with the direct removal of phospholipids via Phree™ tubes. This streamlined approach eliminates the need to transfer supernatants to separate vials. Analytes are separated using a liquid chromatography system and detected by high-resolution accurate-mass Orbitrap mass spectrometry with positive/negative polarity switching and parallel reaction monitoring acquisition mode, ensuring highly specific detection and sensitive quantification.

This study describes a method for the analysis of cocaine, its metabolites, and opioids in whole blood samples using protein precipitation, followed by detection with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method utilizes low sample volumes (0.1 mL) and minimizes matrix effects for enhanced sensitivity and reproducibility. Samples are prepared straightforwardly, ensuring a reliable and fast workflow for forensic toxicological applications.

A sensitive and rapid method for the simultaneous determination of Δ9-tetrahydrocannabinol (THC), its hydroxylated metabolite (THCOH), and its carboxylated metabolite (THCCOOH) in whole blood and urine has been developed using solid-phase extraction (SPE) followed by gas chromatography-mass spectrometry (GC-MS). A microwave-assisted derivatization step was implemented to enhance analyte volatility and chromatographic performance, thereby improving detection sensitivity and reducing overall analysis time compared with conventional heating techniques. In this methodology, sample preparation is carried out by applying SPE to efficiently isolate the target cannabinoids from blood and urine matrices. Subsequent derivatization under microwave irradiation ensured complete conversion of the analytes to their more volatile derivatives, optimizing their separation and detection by GC-MS. The simplicity of the presented protocol, combined with the use of instrumentation accessible to most forensic toxicology laboratories, justifies its adoption in routine analyses.

This chapter describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of drugs of abuse and their metabolites in human urine. The target analytes include 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), benzoylecgonine, 6-acetylmorphine, morphine, codeine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine, and methamphetamine. Sample preparation involves enzymatic hydrolysis with β-glucuronidase, followed by dilution with methanol and centrifugation, enabling direct analysis without derivatization. Chromatographic separation is achieved within 4 min, and detection is performed in multiple reaction monitoring mode with deuterated internal standards for reliable quantification. This simple and robust approach fulfills international requirements for confirmatory urine drug testing and can be readily implemented in forensic and workplace toxicology laboratories.