Forensic Narcotics Drug Analysis: State-of-the-Art Developments and Future Trends

Abstract

Narcotics trafficking is a fundamental part of organized crime, posing significant and evolving challenges for forensic investigations. Addressing these challenges requires rapid, precise, and scientifically validated analytical methods for reliable identification of illicit substances. Over the past five years, forensic drug testing has advanced considerably, improving detection of traditional drugs—such as tetrahydrocannabinol, cocaine, heroin, amphetamine-type stimulants, and lysergic acid diethylamide—as well as emerging new psychoactive substances (NPS), including synthetic cannabinoids (e.g., 5F-MDMB-PICA), cathinones (e.g., α-PVP), potent opioids (e.g., carfentanil), designer psychedelics (e.g., 25I-NBOMe), benzodiazepines (e.g., flualprazolam), and dissociatives (e.g., 3-HO-PCP). Current technologies include colorimetric assays, ambient ionization mass spectrometry, and chromatographic methods coupled with various detectors, all enhancing accuracy and precision. Vibrational spectroscopy techniques, like Raman and Fourier transform infrared spectroscopy, have become essential for non-destructive identification. Additionally, new sensors with disposable electrodes and miniaturized transducers allow ultrasensitive on-site detection of drugs and metabolites. Advanced chemometric algorithms extract maximum information from complex data, enabling faster and more reliable identifications. An important emerging trend is the adoption of green analytical methods—including direct analysis, solvent-free extraction, miniaturized instruments, and eco-friendly chromatographic processes—that reduce environmental impact without sacrificing performance. This review provides a comprehensive overview of innovations over the last five years in forensic drug analysis based on the ScienceDirect database and highlights technological trends shaping the future of forensic toxicology.

1. Introduction

1.1. Background About the Most Common Narcotic Drugs in EU

Illegal activities related to narcotics represent one of the fundamental areas of organized crime. The most active criminal organizations in the world are, among sorts of crime, involved in the production and trafficking of drugs, and for some of them, this is their main or even sole criminal activity [1]. This has been confirmed by the European Drug Report from the EU Drugs Agency (EUDA), which points to the high availability of all types of psychoactive substances on the EU drug market, with banned substances appearing in high purity and in new forms, mixtures, and combinations. Large drug seizures, especially of cocaine, have been discovered in recent years, mainly in countries with major seaports, such as Spain, Portugal, the Netherlands, and Belgium. Almost 70% of customs seizures occur in EU ports. For example, in just one operation during 2025 in Belgium, 780 kg of cocaine was seized, along with an illegal laboratory for cocaine conversion and a larger quantity of cocaine paste intended for processing in that laboratory [1,2].

When it comes to the European drug market, the report further states that amphetamine-type stimulants are particularly present in Western European countries, due to the fact that some of these countries are active producers of amphetamines and smuggling into other EU countries is facilitated by the absence of national borders. In 2022 alone, 108 dismantled laboratories for amphetamine production were reported in the EU, while the quantity of seized drugs remained stable at around 7 tons between 2021 and 2022. In Southern Europe, cannabis is predominantly present, while heroin is more commonly found in Eastern Europe—though its presence in Western Europe has significantly declined. Nevertheless, heroin remains the most commonly used illegal opioid in Europe and continues to cause the most frequent health issues related to drug use [2]. A 95% drop in opium production in Afghanistan in 2023 led to a slight decrease in the overall quantity of heroin in the EU. Still, these trends need to be monitored in the coming years. The European drug market is also seeing the emergence of new psychoactive substances (NPS), for which scientific knowledge is still very limited, as research is ongoing. Moreover, user experiences have yet to be widely documented, and additional complications arise from the diversity of these substances and their frequent replacement by new generations of compounds on the illegal market [1,2,3]. The chemical structures of commonly seized narcotic drugs are presented in Figure 1. Cannabis sativa L. remains by far the most commonly used illegal drug in Europe [2]. National surveys on C. sativa use indicate that an estimated 8% of European adults (22.8 million people aged 15 to 64) used C. sativa in the past year. Seizures of cannabis products in the EU remained at historically high levels in 2022, indicating high availability of this drug [1]. Understanding the drug–crime nexus demands an interdisciplinary approach.

1.2. Analytical Approaches for Narcotics Detection in Forensic Science

Analytical techniques play a vital role in forensic science, as identifying a suspect substance is often the initial step in an investigation. While immediate lab testing is ideal, field investigations usually start with rapid, easy to use screening kits meant for non-chemists. Positive screens must then be confirmed in the laboratory with instrumental analyses that precisely determine the drug in the seized samples [3]. However, a negative preliminary test is not the final result showing that any active drug component is absent in the sample due to possible chemical camouflage and concealment of the drug [4].

Novel psychoactive substances (NPSs) are a group of substances comprised of either the known substances that have recently become available, have been abused in the recent period, administered in a new way, a variety and number of new compounds, or chemically altered versions of known drugs, which pose challenges for detection, often requiring advanced, sensitive analytical techniques and usually their appropriate combination. Standard tests may miss these compounds, making clear, verifiable identification standards essential. Beyond confirming a substance is a drug, investigations often seek to determine chemical links among seized samples. This helps law enforcement link sources and trace distribution routes. Expert analysis should ideally reveal a drug’s composition, purity, and profile. Comparative profiling of active compounds and cutting agents provides valuable intelligence on illicit market patterns [5,6]. Purity levels and the number of cutting agents indicate whether seized material comes from bulk production or street-level sales: clandestine wholesale batches may contain one or few diluents, whereas retail drugs are heavily adulterated to boost profit. Links among samples and comparison of samples within different batches is usually performed by analyzing major alkaloids, minor components, or trace compounds in the samples [7,8], while isotopic analysis is used for origin assessment [9]. Forensic profiles also reveal market trends and health risks. EU syringe studies, for instance, show frequent co-use of stimulants and opioids, as noted in European Monitoring Centre for Drugs and Drug Addiction (EMCDDA’s) report—insights crucial for both enforcement and prevention [10]. To use such intelligence effectively, police, prosecutors, and judges must understand what modern forensic analyses can (and cannot) deliver. Clear, well-framed requests ensure that the right tests are ordered, conserve resources, and safeguard vital evidence [11].

The Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG), United Nations Office on Drugs and Crime (UNODC), and European Network of Forensic Science Institutes (ENFSI) have reported a range of recommended analytical techniques as reliable methods for identifying unknown substances and pharmaceuticals, particularly in forensic contexts. Figure 2 depicts a common analytical scheme applied in forensic drug laboratories, with recommendation for the application of relevant ENFSI Drugs Working Group documents [12]. Colorimetric spot tests and thin-layer chromatography (TLC) have long been widely used for preliminary screening. Despite their limitations in quantification, precision, and selectivity, TLC, which enables compound separation, is frequently employed as a complementary tool in initial drug screening [13]. Recently, high-performance thin layer chromatography (HPTLC) has overcome many TLC limitations, namely automated sample application, development, derivatization, densitometric scanning yield sub-nanogram detection limits, and linear calibration curves, enabling true quantification rather than mere presence/absence screening. When coupled with multivariate chemometric tools (e.g., principal component analysis or partial least squares analysis), HPTLC profiles can resolve co-eluting analogues and even fingerprint cutting agent patterns in a single run. Colorimetric testing is likewise being upgraded for accurate drug testing via smartphone cameras or portable spectrophotometers, capturing full Red Green Blue or hyperspectral data, which chemometrics algorithms then classify, turning a once subjective spot test into a semi-quantitative, statistically validated assay. Automated image analysis reduces operator bias, while chemometric modeling correlates color intensity with concentration, allowing rapid potency estimates of seized pills or powders. Together, HPTLC and advanced colorimetry extend the reach of low-cost screening methods, providing quantitative, court-defensible results that bridge the gap to confirmatory instrumental techniques. Several studies and reports have been published related to the application of analytical technique in forensics [3,12,14,15,16]. Unlike Ahmed et al. [14], this review emphasizes recent advances in miniaturized and green analytical methods, including electrochemical techniques, FTIR, Raman spectroscopy, green metrics, and multivariate analysis. It also highlights emerging tools, such as microsampling and portable sensors, for rapid, on-site drug detection.

Qualitative sampling is the process with the aim of choosing samples from the whole seizure that reflects its composition. Sampling methods for qualitative analysis (arbitrary or statistical) should fit the purpose of the national law and the request of domestic court, prosecutions, or police [15,16]. Quantitative sampling is the process that should enhance the average concentration of the active psychoactive substance in the whole seizure. The sampling strategy for quantitative analysis should consider the following: the heterogeneity of the samples, particle size, and large number of samples in the seizures. For these reasons, quantitative sampling problems are solved by the implementation of an incremental sampling protocol, with the choice of the relevant number of increments that meet the specific requirements of the particular legal system in the country. The proper recommendations for sampling strategies for most common illicit drugs were published by the ENFSI Drugs Working Group in two different guidelines dedicated to qualitative and quantitative sampling of seized drugs [15,16].

High-resolution techniques, such as gas chromatography (GC) coupled with mass spectrometry (MS) and flame ionization detector (FID) and liquid chromatography–tandem mass spectrometry (LC-MS/MS), provide highly accurate compound separation, identification, and quantification, making them essential for the analysis of complex mixtures and NPSs [14]. Nuclear Magnetic Resonance (NMR) systems with different capabilities) are particularly valuable for elucidating molecular structures and confirming the identity of unknown compounds through detailed information on atomic environments. Additionally, direct analysis in real time-mass spectrometry (DART-MS) offers rapid, solvent-free screening with minimal sample preparation, making it highly suitable for real-time or high-throughput analysis of drugs [17,18,19]. Vibrational spectroscopic methods, such as Fourier transform infrared (FTIR) and Raman spectroscopy (RS), are increasingly favored for their rapid, non-destructive, and eco-friendly analysis of solid samples [20,21]. In addition, modern electrochemical sensors and lab-on-a-chip platforms are gaining traction for their portability, high sensitivity, and capability to detect trace levels of illicit substances in diverse matrices, supporting on-site forensic applications and expanding the scope of preliminary drug screening. Alongside technological progress, forensic science has increasingly embraced green analytical methods to reduce environmental impact: minimize solvent and reagent use, lower energy consumption, and favor safer materials. Techniques, like microextraction, portable spectrometers, and ambient ionization mass spectrometry, offer lower waste and operational costs. Solvent-free or reduced-solvent methods, such as supercritical fluid chromatography (SFC), are also gaining interest as eco-friendly alternatives. Integrating green principles enhances sustainability and supports field deployment in resource-limited settings [22]. Additionally, chemometrics—the application of multivariate statistical and machine learning techniques—has become an essential tool in forensic drug analysis. It enhances the interpretation of complex data from spectroscopic and chromatographic methods, improves compound classification, and supports the rapid identification of novel substances. The integration of green principles and chemometric approaches not only advances sustainability but also increases analytical efficiency and reliability in both laboratory and field settings.

Database searches in Scopus and ScienceDirect using keywords, such as Cannabis sativa, marijuana, MDMA, LSD, cocaine, heroin, as well as terms related to analytical techniques and methods, revealed a high volume of publications, underscoring the significant research interest in this area. Over the past five years, significant advancements have been made in both laboratory-based and field-deployable methods. The aim of this paper is to provide an overview of current forensic drug analysis methods, reviewing both traditional and emerging techniques, with a focus on their application in recent forensic practice. Special attention is given to developments from the past five years, highlighting innovations in detection, miniaturization, and data interpretation that have enhanced the reliability and accessibility of forensic drug testing.

2. Cannabis sativa

C. sativa (Cannabis sativa L.) is one of the oldest known cultivated plants, with historical evidence of its use dating back thousands of years for medicinal, industrial, and recreational purposes. Among its numerous bioactive compounds, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is the primary psychoactive constituent responsible for the plant’s intoxicating effects. However, cannabis produces over 100 distinct cannabinoids, with cannabidiol (CBD) being among the most studied due to their pharmacological significance. In Europe, Δ⁹-THC limits for industrial hemp were initially set at 0.5% in 1984, reduced to 0.3% in 1987, and further lowered to 0.2% in 1999 to prevent illicit cannabis cultivation. EU hemp subsidies are granted only for certified seed varieties listed in the “Common catalogue of varieties of agricultural plant species,” provided they meet the 0.2% Δ⁹-THC threshold—a requirement also applied to imported hemp [23]. High Δ⁹-THC levels are linked to increased risks of anxiety, depression, psychosis, and negative impacts on respiratory and cardiovascular health. Accurate quantification of Δ⁹-THC in cannabis flowers is crucial for regulatory compliance and quality control. As legalization and decriminalization efforts expand globally, law enforcement and forensic laboratories face growing challenges in accurately identifying, quantifying, and differentiating cannabis samples for legal and regulatory purposes. Recent advancements in analytical techniques have significantly improved the ability to characterize C. sativa samples with greater precision, sensitivity, and efficiency. Modern analytical techniques offer high sensitivity and specificity, enabling the accurate detection and quantification of the main cannabinoids—such as Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN)—along with their acidic precursors (tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA)).

Rapid screening in situ methods, particularly colorimetric tests, have gained prominence for field applications due to their simplicity, speed, and minimal reagent consumption, proving especially valuable in forensic settings. A miniaturized 4-aminophenol (4-AP)/Fast Blue BB Salt (FBBB)) test combined with image analysis demonstrated high accuracy in distinguishing THC-rich marijuana from hemp samples [24]. A related study developed a FBBB/Polydimethylsiloxane (PDMS)-based assay to quantify total cannabinoids (Δ⁹-THC, CBD, CBN) under basic conditions, producing a red-brown solution. The test leverages FBBB’s selective reactivity—forming red with Δ⁹-THC, orange with CBD, and fluorescing only with Δ⁹-THC—enabling rapid discrimination [25]. A miniaturized FBBB color test on a solid substrate (~10 mg plant extract) effectively classified 25 samples—including hemp, marijuana, and herbs/spices—using RGB data and LDA. The test accurately distinguished hemp from marijuana, with occasional misclassification of low-THC, high-CBD samples [26]. HPTLC has re-emerged as a valuable tool due to its cost-effectiveness, simplicity, and ability to analyze 20 samples simultaneously under the same experimental conditions. Liu and co-workers evaluated eight different mobile phases and found that two systems—xylene–hexane–diethylamine (25:10:1, v/v/v) and 6% diethylamine in toluene—provided the most effective separation of the major cannabinoids (Δ⁹-THC, CBD, and CBN) from other phytocannabinoids [27]. In a recent application, HPTLC combined with image analysis and multivariate tools was used to classify C. sativa samples (drug, fiber, and intermediate types). Cannabinoid peak areas from HPTLC images were analyzed using Xfactor = [Δ⁹-THC + CBN]/CBD, producing results consistent with GC-FID (Table 1). Pattern recognition techniques confirmed accurate classification, demonstrating HPTLC’s efficacy for rapid forensic screening [28]. A follow-up study further validated HPTLC’s utility by incorporating multivariate classification (Partial Least Squares-Discriminant Analysis, PLS-DA) and assessing the environmental impact of HPTLC methods using AGREE tool. The method successfully differentiated drug- and fiber-type seized C. sativa samples while minimizing toxic solvent use and energy consumption, aligning with green chemistry principles [29]. Structural differences in cannabinoid double bonds influence their affinity for Ag(I) ions. By modifying the lower third of silica TLC plates with Ag(I), rapid separation of THC and CBD analogues was achieved. Detection using FBBB combined with smartphone-based imaging enabled semiquantitative analysis. The method is green, rapid, portable, and well-suited for on-site THC screening, with Ag(I)-TLC plates remaining stable for up to three months [30].

High-performance liquid chromatography (HPLC) combined with different detection methods have emerged as gold-standard techniques for C. sativa research due to their high sensitivity, selectivity, and ability to quantify cannabinoids in their decarboxilized or acidic forms, terpenes, and contaminants with precision. Among these, liquid chromatography-photodiode array detector (LC-PDA) has emerged as a versatile tool; Wilson and co-workers developed a high-throughput method that reduced analysis time for Δ⁹-THC and Δ⁹-THCA from over 70 min to under 30 min [31]. A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI/MS/MS) method with QTOF MS was developed to separate and quantify 18 cannabinoids, including Δ⁸-/Δ⁹-THC isomers, which require baseline separation due to multiple reaction monitoring (MRM) limitations in various hemp products and hemp-derived products, including drinks, water-soluble oils, topical serum, body lotion, face cream, lip balm, gummies, hard candy, coffee, snacks, and pet treats. Similarly, Song et al. advanced LC-Diode Array Detection (DAD) methodologies for quantification of 18 phytocannabinoids in 9 strains of hemp flowers that were extracted using methanol between 0.04 and 50% (w/w). Method selectivity was confirmed by ESI/TOFMS, showing minimal interference and identifying five unknown cannabinoids, including isomers of Δ⁹-THC, Δ⁹-THCA, and Δ⁹-THC acetate (Table 1) [32,33]. In another study, a simplified isocratic LC-UV method using methanol for both extraction and separation was developed for hemp compliance testing, achieving separation of Δ⁹-THC and Δ⁹-THCA among 19 cannabinoids in under 10 min [34]. Wilson et al. developed an LC-PDA method that identified CBNA and Δ⁸-THC as chromatographic interference in Δ⁹-THC analysis, risking the misclassification of hemp as marijuana. Analysis of 7448 samples confirmed that these interferences are widespread, highlighting a critical need for improved analytical specificity in C. sativa testing [35]. Recent advances in extraction and chiral chromatographic separation have improved the detection and differentiation of cannabinoids in complex samples using HPLC. A Polymeric Ionic Liquid (PIL)-based Solid Phase Microextraction (SPME) method further enhanced this by reducing cannabinoid interference and boosting pesticide recovery, offering better performance than traditional fibers and improving overall quality control [36]. Chiral separation of cannabinoids has been improved using polysaccharide-based chiral stationary phases (CSPs) in reversed-phase LC. De Luca et al. showed that electron-withdrawing groups on CSPs lowered retention and successfully separated cannabichromene (CBC) enantiomers in hemp extract without pure standards (Table 1) [37].

Complementary to LC-based approaches, GC-FID and GC-MS have been essential for regulatory compliance, particularly in distinguishing hemp from marijuana. Cheng and Kerrigan [38] proposed a qualitative decision-point assay using GC-MS with a 1% Δ⁹-THC threshold, which was further validated in an interlaboratory study. This method demonstrated high specificity and positive predictive value, although some false negatives were observed. Ishii et al. introduced a phenylboronic acid (PBA) solid phase extraction (SPE) as an innovative sample’s preparation method for selective THC-COOH glucuronide extraction, reducing urine preparation time to 10–25 min. In addition, subsequent derivatization (silylation or methylation) is certainly necessary for GC-MS analysis (Table 1) [39]. Mulloor et al. developed a GC–MS method for total Δ⁹-THC in C. sativa using analyte protectants to minimize active site interference, yielding results consistent with LC–PDA and ensuring compliance with the 2018 Agriculture Improvement Act [40]. Slosse and co-workers improved cannabis profiling by analyzing 46 samples from 9 Belgian indoor plantations using GC-MS. Normalization to an internal standard combined with a fourth root transformation reduced false positives, and cross-validation confirmed the model’s reliability in helping law enforcement trace seized cannabis to its source [41]. Micalizzi et al. developed a heart-cutting multidimensional GC (MDGC) approach to profile volatile compounds in seized C. sativa samples. The THC content was measured to distinguish hemp from marijuana. Statistical analysis identified key parameters for classifying seized C. sativa, aiding law enforcement in drug trafficking investigations [42]. Additionally, Kim et al. investigated cannabinoid degradation during e-cigarette vaping using GC–MS, revealing novel byproducts, like Δ⁸-THC isomers and CBDQ (Cannabidiol Hydroxy Quinone), which raise safety concerns (Table 1) [43].

For simultaneous analysis of synthetic and natural cannabinoids, micellar electrokinetic chromatography (MEKC) was applied as a green and efficient alternative. Pille-Riin Laanet et al. [44] optimized a chelate buffer system to separate four synthetic cannabinoids alongside THC and CBD, with high precision and complete recovery. This method provides a selective solution for complex plant matrices without extensive sample preparation (Table 1).

Table 1. Experimental conditions for the chromatographic and spectroscopic analysis of illicit drugs.

Chromatographic Methods
Method	Mobile Phase	Detector	Selectivity	Specificity	Ref.
	Cannabis sativa
HPTLC	10 different mp	254 nm and 366 nm	/	/	[27]
HPTLC	Cyclohexane: Diethyl ether: Diethylamine (7:3:1, v/v/v)	Fujifilm X-S10	/	/	[28]
HPLC	A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile	PAD	/	/	[30]
LC-ESI/MS/MS	Solvent A: 2 mM ammonium formate with 0.011% (v/v) formic acid, pH 3.6; Solvent B: Acetonitrile	Mass Spectrometer	/	/	[32]
LC	Solvent A: 0.015% (v/v) formic acid in water; Solvent B: 75:25 (v/v) methanol/acetonitrile	DAD	Good	/	[33]
LC	Solvent A: 0.1% trifluoroacetic acid (TFA) in water; Solvent B: methanol	DAD	High	Good	[34]
LC	Solvent A: water (H2O) with 0.085% phosphoric acid (PA); Solvent B: acetonitrile (ACN) with 0.085% PA	PDA	High	/	[35]
HPLC	Acetonitrile (ACN): orthophosphoric acid (pH = 2.2)	DAD	/	/	[37]
GC-MS	/	Mass Spectrometer	High	High	[40]
GC-MS; GC-FID	Carrier Gas: Helium	Mass Spectrometer; FID	/	/	[42]
GC-MS	Carrier Gas: Helium, purity 99.999%	Mass Spectrometer	/	/	[43]
	Heroin
GC-MS	Carrier Gas: Helium	MS	High	/	[45]
GC-FID	Carrier Gas: Helium	FID system	/	/	[46]
GC-FID	Nitrogen/Helium mixture	FID	/	/	[47]
	Cocaine
LC-MS/MS	Mobile Phase A: 0.1% formic acid in Milli-Q water Mobile Phase B: 10% Mobile Phase A in acetonitrile (i.e., 90% acetonitrile)	Mass Spectrometer	/	/	[48]
GC-MS	Carrier Gas: Helium	Mass Spectrometer	Good	Good	[49]
GC-MS/MS	Carrier Gas: Helium	Quadrupole Mass Spectrometer	High (0.01 ng/mg)	/	[50]
	Lysergic acid diethylamide
LC-MS/MS	Solvent A: 5 mM aqueous formic acid; Solvent B: Acetonitrile (ACN)	Mass Spectrometer	0.025 ng/mL for LSD	/	[51]
LC-MS/MS	0.1% formic acid in H2O (A) and ACN (B), gradient from 10% to 50% B	Mass Spectrometer in MRM mode	/	/	[52]
	Amphetamine-type
GC-MS	Carrier Gas: Helium, 1 mL/min	MSD with EI ionization, SIM acquisition mode	/	/	[53]
	Spectroscopic methods
Method	Excitation wavelength	Detector	Sens.	Spec.	Ref.
	Cannabis sativa
RS	1064 nm	BWTEK and-Raman Ex	/	/	[54]
Fourier transform near-infrared spectroscopy (FT-NIR)	1350–2560 nm	InGaAs photodetector array	/	/	[21]
FT-NIR	700–2500 nm	InGaAs photodetector	/	1	[55]
RS	830 nm	PIXIS:400BR CCD	/	/	[20]
	Heroin
LIF spectroscopy	405 nm	/	/	/	[56]
	New psychoactive substances
NIR	1350–2600 nm	Mobile detection	/	/	[57]
Surface-enhanced RS (SERS)	785 nm	/	High	/	[58]

Table 1. Experimental conditions for the chromatographic and spectroscopic analysis of illicit drugs.

Chromatographic Methods
Method	Mobile Phase	Detector	Selectivity	Specificity	Ref.
	Cannabis sativa
HPTLC	10 different mp	254 nm and 366 nm	/	/	[27]
HPTLC	Cyclohexane: Diethyl ether: Diethylamine (7:3:1, v/v/v)	Fujifilm X-S10	/	/	[28]
HPLC	A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile	PAD	/	/	[30]
LC-ESI/MS/MS	Solvent A: 2 mM ammonium formate with 0.011% (v/v) formic acid, pH 3.6; Solvent B: Acetonitrile	Mass Spectrometer	/	/	[32]
LC	Solvent A: 0.015% (v/v) formic acid in water; Solvent B: 75:25 (v/v) methanol/acetonitrile	DAD	Good	/	[33]
LC	Solvent A: 0.1% trifluoroacetic acid (TFA) in water; Solvent B: methanol	DAD	High	Good	[34]
LC	Solvent A: water (H2O) with 0.085% phosphoric acid (PA); Solvent B: acetonitrile (ACN) with 0.085% PA	PDA	High	/	[35]
HPLC	Acetonitrile (ACN): orthophosphoric acid (pH = 2.2)	DAD	/	/	[37]
GC-MS	/	Mass Spectrometer	High	High	[40]
GC-MS; GC-FID	Carrier Gas: Helium	Mass Spectrometer; FID	/	/	[42]
GC-MS	Carrier Gas: Helium, purity 99.999%	Mass Spectrometer	/	/	[43]
	Heroin
GC-MS	Carrier Gas: Helium	MS	High	/	[45]
GC-FID	Carrier Gas: Helium	FID system	/	/	[46]
GC-FID	Nitrogen/Helium mixture	FID	/	/	[47]
	Cocaine
LC-MS/MS	Mobile Phase A: 0.1% formic acid in Milli-Q water Mobile Phase B: 10% Mobile Phase A in acetonitrile (i.e., 90% acetonitrile)	Mass Spectrometer	/	/	[48]
GC-MS	Carrier Gas: Helium	Mass Spectrometer	Good	Good	[49]
GC-MS/MS	Carrier Gas: Helium	Quadrupole Mass Spectrometer	High (0.01 ng/mg)	/	[50]
	Lysergic acid diethylamide
LC-MS/MS	Solvent A: 5 mM aqueous formic acid; Solvent B: Acetonitrile (ACN)	Mass Spectrometer	0.025 ng/mL for LSD	/	[51]
LC-MS/MS	0.1% formic acid in H2O (A) and ACN (B), gradient from 10% to 50% B	Mass Spectrometer in MRM mode	/	/	[52]
	Amphetamine-type
GC-MS	Carrier Gas: Helium, 1 mL/min	MSD with EI ionization, SIM acquisition mode	/	/	[53]
	Spectroscopic methods
Method	Excitation wavelength	Detector	Sens.	Spec.	Ref.
	Cannabis sativa
RS	1064 nm	BWTEK and-Raman Ex	/	/	[54]
Fourier transform near-infrared spectroscopy (FT-NIR)	1350–2560 nm	InGaAs photodetector array	/	/	[21]
FT-NIR	700–2500 nm	InGaAs photodetector	/	1	[55]
RS	830 nm	PIXIS:400BR CCD	/	/	[20]
	Heroin
LIF spectroscopy	405 nm	/	/	/	[56]
	New psychoactive substances
NIR	1350–2600 nm	Mobile detection	/	/	[57]
Surface-enhanced RS (SERS)	785 nm	/	High	/	[58]

DART-MS (direct analysis in real time-mass spectrometry) offers rapid, high-throughput analysis of C. sativa samples, enabling real-time and in situ detection of cannabinoids, without extensive sample preparation. DART-MS preserves molecular integrity, providing accurate, and sensitive results for quality control and compliance testing in the cannabis industry. Dong et al. introduced a rapid DART-MS method with flash derivatization and an Ag-phosphine assay to differentiate hemp and marijuana by ∆⁹-THC, noting ∆⁸-THC as a potential confounder [17]. Among these, DART-High Resolution (HR)MS has emerged as a powerful tool for high-throughput analysis, enabling the detection of cannabinoid acids, neutral cannabinoids, and terpenes in retail cannabis products—including edibles, concentrates, tinctures, and flower—without extensive sample preparation. Direct introduction of bulk material into the DART stream enables rapid THC and CBD identification, minimizing reliance on traditional chromatography and streamlining forensic triage [18,19].

Complementary to mass spectrometry, molecular fingerprint techniques, such as Raman spectroscopy (RS) and FT-NIR spectroscopy, offer non-destructive and rapid alternatives for cannabinoid discrimination and quantification. The qualitative RS, in particular, leverages distinct spectral signatures (e.g., band ratios at 1295/1440 and 1623/1663 cm⁻¹) to differentiate cannabis chemovars (high-THC, CBD-rich hemp, and industrial hemp) [20,54]. A rapid 15-s FT-NIR method was developed as an on-site quantitative method for analysis of cannabinoids in hemp. Testing 91 samples showed that some exceeded Δ⁹-THC limits, and many mislabeled the CBD content. The method demonstrates high accuracy and offers a fast alternative for hemp quality testing [21]. M. Birenboim et al. [55] showed that FT-NIRS with PLS-DAand PLS regression (PLS-R) achieved perfect classification of major chemovars (high-THC, high-CBD, hybrid, high-CBG) using only three latent variables, with minimal overfitting, thus enabling rapid cannabinoid analysis in C. sativa. RS offers a green alternative for distinguishing C. sativa, CBD-rich hemp, and industrial hemp by identifying unique spectral signatures of key cannabinoids. This study shows that RS can also detect CBD and CBG, distinguishing C. sativa, CBD-rich plants, and hemp. Key spectroscopic signatures of major cannabinoids enable Raman-based quantitative analysis in plant material [20] (Table 1).

Recently, electrochemical sensors have emerged as powerful tools for green, rapid and cost-effective cannabinoid detection, offering significant advantages over traditional chromatographic techniques (Table 2). These methods enable on-site analysis with minimal sample preparation, high sensitivity, and real-time results, making them ideal for law enforcement and forensics applications. An electrochemical sensor, combined with multivariate analysis, accurately classified C. sativa samples by the total Δ⁹-THC content. Using voltammetric data, PLS-DA models achieved 85% and 100% prediction accuracy for EU/US (0.3%) and Italian (0.6%) legal limits, respectively [59]. A separate surfactant-assisted sensor allowed on-site identification and semi-quantification of six major cannabinoids (THCA, Δ⁹-THC, CBDA, CBD, CBC, CBN) in seized cannabis. Validated with forensic samples, it achieved 100% accuracy in THC/CBD discrimination [60]. A stable, biomolecule-free roadside sensor was designed for the detection of ultra-low concentrations of Δ⁹-THC. Given THC’s susceptibility to oxidation, optimal storage conditions—controlling temperature, humidity, airflow, and light—were investigated to enhance electrode stability and signal reliability. The results showed that frozen storage combined with acidic pH modification maintains electrode performance for up to six months, offering a promising solution for long-term Δ⁹-THC detection [61]. A low-cost, capillary-driven microfluidic device was developed to distinguish Δ⁹-THC from CBD, combining electrochemical and colorimetric detection in a portable, pump-free PET film platform for regulatory-compliant verification of THC-free products [62]. A rapid time-resolved fluoroimmunoassay test strip was developed for on-site CBD detection using a high-affinity monoclonal antibody from a novel CBD conjugate, showing no cross-reactivity with nine related cannabinoids [62].

Table 2. Experimental conditions for the electrochemical analysis of illicit drugs.

Electrode	Modifying Agent	Detection Method	Linear Range	Sensitivity	LOD	Ref.
Cannabis sativa
Screen-printed electrode (SPE)	Carbon Black (CB N220)	Differential Pulse Voltammetry (DPV)	/	/	/	[59]
Unmodified carbon SPEs	CTAB	Square Wave Voltammetry (SWV)	/	/	/	[60]
Sensor pristine electrodes (screen-printed)	/	SWV	/	/	0.85 ng mL−1 Δ9-THC (THC)	[61]
Capillary-driven microfluidic electrochemical device (CDMFE)	/	DPV (for total Δ9-THC + CBD);	0–120 μg/mL (Δ9-THC and CBD.	0.16 μA/μg mL (Δ9-THC + CBD)	0.26 μg/mL (Δ9-THC + CBD)	[62]
Heroin
Flexible screen-printed electrode (SPE)	Nitrogen (N)- and tungsten boride (WB)-doped carbon nanotubes (CNTs)	CV	/	/	100 nM	[63]
Glassy carbon electrode (GCE)	Graphene oxide/Carboxymethylcellulose/Magnesium oxide	DPV; CV	/	/	1 × 10−7 µM	[64]
Electrode modified with ZnO/Fe3O4/carbon MHNTA	ZnO/Fe3O4/Carbon composite nanotubes	Voltammetry	0.01–500.0 μM	/	4.7 nM	[65]
Rotating GCE	Chitosan-ionic liquid (Ch-IL) composite film	DPV; CV	MO: 1–20 pM COD: 0.5–12 pM	/	0.81 fM MO; 0.22 fM COD	[66]
Electrochemically pretreated (p-SPE)	Anodic electrochemical pretreatment	Voltammetry	/	0.019 μA μM−1	5.2 μM	[67]
Cocaine
Ion-selective electrode with potentiometric transducer	Molecularly imprinted polymer nanoparticles (nanoMIPs) incorporated in PVC matrix	Potentiometry	1 nM–1 mM	/	/	[68]
/	/	TdT and CRISPR-Cas12a	40 pM–150 nM	/	15 pM	[69]
Carbon paste electrode	Multi-walled carbon nanotubes	SWV	2.36 × 10−6 to 1.38 × 10−5 mol/L	0.99	5.75 × 10−7 mol/L	[70]
Electrochemical aptamer-based biosensor	/	/	0.5 fM–30 fM	/	0.16 fM	[71]
Lysergic acid diethylamide
Carbon-SPE	/	Voltammetric	>0.99	/	0.69 μmol/L	[72]
Boron-doped diamond electrode (BDDE)	/	SWV	5.0–100 μmol L−1	0.45 μmol L− 1	0.5 μmol L−1	[73]
Graphite screen-printed electrode (SPE-Gr)	/	Differential Pulse Stripping Adsorptive Voltammetry (AdSDPV)	10–1000 μg mL−1	/	0.3 μg mL−1	[74]
Paper-based electrode drawn with 8B graphite pencil + silver paint	/	SWV	/	/	0.38 μmol/L	[75]
3,4-Methylenedioxy- Methamphetamine
SPE	/	SWV	/	/	/	[76]
Carbon-SPE	/	CV; SWV	1.75–19.98 µg mL−1	/	1.75 µg mL−1	[77]

Table 2. Experimental conditions for the electrochemical analysis of illicit drugs.

Electrode	Modifying Agent	Detection Method	Linear Range	Sensitivity	LOD	Ref.
Cannabis sativa
Screen-printed electrode (SPE)	Carbon Black (CB N220)	Differential Pulse Voltammetry (DPV)	/	/	/	[59]
Unmodified carbon SPEs	CTAB	Square Wave Voltammetry (SWV)	/	/	/	[60]
Sensor pristine electrodes (screen-printed)	/	SWV	/	/	0.85 ng mL−1 Δ9-THC (THC)	[61]
Capillary-driven microfluidic electrochemical device (CDMFE)	/	DPV (for total Δ9-THC + CBD);	0–120 μg/mL (Δ9-THC and CBD.	0.16 μA/μg mL (Δ9-THC + CBD)	0.26 μg/mL (Δ9-THC + CBD)	[62]
Heroin
Flexible screen-printed electrode (SPE)	Nitrogen (N)- and tungsten boride (WB)-doped carbon nanotubes (CNTs)	CV	/	/	100 nM	[63]
Glassy carbon electrode (GCE)	Graphene oxide/Carboxymethylcellulose/Magnesium oxide	DPV; CV	/	/	1 × 10−7 µM	[64]
Electrode modified with ZnO/Fe3O4/carbon MHNTA	ZnO/Fe3O4/Carbon composite nanotubes	Voltammetry	0.01–500.0 μM	/	4.7 nM	[65]
Rotating GCE	Chitosan-ionic liquid (Ch-IL) composite film	DPV; CV	MO: 1–20 pM COD: 0.5–12 pM	/	0.81 fM MO; 0.22 fM COD	[66]
Electrochemically pretreated (p-SPE)	Anodic electrochemical pretreatment	Voltammetry	/	0.019 μA μM−1	5.2 μM	[67]
Cocaine
Ion-selective electrode with potentiometric transducer	Molecularly imprinted polymer nanoparticles (nanoMIPs) incorporated in PVC matrix	Potentiometry	1 nM–1 mM	/	/	[68]
/	/	TdT and CRISPR-Cas12a	40 pM–150 nM	/	15 pM	[69]
Carbon paste electrode	Multi-walled carbon nanotubes	SWV	2.36 × 10−6 to 1.38 × 10−5 mol/L	0.99	5.75 × 10−7 mol/L	[70]
Electrochemical aptamer-based biosensor	/	/	0.5 fM–30 fM	/	0.16 fM	[71]
Lysergic acid diethylamide
Carbon-SPE	/	Voltammetric	>0.99	/	0.69 μmol/L	[72]
Boron-doped diamond electrode (BDDE)	/	SWV	5.0–100 μmol L−1	0.45 μmol L− 1	0.5 μmol L−1	[73]
Graphite screen-printed electrode (SPE-Gr)	/	Differential Pulse Stripping Adsorptive Voltammetry (AdSDPV)	10–1000 μg mL−1	/	0.3 μg mL−1	[74]
Paper-based electrode drawn with 8B graphite pencil + silver paint	/	SWV	/	/	0.38 μmol/L	[75]
3,4-Methylenedioxy- Methamphetamine
SPE	/	SWV	/	/	/	[76]
Carbon-SPE	/	CV; SWV	1.75–19.98 µg mL−1	/	1.75 µg mL−1	[77]

3. Heroin

Heroin (3,6-diacetylmorphine) is a semisynthetic opioid first synthesized in 1874 and later marketed as a non-addictive analgesic, though its high toxicity and addictive potential quickly became apparent [45]. Derived from morphine, it remains one of the most widely abused narcotics globally, with a particularly high prevalence in Asia and the Middle East [78,79]. The opioid crisis has intensified in recent years, with an 82% increase in overdose deaths recorded in 2020 alone, mostly due to the synthetic opioids in the illegal drug market [80]. The growing severity of these trends highlights a pressing demand for sophisticated analytical techniques capable of highly sensitive and selective heroin detection and characterization [63]. Sripratumwong et al. developed a GC-FID method for analyzing heroin hydrochloride in seized drug samples from Ratchaburi Province in Thailand. The method exhibited high precision and revealed heroin purities ranging from 70% to 88%, confirming its suitability for forensic drug profiling [46]. Recent work by Baheri and co-workers used GC-FID and GC-MS to evaluate medicinal impurities in 440 heroin samples. Caffeine was present in 75% of cases, followed by acetaminophen (41%) and dextromethorphan (28%). In another study, 440 heroin samples seized in 2023 were analyzed to identify pharmaceutical adulterants and assess changes in impurity trends using GC-FID and GC-MS. Caffeine (75%), acetaminophen (41%), and dextromethorphan (28%) were the most common additives, primarily used to increase bulk. Minor adulterants, like diazepam, lidocaine, and clotrimazole, were added to enhance effects. Compared to earlier years, the impurity profile has shifted, with older additives, such as tramadol and clonazepam, no longer detected. Substances, like fentanyl and xylazine, reported elsewhere, were absent from the analyzed samples [47]. Chandra et al. [81] analyzed 67 samples from Delhi, finding consistent adulteration with caffeine (78%), acetaminophen (62%), and phenobarbital. TLC was used for qualitative analysis, while GC-MS confirmed and quantified constituents. Only 3% of samples contained high-purity heroin HCl, and the presence of secondary opioids indicated incomplete acetylation, suggesting organized manufacturing [81].

Due to the rapid metabolism of 6-acetylmorphine, stable biomarkers are needed to confirm heroin use. Karakasi et al. developed a GC-MS method to detect markers, like meconin and papaverine. In 34 cases, meconin was the most frequently found even when 6-acetylmorphine was absent, highlighting its value as a complementary heroin indicator [45]. In a complementary study, Li et al. [82] applied ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) metabolomics to examine liver metabolism in heroin-addicted mice. Through OPLS-DA analysis and Variable Importance in Projection (VIP) scoring, they identified altered metabolic pathways associated with heroin exposure, advancing our understanding of its systemic effects [82]. Khazaei and co-workers implemented laser-induced fluorescence spectroscopy (LIFS) coupled with solvent densitometry using modified Beer–Lambert (MBL) formalism. Four unique parameters (Cp, K, α, SS) differentiated heroin from opium and hashish, offering a quantitative fingerprint for street-level substance identification [56].

Electrochemical sensing has also emerged as a powerful approach. Javed et al. created a GO/CMC-MgO nanohybrid membrane for dual heroin and dopamine detection via DPV, achieving ultra-low detection limits and enabling real-time neurochemical monitoring [64]. Electrochemical detection was further refined by Foroughi et al., who developed a ZnO/Fe₃O₄/C MHNTA-modified electrode for simultaneous heroin and oxymorphone detection. The sensor exhibited excellent stability, significant peak separation, and reduced overpotential, allowing accurate detection in complex mixtures without sample pretreatment [65]. Jalalvand et al. developed an advanced biosensor using a chitosan–ionic liquid composite with AuPtPd nanoparticles and molecularly imprinted polymers (MIPs) for morphine and codeine detection. The system achieved picomolar-level sensitivity with minimal interference and showed strong correlation with HPLC-UV in serum samples, making it highly applicable to forensic and clinical settings [66]. Montiel et al. introduced a rapid, field-deployable method using pretreated p-SPE for heroin detection. By resolving paracetamol interference through anodic pretreatment, the system detected heroin and its metabolite 6-MAM with high reproducibility and low detection limits, validated against 14 street samples [67]. Similarly, Zhou et al. [63] developed a portable electrochemical platform utilizing nitrogen and tungsten boride-doped carbon nanomaterials. The sensor showed enhanced catalytic activity and robustness, suitable for real-time forensic analysis and drug monitoring in the environment [63] (Table 2).

Modern heroin analysis relies on a diverse set of advanced analytical techniques, including chromatography, spectroscopy, and innovative electrochemical sensors. These methods provide high sensitivity, selectivity, and rapid performance even in complex matrices, such as biological fluids or street samples. Particularly valuable are portable electrochemical platforms, which offer on-site detection capabilities critical for law enforcement and harm reduction. Continued innovation in these analytical tools is essential for improving forensic investigations and public health responses to heroin abuse.

4. Cocaine

Among illegal drugs, cocaine—an alkaloid naturally derived from the leaves of Erythroxylum coca—has been consumed in South America for over 2000 years. Its isolation in the 19th century led to medical use as a local anesthetic, but it was later banned due to its severe physiological and psychological impacts [83]. Prolonged cocaine use results in extensive system-wide damage, including anxiety disorders, heart toxicity, cerebrovascular incidents, and acute cardiovascular failure, thereby posing substantial public health and economic burdens [68]. Recent advancements in analytical methodologies have significantly enhanced the detection and quantification of cocaine and its metabolites across various biological and non-biological matrices. Key developments have focused on increasing sensitivity, selectivity, and efficiency while preserving sample integrity—crucial factors for both forensic and clinical applications [84].

Chromatographic techniques, such as liquid and gas chromatography coupled with mass spectrometry, remain indispensable, offering robust platforms for confirmatory testing and pharmacokinetic investigations of cocaine. In the context of polysubstance use, Wei et al. (2024) validated a highly sensitive LC-MS/MS protocol, enabling the simultaneous quantification of cocaine and heroin metabolites in rat blood [48]. Bilko et al. [85] refined a non-destructive, sonication-assisted aqueous extraction technique for quantifying cocaine on banknotes. Chromatographic methods continue to serve as the backbone for confirmatory cocaine testing, especially when paired with mass spectrometry [85]. Menotti et al. [49] developed a GC-MS approach for the detection of cocaine, benzoylecgonine, and cocaethylene in urine following solid-phase extraction. This strategy was extended by La Maida et al. (2021), who adapted a GC-MS/MS method for hair analysis, enabling the detection of cocaine and four metabolites using electron impact ionization and MRM, thus improving selectivity and sensitivity in long-term drug use assessment [50]. Dalem et al. [86] optimized a GC-MS protocol for cocaine detection in blood samples. Non-destructive spectroscopic techniques, including attenuated total reflectance Fourier transform infrared (ATR-FTIR), RS, and NIR spectroscopy, are gaining momentum when coupled with chemometric analysis. Kochenborger John et al. [87] demonstrated how advanced spectral processing can overcome traditional barriers, enabling reliable analysis of seized samples and unconventional matrices without requiring extensive sample clean up.

Electrochemical methods are evolving through miniaturization and interface engineering, while efforts to democratize access to reliable diagnostics are driving the development of cost-effective solutions. Borgul et al. [88] applied ion transfer voltammetry at electrified liquid–liquid interfaces (eLLI) using miniaturized fused silica capillaries to quantify cocaine metabolites in urine. Their system explored diffusion dynamics and pH-dependent partitioning, illustrating the feasibility of portable electrochemical devices for point-of-care diagnostics [88]. Similarly, CV at water/1,2-dichloroethane interfaces has been employed to characterize eight common cocaine adulterants, establishing detection limits between 0.1 and 5 μM—critical for forensic profiling of street samples [89]. Burton et al. [90] developed a nanohybrid sensor composed of aptamer-functionalized ZnSe/In₂S₃ quantum dots and cetyltrimethylammonium bromide (CTAB)-capped gold nanoparticles. By exploiting localized surface plasmon resonance-induced fluorescence enhancement, this sensor achieved a detection with high selectivity for cocaine [90]. Taking sensitivity even further, Abnous et al. [69] integrated CRISPR-Cas12a with electrochemical readout, enabling attomole-level detection of cocaine in serum samples. Their design employs aptamer-complex stabilization on screen-printed gold electrodes, marking one of the most sensitive and field-deployable systems to date [69]. Conceição et al. [70] developed a SWV method for the quantification of cocaine in seized samples using multi-walled carbon nanotube (MWCNT)-modified carbon paste electrodes. With a detection limit of 0.58 µM and a per-test cost under USD 1.42; this method is particularly suitable for low-purity cocaine samples in resource-constrained settings [70]. For clinical applications, especially in treatment compliance monitoring, ultra-sensitive detection in biofluids is essential. Hamdi et al. [71] reported a femtomolar-level aptasensor using hydrothermally synthesized CuFe₂O₄ integrated with electroreduced graphene oxide (ErGO). This platform exhibited exceptional electron transfer efficiency and a high surface area, achieving a detection limit of 0.16 fM in serum [71] (Table 2). Conventional chromatographic and spectroscopic techniques remain essential for their accuracy and robustness, yet they often require labor-intensive sample preparation, are vulnerable to matrix interferences, and lack portability. In contrast, emerging technologies—especially those leveraging electrochemistry, nanomaterials, and synthetic biology—offer transformative improvements in sensitivity, selectivity, cost-effectiveness, and field-readiness.

5. Lysergic Acid Diethylamide

Lysergic acid diethylamide (LSD) is a semi-synthetic psychedelic compound derived from lysergic acid, a naturally occurring ergoline alkaloid biosynthesized by the fungus Claviceps purpurea, which parasitizes cereal crops such as rye. Through chemical modification involving diethylamide substitution, lysergic acid is transformed into LSD, a compound known for its exceptional psychoactive potency. LSD poses risks because it can trigger episodes of panic, confusion, and unusual behavior, occasionally leading to irrational or harmful actions. Initially, LSD was utilized as a research aid to explore brain function and as a tool in psychotherapy. Over time, especially during the 1960s, its use expanded as a recreational drug across Europe and the United States, becoming particularly popular among younger populations. Even small doses, between 25 and 200 micrograms, are capable of causing significant changes in perception and awareness [91,92]. These effects pose challenges in both clinical and forensic contexts, necessitating the development of rapid, reliable, and selective analytical methods for its detection, particularly in trace amounts and unstable matrices. The UNODC endorses a colorimetric LSD test with Ehrlich’s reagent, where p-dimethylaminobenzaldehyde (p-DMAB) undergoes electrophilic aromatic substitution at the indole α-carbon to yield a diagnostic color. The resulting color change depends on various factors, including the concentration of LSD, reagent proportions, temperature, and other variables [93]. However, this method suffers from a lack of selectivity and may yield false positives or ambiguous results due to interference from compounds with similar structures.

Recently, LC combined with MS and electrochemical methods have emerged as a promising alternative. Dimitrova et al. [51] developed two LC-MS/MS methods for detecting LSD and its metabolite in clinical samples, such as blood. Method 1 offers broad screening of 165 compounds with protein precipitation, while Method 2 uses liquid–liquid extraction and a 7.5-min run for sensitive quantification. Combined, they provide efficient screening and precise analysis in forensic cases [51]. Patton et al. [52] developed an automated LC-MS/MS method for the determination of LSD and O-H-LSD detection in urine with a 0.05 ng/mL limit. Using Dispersive Pipette XTR action on robotic systems, the proposed method meets U.S. DoD standards and suits high-throughput forensic and workplace testing [52]. 1-Propionyl-LSD (1 P-LSD), a controlled LSD derivative and prodrug, is rapidly converted to LSD in humans. The LC–MS/MS method was developed to detect LSD, iso-LSD, 2-oxo-3-hydroxy-LSD, and 1 P-LSD in hair samples. In 18 suspect samples, LSD levels decreased from proximal to distal hair segments; 1 P-LSD was not detected. This is the first hair analysis monitoring both LSD and 1 P-LSD [94].

Emphasizing portability and sustainability, Arrieiro et al. [72] developed a hybrid electrochemical–colorimetric method using carbon electrodes to enhance the Ehrlich’s reagent assay for LSD detection. The system produced three LSD-specific signals and showed near-100% recovery in seized samples, confirmed by LC-QTOF/MS, demonstrating forensic reliability [72]. Pimentel et al. (2021) introduced a fast and simple square-wave voltammetry (SWV) method employing a boron-doped diamond electrode (BDDE) for direct and sensitive analysis of LSD in blotter papers in seized forensic samples [73]. A combined colorimetric-electrochemical method was developed for the rapid identification of LSD and related compounds in blotter papers. It uses Emerson’s reagent and voltammetry to generate three distinct signals, enabling clear differentiation between common phenylethylamines. The method is simple, stable, and effective for forensic screening [74].

Ribeiro et al. [75] developed a paper-based electrochemical sensor using graphite pencil traces on watercolor paper. Results obtained from seized LSD samples showed less than 10% deviation from GC-MS reference values. The device also demonstrated strong selectivity against common psychoactive interferents, such as 3,4-Methylenedioxymethamphetamine (MDMA) and methamphetamine. Its low cost, environmental friendliness, and rapid response (<5 min) render it highly suitable for field applications in forensic and harm reduction settings [75]. Lysergic acid diethylamide (LSD) and phenylethylamine derivatives (NBOHs and NBOMes) are common in seized blotter papers, requiring rapid forensic screening. Another study presents a 3D-printed electrochemical double cell (3D-EDC) for their selective detection using dual electrodes or pH conditions. Two strategies—different electrodes or pH levels—enabled stable and consistent signals. The 3D-EDC provides a fast, robust, and cost-effective tool for on-site drug screening (Table 2).

6. Amphetamine-Type Stimulants

Amphetamine-type stimulants (ATSs), as defined by the UNODC, comprise a group of synthetic stimulants, including amphetamine, methamphetamine, and substances from the ecstasy group, such as MDMA and its analogues. Acute effects of these stimulants are alertness, euphoria, enhanced motivation, anxiety, paranoia, reduced appetite, and sleep addiction. They speed up the nervous system and produce effects similar to adrenaline [95]. Globally, ATSs are the third most commonly abused class of drugs, following cannabis and opioids. In 2021, an estimated 36 million people used amphetamines, while around 20 million engaged in the misuse of ecstasy-type substances. Its variable purity and frequent adulteration further elevate health risks. Bouzoukas and colleagues developed and validated a rapid GC-MS method for the simultaneous detection of 9 amphetamine-type stimulants, 7 synthetic cathinones (SCs), and 5 phenethylamines (PEAs) in blood and urine. The method, which employs solid-phase extraction followed by derivatization, achieved complete separation of all analytes within 11 min. When applied to 46 forensic case samples, it successfully identified substances, such as methamphetamine, amphetamine, and MDMA, in 14 cases [53]. One study introduced a two-step sensor to distinguish MDMA from 2C-B, a common false positive. It accurately identified all 39 MDMA and 10 of 11 2C-B tablets [76]. Another study of 65 samples across 71 labs showed that multi-technique analysis (ASTM E2329-17) reliably detected methamphetamine and cocaine with minimal false positives, unlike inconsistent single-method results. These findings support using advanced, multi-approach systems to detect drugs in complex cases [96]. This study quantified MDMA in 20 ecstasy tablets using both manual and automated ¹H NMR, alongside GC-MS without internal standards. Manual NMR measured an average MDMA content of 42.6% w/w, while the automated method yielded 45.9% w/w. Both NMR techniques offered rapid, reliable, and standard-free quantification, demonstrating strong agreement with GC-MS results [97]. With growing MDMA use and drug-impaired driving concerns, this study highlights electrochemical detection as a promising alternative to drug screening tests Using voltammetry with carbon paste and screen-printed electrodes, MDMA was successfully detected in saliva, supporting the method as fast, simple, and reliable for forensic use [77] (Table 1).

7. New Psychoactive Substances

The rapid evolution and structural diversity of NPSs pose significant global public health and safety risks. Understanding their chemical composition is crucial for emergency treatment and forensic analysis. Leal Cunha et al. analyzed 121 drug samples (mostly ecstasy) from Bahia and Sergipe (2014–2019). GC-MS and 1D NMR identified 19 substances in the investigated samples, including MDA, synthetic cathinones, and caffeine, while MDMA was found in 57% of tablets, Brazil [98]. In another study, a novel screening strategy of NPSs was developed using LC-HRMS based on fragmentation patterns to identify both known and novel NPS analogs. Analysis of 78 seized samples revealed four ketamine-based NPSs, three of which were newly identified: the predicted positions of the phenyl substituents, based on substituent effects, were confirmed by NMR analysis [99]. Hwang et al. developed a magnetic solid phase extraction (m-SPE) method using COOH-functionalized magnetic carbon nanotubes to extract 40 NPSs (cannabinoids, phenethylamines, tryptamines) from human plasma. Analyzed by LC-QTOF-MS, it was a faster and less labor-intensive analytical approach than conventional SPE [100]. Yu Fan developed and validated a LC-MS/MS method for screening 74 phenethylamines in urine using dilute and shoot. Twenty samples tested positive for seven phenethylamines from 67 samples [101]. Birk et al. reported the first detection of semi-synthetic hexahydrocannabinol (HHC) in Scottish prison seizures (period from November 2023 to April 2024). GC-MS analysis identified HHC alongside CBD, CBN, Δ⁹-THC, and Δ⁸-THC. This study marks the first detections of the semi-synthetic cannabinoid, HHC, demonstrating its emergence within the illicit drug market [102]. ATR-FTIR rapidly screens NPS but faces challenges with structural variability and complex mixtures of NPS with adulterants or NPS on plant materials. Six machine learning models (k-Nearest Neighbors, Support Vector Machine, Random Forest, extra trees, voting, and artificial neural networks) categorized 362 NPSs into eight classes using 1099 IR spectra from lab and portable FTIR systems. HCA grouped 100 synthetic cannabinoids into eight subclasses by structural features, with ML models sub-classifying them. This work pioneers reference-free NPS classification for diverse analytical platforms [103].

Portable NIR spectroscopy (1350–2600 nm) rapidly distinguishes cathinone and phenethylamine NPS isomers in 2 s. A validated model accurately identified methylmethcathinone (MMC) and its isomers, such as 2-MMC, 3-MMC, and 4-MMC, in 51 mixtures and 22 seized samples, except at low concentrations (10 wt%). This demonstrates portable NIR’s potential for on-site NPS screening, complementing GC-MS confirmation [57]. A rapid SERS method using silver colloids detected six synthetic cathinones, combining DFT-predicted Raman spectra with experimental validation. Optimized with MgCl₂/KBr aggregating agents, it achieved sub-minute detection while identifying core peaks and analog-specific groups. This field-deployable approach offers law enforcement the practical on-site screening tool for seized drugs [58]. ⁹F NMR identified 83 fluorinated NPSs (cannabinoids, cathinones, fentanyls), with distinct chemical shifts serving as structural fingerprints. The method quantified NPSs in 17 seized products (herbal blends, e-liquids), demonstrating its specificity for fluorinated compounds [104].

An optimized synthesis using indazole-3-carboxylic acid achieved selective N1-alkylation of indazole-based synthetic cannabinoids (51–96% yields), producing nine SCs (five novel) and six known metabolites. Characterized by NMR/LC-QTOF-HRMS, this method enhances forensic standard production and drug monitoring with improved selectivity and yields over traditional approaches [105]. Additionally, the DART-MS method was applied for analyzing drug residues on discarded filter and glassine paper. Validated with 40 samples (phencyclidine-PCP, heroin, fentanyl, methylphenidate, phentermine, cocaine, methamphetamine) against glass capillaries using the NIST DART-MS Forensics Database, the method showed 90% accuracy, with errors only in tablets. It offers rapid, prep-free screening to improve lab efficiency [106].

8. Microsampling

Microsampling refers to techniques that collect less than 100 μL of human body fluids, aiming to reduce the invasiveness of sample collection [107,108]. In forensic applications, these methods offer several advantages, including simplified and minimally invasive sampling, easier transport and storage under ambient conditions, reduced costs, and minimal sample preparation, making them particularly suitable for field-based and retrospective analyses. Applications of microsampling have significantly expanded with the development of advanced techniques, such as SPME, dried blood spot (DBS), Volumetric Absorptive Microsampling (VAMS), dried matrix spotting (DMS), Biofluid Samplers (BFS), and Fabric Phase Sorptive Membrane (FPSM) arrays.

8.1. Dried Blood Spot (DBS)

DBS sampling involves collecting small volumes of whole blood, typically via a finger or heel prick, onto specially designed filter paper. Commercial DBS cards feature pre-marked circles for consistent sample collection. The process is simple, rapid, and minimally invasive, making it ideal for neonatal screening and patient self-sampling. After drying at room temperature, DBS samples can be stored and transported without refrigeration. Originally used in newborn metabolic disorder screening, DBS has expanded into therapeutic drug monitoring (TDM), especially when combined with LC-MS techniques. DBS spots typically contain ~50 µL of blood, and low LOQs are needed due to the small sample volume [109,110,111].

8.2. Volumetric Adsorptive Microsampling (VAMS)

VAMS was developed to reduce sample variability in volume and composition, while addressing the hematocrit effect often encountered in DBS analysis. VAMS uses a hydrophilic polymeric tip attached to a plastic handle to absorb a fixed volume of biological fluids, such as blood, urine, saliva, and others. Accurate collection requires the tip to contact the sample surface at a 45° angle, which is especially important for patient self-sampling. A key advantage of VAMS is its ability to consistently collect predefined volumes (e.g., 10–50 μL), enhancing reproducibility. This fixed volume influences parameters such as contact time and drying duration. Devices are usually dried for at least two hours under controlled conditions to prevent contamination. As with DBS, analyte stability on VAMS devices depends on factors like the compound’s properties and storage conditions. Due to the small sample volume, high analytical sensitivity is essential, often requiring advanced instrumentation and trained personnel for accurate quantification [112,113,114,115].

8.3. Fabric Phase Sorptive Extraction (FPSE)

FPSE, introduced in 2014, addresses key challenges of microsampling, such as matrix interference and complex cleanup. It uses sol-gel-derived membranes tailored to analyte properties, enabling high selectivity and recovery. FPSE led to the development of the Biofluid Sampler (BFS), a device compatible with various biological fluids and LC-based analysis. BFS allows uniform sample distribution, ambient storage, and solvent-efficient back-extraction, reducing costs and enhancing sensitivity. It also supports use with less sensitive instruments (e.g., HPLC-DAD) and minimizes hematocrit-related issues [116,117].

8.4. Dried Matrix Spot (DMS)

DMS techniques have evolved from the success of dried blood spot (DBS) sampling, enabling microsampling of alternative biological fluids, such as urine and saliva. Dried urine spots (DUS) were among the first adaptations, offering a simple and cost-effective alternative to conventional “dilute and shoot” methods. DMS formats offer key advantages, including simplified handling, ambient storage, and elimination of cold-chain transport [118].

9. Green Aspects of Forensic Methods

The incorporation of green analytical methods in forensic practice is gaining significant attention as laboratories seek to reduce environmental impact while maintaining rigorous analytical standards required for legal proceedings (Figure 3). Solvent-free extraction techniques, like solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE), minimize hazardous solvent use but still effectively drugs from complex matrices [119], while microwave-assisted and ultrasound-assisted extraction methods reduce both solvent consumption and processing time compared to traditional approaches [120].

Miniaturized systems, including microfluidic devices and portable spectroscopic tools, like handheld Raman and IR spectrometers, enable rapid on-site analysis with minimal reagent waste [121,122]. Chromatographic methods have also evolved, with green HPLC employing water-ethanol systems, significantly reducing toxic waste. Green GC and HPLC are increasingly being applied in forensic analysis for the detection of drugs, explosives, and toxicants, offering reliable results while minimizing hazardous solvent use and environmental impact [123]. DESI-MS and DART-MS allow solvent-free drug detection from various surfaces. In drug analysis, DART-MS offers key advantages, such as rapid, real-time, in situ identification of illicit substances without the need for sample preparation, high sensitivity to trace levels, and the ability to analyze complex mixtures directly from tablets, powders, or biological matrices, making it a powerful tool in forensic drug screening and field investigations [124]. Portable GC–MS has been employed in drug checking to identify highly potent synthetic opioids [125], including distinguishing between fentanyl analogs—something not always achievable with fentanyl test strips or other portable instruments, like FTIR in field settings. Similar techniques have also been applied to detect illicit drugs and identify adulterants in seized samples [126]. This study assessed and compared the performance of the portable GC–MS system against traditional benchtop GC–MS instrumentation, a standard procedure to evaluate the viability of portable alternatives [127].

Smartphone applications, like PhotoMetrix, improve the portability and convenience of analytical systems by converting RedGreenBlue coordinates into histograms and applying chemometric techniques, such as simple linear correlation for univariate analysis and Principal Component Analysis (PCA) for multivariate analysis [128]. These image-based methods are also greener alternatives, requiring fewer reagents, minimal sample preparation, and producing little to no hazardous waste. A typical colorimetric system includes a sample holder, light source, image-capturing device, and a computer for histogram extraction and model construction [129].

These green methods have demonstrated comparable or superior performance to conventional techniques while aligning with standards for testing laboratories (like ISO 17025), proving that environmental sustainability and forensic reliability can be successfully integrated in modern drug analysis [130,131].

Several green metrics have been developed to assess the greenness of analytical methods, including NEMI, Analytical Eco-Scale, and GAPI [132,133]. These metrics evaluate various aspects of analytical procedures, such as reagent selection, waste disposal, and energy requirements. However, existing metrics often assess the entire analytical process, making it challenging to evaluate specific steps like sample preparation. To address this, González-Martín et al. [134] proposed a new metric specifically for assessing the sustainability of sample preparation techniques. These advancements in green metrics aim to promote environmentally friendly practices in forensic analysis and analytical chemistry [134].

10. Application of Chemometrics in Forensic Analysis

Chemometrics, the application of statistical and mathematical methods to chemical data, is increasingly utilized in forensic science for more objective evidence interpretation and analysis optimization. It offers powerful tools for handling complex data in various forensic disciplines, including drug profiling, arson debris analysis, and spectral imaging. Common pattern recognition and regression prediction techniques are presented in Figure 4.

Chemometric techniques, such as exploratory analysis, supervised classification, multivariate calibration, and curve resolution methods, are commonly employed in forensic applications [135]. These methods can enhance productivity and provide additional information in complex cases. However, their implementation in routine forensic work faces challenges due to a lack of expertise among forensic scientists. UPLC-PDA, GC-MS, and HPTLC were used to profile C. sativa extracts and assess solvent effects. Multivariate analysis enabled sample classification by region and cultivar. NMR supported metabolite profiling and biomarker identification [136]. Two-dimensional gas chromatography combined with pixel-based chemometric processing has been used for chemical profiling of heroin and cannabis samples [137]. Infrared and Raman spectroscopy, coupled with chemometrics, have shown promise for on-site drug analysis and characterization, particularly in the context of harm reduction and forensic applications [138]. A comprehensive model system using Raman spectroscopy and chemometrics demonstrated the potential for reliable identification of target analytes in complex mixtures, achieving classification rates of ~90% [139]. Research based on the gas chromatography analysis of heroin samples seized from three different locations in Serbia involved chemometric approach with appropriate statistical tools (multiple-linear regression (MLR), hierarchical cluster analysis (HCA), and Wald–Wolfowitz run (WWR test) on chromatographic data of heroin samples in order to correlate and examine the origin of seized heroin samples [140]. These studies highlight the growing importance of combining advanced analytical techniques with chemometric methods for improved illicit drug analysis and profiling.

11. Conclusions

The analysis of illicit drugs, as described in this review for C. sativa, heroin, cocaine, LSD, amphetamine-type stimulants, and NPSs, has evolved significantly with advancements in analytical techniques. Traditional chromatographic methods, such as GC-MS, HPLC with different detectors, and HPTLC together with infrared spectroscopy remain foundational for their accuracy and reliability in forensic and regulatory settings. However, emerging technologies—such as portable spectroscopic tools (RS, FT-NIR), electrochemical sensors, and ambient mass spectrometry (DART-MS)—offer rapid, on-site detection with minimal sample preparation, enhancing harm reduction efforts for laboratory personnel and law enforcement. Good selectivity and low detection limits contribute to the advantages of these techniques not only for seized drugs analysis but also for the detection of drugs in body fluids. Furthermore, the application of 1D, 2D NMR, and NMR-on-a-chip system becomes more often the routine procedure either for identification of seized compounds or for their quantification given that no reference material is needed. The growing emphasis on green analytical chemistry has led to the adoption of solvent-free extraction methods (SPME, Headspace-Solid Phase Microextraction, supercritical fluid extraction), miniaturized systems, and low-energy techniques, ensuring sustainability without compromising forensic rigor. Furthermore, chemometrics has become indispensable in interpreting complex chromatographic or spectroscopic datasets, enabling precise classification, comparison of samples, drug profiling and source tracking through multivariate analysis and machine learning. Despite these advancements, challenges persist, including the need for standardized forensic protocols, improved sensitivity for trace detection, and broader accessibility of advanced tools in resource-limited settings. Future research should focus on integrating artificial intelligence for real-time data analysis, expanding databases for emerging novel psychoactive substances, and refining portable technologies for field applications. Ultimately, the convergence of analytical innovation, green chemistry principles, and computational modeling will strengthen forensic capabilities, ensuring accurate, efficient, and environmentally responsible drug analysis to face an ever-evolving illicit drug market.

12. Future Directions in Forensic Drug Analysis

The future of forensic drug analysis is poised for transformative advancements across multiple key areas, with enhanced detection of novel psychoactive substances leveraging AI-driven predictive modeling to anticipate emerging drug trends, expanded spectral libraries for comprehensive identification of new compounds and advanced separation techniques, e.g., 2D-LC and ion mobility spectrometry for isomer-specific differentiation. An overview of the advantages and disadvantages of commonly used analytical methods for the detection and characterization of narcotic drugs is provided, highlighting their strengths, limitations, and practical considerations in forensic science (

Table 3. Advantages and disadvantages of analytical methods in forensic drug analysis.

Method	Advantages	Disadvantages
FTIR	Rapid and non-destructive, minimal sample preparation.	Limited sensitivity, less effective in mixtures, requires reference spectra.
RS	Non-destructive and portable, effective through containers (e.g., glass/plastic), minimal sample preparation.	Fluorescence interference, limited quantitative ability, expensive equipment.
GC-MS	High sensitivity and specificity, excellent for complex mixtures	Requires volatile/thermally stable compounds, long analysis time, requires skilled operator.
GC-FID	Sensitive for organic compounds, quantitative analysis, cost-effective	Lacks compound identification, requires standards, not suitable for non-volatile drugs.
HPLC-DAD	Suitable for thermally labile and non-volatile drugs, high resolution and precision, good reproducibility.	Limited compound identification, requires extensive method development, solvent use and disposal issues.
HPLC-MS	High sensitivity and selectivity, capable of structural elucidation, detects low concentrations.	High cost, requires expert handling, matrix effects may interfere
Sensors	Low-cost and portable, rapid response, good for field testing.	Lower selectivity and sensitivity, limited lifetime and stability, calibration challenges
CV	Sensitive to redox-active drugs, small sample volume, Fast analysis	Limited selectivity, not suitable for complex mixtures, complex interpretation of data
HPTLC	Fast and inexpensive, parallel analysis of multiple samples, minimal solvent use	Lower sensitivity and resolution, not ideal for complex matrices
NMR	Excellent structural elucidation, non-destructive, capable for complex mixtures	High cost, Low sensitivity, Requires large sample amounts and trained staffs

).

Portable and on-site analytical technologies will see significant development through next-generation handheld devices, such as miniaturized GC-MS and NMR-on-a-chip, alongside non-destructive rapid screening methods, like surface-enhanced Raman spectroscopy (SERS) and hyperspectral imaging, as well as biosensors and wearable detectors for continuous drug monitoring. Green and sustainable forensic methods will gain traction with solvent-free techniques, like supercritical fluid chromatography (SFC) and DART-MS, biodegradable sorbents for eco-friendly extraction, and energy-efficient portable analyzers for remote applications. The integration of chemometrics and big analytical datasets will favor drug profiling through automated data fusion of multiple analytical techniques. The exchange of data derived from profiling processes can contribute to global tracking of trafficking networks. This integration contributes to recovering hidden features and more comprehensive conclusions in the forensic analysis of seized drugs. Forensic toxicology and metabolomics will advance with high-resolution mass spectrometry (HRMS) for metabolite discovery, microsampling techniques, like dried blood spots (DBS), and personalized toxicology through genetic profiling. Finally, combating drug adulteration and counterfeits may involve rapid screening technologies, such as portable X-ray diffraction (pXRD) and 3D-printed drug standards for improved calibration, collectively driving the field toward more efficient, sustainable, and precise drug analysis.