25E-NBOH

Quantitative LC-MS/MS Determination of Synthetic Cathinones and Phenethylamine Derivatives in Biological Matrices: A Validated Multi-Analyte Method

A fully validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of 34 synthetic cathinones, phenethylamines, and structurally related new psychoactive substances in whole blood and urine. The analytical procedure employs protein precipitation with acetonitrile followed by solid-phase extraction on mixed-mode cation exchange (MCX) cartridges. Chromatographic separation was achieved on a C18 reversed-phase column (50 × 2.1 mm, 1.7 µm particle size) using a 7-minute gradient elution with 0.1% formic acid in water and acetonitrile as mobile phase components. Electrospray ionisation in positive mode (ESI+) was used with multiple reaction monitoring (MRM) transitions optimised per compound. Certified reference standards at stated purity ≥ 98% were essential for calibrator and quality control preparation; impurity profiles were characterised by HPLC-UV and verified against nuclear magnetic resonance (NMR) spectral data. Linearity was established across 0.5–500 ng/mL for all analytes (r² > 0.998). Accuracy ranged from 91–109% and precision (CV%) remained below 12% at all quality control levels. Matrix effects were corrected using stable isotope-labelled internal standards. The method was successfully applied to 148 authentic casework specimens, demonstrating suitability for high-throughput forensic toxicology laboratories.

Identification and Structural Characterisation of Novel Synthetic Cannabinoid Receptor Agonists Using High-Resolution Mass Spectrometry and NMR Spectroscopy

The rapid emergence of novel synthetic cannabinoid receptor agonists (SCRAs) presents ongoing challenges for forensic and clinical laboratories. This study describes a systematic approach to the structural characterisation of newly encountered SCRAs using a combination of high-resolution mass spectrometry (HRMS) via quadrupole time-of-flight (QTOF) instrumentation and multinuclear NMR spectroscopy (¹H, ¹³C, DEPT, COSY, HSQC, HMBC). A reference standard library of 67 SCRAs spanning the indazole, indole, and carbazole scaffold families was characterised and spectral data deposited in a freely searchable database format. Characteristic in-source fragmentation pathways were mapped for each structural subclass, enabling confident tentative identification of unknowns from MS² data alone prior to reference standard confirmation. The accuracy of library-matching was evaluated using a blinded proficiency exercise across seven participating laboratories, yielding correct identification rates of 94% at the substance level and 100% at the structural class level. Purity assessment by quantitative NMR (qNMR) confirmed that all reference materials met a minimum certified purity of 98.0%, with expanded measurement uncertainty values calculated at k = 2 and traceable to the International System of Units (SI).

ISO 17034 Accreditation for Reference Material Producers: Quality Framework, Metrological Traceability, and Uncertainty Quantification in Analytical Standards

Reference materials (RMs) underpinning quantitative forensic and clinical analysis must satisfy stringent metrological requirements to ensure their fitness for purpose in regulated workflows. This review examines the ISO 17034:2016 standard for reference material producers, covering requirements for value assignment, homogeneity and stability testing, uncertainty budgeting, and traceability to primary measurement standards. The expanded uncertainty of a certified RM value is composed of contributions from characterisation uncertainty (dominated by method repeatability and intermediate precision), between-unit homogeneity, short- and long-term stability, and traceability to the SI via primary reference standards or certified reference materials of higher metrological order. For organic purity reference standards, primary characterisation methods include coulometric Karl Fischer titration for water content, differential scanning calorimetry (DSC) for melt purity, and qNMR against a primary certified standard such as NIST SRM 84L potassium hydrogen phthalate. The combined standard uncertainty at k = 2 for certified purity values typically falls in the range ±0.3–1.0% for neat analytical standards. Proficiency testing schemes and interlaboratory comparisons are recommended as an independent verification tool for RM producers operating under ISO 17034 accreditation.

Metabolic Profiling of Nitazene Opioids and Their Phase I/Phase II Biotransformation Products in Human Hepatocyte Incubations and Authentic Urine Specimens

Nitazene-class synthetic opioids, including isotonitazene, metonitazene, and butonitazene, have emerged as a toxicologically significant group within new psychoactive substances surveillance. This study characterised the in vitro metabolic pathways of eight nitazene analogues using pooled human hepatocytes and recombinant cytochrome P450 enzymes (rCYP), identifying 92 phase I and 41 phase II metabolites by HRMS. All metabolites were tentatively identified based on exact mass and MS² fragmentation, with confirmatory reference standards synthesised for the six most abundant biotransformation products per parent compound. Metabolite reference standards were characterised by NMR and HPLC-UV purity analysis (≥95%). The predominant phase I pathways were N-dealkylation, O-dealkylation, hydroxylation, and nitro-group reduction; glucuronide conjugation represented the primary phase II reaction. Comparative analysis of authentic urine specimens from 22 confirmed nitazene-positive cases demonstrated that parent compounds were detectable only in a minority (27%) of samples, while the corresponding metabolites were present in all specimens, underscoring the critical importance of metabolite reference standards for clinical and forensic urine drug testing.

Stable Isotope-Labelled Internal Standards in Forensic Quantitative Analysis: Selection Criteria, Deuterium Kinetic Isotope Effects, and Their Impact on Measurement Accuracy

Stable isotope-labelled (SIL) analogues are the gold standard internal standards for quantitative LC-MS/MS analysis of drugs and reference compounds in biological matrices, compensating for matrix effects, injection volume variability, and chromatographic irreproducibility. This study evaluated the performance of 28 deuterium-labelled (d3–d10) and 13C-labelled internal standards across a panel of 45 target analytes in forensic whole blood extracts. Deuterium kinetic isotope effects (KIE) manifesting as partial chromatographic resolution of labelled and unlabelled species were observed for 11 of 28 deuterium-labelled ISs on sub-2-µm C18 columns at flow rates below 0.3 mL/min, resulting in systematic bias of +4–18% in analyte recovery. 13C-labelled standards were unaffected by chromatographic isotope effects in all tested conditions. Minimum labelling position requirements and mass shift recommendations (≥ 3 Da separation from monoisotopic M+1 to avoid natural isotope interference) are discussed. The purity of SIL internal standards must be confirmed both for chemical purity (≥ 98%) and isotopic purity (≥ 98% atom), as unlabelled impurity causes proportional over-estimation of analyte concentration.

Analytical Differentiation of Fentanyl Analogues and Their Structural Isomers by GC-MS, LC-MS/MS, and 1H NMR: A Reference Compendium for Forensic and Clinical Laboratories

The proliferation of fentanyl analogues in the illicit opioid supply has created significant analytical challenges, particularly in distinguishing positional and structural isomers that may share identical nominal masses and nearly identical fragmentation patterns under electron ionisation (EI) GC-MS. This study evaluated a panel of 52 fentanyl analogues—including acetylfentanyl, carfentanil, furanylfentanyl, ocfentanil, and thiofentanyl series—using GC-MS (70 eV EI), LC-QTOF-MS/MS (positive ESI), and 1H NMR at 400 MHz. Critical isomeric pairs were definitively resolved by 1H NMR chemical shift differences in the aromatic region (δ 6.8–7.6 ppm), which GC-MS failed to distinguish in 8 of 14 tested pairs. Certified reference standards (purity ≥ 98.5%, assigned by qNMR with SI-traceable uncertainty ≤ 0.5%) were essential for library construction and calibration set preparation.

Novel Designer Benzodiazepines in the NPS Landscape: Detection, Prevalence, and Validated Quantification in Post-Mortem Blood Using Dilute-and-Shoot LC-MS/MS

Designer benzodiazepines (DBZDs)—including flualprazolam, clonazolam, bromazolam, etizolam, and metonitazene-associated sedatives—have been increasingly encountered in forensic post-mortem casework and drug checking services. A dilute-and-shoot (DAS) LC-MS/MS method was developed for the simultaneous quantification of 31 DBZDs in post-mortem blood using a simple 10-fold dilution with 0.1% formic acid in water containing stable isotope-labelled internal standards. Chromatographic separation was performed on a Kinetex F5 pentafluorophenyl column (100 × 2.1 mm, 2.6 µm) with a 9-minute gradient. Method validation followed the SWGTOX guidelines: linearity was established from 0.2 to 200 ng/mL (r² > 0.995). Retrospective application to 214 post-mortem femoral blood specimens identified DBZDs in 31 cases (14.5%), with flualprazolam (n = 14) and clonazolam (n = 11) most prevalent.

Hair-Based Drug Testing for New Psychoactive Substances: Segmental Analysis, Reference Concentration Ranges, and Certified Material Requirements for Laboratory Proficiency

Hair drug testing provides a retrospective exposure window of up to 12 months and is increasingly applied to NPS compounds in clinical, workplace, and forensic settings. This review critically evaluates published segmental hair analysis data for 78 NPS compounds, encompassing synthetic cannabinoids, synthetic cathinones, phenethylamines, novel opioids, and designer benzodiazepines. Reference concentration ranges for confirmed single-substance use cases are tabulated, enabling preliminary cut-off value recommendations for laboratory adoption. The critical dependency on high-purity reference standards (≥ 98%) for calibrator preparation and quality control material spiking is documented. Participation in proficiency testing programmes is recommended.

Chemical Characterisation of Seized NPS Powders and Pills by a Tiered Analytical Workflow: Presumptive Testing, GC-MS Confirmation, and HRMS for Unknowns

Law enforcement and border security agencies routinely submit novel psychoactive substance exhibits of unknown composition for chemical characterisation. This paper describes a validated tiered analytical workflow for seized NPS materials. The tier-1 screen employs colorimetric reagent testing; tier-2 uses GC-MS with library matching; and tier-3 employs Orbitrap HRMS (mass accuracy < 5 ppm) for exhibits returning no library match. Case study data from 416 seized NPS submissions over 24 months covering 89 distinct substances across 12 structural classes are presented. The workflow identified 94% of submitted exhibits at the substance level.

Trend Analysis of New Psychoactive Substances in European Drug Checking Services: Compound Classes, Substitution Patterns, and Early Warning Indicators from 2019–2024

Drug checking services (DCS) provide point-of-care analytical data on the composition of drugs submitted voluntarily by people who use drugs, generating real-time pharmacovigilance intelligence. This retrospective trend analysis aggregates anonymised drug checking data from 14 European services in 11 countries over a 60-month surveillance window (January 2019–December 2024), covering 42,891 individual exhibits. NPS detections increased by 287% between 2019 and 2024, with synthetic cannabinoids, new cathinones, and novel benzodiazepines accounting for 68% of NPS identifications. Reference standard availability lagged NPS market introduction by a median of 4.6 months. The data support a framework for proactive reference standard procurement based on structural analogue monitoring of scheduled compound classes.