Drug Clozapine Research Articles

Metabolic Side Effects from Antipsychotic Treatment with Clozapine Linked to Aryl Hydrocarbon Receptor (AhR) Activation.

Metabolic syndrome (MetS) is the most common adverse drug reaction from psychiatric pharmacotherapy. Neuroreceptor blockade by the antipsychotic drug clozapine induces MetS in about 30% of patients. Similar to insulin resistance, clozapine impedes Akt kinase activation, leading to intracellular glucose and glutathione depletion. Additional cystine shortage triggers tryptophan degradation to kynurenine, which is a well-known AhR ligand. Ligand-bound AhR downregulates the intracellular iron pool, thereby increasing the risk of mitochondrial dysfunction. Scavenging iron stabilizes the transcription factor HIF-1, which shifts the metabolism toward transient glycolysis. Furthermore, the AhR inhibits AMPK activation, leading to obesity and liver steatosis. Increasing glucose uptake by AMPK activation prevents dyslipidemia and liver damage and, therefore, reduces the risk of MetS. In line with the in vitro results, feeding experiments with rats revealed a disturbed glucose-/lipid-/iron-metabolism from clozapine treatment with hyperglycemia and hepatic iron deposits in female rats and steatosis and anemia in male animals. Decreased energy expenditure from clozapine treatment seems to be the cause of the fast weight gain in the first weeks of treatment. In patients, this weight gain due to neuroleptic treatment correlates with an improvement in psychotic syndromes and can even be used to anticipate the therapeutic effect of the treatment.

B-303 Stability of Antipsychotic Drugs in Patient Serum and Plasma Clinical Samples

Abstract Background Therapeutic drug monitoring (TDM) of antipsychotic blood levels is strongly recommended (clozapine and olanzapine) and recommended (risperidone, aripiprazole, and quetiapine) by ASCP/AGNP Consensus Statement. For effective TDM, it is paramount to understand both the stability of the antipsychotic drugs in serum and plasma, as well as any differences in levels due to sample matrix to ensure robust concentration determinations. While clozapine levels are considered equivalent in serum and plasma, we included in our study four additional antipsychotics. Methods Whole blood samples from ten patients taking clozapine, risperidone, aripiprazole, olanzapine, or quetiapine were collected in one red top tube and in one purple top tube according to an IRB-approved protocol. Samples were then processed within 2 hours of collection to obtain matched serum and K2EDTA plasma samples from each of the patients. CE marked immunoassays for the measurement of clozapine (also FDA cleared), total risperidone/paliperidone, total aripiprazole, olanzapine, and quetiapine were used to quantitate drug concentrations. Stability of the samples was assessed at 4°C, ambient room temperature (ART), and frozen at -80°C, with respect to their Day 0 concentration by measurement of six replicates at each timepoint using the immunoassays. Results Comparison of Day 0 values between serum and plasma for the same patient showed ±9% bias between sample matrices for all drugs, except olanzapine which showed differences up to ±15%. Average bias between serum and plasma was ≤-5% (olanzapine: -9%). All drugs were stable (±15% deviation from Day 0; olanzapine up to ±23%) in serum and in plasma for 7 days at both 4°C and ART and for up to 8 months frozen. Conclusions This study demonstrated that (1) the antipsychotic drugs clozapine, risperidone, aripiprazole, olanzapine, and quetiapine have sufficient stability in both serum and plasma for effective TDM and (2) for TDM, serum or plasma may be used.

Highly sensitive MXene-based voltammetric sensor for nanomolar clozapine detection

Clozapine is vital for managing schizophrenia and other psychiatric disorders. As a potent medication with risks of severe side effects, accurate detection ensures effective treatment monitoring, patient safety, and optimization of therapeutic outcomes. Herein, niobium carbide two-dimensional transition metal carbides (Nb2CTx MXene) was prepared by etching niobium aluminum carbide (Nb2AlC), which was next partially oxidized by a hydrothermal method to niobium oxide (Nb2O5)/Nb2CTx and ultrasonically bound to carboxylated multi-walled carbon nanotubes (MWCNTs-COOH). It was modified onto a glassy carbon electrode for the detection of the antipsychotic drug clozapine ingested by humans. The morphology and electrochemical properties of MXene-based nanocomposite electrode were verified by scanning electron microscopy, X-ray diffractometry, Raman, cyclic voltammetry and electrochemical impedance characterization methods. The results showed that electrode responded well to clozapine solutions, with good linearity (R2 = 0.9979 and 0.9995) of the peak currents with clozapine concentrations (0.01–1.0 μM and 1.0–10.0 μM) and that this sensor had good immunity, repeatability, reproducibility and stability. The limit of detection was 8.38 nM and the recovery was 92.5 % ~ 106.5 % in real serum and urine samples. The MXene-based sensors offer excellent analytical performance and hold promise for cost-effective generalisation to the detection of nanomolar clozapine in a variety of clinical applications.

Molecular Docking Study of Various Protein on Clozapine

The antipsychotic drug Clozapine is widely recognized for its efficacy in treating resistant schizophrenia. This study aims to elucidate the binding interactions of Clozapine with four key proteins: 1GQ1, 1MKF, 4OAJ, and 7WU2. These proteins represent diverse biological targets, including receptors and enzymes relevant to neurological function and drug metabolism. Utilizing molecular docking simulations, we compared the binding affinities and interaction profiles of Clozapine with these proteins.

Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

Antipsychotic drugs selectively decorrelate long-range interactions in deep cortical layers.

Psychosis is characterized by a diminished ability of the brain to distinguish externally driven activity patterns from self-generated activity patterns. Antipsychotic drugs are a class of small molecules with relatively broad binding affinity for a variety of neuromodulator receptors that, in humans, can prevent or ameliorate psychosis. How these drugs influence the function of cortical circuits, and in particular their ability to distinguish between externally and self-generated activity patterns, is still largely unclear. To have experimental control over self-generated sensory feedback, we used a virtual reality environment in which the coupling between movement and visual feedback can be altered. We then used widefield calcium imaging to determine the cell type-specific functional effects of antipsychotic drugs in mouse dorsal cortex under different conditions of visuomotor coupling. By comparing cell type-specific activation patterns between locomotion onsets that were experimentally coupled to self-generated visual feedback and locomotion onsets that were not coupled, we show that deep cortical layers were differentially activated in these two conditions. We then show that the antipsychotic drug clozapine disrupted visuomotor integration at locomotion onsets also primarily in deep cortical layers. Given that one of the key components of visuomotor integration in cortex is long-range cortico-cortical connections, we tested whether the effect of clozapine was detectable in the correlation structure of activity patterns across dorsal cortex. We found that clozapine as well as two other antipsychotic drugs, aripiprazole and haloperidol, resulted in a strong reduction in correlations of layer 5 activity between cortical areas and impaired the spread of visuomotor prediction errors generated in visual cortex. Our results are consistent with the interpretation that a major functional effect of antipsychotic drugs is a selective alteration of long-range layer 5-mediated communication.

Antipsychotic drugs selectively decorrelate long-range interactions in deep cortical layers

Psychosis is characterized by a diminished ability of the brain to distinguish externally driven activity patterns from self-generated activity patterns. Antipsychotic drugs are a class of small molecules with relatively broad binding affinity for a variety of neuromodulator receptors that, in humans, can prevent or ameliorate psychosis. How these drugs influence the function of cortical circuits, and in particular their ability to distinguish between externally and self-generated activity patterns, is still largely unclear. To have experimental control over self-generated sensory feedback, we used a virtual reality environment in which the coupling between movement and visual feedback can be altered. We then used widefield calcium imaging to determine the cell type-specific functional effects of antipsychotic drugs in mouse dorsal cortex under different conditions of visuomotor coupling. By comparing cell type-specific activation patterns between locomotion onsets that were experimentally coupled to self-generated visual feedback and locomotion onsets that were not coupled, we show that deep cortical layers were differentially activated in these two conditions. We then show that the antipsychotic drug clozapine disrupted visuomotor integration at locomotion onsets also primarily in deep cortical layers. Given that one of the key components of visuomotor integration in cortex is long-range cortico-cortical connections, we tested whether the effect of clozapine was detectable in the correlation structure of activity patterns across dorsal cortex. We found that clozapine as well as two other antipsychotic drugs, aripiprazole and haloperidol, resulted in a strong reduction in correlations of layer 5 activity between cortical areas and impaired the spread of visuomotor prediction errors generated in visual cortex. Our results are consistent with the interpretation that a major functional effect of antipsychotic drugs is a selective alteration of long-range layer 5-mediated communication.

Chromatographic Methods for the Analysis of the Antipsychotic Drug Clozapine and Its Major Metabolites: A Review.

Clozapine (CLZ), a second-generation antipsychotic, can effectively reduce schizophrenia, bipolar disorder and major depression symptoms. This review provides an overview of all reported chromatographic methods (62 references) for the quantification of CLZ and its two main metabolites, norclozapine and clozapine N-oxide in pharmaceutical formulations, biological matrices and environmental samples.

Probes for the heterogeneity of muscimol binding sites in rat brain.

Introduction: The plant-based alkaloid muscimol is a potent agonist of inhibitory GABAA-neurotransmitter receptors. GABAA receptors are a heterogeneous family of pentameric complexes, with 5 out of 19 subunits assembling around the central anion pore. Muscimol is considered to bind to all receptor subtypes at the orthosteric drug binding site at the β+/α- interface. Recently, we observed that the antipsychotic drugs clozapine (CLZ), loxapine (LOX) and chlorpromazine (CPZ) although exerting functional inhibition on multiple GABAA receptor subtypes showed diverging results in displacing 3H-muscimol. While a complete displacement could be observed in hippocampal membranes by bicuculline (BIC), and no displacement with CPZ, the compounds CLZ and LOX competed partially. Non-sigmoidal, complex dose response curves were indicative of multiple sites. In the current study we now aimed to investigate more extensively this heterogeneity of bicuculline sensitive muscimol sites in rat brain. Methods: We tested membranes from four different brain regions (hippocampus, cerebellum, thalamus and striatum) and selected recombinantly expressed subunit combinations with displacement assays. 3H-muscimol displacement was tested with BIC, LOX, CLZ and CPZ. In silico ligand structural analysis and computational docking was performed. Results: We observed a unique pharmacology of each tested compound in the studied brain regions. Combining two of the tested ligands suggests that in striatum all CLZ sites are contained in the pool of LOX sites, while the CPZ sites may in part be non-overlapping with LOX sites. Experiments on recombinantly expressed receptors indicate, that BIC can displace 3H-muscimol from all tested receptors, while LOX and CLZ display different and variable competition indicative of multiple sites. Molecular docking produced structural correlates of the observed diversity of muscimol sites on the basis of bicuculline bound experimental structures. Discussion: These findings indicate that 3H-muscimol binding sites in rat brain are heterogeneous, with different populations of receptors, which are CPZ, LOX or CLZ sensitive or insensitive. These binding sites show a varying distribution in different rat brain regions. Molecular docking suggests that the so-called loop F region of α subunits drives the observed differences.

Therapeutic drug monitoring of clozapine in human serum by high-throughput paper spray mass spectrometry

IntroductionMonitoring the atypical antipsychotic drug clozapine is crucial to ensure patient safety. This article showcases a high-throughput analytical method for measuring clozapine and its primary metabolite norclozapine (N-desmethylclozapine) in serum using paper spray mass spectrometry (PS-MS). ObjectivesThis study aimed to assess the viability of a PS-MS method for the rapid measurement of clozapine and norclozapine in human serum samples as an alternative to liquid chromatography mass spectrometry (LC-MS). MethodsSerum samples were processed by protein precipitation followed by deposition of the supernatant containing labelled internal standards onto paper spray substrates mounted in cartridges. Analytes were then analyzed using a triple quadrupole mass spectrometer equipped with a commercial paper spray ionization source. The results obtained from the patient samples were compared to those from a validated LC-MS assay. ResultsPS-MS calibrations for clozapine and norclozapine were linear (R2 > 0.99) over five days. Between-run precision was below 8 %, and within-run precision did not exceed 10 %. When compared to a validated LC-MS method, the mean bias for 39 patient samples was −9% for clozapine and −1% for norclozapine, with no outliers. Mass spectrometry ion ratio comparisons indicated no interference for patient samples above the lower limit of quantification. There was less than 7 % change in the measured concentrations of both analytes over five days for samples dried on paper substrates. Notably, virtually no maintenance of the MS source was required during this study. ConclusionThis study illustrates the potential of PS-MS for serum drug monitoring in the clinical laboratory.

Removal of clozapine from polluted waters via C₃N₄ based materials

New photocatalysts with a g-C₃N₄-ZnO heterojunction were prepared following different synthetic routes, fully characterized, and evaluated for the abatement of the antipsychotic drug clozapine. Experiments were performed with clozapine standard and clozapine based commercial tablet solutions in a sun simulator and the evaluation of clozapine removal was assessed in different aqueous media: Milli-Q and natural water (Po River) to evaluate the role of natural occurring components on the drug removal efficiency. The photocatalyst prepared via hydrothermal method (CA_1) exhibited the highest efficiency, allowing to achieve the complete abatement of clozapine within 30 min, in Milli-Q water. Its photocatalytic performance decreased in river water, where the total drug degradation was obtained after1h, and is notably inhibited on Leponex tablets (80% removal after 1 h of irradiation). Clozapine degradation proceeds through the formation of nineteen (19) transformation products, all less hazardous that the parent compound, as proved by the acute and chronic toxicity valued by ECOSAR. Mechanistic studies evidenced that OH⦁ radicals are the key reactive species involved in the pollutant removal, while other species such as superoxide radical anion (O₂•⁻) and especially singlet oxygen (O₂¹) played a marginal role in the degradation process.

Metal-catalyzed coupling/carbonylative cyclizations for accessing dibenzodiazepinones: an expedient route to clozapine and other drugs.

A sequential strategy to access 10,11-dihydro-5H-dibenzo[b,e][1,4]diazepinones (DBDAPs) is disclosed in this article through a palladium and copper-catalyzed amination (Buchwald-Hartwig (B-H) or Chan-Lam (C-L)) followed by a palladium-catalyzed intramolecular aminocarbonylation with Mo(CO)6 as CO surrogate (to avoid toxic CO handling) of readily available o-phenylenediamines and either 1,2-dibromobenzene or 2-bromophenylboronic acid. The 10,11-dihydro-5H-dibenzo[b,e][1,4]diazepinone could be synthezised in good yield using a sequential catalytic procedure, using both C-L and B-H approaches. Gratifingly, the use of the C-L reaction was more impressive, and afforded the dibenzodiazepinones in good yields (up to 45%; 2 steps) and much milder conditions using copper as the catalyst. The synthetic utility of this novel strategy was showcased by demonstrating a formal synthesis for the antipsychotic drug clozapine and to an anticancer triazole-DBDAP hybrid.

The effect of lithium on clozapine-induced neutropenia reduction in refractory schizoaffective disorder

Resistant schizoaffective disorder has persistent psychotic symptoms and intermittent mood swings. A satisfying method for treating these patients is the use of the antipsychotic drug clozapine, but its use is limited due to significant side effects such as leukopenia and neutropenia. Lithium carbonate is another psychiatric drug that reduces these side effects. We hypothesized that lithium, along with clozapine, in addition to controlling side effects, could have a dual effect on mood episodes in schizoaffective disorder, and used it in a chronic case. The patient was a 48-year-old man with a 20-year history of the disease. Therefore, the use of lithium along with clozapine is recommended in refractory schizoaffective patients.

Human Hemoglobin and Antipsychotics Clozapine, Ziprasidone and Sertindole: Friends or Foes?

Packed with hemoglobin, an essential protein for oxygen transport, human erythrocytes are a suitable model system for testing the pleiotropic effects of lipophilic drugs. Our study investigated the interaction between antipsychotic drugs clozapine, ziprasidone, sertindole, and human hemoglobin under simulated physiological conditions. Analysis of protein fluorescence quenching at different temperatures and data obtained from the van't Hoff diagram and molecular docking indicate that the interactions are static and that the tetrameric human hemoglobin has one binding site for all drugs in the central cavity near αβ interfaces and is dominantly mediated through hydrophobic forces. The association constants were lower-moderate strength (~104 M-1), the highest observed for clozapine (2.2 × 104 M-1 at 25 °C). The clozapine binding showed "friendly" effects: increased α-helical content, a higher melting point, and protein protection from free radical-mediated oxidation. On the other hand, bound ziprasidone and sertindole had a slightly pro-oxidative effect, increasing ferrihemoglobin content, a possible "foe". Since the interaction of proteins with drugs plays a vital role in their pharmacokinetic and pharmacodynamic properties, the physiological significance of the obtained findings is briefly discussed.

Electrochemistry Test Strip as Platform for In Situ Detection of Blood Levels of Antipsychotic Clozapine in Finger-Pricked Sample Volume

With the increasing number of patients suffering from Parkinson's disease, the importance of measuring drug levels in patient body fluids has increased exponentially, particularly for the drug clozapine. There is a growing demand for real-time analysis of biofluids on a single low-cost platform in ultra-low fluid volumes with robustness. This study aims to measure the level of clozapine (Clz) with a portable potentiostat using a practical approach. For this purpose, we developed an inexpensive, portable platform via electrochemistry on a commercial glucose test strip (CTS). CTSs were first modified by removing the enzyme mixture from the surface of the sensing zone, which was followed by modification with Multi walled carbon nanotube (MWCNT) and Nafion. The electrochemical characteristics of CTS electrodes were investigated using cyclic voltammetry (CV) and differential voltammetry (DPV) techniques. The designed sensor displayed decent linear range, detection limit, reproducibility, and reusability results. A linear dynamic range of 0.1-5 μM clozapine was observed under optimized conditions with a good sensitivity (1.295 μA/μM) and detection limit (83 nM). Furthermore, the designed sensing electrode was used to measure the amount of Clz in real samples.

Application of nickel-doped graphene nanotubes to modified GCE as a sensitive electrochemical sensor for the antipsychotic drug clozapine in spiked human blood serum samples

Clozapine (CLZ) is one of the most vital medications for managing schizophrenia, and the timely measurement of CLZ levels has been recognized as an obstacle to the wider use of CLZ. Herein, for the first time, nickel-doped graphene nanotubes (Ni@GRNT) were used to construct an electrochemical CLZ sensor by drop coating Ni@GRNT suspension on a glassy carbon electrode. The Ni@GRNT was synthesized and characterized using X‐ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. The electrochemical behavior and influence of different physicochemical factors of sensing electrodes were investigated by using cyclic voltammetry, EIS technique, and differential pulse voltammetry techniques. Also, the catalytic rate constant (kcat) and the transfer coefficient (α) were calculated. The modified electrode illustrated satisfactory linear range, detection limit (LOD), reusability, and reproducibility results. At optimal experimental conditions, measurements can be performed at a broad linear dynamic range of 0.3 nmol L−1–60.0 μmol L−1 CLZ and with a LOD of 0.1 nmol L−1. The sensitivity value was estimated to be 3.06 μA µmol L−1 cm−2. Ultimately, this platform was successfully used for CLZ sensing in spiked human blood serum and tablet samples with an accuracy of > 93%.Graphical abstract

Identification of the medial prefrontal circuit responsive to the atypical antipsychotic drug clozapine.

Identification of the medial prefrontal circuit responsive to the atypical antipsychotic drug clozapine.

P10.13.B Repurposing neuroleptics to treat brain metastases: Clozapine as a novel therapy for melanoma brain metastases

Abstract Background Around half of advanced melanoma patients develop brain metastases (BM) and survival is still measured in months. Current treatments, although increasingly effective, are not sufficient for this patient group, necessitating development of new therapeutic options. Drugs targeting BM specifically are a scarcity. However, utilizing drug repurposing, i.e. novel uses of drugs already approved for other medical conditions, may broaden available therapy options. Medications used to treat psychosis can penetrate an intact blood-brain barrier and it has been shown that patients with schizophrenia have lower incidences of certain cancer types than the rest of the population. Still, little is known on antitumor effects of neuroleptics on BM. We have preliminary data showing that the antipsychotic drug clozapine effectively inhibits growth of human melanoma brain metastasis (MBM) cell lines in vitro. Material and Methods We used the H1, H2, H3 and H10 MBM cell lines, as well as normal human astrocytes (NHA). Monolayer viability and IncuCyte studies were performed to determine IC50 doses. Cell migration was measured by a scratch wound assay. Apoptosis was studied by flow cytometry. Colony formation assays were performed using 2D clonogenic assays and 3D soft agar models. A protein array was used to find signaling proteins potentially affected by clozapine. The effect of clozapine on the normal brain was assessed, using a novel brain organoid model developed from fetal rat brains. Results A viability screening using 6 different neuroleptics showed that clozapine was most effective in inhibiting MBM cell viability, while NHA tolerated the same doses. MBM cells exhibited a dose-dependent inhibition of cell proliferation, migration, clonogenic formation and 3D growth. Flow cytometry demonstrated that apoptosis was a major mechanism of cytotoxicity. The protein array showed that CCL3, IL8 and VEGF were downstream targets of clozapine. These results are currently being confirmed by western blots. Doses significantly above MBM IC50 doses did not affect viability of brain organoids. Currently, a partnership with the Dept. of Chemistry (UiB) has been initiated to develop and test novel clozapine derivates, to find a more effective derivative with a larger therapeutic window and lesser side effects. Conclusion Clozapine shows promise as a novel agent for use in cancer therapy due its favorable tolerability, small molecular size and established clinical safety.

Clozapine reduces chemokine-mediated migration of lymphocytes by targeting NF-κB and AKT phosphorylation

Multiple sclerosis is a disease characterised by demyelination of axons in the central nervous system. The atypical antipsychotic drug clozapine has been shown to attenuate disease severity in experimental autoimmune encephalomyelitis (EAE), a mouse model that is useful for the study of multiple sclerosis. However, the mechanism of action by which clozapine reduces disease in EAE is poorly understood. To better understand how clozapine exerts its protective effects, we investigated the underlying signalling pathways by which clozapine may reduce immune cell migration by evaluating chemokine and dopamine receptor-associated signalling pathways. We found that clozapine inhibits migration of immune cells by reducing chemokine production in microglia cells by targeting NF-κB phosphorylation and promoting an anti-inflammatory milieu. Furthermore, clozapine directly targets immune cell migration by changing Ca2+ levels within immune cells and reduces the phosphorylation of signalling protein AKT. Linking these pathways to the antagonising effect of clozapine on dopamine and serotonin receptors, we provide insight into how clozapine alters immune cells migration by directly targeting the underlying migration-associated pathways.

Improving analysis of urine samples from drug-facilitated sexual assault cases

In drug-facilitated sexual assaults (DFSA) the victim is incapacitated by involuntary or voluntary substance consumption. Unfortunately, drug analysis of samples from DFSA is often complicated by late reporting and rapid drug metabolism. Thus, it can be challenging to detect relevant drugs in samples collected more than 24 hours after the assault and sensitive analytical methods are therefore essential. Urine samples provides a longer detection window compared to blood samples. However, many drugs are excreted in urine as conjugated metabolites and sample pretreatment commonly includes an overnight enzymatic hydrolysis under heating conditions. Investigations on the pretreatment conditions and the effects on drug stability provides valuable knowledge in these settings. The aim of this study was to improve enzymatic pretreatment of urine samples and investigate efficiency of a β-glucuronidase/arylsulfatase enzyme using morphine-6-glucuronide as test compound. Additionally, we investigated the stability of 110 drugs and metabolites relevant to DFSA under the optimal pretreatment conditions. To test enzyme efficiency, drug-free urine samples were spiked with morphine-6-glucuronide (M6G) and enzymatically hydrolyzed with β-glucuronidase/arylsulfatase (Helix pomatia, Roche) for 2, 4, 6 and 18 hours at 37 °C. To test drug stability, additional samples spiked with morphine were incubated for 18 hours at 37 °C without enzymatic treatment. After incubation, the enzymatic hydrolysis was stopped by protein precipitation before the extracts were analyzed by UHPLC–MS/MS. To test drug stability for the optimal enzyme incubation time, drug-free urine was collected and spiked with 110 DFSA relevant drugs and metabolites. Samples were either 1) enzymatically hydrolyzed with a β-glucuronidase/arylsulfatase and incubated overnight at 37 °C, 2) incubated overnight at 37 °C without enzymatical hydrolysis or 3) not treated with enzyme or heat. Following overnight incubation, samples were centrifuged twice and diluted with methanol and acetonitrile (50:50) before analysis by UHPLC–MS/MS. Initial results showed that urine samples spiked with morphine and M6G had decreasing concentration of M6G following incubation at 2 h (91% of spiked concentration), 4 h (56%), 6 h (44%), and 18 h (18%). Juxtaposed, morphine concentration increased with incubation at 2 h (19% of theoretical concentration), 4 h (38%), 6 h (47%), and 18 h (92%). This indicated an expected M6G to morphine turnover where the optimal enzyme efficiency was at incubation for 18 h. Results further showed that morphine stability was not affected (recovery: 102%) by heating conditions. In the multi-drug experiment, most drugs were stable under the heating conditions with recoveries above 80%. For some drugs, a decline in concentration was observed after incubation at 37 °C for 18 h, indicating drug instability. For the drug clozapine, the recovery decreased by ¼ while the recovery of the metabolite N-demethylclozapine increased. This demonstrates the importance of including both parent drug and metabolites in the analytical method of urine samples. To prevent this drug instability during pretreatment of urine samples, further experiments will be conducted to examine the use of other enzymes that requires shorter incubation and lower temperatures. The preliminary results showed that the efficiency of a β-glucuronidase/arylsulfatase improved with increasing incubation time. Furthermore, the enzymatical hydrolysis and incubation at 37 °C did not affect the stability of most drugs (75%) relevant to DFSA investigations. This work is financially supported by the Danish Victims Fund (grant number 20-610-0092).