Proteomic analysis of STEAP1 knockdown in human LNCaP prostate cancer cells.

Prostate cancer (PCa) continues to be one of the most common cancers in men worldwide. The six transmembrane epithelial antigen of the prostate 1 (STEAP1) protein is overexpressed in several types of human tumors, particularly in PCa. Our research group has demonstrated that STEAP1 overexpression is associated with PCa progression and aggressiveness. Therefore, understanding the cellular and molecular mechanisms triggered by STEAP1 overexpression will provide important insights to delineate new strategies for PCa treatment. In the present work, a proteomic strategy was used to characterize the intracellular signaling pathways and the molecular targets downstream of STEAP1 in PCa cells. A label-free approach was applied using an Orbitrap LC-MS/MS system to characterize the proteome of STEAP1-knockdown PCa cells. More than 6700 proteins were identified, of which a total of 526 proteins were found differentially expressed in scramble siRNA versus STEAP1 siRNA (234 proteins up-regulated and 292 proteins down-regulated). Bioinformatics analysis allowed us to explore the mechanism through which STEAP1 exerts influence on PCa, revealing that endocytosis, RNA transport, apoptosis, aminoacyl-tRNA biosynthesis, and metabolic pathways are the main biological processes where STEAP1 is involved. By immunoblotting, it was confirmed that STEAP1 silencing induced the up-regulation of cathepsin B, intersectin-1, and syntaxin 4, and the down-regulation of HRas, PIK3C2A, and DIS3. These findings suggested that blocking STEAP1 might be a suitable strategy to activate apoptosis and endocytosis, and diminish cellular metabolism and intercellular communication, leading to inhibition of PCa progression.

Development of a novel electrochemical biosensor based on plastic antibodies for detection of STEAP1 biomarker in cancer.

STEAP1 is a cell surface protein of the STEAP family whose main function focuses on intercellular communication and cell growth. STEAP1 is considered a promising putative biomarker and a candidate target for prostate cancer treatment. For specific and selective detection of STEAP1, a molecularly imprinted polymers (MIP) was developed on a screen-printed electrode (C-SPE) whose surface was modified with a nanocomposite based on carbon nanotubes decorated with dendritic platinum nanoparticles (CNTs- PAH /Pt). Then, the MIPs were produced on the modified C-SPE by electropolymerization of a mixture of STEAP1 and a monomer (pyrrole-2-carboxylic acid). Then, the protein was removed from the polymeric network by enzymatic treatment with trypsin, which created the specific template cavities for further STEAP1 detection. Electrochemical techniques such as EIS and CV were used to follow the chemical modification steps of C-SPE. The analytical performance of the biosensor was evaluated by SWV in PBS buffer and in lysates of neoplastic prostate cancer cells (LNCaP) extracts. The MIP material showing a linear range from 130 pg/ml to 13 µg/ml. Overall, the biosensor exhibits essential properties such as selectivity, sensitivity and reproducibility for its application in medical and clinical research diagnosis and/or prognosis of prostate cancer.

STEAP1 Knockdown Decreases the Sensitivity of Prostate Cancer Cells to Paclitaxel, Docetaxel and Cabazitaxel.

The Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) protein has been indicated as an overexpressed oncoprotein in prostate cancer (PCa), associated with tumor progression and aggressiveness. Taxane-based antineoplastic drugs such as paclitaxel, docetaxel, or cabazitaxel, have been investigated in PCa treatment, namely for the development of combined therapies with the improvement of therapeutic effectiveness. This study aimed to evaluate the expression of STEAP1 in response to taxane-based drugs and assess whether the sensitivity of PCa cells to treatment with paclitaxel, docetaxel, or cabazitaxel may change when the STEAP1 gene is silenced. Thus, wild-type and STEAP1 knockdown LNCaP and C4-2B cells were exposed to paclitaxel, docetaxel or cabazitaxel, and STEAP1 expression, cell viability, and survival pathways were evaluated. The results obtained showed that STEAP1 knockdown or taxane-based drugs treatment significantly reduced the viability and survival of PCa cells. Relatively to the expression of proliferation markers and apoptosis regulators, LNCaP cells showed a reduced proliferation, whereas apoptosis was increased. However, the effect of paclitaxel, docetaxel, or cabazitaxel treatment was reversed when combined with STEAP1 knockdown. Besides, these chemotherapeutic drugs may stimulate the cell growth of PCa cells knocked down for STEAP1. In conclusion, this study demonstrated that STEAP1 expression levels might influence the response of PCa cells to chemotherapeutics drugs, indicating that the use of paclitaxel, docetaxel, or cabazitaxel may lead to harmful effects in PCa cells with decreased expression of STEAP1.

E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate.

Cervical cancer has been extensively associated with human papillomavirus (HPV) due to the expression of oncoproteins such as E6. This protein can interfere with p53 tumor suppressor activity, blocking apoptosis of abnormal cells. The functional inhibition of E6 protein is a promising therapeutic strategy for HPV-induced cancers. Conducting biointeraction and characterization studies between E6 protein and potential anti-HPV drugs is necessary to obtain large quantities of high-purity and soluble E6 protein. The recombinant production of E6 protein is particularly challenging because it tends to aggregate. One way to circumvent this problem is to use a dual MBP-His6 tag that can facilitate the expression, proper folding, and solubility of the E6 protein. This chapter outlines effective methods for expressing and obtaining E6 protein with a dual affinity tag by combining different chromatographic methods.

Mini-Bioreactor Platform for Membrane Protein Production in Komagataella pastoris.

Membrane proteins (MPs) play vital roles across various cellular functions, biological processes, physiological signaling pathways, and human-related disorders. Considering the clinical relevance of MPs and their application as therapeutic targets, it is crucial to explore highly effective production platforms and purification approaches to ultimately obtain a high-resolution structure of the target. Therefore, it would be possible to gather detailed knowledge on their mechanism of action which will be the basis for the rational design of novel and stronger drugs. Unfortunately, when compared to their soluble counterparts, 3D structures of MPs are really scarce (<2%), mainly due to poorly natural abundance, challenges associated with protein solubility and stability, and difficulties in producing bioactive and properly structural folded targets. These drawbacks could significantly impair the use of MPs as therapeutic targeting and demand efforts to develop tailor-made strategies for their appropriate handling. Therefore, this chapter is focused on describing a detailed and high-throughput procedure for the biosynthesis of MPs using Komagataella pastoris cell cultures as expression system in a mini-bioreactor platform. Additionally, insights on a purification strategy that combines immobilized-metal affinity and ion-exchange chromatography are described to further obtain the target protein with a significant degree of purity.

Development and Characterization of Quercetin-Loaded Delivery Systems for Increasing Its Bioavailability in Cervical Cancer Cells.

Quercetin is a natural flavonoid with high anticancer activity, especially for related-HPV cancers such as cervical cancer. However, quercetin exhibits a reduced aqueous solubility and stability, resulting in a low bioavailability that limits its therapeutic use. In this study, chitosan/sulfonyl-ether-ß-cyclodextrin (SBE-ß-CD)-conjugated delivery systems have been explored in order to increase quercetin loading capacity, carriage, solubility and consequently bioavailability in cervical cancer cells. SBE-ß-CD/quercetin inclusion complexes were tested as well as chitosan/SBE-ß-CD/quercetin-conjugated delivery systems, using two types of chitosan differing in molecular weight. Regarding characterization studies, HMW chitosan/SBE-ß-CD/quercetin formulations have demonstrated the best results, which are obtaining nanoparticle sizes of 272.07 ± 2.87 nm, a polydispersity index (PdI) of 0.287 ± 0.011, a zeta potential of +38.0 ± 1.34 mV and an encapsulation efficiency of approximately 99.9%. In vitro release studies were also performed for 5 kDa chitosan formulations, indicating a quercetin release of 9.6% and 57.53% at pH 7.4 and 5.8, respectively. IC50 values on HeLa cells indicated an increased cytotoxic effect with HMW chitosan/SBE-ß-CD/quercetin delivery systems (43.55 µM), suggesting a remarkable improvement of quercetin bioavailability.

Evaluation of Antipsychotic Drugs' Stability in Oral Fluid Samples.

Antipsychotics have narrow therapeutic windows, and their monitoring in biological fluids is therefore important; consequently, stability in those fluids must be investigated during method development and validation. This work evaluates the stability of chlorpromazine, levomepromazine, cyamemazine, clozapine, haloperidol, and quetiapine in oral fluid (OF) samples, using the dried saliva spots (DSS) sampling approach and gas chromatography coupled to tandem mass spectrometry. Since many parameters can influence the stability of the target analytes, design of experiments was adopted to check the crucial factors that affect that stability in a multivariate fashion. The studied parameters were the presence of preservatives at different concentrations, temperature, light, and time. It was possible to observe that antipsychotic stability improved when OF samples in DSS were stored at 4 °C, with a low ascorbic acid concentration, and in the absence of light. With these conditions, chlorpromazine and quetiapine were stable for 14 days, clozapine and haloperidol were stable for 28 days, levomepromazine remained stable for 44 days, and cyamemazine was stable for the entire monitored period (146 days). This is the first study that evaluates the stability of these antipsychotics in OF samples after application to DSS cards.

Proteomics profiling of vitreous humor reveals complement and coagulation components, adhesion factors, and neurodegeneration markers as discriminatory biomarkers of vitreoretinal eye diseases.

Introduction: Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and proliferative DR (PDR), no treatment options exist for the highly prevalent dry form of AMD. Methods: To unravel the biological processes underlying these pathologies and to find new potential biomarkers, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR (n=4), AMD (n=4) compared to idiopathic epiretinal membranes (ERM) (n=4). Results and discussion: Post-hoc tests revealed 96 proteins capable of differentiating among the different groups, whereas 118 proteins were found differentially regulated in PDR compared to ERM and 95 proteins in PDR compared to dry AMD. Pathway analysis indicates that mediators of complement, coagulation cascades and acute phase responses are enriched in PDR vitreous, whilst proteins highly correlated to the extracellular matrix (ECM) organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development were found underexpressed. According to these results, 35 proteins were selected and monitored by MRM (multiple reaction monitoring) in a larger cohort of patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and retinal detachment (n=13). Of these, 26 proteins could differentiate between these vitreoretinal diseases. Based on Partial least squares discriminant and multivariate exploratory receiver operating characteristic (ROC) analyses, a panel of 15 discriminatory biomarkers was defined, which includes complement and coagulation components (complement C2 and prothrombin), acute-phase mediators (alpha-1-antichymotrypsin), adhesion molecules (e.g., myocilin, galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2).

Specific Six-Transmembrane Epithelial Antigen of the Prostate 1 Capture with Gellan Gum Microspheres: Design, Optimization and Integration.

This work demonstrates the potential of calcium- and nickel-crosslinked Gellan Gum (GG) microspheres to capture the Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) directly from complex Komagataella pastoris mini-bioreactor lysates in a batch method. Calcium-crosslinked microspheres were applied in an ionic exchange strategy, by manipulation of pH and ionic strength, whereas nickel-crosslinked microspheres were applied in an affinity strategy, mirroring a standard immobilized metal affinity chromatography. Both formulations presented small diameters, with appreciable crosslinker content, but calcium-crosslinked microspheres were far smoother. The most promising results were obtained for the ionic strategy, wherein calcium-crosslinked GG microspheres were able to completely bind 0.1% (v/v) DM solubilized STEAP1 in lysate samples (~7 mg/mL). The target protein was eluted in a complexed state at pH 11 with 500 mM NaCl in 10 mM Tris buffer, in a single step with minimal losses. Coupling the batch clarified sample with a co-immunoprecipitation polishing step yields a sample of monomeric STEAP1 with a high degree of purity. For the first time, we demonstrate the potential of a gellan batch method to function as a clarification and primary capture method towards STEAP1, a membrane protein, simplifying and reducing the costs of standard purification workflows.

Volatilomics as an Emerging Strategy to Determine Potential Biomarkers of Female Infertility: A Pilot Study.

Due to its high prevalence, infertility has become a prominent public health issue, posing a significant challenge to modern reproductive medicine. Some clinical conditions that lead to female infertility include polycystic ovary syndrome (PCOS), endometriosis, and premature ovarian failure (POF). Follicular fluid (FF) is the biological matrix that has the most contact with the oocyte and can, therefore, be used as a predictor of its quality. Volatilomics has emerged as a non-invasive, straightforward, affordable, and simple method for characterizing various diseases and determining the effectiveness of their current therapies. In order to find potential biomarkers of infertility, this study set out to determine the volatomic pattern of the follicular fluid from patients with PCOS, endometriosis, and POF. The chromatographic data integration was performed through solid-phase microextraction (SPME), followed by gas chromatography-mass spectrometry (GC-MS). The findings pointed to specific metabolite patterns as potential biomarkers for the studied diseases. These open the door for further research into the relevant metabolomic pathways to enhance infertility knowledge and diagnostic tools. An extended investigation may, however, produce a new mechanistic understanding of the pathophysiology of the diseases.

Development of WRAP5 Peptide Complexes for Targeted Drug/Gene Co-Delivery toward Glioblastoma Therapy.

Despite the great progress over the past few decades in both the diagnosis and treatment of a great variety of human cancers, glioblastoma remains the most lethal brain tumor. In recent years, cancer gene therapy focused on non-viral vectors which emerged as a promising approach to glioblastoma treatment. Transferrin (Tf) easily penetrates brain cells of the blood-brain barrier, and its receptor is highly expressed in this barrier and glioblastoma cells. Therefore, the development of delivery systems containing Tf appears as a reliable strategy to improve their brain cells targeting ability and cellular uptake. In this work, a cell-penetrating peptide (WRAP5), bearing a Tf-targeting sequence, has been exploited to condense tumor suppressor p53-encoding plasmid DNA (pDNA) for the development of nanocomplexes. To increase the functionality of developed nanocomplexes, the drug Temozolomide (TMZ) was also incorporated into the formulations. The physicochemical properties of peptide/pDNA complexes were revealed to be dependent on the nitrogen to phosphate groups ratio and can be optimized to promote efficient cellular internalization. A confocal microscopy study showed the capacity of developed complexes for efficient glioblastoma cell transfection and consequent pDNA delivery into the nucleus, where efficient gene expression took place, followed by p53 protein production. Of promise, these peptide/pDNA complexes induced a significant decrease in the viability of glioblastoma cells. The set of data reported significantly support further in vitro research to evaluate the therapeutic potential of developed complexes against glioblastoma.

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase.

Catechol-O-methyltransferase (COMT) has been involved in a number of medical conditions including catechol-estrogen-induced cancers and a great range of cardiovascular and neurodegenerative diseases such as Parkinson's disease. Currently, Parkinson's disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography-IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 °C and -80 °C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.

The Determination of Cannabinoids in Urine Samples Using Microextraction by Packed Sorbent and Gas Chromatography-Mass Spectrometry.

Cannabis is the most consumed illicit drug worldwide, and its legal status is a source of concern. This study proposes a rapid procedure for the simultaneous quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabidiol (CBD), and cannabinol (CBN) in urine samples. Microextraction by packed sorbent (MEPS) was used to pre-concentrate the analytes, which were detected by gas chromatography-mass spectrometry. The procedure was previously optimized, and the final conditions were: conditioning with 50 µL methanol and 50 µL of water, sample load with two draw-eject cycles, and washing with 310 µL of 0.1% formic acid in water with 5% isopropanol; the elution was made with 35 µL of 0.1% ammonium hydroxide in methanol. This fast extraction procedure allowed quantification in the ranges of 1-400 ng/mL for THC and CBD, 5-400 ng/mL for CBN and 11-OH-THC, and 10-400 ng/mL for THC-COOH with coefficients of determination higher than 0.99. The limits of quantification and detection were between 1 and 10 ng/mL using 0.25 mL of sample. The extraction efficiencies varied between 26 and 85%. This analytical method is the first allowing the for determination of cannabinoids in urine samples using MEPS, a fast, simple, and low-cost alternative to conventional techniques.

Advances in Membrane-Bound Catechol-O-Methyltransferase Stability Achieved Using a New Ionic Liquid-Based Storage Formulation.

Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson's disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs' design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at -80 °C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.

Unveiling the biopathway for the design of novel COMT inhibitors.

Catechol-O-methyltransferase (COMT) is an enzyme responsible for the O-methylation of biologically active catechol-based molecules. It has been associated with several neurological disorders, especially Parkinson's disease (PD), because of its involvement in catecholamine metabolism, and has been considered an important therapeutic target for central nervous system disorders. In this review, we summarize the biophysical, structural, and therapeutical relevance of COMT; the medicinal chemistry behind the development of COMT inhibitors and the application of computer-aided design to support the design of novel molecules; current methodologies for the biosynthesis, isolation, and purification of COMT; and revise existing bioanalytical approaches for the assessment of enzymatic activity in several biological matrices.

Follicular Fluid: A Powerful Tool for the Understanding and Diagnosis of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) represents one of the leading causes of anovulatory infertility and affects 5% to 20% of women worldwide. Until today, both the subsequent etiology and pathophysiology of PCOS remain unclear, and patients with PCOS that undergo assisted reproductive techniques (ART) might present a poor to exaggerated response, low oocyte quality, ovarian hyperstimulation syndrome, as well as changes in the follicular fluid metabolites pattern. These abnormalities originate a decrease of Metaphase II (MII) oocytes and decreased rates for fertilization, cleavage, implantation, blastocyst conversion, poor egg to follicle ratio, and increased miscarriages. Focus on obtaining high-quality embryos has been taken into more consideration over the years. Nowadays, the use of metabolomic analysis in the quantification of proteins and peptides in biological matrices might predict, with more accuracy, the success in assisted reproductive technology. In this article, we review the use of human follicular fluid as the matrix in metabolomic analysis for diagnostic and ART predictor of success for PCOS patients.

Advances on Bioanalysis: Recent Approaches in the Determination of Biomarkers, Drugs of Abuse and Medicines.

New developments in instrumental approaches, for instance, hyphenated techniques, have allowed great advances in the bioanalytical field over the last half century, and there is no doubt that toxicology was one of the most improved areas [...].

Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD.

Nucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptor-binding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 °C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (p-value < 0.05) and presented a non-significant lack of fit (p-value > 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30 °C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, the production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO2 in the fermentation step during 5 h and 30% pO2 in the induction step, with 0.01% L-arabinose throughout 5 h. The yield of mcDNA-RBD was increased to a concentration of 1.15 g/L, when compared to the orbital shaker studies (16.48 mg/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry.

An Update on the Implications of New Psychoactive Substances in Public Health.

The emergence of new psychoactive substances has earned a great deal of attention, and several reports of acute poisoning and deaths have been issued involving, for instance, synthetic opiates. In recent years, there have been profound alterations in the legislation concerning consumption, marketing, and synthesis of these compounds; rapid alert systems have also been subject to changes, and new substances and new markets, mainly through the internet, have appeared. Their effects and how they originate in consumers are still mostly unknown, primarily in what concerns chronic toxicity. This review intends to provide a detailed description of these substances from the point of view of consumption, toxicokinetics, and health consequences, including case reports on intoxications in order to help researchers and public health agents working daily in this area.

Stability of Cocaine, Opiates, and Metabolites in Dried Saliva Spots.

Drug abuse still represents a global problem, and it is associated with an increased risk of diseases, injuries, and deaths. Cocaine (COC) and opiates are the most abused drugs and account for a significant number of fatalities. Therefore, it is important to develop methods capable of effectively identifying and quantifying these substances. The present study aims to evaluate the long-term stability of COC, ecgonine methylester (EME), benzoylecgonine (BEG), cocaethylene (COET), norcocaine (NCOC), morphine (MOR), codeine (COD) and 6-monoacetylmorphine (6-MAM) in oral fluid samples. The analytes of interest were isolated from the matrix (50 µL) using the dried saliva spots (DSS) sampling approach and were subsequently analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The parameters that could influence the stability of the target compounds were studied, and these were storage temperature, light, use of preservatives (and respective concentrations), and time. The effects of each parameter were evaluated using the design of experiments (DOE) approach. The stability of the target analytes was improved when the DSS were stored at room temperature, in the presence of light and using 1% sodium fluoride. The best conditions were then adopted for the DSS storage and long-term stability was assessed. COD was only stable for 1 day, EME was stable for 3 days, COC, COET, NCOC and 6-MAM were stable for 7 days, MOR for 14 days and BEG remained stable throughout the study (136 days). This is the first study that evaluates the stability of these compounds in oral fluid samples after application in DSS cards, and optimizes the conditions in order to improve their stability.