Development of an innovative analytical method for forensic detection of cocaine, antidepressants, and metabolites in postmortem blood using magnetic nanoparticles.

Cocaine and antidepressants rank high globally in substance consumption, emphasizing their impact on public health. The determination of these compounds and related substances in biological samples is crucial for forensic toxicology. This study focused on developing an innovative analytical method for the determination of cocaine, antidepressants, and their related metabolites in postmortem blood samples, using unmodified commercial Fe3O4 nanoparticles as a sorbent for dispersive magnetic solid-phase extraction (m-d-SPE), coupled with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. An aliquot of 100 µL of whole blood and 5 µL of the internal standard pool were added to 30 mg of nanoparticles. The nanoparticles were separated from the sample using a neodymium magnet inserted into a 3D-printed microtube rack. The liquid was then discarded, followed by desorption with 300 µL of 1/1/1 acetonitrile/methanol/ethyl acetate. The sample was vortexed and separated, and 1.5 µL of the organic supernatant was injected into the LC-MS/MS. The method was acceptably validated and successfully applied to 263 postmortem blood samples. All samples evaluated in this study were positive for at least one substance. The most frequent analyte was benzoylecgonine, followed by cocaine and cocaethylene. The most common antidepressants encountered in the analyzed samples were citalopram and fluoxetine, followed by fluoxetine's metabolite norfluoxetine. This study describes the first report of this sorbent in postmortem blood analysis, demonstrating satisfactory results for linearity, precision, accuracy, and selectivity for all compounds. The method's applicability was confirmed, establishing it as an efficient and sustainable alternative to traditional techniques for forensic casework.

A validated dilute-and-shoot LC-MS/MS urine screening for the analysis of 95 illicit drugs and medicines: insights from clinical and forensic Brazilian cases.

Urine toxicological analysis is a relevant tool in both clinical and forensic scenarios, enabling the diagnosis of acute poisonings, elucidation of deaths, verification of substance use in the workplace, and identification of drug-facilitated crimes. For these analyses, the dilute-and-shoot technique associated with liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) is a promising alternative since it has demonstrated satisfactory results and broad applicability. This study developed and validated a comprehensive LC-MS-MS screening method to analyze 95 illicit drugs and medicines in urine samples and application to clinical and forensic Brazilian cases. The dilute-and-shoot protocol was defined through multivariate optimization studies and was set using 100 µL of sample and 300 µL of solvent. The total chromatographic run time was 7.5 minutes. The method was validated following the recommendations of the ANSI/ASB Standard 036 Guideline. The lower limits of quantification varied from 20 to 100 ng/mL. Within-run and between-run precision CVs% were lower than 20%, and bias was within ± 20%. Only four of the 95 analytes presented significant ionization suppression or enhancement (> 25%). As proof of applicability, 839 urine samples from in vivo and postmortem cases were analyzed. In total, 90.9% of the analyzed samples were positive for at least one substance, and 78 of the 95 analytes were detected. The most prevalent substances were lidocaine (40.2%), acetaminophen (38.0%), and benzoylecgonine (31.5%). The developed method proved to be an efficient and simplified alternative for analyzing 95 therapeutic and illicit drugs in urine samples. Additionally, the results obtained from sample analysis are essential to understanding the profile of Brazilian substance use, serving as a valuable database for the promotion of health and safety public policies.

Cork sheet as an efficient biosorbent for forensic toxicology: Application to vitreous humor analysis.

There is an increasing number of people affected worldwide by mental health disorders, such as depression and anxiety. One of the main courses of treatment, along with psychotherapy, is the use of psychoactive medications, like antidepressants and benzodiazepines. Also, the unprescribed use of these substances is a concerning public health issue. Hence, the analysis of psychotropic medications is mandatory in postmortem toxicology and various biological samples can be used for this detection, among them the vitreous humor (VH) stands out. Also, there is a demand for more sustainable and more efficient extraction methodologies according to green chemistry. An example is solid phase microextraction techniques (SPME), which use a solid sorbent and small solvent amounts. Biosorbents are substances of natural origin with sorptive properties, and they have been successfully used in SPME in environmental toxicology for water analysis, mainly. This study aimed to develop a sustainable, fast, cheap and simple SPME methodology using cork sheet strips as a biosorbent, to extract antidepressants, benzodiazepines and others from VH samples by liquid chromatography coupled to tandem mass spectrometry. The extraction was conducted in a 96-well plate using 200 µL of VH and optimization of relevant parameters for extraction was performed. For solvent optimization, two simplex-centroid experiments were planned for extraction and desorption and to evaluate time and pH, a Doehlert design experiment was performed. The analytical method for the determination and quantification of 17 substances was validated. The quantification limits were 5 ng/mL for all analytes and the calibration curves were linear between 5 and 30 ng/mL. This study was able to develop an efficient, cheap, simple and fast microextraction method for 17 analytes in VH, using strips of cork sheet for extraction and a 96-well plate as a container. Furthermore, this approach system could be automated for routine toxicology laboratories.

Development and validation of a dried plasma spot LC-MS/MS method for therapeutic monitoring of vancomycin and comparison with enzyme-multiplied immunoassay.

Vancomycin is used as an antimicrobial agent for the treatment of severe gram-positive infections. The importance of therapeutic monitoring of antimicrobials has led to the development of more specific sample preparation techniques capable of identifying with accuracy the concentration of this substance in the organism. An aliquot of 10 µl of plasma was transferred to Whatman 903 paper and dried at room temperature. The extraction method was performed by cutting and transferring the paper to a microtube and adding sodium phosphate buffer and internal standard. The mixture was shaken and centrifuged, and a 5-µl aliquot was injected into the analytical system. The optimization of the main parameters that can influence the extraction efficiency was performed using multivariate approaches to obtain the best conditions. The method developed was validated, providing coefficients of determination higher than 0.994 and a lower limit of quantification of 1 mg/L. Within- and between-run precision ranged from 11.4 to 17.30% and from 6.65 to 13.51%, respectively. This method was successfully applied to 75 samples of patients undergoing vancomycin therapy. The method was rapid, simple, and environmentally friendly with satisfactory analytical performance and was advantageous over the laborious and time-consuming methodologies used in therapeutic drug monitoring routine analyses.

Bioanalytical method for simultaneous determination of benzodiazepines in vitreous humor using liquid chromatography-tandem mass spectrometry.

The use of vitreous humor (VH) in forensic casework has been growing in the last years due to numerous advantages. Several compounds can be evaluated in this matrix, including benzodiazepines whose determination is essential due to their great availability and potential of dependance and misuse. Postmortem toxicological analyses are required to determine the influence of benzodiazepines in deaths. However, most of the analytical methods which determine these drugs in VH are laborious and time consuming. This article describes a simple method based on protein precipitation for the determination of eight benzodiazepines in VH samples. Samples were prepared through a protein precipitation method and analyzed by liquid chromatography tandem mass spectrometry. Solvent choice and sample and solvent volumes for precipitation were optimized using chemometric approaches. The method was validated for selectivity, lower limit of quantification (LLOQ), linearity, carryover, precision, bias, matrix effect and dilution integrity. In order to verify the applicability, 62 vitreous humor samples were analyzed. LLOQs were 1 ng/mL and calibration curves were linear from 1 to 25 ng/mL (r2 > 0,99) for all analytes. Bias, precision and dilution integrity results were satisfactory according to proper guidelines. Ionization suppression was significant with values ranging from 8 to 37%. Two samples from real cases were positive for diazepam with the following concentrations: 6.80 ng/mL and 47.68 ng/mL, approximately 10 times lower than those found in peripheral blood. The procedure described here can be used as a straightforward and low cost method for the quantitation of multiple benzodiazepines in VH.

A rapid analytical strategy for the determination of ayahuasca alkaloids in non-ritualistic approaches by UHPLC-MS/MS.

INTRODUCTION: Ayahuasca is a beverage composed by a mixture of herbs which contain the compound N,N-dimethyltriptamine (DMT) and the ß-carbolines. Although its use is legalized in Brazil only for religious and spiritual ceremonies, there is a growing black market specialized in the distribution of these compounds in form of herbal material through internet and mail. The purpose of this work was the development of an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the determination of ayahuasca alkaloids and its application in seized ayahuasca products. METHODS: An aliquot of seized products was weighted and diluted with methanol. An aliquot of this solution was added with internal standard (DMT-d6), followed by injection in the analytical system. RESULTS: The limit of quantitation was 10ng/mL for DMT and 25ng/mL for harmine, harmaline and tetrahydroharmine. The concentration ranges used were 10-100ng/mL for DMT, harmine and harmaline and all analytes presented a coefficient of determination (r2)≥0,99. Analysis of four seized samples presented concentrations of DMT ranging between 31.5 and 46.5mg/g. Presence of ß-carbolines was not detected in the products. The variability of DMT concentrations can be correlated with the potential intoxications described in the literature. CONCLUSION: This work successfully established a determination method for ayahuasca alkaloids in herbal material. In addition, the workflow proved to be simple, rapid and useful to estimate the concentration of psychoactive compounds in seized materials, leading to further investigation of ayahuasca ritualistic or recreational exposure.

A straightforward and semiautomated membrane-based method as efficient tool for the determination of cocaine and its metabolites in urine samples using liquid chromatography coupled to quadrupole time-of-flight-mass spectrometry.

In this study, a novel and straightforward analytical methodology was proposed for the determination of cocaine (COC) and its main metabolites benzoylecgonine (BZE) cocaethylene (CE) and hydroxy­cocaine (COCOH) in urine samples. This approach consisted of a high-throughput and semiautomated configuration based on hollow-fiber renewal liquid membrane extraction (HFRLM) coupled to a 96-well plate system, which was proposed for the first time to analyze complex biological samples such as urine. The analytical determinations were performed using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight-mass spectrometry (LC-ESI-QTOF-MS). The analytical methodology was fully optimized through Doehlert and simplex-centroid designs, and univariate approaches. Polypropylene membranes of 1 cm length were inserted in the pins of an extraction blade combined with a 96-well plate system and its pores were filled with hexane:dichloromethane:ethyl acetate (1:1:1 v/v/v) for 180 s; moreover, 20 µL of this mixture was added to the sample to allow for a renewable liquid membrane. The extraction step was carried out by keeping the blades immersed in vials containing 1.5 mL of diluted urine adjusted at pH 10 with 10% (w/v) of Na2CO3 during 20 min, followed by liquid desorption with 100 µL of acetonitrile. Finally, the extract was dried under N2 stream and resuspended with 20 µL of ultrapure water. Satisfactory analytical performance was obtained with coefficients of determination ranging from 0.9875 for BZE to 0.9986 for CE; intra-day precision ranged from 1.6 to 13.5%, and inter-day precision varied from 2.2 to 17.5%. Limits of detection ranged from 1.5 to 15.1 ng mL-1, and limits of quantification varied from 5 to 50 ng mL-1, with relative recoveries varied from 70.7 to 124.1%.

A green and low-cost method employing switchable hydrophilicity solvent for the simultaneous determination of antidepressants in human urine by gas chromatography - mass spectrometry detection.

In this study, the use of switchable hydrophilicity solvent with a simple and low-cost lab-made device for the extraction procedure in homogeneous liquid-liquid microextraction is proposed for the first time in the determination of antidepressants in human urine. The antidepressants studied consisted of fluoxetine, amitriptyline, nortriptyline, imipramine, desipramine and sertraline. The optimization of the main parameters that can influence on the extraction efficiency was performed through multivariate approaches. The analytes were separated and identified by gas chromatography coupled to mass spectrometry (GC-MS). The optimal extraction conditions consisted of using N,N-dimethylcyclohexylamine (DMCHA) as the switchable hydrophilicity solvent (SHS), 500 µL of urine sample previously diluted with ultrapure water at 1:1 ratio (v/v), 200 µL of a mixture of SHS:HCl 6 mol L-1 (1:1 v/v), 600 µL of NaOH 10 mol L-1 and 3 min of extraction time. A volume of 40 µL of diphenylamine at concentration of 500 µg L-1 (20 ng) was used as internal standard. The method developed was in-house validated, providing coefficients of determination higher than 0.995 for all analytes, limits of detection (LOD) from 0.02 to 0.88 µg L-1, limits of quantification (LOQ) from 0.05 to 2.92 µg L-1, relative recoveries of 68 to 102%, intra-day precision from 0.5 to 15.9%, inter-day precision from 4.2 to 19.3%, selectivity and robustness. The method proposed was successfully applied in five human urine samples from a Toxicological Information Center located in Porto Alegre (Brazil). The results demonstrated that the µP-SHS-HLLME approach is highly cost-effective, rapid, simple and environmentally-friendly with satisfactory analytical performance.