Paper-supported polystyrene membranes for micro-solid phase extraction of date-rape drugs from urine: A sustainable analytical approach.

BACKGROUND: The rapid development of micro-solid phase extraction (µ-SPE) procedures with new sorption materials, in particular, based on using natural materials, is currently reported. The production of these sorbents and the entire extraction procedure should support the implementation of Green Analytical Chemistry (GAC) principles. Promising materials are sorbents based on paper, which can be relatively easily modified, among others: by covering it with a polymer membrane. In this work, the practical application of paper-supported polystyrene used in the analysis of urine samples containing selected date-rape drugs (DRD) substances, and evaluation of the entire procedure using GAC metrics is presented. RESULTS: The paper-supported polystyrene membranes were successfully fabricated and characterized. The successful polystyrene coating on the paper was confirmed through ATR-FTIR measurements, ensuring even coverage. The µ-SPE procedure using this material facilitated extraction with a throughput of approximately 120 samples per hour in just a few steps. Throughout the research, a mixture of 100 mM acetic acid:methanol:acetonitrile (70:15:15, v/v/v) was selected as an optimal background electrolyte for capillary electrophoresis - mass spectrometry analysis. Validation results of this method demonstrated its suitability, exhibiting good linearity (R2 > 0.95), low limits of detection (3.1-15 ng mL-1), acceptable precision (<15 %), and recovery for all tested analytes. Furthermore, the greenness evaluation conducted with six different metrics: AGREEprep, AGREE, ComplexGAPI, SPMS, hexagonal metric, and WAC indicated the overall eco-friendliness and sustainability of the method, with minor concerns regarding energy consumption. SIGNIFICANCE: The use of cellulose paper with polystyrene membranes for µ-SPE provides a versatile and eco-friendly extraction method for detecting DRDs in urine samples. The presented work is an example of the use of GAC metrics in the evaluation of the analytical procedure. The optimized PT-µ-SPE/CE-MS method allows for minimized reagent usage and waste production. Moreover, the method proves to be sustainable and efficient for forensic toxicology analysis.

Raman Spectroscopy for the Time since Deposition Estimation of a Menstrual Bloodstain.

Forensic chemistry plays a crucial role in aiding law enforcement investigations by applying analytical techniques for the analysis of evidence. While bloodstains are frequently encountered at crime scenes, distinguishing between peripheral and menstrual bloodstains presents a challenge. This is due to their similar appearance post-drying. Raman spectroscopy has emerged as a promising technique capable of discriminating between the two types of bloodstains, offering invaluable probative information. Moreover, estimating the time since deposition (TSD) of bloodstains aids in crime scene reconstruction and prioritizing what evidence to collect. Despite extensive research focusing on TSD estimations, primarily in peripheral bloodstains, a crucial gap exists in determining the TSD of menstrual bloodstains. This study demonstrates how Raman spectroscopy effectively analyzes biological samples like menstrual blood, showing similar aging patterns to those of peripheral blood and provides proof-of-concept models for determining the TSD of menstrual blood. While this work shows promising results for creating a universal model for bloodstain age determination, further testing with more donors needs to be conducted before the implementation of this method into forensic practice.

Direct Immersion-Solid Phase Microextraction for Therapeutic Drug Monitoring of Patients with Mood Disorders.

This article discusses a new method for monitoring drug concentrations in blood samples from patients with mood disorders. The method uses solid-phase microextraction to extract analytes directly from blood samples. It has been adapted to identify the most commonly used drugs in mood disorders, including amitriptyline, citalopram, fluoxetine, paroxetine, sertraline, trazodone, duloxetine, venlafaxine, lamotrigine, quetiapine, olanzapine, and mirtazapine. The analysis is carried out using high-performance liquid chromatography coupled with mass spectroscopy. The proposed DI-SPME/LC-MS method allows for a simple and quick screening analysis while minimizing the volume of the tested sample and solvent, in line with the principles of green analytical chemistry. The method was used to analyze 38 blood samples taken from patients with mood disorders, and drug concentrations were determined and compared with therapeutic and toxic dose ranges. This allowed for better control of the course of treatment.

Comparison of ZrO2 Particles and Polyaniline as Additives in Polystyrene-Based Sorbents for the Micro-Solid Phase Extraction of Psychoactive Drugs from Biofluids.

The intensive development of extraction methods based on µ-SPE extraction contributes to the increased interest in the synthesis of new sorption materials. This work presents the characterization of polystyrene fibers and polystyrene fibers blended with ZrO2 particles or polyaniline obtained by electrospinning and their use in the extraction of selected psychoactive drugs from biological samples. The characteristic of produced fibers is made by performing SEM images, measuring average fiber diameter, and examining their sorption abilities. Among the fibers based on pure polystyrene, tested in the first stage, the best sorption properties are demonstrated for the fibers obtained from a polystyrene solution in DMF with a concentration of 17.5 wt%. In the next stage, this material was modified with synthesized ZrO2 particles and polyaniline. Among the tested materials, the sorbent based on polystyrene with polyaniline shows the best sorption properties of the tested substances. The use of this material in the µ-SPE in a needle enables the extraction of selected compounds from aqueous and biological samples such as urine and human plasma.

Electroanalytical characterization of clozapine at the electrified liquid-liquid interface and its detection in soft and hard drinks.

Clozapine (CZ) is a prescribed benzodiazepine psychiatric drug that is often possessed as an illicit drug and is associated with drug-facilitated sexual assaults (DFSA) due to its strong sedative capabilities. Hence, we propose an electrified liquid-liquid interface (eLLI) based transducing element as an alternative electroanalytical platform for rapid screening of CZ in soft and hard drinks which is habitually associated with DFSA crimes. First, molecular partitioning and the effect of chemical composition, pH, and the presence of ethanol in the biphasic configuration of the aqueous phase on the interfacial behaviour and analytical performance of the CZ at the eLLI have been investigated with voltammetry. Next, the electrochemical profiles of various soft and hard drinks were studied at the eLLI. The eLLI-based CZ sensor has shown a broad dynamic range (15-150 µM), lower detection limits (1µM), and adequate reliability towards rapid CZ screening in spiked soft and hard drink samples with reference to the standard chromatographic analysis.

Nitrazepam and 7-aminonitrazepam studied at the macroscopic and microscopic electrified liquid-liquid interface.

Two benzodiazepine type drugs, that is, nitrazepam and 7-aminonitrazepam, were studied at the electrified liquid-liquid interface (eLLI). Both drugs are illicit and act sedative in the human body and moreover are used as date rape drugs. Existence of the diazepine ring in the concerned chemicals structure and one additional amine group (for 7-aminonitrazepam) allows for the molecular charging below their pKa values, and hence, both drugs can cross the eLLI interface upon application of the appropriate value of the Galvani potential difference. Chosen molecules were studied at the macroscopic eLLI formed in the four electrode cell and microscopic eLLI formed within a microtip defined as the single pore having 25 µm in diameter. Microscopic eLLI was formed using only a few µL of the organic and the aqueous phase with the help of a 3D printed cell. Parameters such as limit of detection and voltammetric detection sensitivity are derived from the experimental data. Developed methodology was used to detect nitrazepam in pharmaceutical formulation and both drugs (nitrazepam and 7-aminonitrazepam) in spiked biological fluids (urine and blood).

Fast and Noninvasive Hair Test for Preliminary Diagnosis of Mood Disorders.

The main objective of this study was to develop a test for the fast and noninvasive prediagnosis of mood disorders based on the noninvasive analysis of hair samples. The database included 75 control subjects (who were not diagnosed with depression) and 40 patients diagnosed with mood disorders such as depression or bipolar disorder. Both women and men, aged 18-65 years, participated in the research. After taking the hair samples, they were washed (methanol-water-methanol by shaking in a centrifuge for two min) and air-dried in a fume hood. Each hair collection was analyzed using Fourier transform infrared spectroscopy attenuated total reflection (ATR-FTIR) spectroscopy. Subsequently, the results obtained were analyzed based on chemometric methods: hierarchical cluster analysis (HCA) and principal component analysis (PCA). As a results of the research conducted, potential differences were noticed. There was a visible change in the spectra intensity at around 2800-3100 cm-1 and smaller differences around 1460 cm-1; the bands can be assigned to protein vibrations. However, these are preliminary studies that provide a good basis for the development of a test for the initial diagnosis of mood disorders.

The DI-SPME Method for Determination of Selected Narcotics and Their Metabolites, and Application to Bone Marrow and Whole Blood Analysis.

The present investigation utilised the quick and easy SPME/LC-MS method to determine selected narcotic substances and their metabolites in whole blood. The study included qualitative analysis and validation of the method. Analytes were determined in the linearity range of 25−300 ng/mL. The precision during and between days (in general CV < 13.41%), and the LOD which results in between 0.36 and 11.08 ng/mL, and the LOQ between 1.20 and 36.90 ng/mL were investigated. The validation results obtained, as well as the results of subsequent in-laboratory tests, confirmed the applicability of the method in the analysis of blood samples. An attempt to apply the method to the analysis of bone marrow samples has yielded promising results; however, more detailed studies are needed in this area.

A sustainable approach for the stability study of psychotropic substances using vitreous humor and liver as alternative matrices.

The stability of psychotropic substances representing various drug groups important from the perspective of forensic chemistry, including benzodiazepines, antidepressants, carbamazepine, cocaine, and their selected metabolites, was investigated for 1 month in two alternative biological matrices, vitreous humor and liver homogenate. Three different thermal storage conditions (-20, 4, and 20 °C) were tested. Liquid chromatography-mass spectrometry (LC-MS) analysis was preceded by an effective solid-phase microextraction (SPME) procedure. The results were statistically analyzed using one-way ANOVA to find significant concentration variations over time. The results obtained allowed for dividing the analytes into four groups: stable under all tested conditions, only at -20 and 4 °C, only at 20 °C, and overall unstable. Nordiazepam, venlafaxine, and cocaine and its metabolites turned out to be the most unstable substances, while fluoxetine showed the highest storage stability in both matrices. The SPME/LC-MS method was comprehensively evaluated according to the principles of white analytical chemistry (WAC), which reconcile the greenness and functionality of the method. A close to 100% whiteness score proves its sustainability and suitability for the intended application.

A Perspective of the Comprehensive and Objective Assessment of Analytical Methods Including the Greenness and Functionality Criteria: Application to the Determination of Zinc in Aqueous Samples.

The recently proposed concept of White Analytical Chemistry (WAC), referring to the Red-Green-Blue color model, combines ecological aspects (green) with functionality (red and blue criteria), presenting the complete method as "white". However, it is not easy to carry out an overall quantitative evaluation of the analytical method in line with the WAC idea in an objective manner. This paper outlines the perspective of the future development of such a possibility by attempting to answer selected questions about the evaluation process. Based on the study consisting in the evaluation of selected model methods by a group of 12 independent analysts, it was shown how well individual criteria are assessed, whether the variability of assessments by different people is comparable for each criterion, how large it is, and whether averaging the scores from different researchers can help to choose the best method more objectively.

Differentiation of isomeric metabolites of carbamazepine based on acid-base properties; Experimental vs theoretical approach.

Metabolism of carbamazepine is complex and leads to the three isomeric derivatives whose occurrence is dependent on the type of sample material. Their unambiguous differentiation is overall important. In this work, the qualitative analysis of 2-hydroxycarbamazepine, 3-hydroxycrbamazepine and carbamazepine-10,11-epoxide was attempted for the first time using capillary zone electrophoresis, based on the models linking electrophoretic mobility with pKa value determining the acidity of the hydroxyl groups. For this purpose, pKa values ​​were determined using electrophoretic and theoretical methods, and then the compliance of the obtained mobility models with the measured values ​​was analyzed. Despite the slight difference in acidity ​​(0.3-0.4 pH unit), the obtained results prove that the correct identification of the metabolites under consideration, and reliable prediction of the selectivity of their separation, are possible on the basis of experimentally determined pKa values, even with highly simplified methods assuming the lack of certain data. However, it is important to choose the optimal pH value, which should be close to pKa. On the other hand, worse results were obtained for the theoretically determined mobilities, which differed significantly from the experimental values. An attempt was also made to explain the acidity of hydroxycarbamazepines and the associated thermodynamic parameters - deprotonation enthalpy and entropy, with respect to their structure. The lack of intramolecular hydrogen bonds and the significant contribution of entropic effects stabilizing the protonated form seems to be significant. The higher pKa value for CBZ-2-OH probably results from the stronger effect of the energetically unfavorable organization of solvent dipoles due to ionization.

The Double Face of Ketamine-The Possibility of Its Identification in Blood and Beverages.

The purpose of this study was to develop and validate a high-sensitivity methodology for identifying one of the most used drugs-ketamine. Ketamine is used medicinally to treat depression, alcoholism, and heroin addiction. Moreover, ketamine is the main ingredient used in so-called "date-rape" pills (DRP). This study presents a novel methodology for the simultaneous determination of ketamine based on the Dried Blood Spot (DBS) method, in combination with capillary electrophoresis coupled with a mass spectrometer (CE-TOF-MS). Then, 6-mm circles were punched out from DBS collected on Whatman DMPK-C paper and extracted using microwave-assisted extraction (MAE). The assay was linear in the range of 25-300 ng/mL. Values of limits of detection (LOD = 6.0 ng/mL) and quantification (LOQ = 19.8 ng/mL) were determined based on the signal to noise ratio. Intra-day precision at each determined concentration level was in the range of 6.1-11.1%, and inter-day between 7.9-13.1%. The obtained precision was under 15.0% (for medium and high concentrations) and lower than 20.0% (for low concentrations), which are in accordance with acceptance criteria. Therefore, the DBS/MAE/CE-TOF-MS method was successfully checked for analysis of ketamine in matrices other than blood, i.e., rose wine and orange juice. Moreover, it is possible to identify ketamine in the presence of flunitrazepam, which is the other most popular ingredient used in DRP. Based on this information, the selectivity of the proposed methodology for identifying ketamine in the presence of other components of rape pills was checked.

Copper and Zinc as Potential Biomarkers of Mood Disorders and Pandemic Syndrome.

The diagnosis of affective disorders has been the subject of constant research by clinicians from all over the world for many years. Making an appropriate diagnosis among patients suffering from mood disorders is sometimes problematic due to the personality-changing nature of patients and the similarity in the clinical picture of episodes in affective disorders. For this reason, there is a need to develop rapid and effective methods of determining biological markers that differentiate these diseases. The research was carried out with blood taken from 15 patients and 15 volunteers. The analysis of biological material for trace concentrations of zinc and copper was carried out with the use of ultrasensitive triple-quadrupole inductively coupled plasma mass spectrometry (TQ ICP-MS). The obtained results prove that the concentration of copper in the test group was lower than in the control group. For the zinc concentrations, the inverse relationship was observed. The group of patients was characterized by a higher concentration of this element than the group of healthy volunteers. Summarizing the obtained results and comparing them with the results of studies by other authors, it was found that zinc and copper may be potential biomarkers of affective disorders and pandemic syndrome.

Probing menstrual bloodstain aging with fluorescence spectroscopy.

Menstrual blood (MB) is a common and important type of forensic evidence, especially in sexual assault cases. MB is composed of peripheral blood (PB), vaginal fluid, and endometrial cells of the uterine wall. In forensic investigations, the differentiation of MB and PB can determine whether the blood present is a result of tissue damage from an assault or a natural cause and thus help to reconstruct the event. Understanding how menstrual blood changes is necessary to develop a method for bloodstain aging. Fluorescence spectroscopy, a promising spectroscopic method for bloodstain analysis, was used to probe the biochemical changes that occur over time in menstrual bloodstains. It was found that steady-state fluorescence spectra underwent significant changes over first nine hours post deposition. The underlying mechanism of fluorescence changes was proposed to involve the kinetic transformation of three fluorophores: tryptophan, nicotinamide adenine dinucleotide and flavins.

Development of a new method for drug detection based on a combination of the dried blood spot method and capillary electrophoresis.

The aim of this study was to develop a new approach to sample preparation of biological material based on a combination of the Dried Blood Spot (DBS) method and capillary electrophoresis coupled with mass spectrometry (CE-MS) for the analysis of blood samples collected in vivo or post-mortem. The proposed approach allowed the identification of typical drugs from different groups, such as tricyclic antidepressants (amitriptyline, imipramine), selective serotonin reuptake inhibitors (citalopram), benzodiazepines (tetrazepam) and hypnotics (zolpidem). In this study, a blood sample was spotted on FTA DMPK C cards, then dried, and 6-mm discs were cut out. The sample preparation procedure involved microwave-assisted extraction (MAE). Various extraction agents, temperatures and durations of extraction were examined in order to achieve the highest efficiency of the process. The method was subjected to a validation procedure. Limits of detection (LOD = 1.76 - 14.7 ng/mL) and quantification (LOQ = 5.25 - 49.0 ng/mL), inter- (CV = 1.31 - 9.43%) and intra- (CV = 3.26 - 18.52%) day precision of the determinations, recovery (RE = 85.0-105.4%) and matrix effect on ionization of analytes (ME = 98.6-105.5%) were determined. Furthermore, the developed DBS/MAE/CM-MS method was selective and analytes present in the blood applied on DBS cards were found to be stable after 7 and after 14 days. Moreover, the developed method was successfully applied to the analysis of both post-mortem samples and blood samples taken from patients treated with the analyzed drugs.

Does selenium fortification of kale and kohlrabi sprouts change significantly their biochemical and cytotoxic properties?

BACKGROUND: The sprouts of Brassica vegetables are known from their nutritional and chemopreventive values. Moreover, sprouts fortification with some trace elements, like selenium, may increase their importance in human diet. Thus, the aim of our study was to examine if selenium enrichment of kale and kohlrabi sprouts may influence their biochemical properties (phenolic acids and L-tryptophan content, antioxidant potential) or cytotoxic activity. Additional aim of the study was to evaluate the profile of selenium compounds and to describe the multidimensional interactions between the mentioned parameters. METHODS: Selenium content in the sprouts was evaluated by double-channel atomic fluorescence spectrometer AFS-230 with the flow hydride-generation system. Separation of selenium species in water soluble fraction was performed by size-exclusion LC-ICP-MS. The identification and quantification of phenolic acids and L-tryptophan was performed by HPLC. For antioxidant activity DPPH and FRAP methods were used. Cytotoxic activity of the sprouts extracts on a panel of human metastatic carcinoma cells was evaluated by MTT test. RESULTS: Selenium content in the fortified sprouts was several orders of magnitude higher than in the unfortified ones. Only small percentage of supplemented selenium (ca. 10 %) was incorporated into the sprouts as seleno-L-methionine, while the other detected selenium species remained unidentified. Selenium fortification differently stimulated the production of phenolic acids (sinapic, chlorogenic, isochlorogenic and caffeic acid) in the tested sprouts, depending on the particular species, selenium dose and the investigated compound. PCA analysis revealed strong correlation between antioxidant parameters and phenolic acids and L-tryptophan, while Se correlated only with caffeic acid. The sprouts extracts (≥1 mg/mL) showed cytotoxic potency to all the studied cancer cell lines (SW480, SW620, HepG2, SiHa), regardless the selenium supplementation. CONCLUSION: Se-fortified kale and kohlrabi sprouts are good candidates for functional food ingredients. Moreover, these results indicate that the sprouts enriched with sodium selenite show higher nutritional value, without significant changes in their cytotoxic activity.

Fast and efficient analyses of the post-mortem human blood and bone marrow using DI-SPME/LC-TOFMS method for forensic medicine purposes.

For the first time the method DI-SPME/LC-TOFMS was used and developed in order to determine the large antidepressant drugs in real forensic cases. The aim of the study was to optimize the new DI-SPME/LC-TOFMS method for the quantification of the large group of psychotropic drugs such as benzodiazepines, selective serotonin reuptake inhibitors, selective serotonin and noradrenaline reuptake inhibitors, tricyclic antidepressants and sleeping pills "Z". The volume of the sample, adsorption time, post-adsorption purification and desorption time were precisely optimized. The validation parameters such as limit of detection and quantification, linearity, precision during and between days and the matrix effect were determined. All obtained values are within the acceptable range for toxicological analyses. The usefulness of the method was confirmed by analyzing the post-mortem samples. Drug concentrations were determined in real samples with high precision, which gives perspectives for the DI-SPME/LC-TOFMS routine application in toxicological and forensic analyses in the future.

Postmortem analysis of human bone marrow aspirate - Quantitative determination of SSRI and SNRI drugs.

For the first time the MAE/UHPLC-TOFMS method was developed and used in order to determine the antidepressant drugs within the human bone marrow aspirate in real forensic cases. The following drugs, belonging to the group of selective serotonin (or serotonin-norepinephrine) reuptake inhibitors, were tested in this study: venlafaxine, citalopram, fluoxetine, sertraline and paroxetine. The sample preparation proposed in the present article included several steps: homogenization in ultrasound bath, liquid-liquid extraction, fat removal and evaporation under nitrogen. The extraction involved microwave-assisted extraction, performed for 15 min at 55 °C, with hexane-isoamyl alcohol (99:1, v/v) mixture, as an extraction solvent. Time and temperature of extraction were optimized using the simplex method. The fat removal step was introduced because of the fatty nature of the bone marrow that resulted in insufficiently purified samples, impossible to analyze using HPLC. It was achieved by adding a mixture of ethanol, water and formic acid to samples consisting of hexane and isoamyl alcohol, so that the analytes could diffuse to polar phase and fat could stay in non-polar phase. The method was validated and parameters such as: LOD, LOQ, linearity, matrix effect, recovery were determined. The validation parameters obtained allowed to recognize the method as quantitative. Due to lack of the data on therapeutic and toxic levels of considered drugs in bone marrow, data regarding serum has been used for reference. Under this assumption, the developed method allows for quantification of all mentioned drugs at therapeutic levels. Moreover this method has been used in real cases. Finally four analytes: venlafaxine (104 ng/mL), fluoxetine (84 ng/mL), paroxetine (about 3.6 µg/mL) and citalopram (68 ng/mL) were found in three case samples.

Dried blood spots sampling in case samples deprived of hematocrit level information - Investigation and calculation strategy.

The application of a new calculation strategy for the psychotropic drug concentration in blood and bone marrow samples in the form of dried blood spots (DBS) was the main aim of the study. The standard DBS method consists of the deposition of the capillary blood onto the dedicated paper cards. Nowadays, the DBS technique is seen as a fast and partly superior microsampling alternative methodology replacing the conventional venous blood and plasma collection. The calculation approach to drug concentration in the limited volume of the case sample, where the hematocrit level cannot be determined, constitutes an important step of this method. The method has been validated and the results of the determination of alprazolam and diazepam previously spiked in the post-mortem blood and bone marrow sample have been satisfactory.

Direct analysis from dried blood spot card surfaces with direct probe mass spectrometry - Evaluation study.

RATIONALE: The Direct Probe Mass Spectrometry (DIPMS) method allows successful analysis of powders, solid and liquid samples. The potential of direct surface analysis could find further application in the examination of surfaces with good absorption properties such as Dried Blood Spot (DBS) cards that constitute a great alternative to the classical blood collection method directly from veins. METHODS: DIPMS was performed with the ionization carried out under atmospheric pressure in an Atmospheric Pressure Chemical Ionization source. Direct analysis of diazepam solutions in methanol and after their deposition onto a DBS card was conducted. Subsequently, images of the DBS cards with and without blood samples were acquired using Scanning Electron Microscopy (SEM). RESULTS: Direct quantitative analysis of diazepam liquid samples by DIPMS was successfully performed. Linear correlation between the concentration of diazepam and the peak intensity with a R2 coefficient of 0.937 was obtained. However, the method failed when the analysis was conducted directly from the surface of the DBS cards and no diazepam peak was observed in the mass spectrum. The SEM images confirmed the good absorption properties of DBS cards and the absence of blood components on the surface. CONCLUSIONS: DIPMS is an excellent technique for the rapid, direct analysis of powders, solid and liquid samples; however, the potential of the method is limited when samples are deposited on surfaces with good absorption properties such as DBS cards.