Post-mortem formation of ethanol: Is 1-propanol a reliable marker? A proof-of-concept study using an in vitro putrefactive environment setup.

Ethanol is the psychoactive substance identified most frequently in post-mortem specimens. Unfortunately, interpreting post-mortem ethanol concentrations can be difficult because of post-mortem alcohol redistribution and the possibility of post-mortem alcohol neogenesis. Indeed, in the time interval between death and sample collection, the decedent may be exposed to non-controlled environments for an extended period, promoting microbial colonization. Many authors report that in the presence of carbohydrates and other biomolecules, various species of bacteria, yeast, and fungi can synthesize ethanol and other volatile substances in vitro and in vivo. The aim of this study was to study the impact of several variables on microbial ethanol production as well as develop a mathematical model that could estimate the microbial-produced ethanol in correlation with the most significant consensual produced higher alcohol, 1-propanol. An experimental setup was developed using human blood samples and cadaveric fragments incubated under strictly anaerobic conditions to produce a novel substrate, "cadaveric putrefactive blood" mimicking post-mortem corpse conditions. The samples were analyzed daily for ethanol and 1-propanol using an HS-GC-FID validated method. The formation of ethanol was evaluated considering different parameters such as putrefactive stage, blood glucose concentration, storage temperature, and storage time. Statistical analysis was performed using the Mann-Whitney non-parametric test and simple linear regression. The results indicate that the early putrefactive stage, high blood glucose concentration, high temperature, and time of incubation increase microbial ethanol production. In addition, the developed mathematical equation confirms the feasibility of using 1-propanol as a marker of post-mortem ethanol production.

Technical Evaluation of a Paper-Based Electrochemical Strip to Measure Nitrite Ions in the Forensic Field.

Nitrite is a compound used as a food additive for its preservative action and coloring capability, as well as an industrial agent for its antifreezing action and for preventing corrosion, and it is also used as a pharmaceutical in cyanide detoxification therapy. However, even recently, because of its high toxicity, it has been used as a murder and suicidal agent due to its affordability and ready availability. In this technical report, we describe an electrochemical paper-based device for selectively determining nitrite in complex biofluids, such as blood, cadaveric blood, vitreous humor, serum, plasma, and urine. The approach was validated in terms of the linearity of response, selectivity, and sensitivity, and the accuracy of the determination was verified by comparing the results with a chromatographic instrumental method. A linear response was observed in the micromolar range; the sensitivity of the method expressed as the limit of detection was 0.4 µM in buffer measurements. The simplicity of use, the portability of the device, and the performance shown make the approach suitable for detecting nitrite in complex biofluids, including contexts of forensic interest, such as murders or suicides in which nitrite is used as a toxic agent. Limits of detection of ca. 1, 2, 4, 5, 3, and 4 µM were obtained in vitreous humor, urine, serum and plasma, blood, and cadaveric blood, also highlighting a satisfactory accuracy comprised between 91 and 112%.

Chitosan derivatives as dynamic coatings for transferrin glycoform separation in capillary electrophoresis.

Chitosan and its derivatives are interesting biopolymers for different field of analytical chemistry, especially in separation techniques. The present study was aimed at testing chitosan water soluble derivatives as dynamic coating agents for application to capillary electrophoresis. In particular, chitosan was modified following three different chemical reactions (nucleophilic substitution, reductive amination, and condensation) to introduce differences in charge and steric hindrance, and to assess the effect of these physico-chemical properties in capillary electrophoresis. The effects were tested on the capillary electrophoretic separation of the glycoforms of human transferrin, an important iron-transporting serum protein, one of which, namely disialo-transferrin (CDT), is a biomarker of alcohol abuse. Chitosan derivatives were characterized by using NMR and 1H NMR, HP-SEC-TDA, DLS, and rheology. The use of these compounds as dynamic coatings in the electrolyte running buffer in capillary electrophoresis was tested assessing the peak resolution of the main glycoforms of human transferrin and particularly of disialo-transferrin. The results showed distinct changes of the peak resolution produced by the different derivatives. The best results in terms of peak resolution were achieved using polyethylene glycol (PEG)-modified chitosan, which, in comparison to a reference analytical approach, provided an almost baseline resolution of disialo-transferrin from the adjacent peaks.

Chitosan Film Sensor for Ammonia Detection in Microdiffusion Analytical Devices.

Chitosan films have attracted increased attention in the field of sensors because of chitosan's unique chemico-physical properties, including high adsorption capacity, filmability and transparency. A chitosan film sensor was developed through the dispersion of an ammonia specific reagent (Nessler's reagent) into a chitosan film matrix. The chitosan film sensor was characterized to assess the film's properties by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). A gas diffusion device was prepared with the chitosan film sensor, enabling the collection and detection of ammonia vapor from biological samples. The chitosan film sensor color change was correlated with the ammonia concentration in samples of human serum and artificial urine. This method enabled facile ammonia detection and concentration measurement, making the sensor useful not only in clinical laboratories, but also for point-of-care devices and wherever there is limited access to modern laboratory facilities.

Application of Paper-Based Microfluidic Analytical Devices (µPAD) in Forensic and Clinical Toxicology: A Review.

The need for providing rapid and, possibly, on-the-spot analytical results in the case of intoxication has prompted researchers to develop rapid, sensitive, and cost-effective methods and analytical devices suitable for use in nonspecialized laboratories and at the point of need (PON). In recent years, the technology of paper-based microfluidic analytical devices (µPADs) has undergone rapid development and now provides a feasible, low-cost alternative to traditional rapid tests for detecting harmful compounds. In fact, µPADs have been developed to detect toxic molecules (arsenic, cyanide, ethanol, and nitrite), drugs, and drugs of abuse (benzodiazepines, cathinones, cocaine, fentanyl, ketamine, MDMA, morphine, synthetic cannabinoids, tetrahydrocannabinol, and xylazine), and also psychoactive substances used for drug-facilitated crimes (flunitrazepam, gamma-hydroxybutyric acid (GHB), ketamine, metamizole, midazolam, and scopolamine). The present report critically evaluates the recent developments in paper-based devices, particularly in detection methods, and how these new analytical tools have been tested in forensic and clinical toxicology, also including future perspectives on their application, such as multisensing paper-based devices, microfluidic paper-based separation, and wearable paper-based sensors.

An origami microfluidic paper device for on-site assessment of urine tampering. First use of Nessler's reagent for the colorimetric determination of creatinine.

The relevance of the problem of urine tampering is well-known in forensic toxicology, with sample dilution being the most used method to cheat toxicological controls. Among the criteria to assess urine integrity, the quantification of creatinine probably represents the most popular method. The present paper presents a simple and low-cost analytical device for on-site creatinine determination as first-line screening for urine dilution. The proposed microfluidic devices were designed as a three-dimensional origami pattern. The device included three colorimetric reactions based on picric acid (PA-based reagent), 3,5-dinitrobenzoic acid (DNBA-based reagent), and Nessler's reagent. The last one, to the best of our knowledge, has never been used before for creatinine determination. In order to assure the highest ease and economy of operation, the color detection and data processing were performed using a built-in smartphone camera and the associated software. The optimized device showed a detection limit of 0.02 g/L. The proposed method was used for the qualitative screening for urine dilution of 48 samples, showing a diagnostic sensitivity and specificity for PA-based, DNBA-based and Nessler's reagent of 83.3%-80.0%, 72.2%-70.0%, and 100.0%-93.3% respectively, versus reference enzymatic method adopting a cut-off of 0.2 g/L. In conclusion, the present preliminary study shows that the proposed device could be a useful tool for on-site screening for urine tampering at the time of sample collection for toxicological testing.

Validation of a New Salt-Assisted HS-GC-FID Method for the Determination of Ethanol in the Vitreous Humor.

Headspace gas chromatography with a flame ionization detector (HS-GC-FID) is a well-established approach for determining blood alcohol concentration, including in cadaveric specimens. Although the integrity of blood specimens can be adequately guaranteed after the sampling, the quantification of ethanol in cadaveric blood can be affected by postmortem fermentative phenomena occurring between the time since death and the sampling of biofluids. The vitreous humor is less affected by putrefactive phenomena allowing compound determination and its use as an alternative biological matrix. The present work aimed to develop and validate a method using the salting-out effect and based on HS-GC-FID for the determination of ethanol in the vitreous humor. The reported analytical method is based on a simple vitreous humor pre-treatment consisting of a dilution (1:9) with a solution of 2.5 mol/L K2CO3 and 0.0012 mol/L tert-butanol (internal standard). After 1 min of incubation, part of the specimen evaporated in the headspace (2,000 µL) is injected into the chromatographic system and analyzed in isothermal mode (40°C), with a chromatographic time of 1.6 min. The method was validated in terms of selectivity, the lowest limit of detection, intraday and total imprecision, and trueness (bias). The determination of ethanol in the vitreous humor and blood was carried out in 75 cases. The correlation between the two matrices was confirmed in 61 cases. However, 14 vitreous humor specimens showed lower ethanol concentrations, and in the related blood specimens, it was possible to identify the signal of n-propanol, a typical product of postmortem microbial fermentation, that justifies the excess of ethanol in the blood specimens.

Development and Validation of a Rapid Method for Identification of New Synthetic Cannabinoids in Hair Based on High-Performance Liquid Chromatography-Ion Trap Mass Spectrometry Using a Simplified User Interface.

The penetration of the new psychoactive substances (NPSs) into the market of clandestine drugs is highly dynamic, causing potentially false-negative results using the current analytical instrumentation, particularly in the screening phase. At present, the suggested approach to perform a comprehensive screening requires the use of high-resolution mass spectrometry (MS) with associated high costs of purchase and maintenance and need of skilled and dedicated personnel. Here we describe the development and validation of a simplified approach based on a high-performance liquid chromatography-ion trap MS system with a user-friendly interface dedicated to toxicological analysis. The system, originally intended for a broad toxicological screening, was tuned to identify new synthetic cannabinoids in hair. After a washing step with dichloromethane, hair (about 50 mg) was incubated for 3 h with 1.5 mL ethanol. One milliliter of this solution was then dried, reconstituted with mobile phase and injected. The peak identification was based on the chromatographic retention times and MS2/MS3 data using a database which included up to 158 NPSs. The method was validated according to international guidelines on a selected panel of NPSs, namely methyl 2-[[1-(5-fluoropentyl)indazole-3-carbonyl]amino]-3,3-dimethylbutanoate (5F-ADB), 1-Pentyfluoro-1H-indole-3-carboxylic acid 8-quinolinyl ester (5F-PB 22), N-[(2S)-1-amino-3-methyl-1-oxobutan-2-yl]-1-(5-chloropentyl)indazole-3-carboxamide (5Cl-AB-PINACA), (S)-N-(1-amino-1-oxo-3-phenylpropan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide [5F-APP-PICA (PX-1)],: (R)-N-(1-amino-1-oxo-3-phenylpropan-2-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide [5F-APP-PINACA (PX-2)], N-[(2S)-1-Amino-3-methyl-1-oxobutan-2-yl]-1-(cyclohexylmethyl)indazole-3-carboxamide (AB-CHMINACA), N-[(2S)-1-Amino-3-methyl-1-oxobutan-2-yl]-1-[(4-fluorophenyl)methyl] indazole-3-carboxamid (AB-FUBINACA), methyl (2S)-2-[[1-(cyclohexylmethyl)indole-3-carbonyl]amino]-3,3-dimethylbutanoate (MDMB-CHMICA), (S)-Methyl 2-(1-(5-fluoropentyl)-1H-indole-3-carboxamido)-3-methylbutanoate (MMB-2201) and (1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone (UR-144). The tested analytical method showed detection limits between 0.065 and 0.125 ng/mg. The intraday imprecision of the method showed average values within the range of 7.3-20%. The estimation of the trueness (bias) of method showed average values within the range of 1.5-12.3%. The analytical performance was also successfully assessed by four proficiency test samples containing NPS. No synthetic cannabinoids were detected in application to 82 hair samples from forensic cases previously analyzed with liquid chromatography-MS triple quadrupole.

Contact Lens Wear Induces Alterations of Lactoferrin Functionality in Human Tears.

The tear film is a complex matrix composed of several molecular classes, from small metal ions to macromolecules. Contact lens (CL) wear can affect the protein homeostasis of the tear film, by accumulating deposits on the CL surface and/or altering their structural and functional properties. This work investigates the effect of CL wear on lactoferrin (Lf), one of the most abundant tear proteins, known as an unspecific biomarker of inflammation. Tears from eight volunteers were collected and analyzed after alternated periods of CL wear and without CL. The experimental approach is to probe Lf into unprocessed human tears by the peculiar fluorescence emission originating from complex formation of Lf with terbium (Tb3+) at the iron-binding sites. The experimental data indicate that CL wear does not significantly affect the total amount of Lf. On the other hand, Lf affinity for Tb3+ is reduced upon CL wear, suggesting relevant changes in Lf structure and possible alterations of protein functionality. Future studies based on this approach will help define CL features (material, lens-care solution, wearing time, etc.) with minimal effects on tear protein activity, in order to obtain more biocompatible and comfortable devices.

New evidence of high association between carbohydrate deficient transferrin (CDT) and alcohol-related road traffic accidents. A retrospective study on 929 injured drivers.

BACKGROUND: It is well known that traffic injuries still represent one of the main causes of death and that high blood alcohol concentrations while driving significantly increase the occurrence of accidents. However, only limited literature on the correlation between chronic alcohol abuse and accident risk is available. The aim of the present study was to investigate the hypothesis of an association between elevated concentrations of carbohydrate deficient transferrin (CDT) and the occurrence of alcohol-related traffic accidents. METHODS: The analytical determinations of BAC and CDT were performed following certified methods in HS-GC-FID and HPLC, respectively. For BAC, 0.50 g/L was used as cut-off, whereas 2.0% was used for CDT, according to the standardisation proposed by IFCC. A total of 929 drivers, tested for BAC at the time of hospital admission after a traffic accident, were classified into two groups: InjDr 1 (BAC ≤ 0.50 g/L) and InjDr 2 (BAC>0.50 g/L); all drivers were also tested for CDT. RESULTS: InjDr 1 included 674 individuals, only 2.5% showing a CDT above the cutoff, whereas InjDr 2 group consisted of 255 subjects, 28.6% testing positive for CDT (Odds Ratio 15.5). When subdividing the InjDr group into increasing classes of CDT, a steady increase in the percentage of BAC-positive drivers was appreciated. Moreover, average BAC was found to parallel each class of CDT. CONCLUSIONS: The reported data strongly support the use of CDT as a biomarker of increased risk of alcohol-related traffic accidents in the procedures of re-granting of the driving license upon confiscation for "drink driving".

A novel high-throughput liquid chromatography assay for Carbohydrate-Deficient transferrin (CDT) based on flow-modulated isocratic elution and terbium-induced fluorescence.

Transferrin is a glycoprotein containing two bi- or tri-antennary carbohydrate chains ending with sialic acid. Its glycosylation is reduced in chronic alcohol abuse and in inborn glycosylation pathologies, where the carbohydrate-deficient fraction of the protein (CDT) increases significantly. The current methods require a gradient chromatographic separation and time-consuming sample preparation. In comparison, the proposed approach uses a novel flow-modulated liquid chromatography technique (fmLC) and a highly selective and sensitive fluorescence derivatization reaction with terbium ion. A fmLC-FLD method using isocratic anion exchange separation was optimized and validated to resolve disialo-transferrin and trisialo-transferrin from other transferrin glycoforms. Detection took place by recording fluorescence at 550 nm wavelength (excitation at 298 nm). The chromatographic separation needed 5 min, allowing seriate injection every 7.5 min. The method was validated according to the current guidelines of analytical chemistry showing adequate accuracy and precision for the quantitative determination of CDT. The proposed method proved also to be suitable to analyse haemolyzed sera which, because of interference by haemoglobin, fail the standard HPLC-Vis analysis. The method was tested in parallel with HPLC-Vis on 131 sera showing an excellent correlation of results proved by a correlation coefficient of 0.995 (Pearson's r). The proposed approach proved much simpler than the current methods and cheaper in terms of instrumental costs offering a ground-breaking analytical tool that could likely make available the characterization of CDT outside specialized laboratories, such as in occupational medicine centres, doctor's offices, small laboratories, alcohol rehabilitation centres, and in developing countries.

Alcohol-associated traffic injuries in Verona territory: A nine-year survey.

According to the World Health Organization, as many as 25% of traffic accidents are linked to alcohol abuse. This study describes the results of a nine-year study performed on injured drivers (N = 12,806) in the Verona area of Northern Italy. Blood samples were mandatorily collected on injured drivers who were admitted to the Emergency Health Care Unit of Verona Hospital between 2009 and 2017, after they had been involved in a traffic accident. Blood alcohol concentration (BAC) determination was then undertaken using a validated head space-gas chromatography-flame ionisation detector (HS-GC-FID) method. We found that 21% of drivers tested positive for alcohol (BAC ≥0.01 g/L), while 16.8% presented with BAC levels above the Italian legal limit (>0.5 g/L). Of those who had positive BACs, about 50% presented with very high BAC levels (>1.5 g/L). Daily time distribution analyses, involving 2031 alcohol-positive drivers, showed a surge between 18:00 hours and 06:00 hours (74.3%), with a specific rise during the weekend (58.9%). The percentage of alcohol-related road accidents was 20.6%, which is lower than results reported in other international studies performed over the last 20 years. However, evidence that around 50% of the positive subjects showed a BAC >1.5 g/L confirms the correlation between BAC and accident risk, which becomes even more significant at progressively increasing levels of BAC. The study highlights the need to implement further strategies to both prevent and deter the use of alcohol while driving.

Simultaneous analysis of potassium and ammonium ions in the vitreous humour by capillary electrophoresis and their integrated use to infer the post mortem interval (PMI).

Post-mortem changes of ions in the body fluids have been proposed as an objective tool for inferring the time of death. In particular, the post-mortem increase of potassium concentrations in the vitreous humour has gained great attention in the literature. On the other hand, ammonium, another ion released in post-mortem processes, has received much less attention, potentially due to unresolved analytical issues using current clinical chemistry methods. This paper presents an application of a new analytical approach based on capillary electrophoresis providing the simultaneous analysis of potassium and ammonium ions in the vitreous humour. In addition, to assess the consistency of the post-mortem increase of ammonium concentrations in the vitreous humour, the determination of this ion in the vitreous humour of the two eyes of the same body at the same post-mortem interval has been verified. Vitreous humour was collected from 33 medico-legal cases where the time of death was known exactly. Prior to analysis, all samples were diluted 1:20 with a 40 µg/mL solution of BaCl2 (internal standard). In the study of the variability of the ammonium concentration between the two eyes, no statistically significant differences were found, supporting the hypothesis of an even post-mortem increase of the ion concentrations in this particular biological fluid. Significant correlations of potassium and ammonium ions with the post-mortem interval were found, with r2 of 0.75 and 0.70, respectively.

A simple and robust method for broad range screening of hair samples for drugs of abuse using a high-throughput UHPLC-Ion Trap MS instrument.

The use of chromatography hyphenated with mass spectrometry is a well-established approach in clinical and forensic toxicology, particularly for the analysis of the so-called alternative matrices (hair, nails, oral fluid, sweat). This procedure for the quantitative determination of targeted analytes has been reported since the early 1980s and today is the golden standard in analytical toxicology. However this technology has not found wide application the broad spectrum preliminarily screening of samples which is still mostly based on immunoassays. The aim of the present work was to test a recent instrumental approach based on UHPLC-Ion Trap-MS (Toxtyper®, Bruker Daltonics) intended to be used in routine contexts for the analysis of drug of abuse applied to hair toxicological analysis. The reported analytical method is based on a simple hair pre-treatment consisting of an overnight acid incubation in 0.1 mol/L HCl, followed by direct injection, after neutralization with equimolar amount of NaOH. The separation was then performed using a reverse phase column with a rapid gradient elution of 11 min (from 1% acetonitrile in 0.1% ammonium formate to 95% acetonitrile in 0.1% ammonium formate). Detection was by a fast ion trap analyzer (32,500 m/z sec-1) operating in the mass range 70-800 m/z. The chromatographic retention time and MS2/MS3 data were used for compound identification using a proprietary database which allowed to screen for up to 987 compounds. The tested analytical method showed limits of detection in the range between 0.01 and 0.09 ng/mg of hair matrix for a panel of 16 drugs of abuse (except for MDA, morphine, 6-MAM and norketamine, which showed limits of detection of 0.25, 0.15, 0.15 and 0.25 ng/mg, respectively). The method was validated according to international guidelines on a selected panel of drugs of abuse. The analytical performance of the instrument was assessed by analyzing 968 hair samples from forensic cases. A good concordance with a reference confirmatory method based on GC-MS was found in terms of classification of both negative and positive samples. Finally, the method was also successfully tested by analyzing 12 proficiency test samples containing not only common drugs of abuse but also new psychoactive substances, including fentanyls and cathinones.

Thanatochemistry at the crime scene: a microfluidic paper-based device for ammonium analysis in the vitreous humor.

Most of the on-site approaches for inferring of the post-mortem interval are still based on observative data from the direct body inspection, whereas, objective and quantitative analyses, such as potassium in the vitreous humor, are require laboratory instrumentation and skilled personnel. The present paper presents a simple and low cost analytical method suitable for use at the crime scene for inferring the time since death. The method uses a microfluidic paper-based device (µPAD) for the determination of ammonium in the vitreous humor (VH) based on the selective interaction between the ammonium and the Nessler's reagent. The color change was measured in terms of "RGB distance" by using a simple and free smartphone application. The optimized device showed a limit of detection of 0.4 mmol L-1, with between days precision less than 9.3% expressed as relative standard deviation, and accuracy between days from 94.5% to 104.5%. The selectivity of the Nessler's reaction was tested towards the main vitreous humor compounds, and no significant interferences were found. This paper-based analytical device was successfully used for the determination of ammonium ion in VH samples from forensic autopsies. The results obtained with the proposed method, although for a limited number of cases (n = 25), showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover, in order to make the evaluation of results as simple as possible, a direct correlation between the color intensity, expressed as RGB distance, and the post-mortem interval was studied and a significant correlation was found (R2 > 0.78). In conclusion, the present preliminary study showes that the proposed device could be an additional tool to the traditional methods for a more accurate, although still presumptive, estimation of the time of death directly at the crime scene.

A novel low-cost approach for the semi-quantitative analysis of carbohydrate-deficient transferrin (CDT) based on fluorescence resonance energy transfer (FRET).

BACKGROUND AND AIM: The increase of the carbohydrate-deficient transferrin (CDT) as results of an heavy intake of alcohol for at least two weeks, is a well-known biochemical modification since the middle '70s. Notwithstanding the first commercial kit for the diagnosis of chronic alcohol abuse based on this biomarker was commercially accessible already thirty years ago, only expensive analytical methods are currently available for its determination. The present paper shows a new approach intrinsically sensitive and specific, based on a specific derivatization of transferrin, and not requiring sophisticated instrumentation. METHODS: The proposed procedure is based on a selective chelation of terbium (III) by transferrin followed by detection using an characteristic Fluorescence Resonance Transfer Energy (FRET) phenomenon (ex 298 nm - em 550 nm). RESULTS: The proposed procedure showed a limit of detection of 2.5 pmol/mL and a reproducibility intra-day and inter-days <15% and 20%, respectively. The results obtained analyzing 40 serum samples using the developed method, were compared with those obtained with HPLC-Vis and an R2 = 0.8854 was found. CONCLUSIONS: Considering its main features (low-cost, ease of operation, minimum need of instrumentation) the present method is suitable for application in screening contexts and in non-strictly regulated environments (e.g. clinical diagnosis) as well as in developing countries or remote areas.

Development of a low cost gas diffusion device for ammonia detection in the vitreous humor and its preliminary application for estimation of the time since death.

A simple and low cost analytical device is described for the determination of ammonium in the vitreous humor suitable for inferring the post mortem interval in forensic cases. The device is based on ammonia formation from ammonium ion by means of NaOH addition to the vitreous humor sample and its detection with a pH chemical indicator in the gas phase above the vitreous humor sample. From the gas phase, ammonia diffuses through a polymeric membrane and it is trapped and detected with a droplet of pH indicator thymol blue. The color change of the droplet is measured using a smartphone camera. Under optimal conditions, the device showed a limit of detection of 0.2mM, with between days precision of ≤ 15% expressed as relative standard deviation, and an accuracy between days from 88.3% to 114.5%. This homemade gas diffusion analytical device was successfully used for the determination of ammonia in vitreous humor samples from forensic autopsies. The results obtained with the proposed method, although for a limited number, showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover a significant correlation was also found between the results of the present method and the time elapsed since death by a simple evaluation of the color intensity. In conclusion, this preliminary study showed that the proposed device, after adequate validation, could be a promising tool for a presumptive estimation of the time since death directly at the crime scene.

Development of an in-house mixed-mode solid-phase extraction for the determination of 16 basic drugs in urine by High Performance Liquid Chromatography-Ion Trap Mass Spectrometry.

The aim of the present work was to develop a novel in-house mixed-mode SPE sorbent to be used for the HPLC-Ion TrapMS determination of 16 basic drugs in urine. By using a computational modelling, a virtual monomer library was screened identifying three suitable functional monomers, methacrylic acid (MAA), itaconic acid (IA) and 2-acrylamide-2-methylpropane sulfonic acid (AMPSA), respectively. Three different sorbents were then synthetized based on these monomers, and using as cross-linker trimethylolpropane trimethacrylate (TMPTMA). The sorbent characterization analyses brought to the selection of the AMPSA based phase. Using this novel in-house sorbent, a SPE-HPLC-Ion TrapMS method for drug analysis in urine was validated proving to be selective and accurate and showing a sensitivity adequate for toxicological urine analysis. The comparison of the in-house mixed-mode SPE sorbent with two analogous commercial mixed-mode SPE phases showed that the first one was better not only in terms of process efficiency, but also in terms of quality-price rate. To the best of our knowledge, this is the first time in which an in-house SPE procedure has been applied to the toxicological analysis of a complex matrix, such as urine.

An aptamer-based paper microfluidic device for the colorimetric determination of cocaine.

A method utilizing paper microfluidics coupled with gold nanoparticles and two anticocaine aptamers has been developed to detect seized cocaine samples. The ready-to-use format involves the use of a paper strip that produces a color change resulting from the salt-induced aggregation of gold nanoparticles producing a visible color change indicating the presence of the drug. This format is specific for the detection of cocaine. The visual LOD for the method was 2.5 µg and the camera based LOD was 2.36 µg. The operation of the device is easy and rapid, and does not require extensive training or instrumentation. All of the materials utilized in the device are safe and environmental friendly. This device should prove a useful tool for the screening of forensic samples.

The alcohol used for cleansing the venipuncture site does not jeopardize blood and plasma alcohol measurement with head-space gas chromatography and an enzymatic assay.

INTRODUCTION: This study aimed to establish whether an alcoholic antiseptic, wiped or not before venipuncture, may jeopardize alcohol testing with a commercial enzymatic assay and a reference head-space gas chromatography (GC) technique. MATERIALS AND METHODS: Venous blood was collected from 23 healthy volunteers, with two sequential procedures. In the first blood collection, 2 mL of alcoholic antiseptic (0.5% chlorhexidine, 70% ethanol) were place on a gauge pad, the venipuncture site of right arm was cleaned but the antiseptic was not let to dry before phlebotomy. In the second blood collection, 2 mL of the same alcoholic antiseptic were placed on another gauge pad, the venipuncture site of left harm was cleaned and the antiseptic was accurately cleansed before phlebotomy. Ethanol was measured with a reference GC technique in whole blood and EDTA plasma, and a commercial enzymatic assay in EDTA plasma. RESULTS: No subject complained about feeling a particular itchy sensation when the alcohol was not wiped before puncturing the vein. The concentration of alcohol in all EDTA plasma samples was always lower than the limit of detection of the enzymatic assay (i.e., 2.2 mmol/L; 0.1 g/L). Similarly, alcohol concentration was also undetectable using a reference GC technique (i.e., < 0.22 mmol/L; 0.01 g/L) in EDTA plasma and whole blood. CONCLUSION: It seems reasonable to conclude that using ethanol-containing antiseptics before venipuncture may not be causes of spurious or false positive results of alcohol measurement at least when ideal venipunctures can be performed.