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

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

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

IntroductionThis 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 methodsVenous 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. ResultsNo 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. ConclusionIt 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

Determination of new psychoactive substances in complex matrices by using methods based on micro-analytical systems

Nonostante il notevole ampliamento della rete di controlli, finalizzati a ridurre la diffusione a livello mondiale di composti illeciti di droga, l'abuso di sostanze psicotrope continua ad essere un fenomeno in aumento. I sequestri di cocaina, eroina, morfina e cannabis hanno mostrato una tendenza costante dal 2003 al 2012. I sequestri di stimolanti amfetamino-simili hanno seguito la stessa tendenza, fino al 2010, mentre 2010-2012 i sequestri sono stati triplicati. Inoltre, alcuni laboratori basano la propria attivit\ue0 sullo sviluppo di nuove sostanze psicoattive (NPSS) che non rientrano nella convenzione internazionale di controllo della droga. Secondo l'Ufficio delle Nazioni Unite contro la Droga e il Crimine (UNODC), le NPSs sono definite come "sostanze d'abuso ... che non sono controllate dalla Convenzione sugli stupefacenti del 1961 o la Convenzione del 1971 sulle sostanze psicotrope .... In questo contesto, il termine 'nuovo' non si riferisce necessariamente a nuove invenzioni, ma alle sostanze che sono recentemente disponibili ". La diffusione dei composti psicotropi non comuni \ue8 gi\ue0 una questione di tener conto. Il numero delle NPSs sul mercato globale \ue8 pi\uf9 che raddoppiato nel periodo 2009-2013. Negli ultimi anni la rilevazione di droghe illecite \ue9 stata diretta verso l'impiego di metodi analitici rapidi e precisi. La drammatica crescita della tecnologia permette di eseguire misurazioni analitiche impiegano volume dell'ordine dei nanolitri e picolitri. Questo progetto di ricerca mira a dimostrare l'affidabilit\ue0 dei sistemi microfluidici, come strumenti di analisi a fini tossicologici forensi. Diversi dispositivi microfluidici possono essere utilizzati per perseguire scopi differenti; nel presente progetto saranno esposti tre diverse strategie finalizzate a rilevare nuove sostanze psicoattive. Qui, il termine nuovo, \ue8 utilizzato per indicare l'uso voluttuario di un farmaco comunemente somministrati per scopi anestetici, la ketamina, e di un composto antitosse che viene utilizzato in molti farmaci da banco, il destrometorfano. La prima strategia \ue8 stata focalizzata sulla sviluppo di un dispositivo microfluidico basato su carta mediante rivelazione colorimetrica per l'analisi di ketamina e altre droghe di abuso. Una variet\ue0 di metodi presuntivi sono stati sviluppati, compresi test tubes, microscopia, TLC, GC e IR, ma molti di questi test richiedono tecnici specializzati, o come nel caso dei test tubes, non sono in grado di determinare contemporaneamente una grande variet\ue0 di composti in un unico step. Per quanto a nostra conoscenza questo lavoro presenta un unico processo per l'esecuzione di pi\uf9 test contemporaneamente. Il test pu\uf2 rilevare un'ampia variet\ue0 di analiti utilizzando pochi microgrammi di campione. Analisi semiquantitativa \ue8 possibile anche utilizzando uno smartphone e software semplice. Il metodo proposto non richiede persone altamente qualificate o strumentazione costosa, e pu\uf2 essere eseguita in loco rendendo possibile una risposta analitica durante le azioni di polizia, servizi di frontiera, e sicurezza negli aeroporti. La seconda parte del progetto ha sottolineato lo sviluppo e la validazione di un semplice metodo di separazione chirale per identificare e quantificare gli enantiomeri del metorfano, e loro principali metaboliti, utilizzando elettroforesi capillare mediante selettori chiarali costituiti da ciclodestrine (CD-CZE). Per quanto a nostra conoscenza, i metodi disponibili sono basati su complessi metodi MEKC e ciclodestrine (CD-MEKC) o metodi che richiedono colonne cromatografiche costosi. Nel 2013, Koo C et al. sviluppato un GC - MS utilizzando colonna achirale, ma il metodo non \ue8 stato applicato a campioni di sangue. Il metodo proposto ha consentito di risolvere destrometorfano da levometorfano in sequestri di eroina e di eseguire l'identificazione chirale e la quantificazione di enantiomeri del metorfano e loro principali metaboliti , destrorfano e levorfanolo, nel sangue prelevato in sede autoptica, con una sensibilit\ue0 accettabile. La sezione finale del progetto \ue8 stato centrato su un studio preliminare finalizzato allo sviluppo di un metodo per rilevare la ketamina, basato su elettroforesi capillare e polimeri a stampo molecolare (MIP-CZE). I risultati preliminari sono stati ottenuti utilizzando la tecnica HPLC-MS per caratterizzare la capacit\ue0 di interazione in termini di recupero estrazione di ketamina e comuni droghe di abuso. La complessit\ue1 del processo ci ha indirizzato a sviluppare un metodo rapido alternativo in grado di valutare la capacit\ue0 di interazione dei polimeri a stampo molecolare da utilizzare per analisi CZE. I risultati preliminari hanno dimostrato l'adeguatezza e l'affidabilit\ue0 del elettroforesi capillare per caratterizzare le propriet\ue0 dei MIP in formato nanoparticellare (NP MIP). Questo studio pone le basi per la possibilit\ue0 di utilizzare NP MIP come selettori ad alta specificit\ue0 da utilizzare in analisi CZE.Notwithstanding the remarkable amplification of the network of controls, finalized to reduce the worldwide spread of illicit drug compounds, the abuse of psychotropic substances continues to be a rising phenomenon. The seizures of cocaine, heroin and illicit morphine and cannabis showed a constant trend from 2003 to 2012. The seizures of amphetamine-type stimulant followed the same trend until to 2010, while from 2010 to 2012 the seizures were trebled. In addition, some laboratories base their activity on developing of New Psychoactive Substances (NPSs) that fall outside international drug control convention. According to the United Nations Office on Drugs and Crime (UNODC), NPSs are defined as \u201csubstances of abuse\u2026that are not controlled by the 1961 Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances\u2026. In this context, the term \u2018new\u2019 does not necessarily refer to new inventions but to substances that have recently become available\u201d. The spreading of uncommon psychotropic compounds is already a matter to take account. The number of NPSs on the global market more than doubled over the period 2009-2013 1. In recent years the detection of illicit drugs has been directed toward a rapid and accurate recognition analytical methods. The dramatic growth of the technology allows performing of analytical measurements employing volume in the range of the nanoliter and picoliter. This research project seeks to demonstrate the reliability of microfluidic systems, as analytical tools for forensic toxicological purposes. Several microfluidic devices can be used to pursue different aims; in the present project will be exposed three different strategies finalized to detect new psychoactive substances. Here, the term new, is utilized to indicate the voluptuary use of a medicine commonly administered for anesthetic aims, the ketamine, and the reveler utilization of an antitussive compound which is used in many over-the-counter drug, the dextromethorphan. The first strategy was focused on the developing of a set of paper microfluidic colorimetric tests for the analysis of ketamine and other common drugs of abuse. A variety of presumptive methods have been developed, including spot tests, chemical microscopy, TLC, GC and IR but many of these tests require skilled handlers, or like spot tests are incapable of simultaneously determining a wide variety of compounds in a single tube analysis step. To the best of our knowledge this work presents a unique process for running multiple assays simultaneously. The test can detect a wide variety of analytes using few micrograms of sample. Semiquantitative analysis is also possible using a smartphone and simple software. The proposed method doesn\u2019t require highly qualified persons or expensive instrumentation, and it can be performed on-site enabling a prompt analytical response during police actions, border services, and airport security. The second part of the project emphasized the development and validation of a simple chiral separation method to identify and quantify the enantiomers of methorphan, and their main metabolites, by using cyclodextrin-assisted capillary zone electrophoresis (CD-CZE). To the best of our knowledge, the available methods are based on complex method cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) 2 or methods which require expensive chromatographic columns 3. In 2013, Koo C et al. developed a GC/MS by using achiral column, but the method was not applied to blood samples 4. The proposed method allowed to resolve dextromethorphan from levomethorphan in seizures of heroin and to perform the chiral identification and the quantification of methorphan enantiomers and their main metabolites, dextrorphan and levorphanol, in post-mortem blood, with an acceptable sensitivity. The final section of the project was centered on preliminary study finalized to the development of a method to detect ketamine, based on molecularly imprinted polymer-assisted capillary zone electrophoresis (MIP-CZE). The preliminary results were obtained by using the technology HPLC-MS to characterize the capacity of interaction in terms of extraction recovery of ketamine and common drugs of abuse non-structure related to ketamine. The tediousness of the process addressed us to develop an alternative rapid method able to evaluate the capacity of interaction of the molecularly imprinted polymers to use in CZE analysis. Preliminary results demonstrated the suitability and the reliability of the capillary electrophoresis to characterize the binding properties of molecularly imprinted polymers nanoparticle format (NP MIP). This study lays the groundwork for the possibility to utilize NP MIP as high-specificity selector to use in CZE analysis

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\ua0\u3bcg and the camera based LOD was 2.36\ua0\u3bcg. 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

Use of synthetic cannabinoids to cheat the toxicological screenings

Use of synthetic cannabinoids to cheat the toxicological screening

Calculation of LogP based on migration data in MEKC: application to the new synthetic cannabinoids.

Since 2004, a number of \u201cherbal blends\u201d containing synthetic cannabinoid analogues have appeared in the market, as a form of \u201clegal\u201d alternatives to Cannabis. These products are available online through the \u201ce-commerce\u201d and in \u201csmart shops\u201d under several forms and various brand names. A particular warning related to the diffusion of synthetic cannabinoids is based on one hand on their high toxicological potential, and on the other hand on the insensitivity of the current screening tests for cannabis towards these molecules. Moreover, because of their recent introduction, the literature regarding these compounds is still limited. In particular any experimental information on their octanol/water partition coefficient (logP) values is still lacking. Indeed, hydrophobicity value is an important parameter to investigate drug absorption, bioavailability and metabolism of molecules, as well as their toxicity. As a measure of molecular hydrophobicity, the logarithm of the partition coefficient between 1-octanol and water (logP) is widely used. The aim of the present study was to calculate the LogP of synthetic cannabinoids by using micellar electrokinetic chromatography (MEKC) with UV detection. Samples were analyzed using a fused silica capillary (30 mm x 40 cm) and a 25 mM sodium borate buffer pH 8, containing30 mMSDS and n-propanol 20%. After having constructed a calibration line using 10 appropriate standards with known LogP, an EOF marker and a micelle marker, a good correlation was found between the MEKC retention data of further 5 compounds with known LogP. The same calibration line was used to calculate the unknown LogP of the new synthetic cannabinoids, namely JWH-200, AM-694, JWH-250, JWH-073, JWH-015, JWH-018, JWH-081, JWH-122, JWH-019, JWH-210. The resulting Log P were in the range from 2.9 to 5.15. An acceptable agreement was found between the experimental data and a few LogP values calculated with XLogP3-AA reported in the literature

Rapid screening of the binding properties of molecularly imprinted nanoparticles via capillary electrophoresis.

Rapid screening of the binding properties of molecularly imprinted nanoparticles via capillary electrophoresis

Capillary electrophoresis screening of the binding affinity of Molecularly Imprinted Nanoparticles.

The fast paced advancement in molecularly imprinted nanoparticles (MIP NPs) synthesis and applications [1] requires straightforward analytical methods suitable for the evaluation of the binding properties of the generated MIP NP libraries. Often the analysis of the MIP NP binding behaviour cannot be easily performed by classical means, because of their nature, i.e. amphipathic, polymeric and their dimensions (10-200 nm), which are greater than biological macromolecules, but smaller than most of the micromaterials. In response to these needs, capillary electrophoresis (CE) was here exploited to screen the binding affinities of MIP NPs targeting the iron regulating hormone hepcidin towards their template peptide, i.e. the N terminal 6-mer of hepcidin [2- 4]. The CE separation method was developed ex novo, after optimization of the background electrolyte (150 mM sodium phosphate pH 7.4) and of the running temperature (35\ub0C), achieving the full separation of the free ligand from the complexed MIP NPs. The CE binding isotherm allowed estimating a micromolar dissociation constant for the 6-mer template/MIP NP complex. The results were found in agreement with independent measures. The CE offers the advantages of a direct analysis of the MIP NP/ligand incubation mix without preliminary fractionation steps, requiring only minimal sample consumption and short analysis times, thus in conclusion, it appeared to be a valid technique for characterizing the interaction of MIP NP libraries for selected target compounds

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