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

Metodo analitico per l'identificazione di almeno una glicoforma della proteina transferrina

L'invenzione riguarda un metodo analitico innovativo che sfrutta una derivatizzazione specifica con terre rare della transferrina per la determinazione ad alta sensibilit\ue0 della Carbohydrate Deficient Transferrin (CDT) il biomarker pi\uf9 affidabile delle patologie della glicosilazione proteica e dell'abuso cronico di alco

Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids

Urinary insulin-like growth factor-I measurement in an actual sport competition, an additional approach in laboratory antidoping tests

BACKGROUND: The insulin-like growth factor hormone (IGF-I) is an important protein hormone under investigation with physical exercise and for doping detection. Urinary IGF-I level in fact represents a relevant measurement when the postexercise proteinuria is under analysis. To verify the IGF-I level variation in the circulation and in urinary excretion in the occasion of a competition, the plasma and urine IGF-I in athletes before and after an actual competitive event were measured. METHODS: Twenty well-trained cyclists took part in a competition (102 km) and concluded the intense physical exercise in approximately 2(1/2) h. Urine and blood samples were collected from each athlete 10-20 min before and at the end of the competition. Plasma and urine total IGF-I (pIGF, uIGF), total urinary proteins (uPr), and creatinine (uCr) concentrations were measured. RESULTS: The uIGF [from 76.2+/-15.8 to 256.9+/-29.1 ng/l (p<0.001)], uPr [from 29.4+/-6.7 to 325.9+/-95.1 mg/l (p<0.005)], and uCr [from 6.3+/-1.0 to 10.0+/-0.8 mmol/l (p<0.005)] significantly increased. The pIGF was 262.6+/-14.3 and 247.3+/-11.8 microg/l before and end-exercise, respectively. A statistical correlation between uIGF and uPr was demonstrated (p<0.001). The pIGF/uIGF ratio was significantly (p<0.05) decreased comparing the end with before the competition. CONCLUSIONS: The pIGF/uIGF significantly decreased at the end, compared with before the competition, suggesting a changed uIGF excretion. This increment appeared to be increased, although not significantly, considering the ratio with uCr

Asialo-transferrin: Biochemical aspects and association with alcohol abuse investigation

Asialo-human transferrin (asialo-hTf) is a glycoform of the human serum protein transferrin characterized by the lack of the sialic acid (SA) terminal unit. It is known that glycosylation micro-heterogeneity and the presence of SA are strongly involved in protein functioning and pathophysiological activities. Some hTf glycoforms are valuable biomarkers for the detection of both genetic defects of glycosylation and/or sialoform distribution changes. The detection of the carbohydrate deficient transferrin (CDT) glycoforms is currently a widely employed method for the diagnosis of chronic alcohol abuse. The physiological significance of asialo-hTf is still unclear, despite its important biological implications. The current knowledge suggests that asialo-hTf may be involved in regulation of iron transport and release at the hepatic level, which, consequently, could strongly be affected by alcohol consumption. For these reasons, a deeper understanding of asialo-hTf structure and its physiological role is required, and an improved method of its analysis would favor the detection of both chronic abuse and other habits of alcohol intake and/or misuse. Thus, suitable analytical methods possessing higher sensitivity and specificity in comparison with the currently available techniques are certainly recommended. The present review summarizes the studies on asialo-hTf structure, roles, and detection techniques mainly in relation to its possible use as a potentially additional useful biomarker of alcohol abuse, and underlines its prospective value as a forensic and diagnostic tool

Separation methods for acyclovir and related antiviral compounds

Acyclovir (ACV) is an antiviral drug, which selectively inhibits replication of members of the herpes group of DNA viruses with low cell toxicity. Valaciclovir (VACV), a prodrug of ACV is usually preferred in the oral treatment of viral infections, mainly herpes simplex virus (HSV). Also other analogues such as ganciclovir and penciclovir are discussed here. The former acts against cytomegalovirus (CMV) in general and the latter against CMV retinitis. The action mechanism of these antiviral drugs is presented briefly here, mainly via phosphorylation and inhibition of the viral DNA polymerase. The therapeutic use and the pharmacokinetics are also outlined. The measurement of the concentration of acyclovir and related compounds in biological samples poses a particularly significant challenge because these drugs tend to be structurally similar to endogenous substances. The analysis requires the use of highly selective analytical techniques and chromatography methods are a first choice to determine drug content in pharmaceuticals and to measure them in body fluids. Chromatography can be considered the procedure of choice for the bio-analysis of this class of antiviral compounds, as this methodology is characterised by good specificity and accuracy and it is particularly useful when metabolites need to be monitored. Among chromatographic techniques, the reversed-phase (RP) HPLC is widely used for the analysis. C Silica columns from 7.5 to 18 30 cm in length are used, the separation is carried out mainly at room temperature and less than 10 min is sufficient for the analysis at 1.0\u20131.5 ml/min of flow-rate. The separation methods require an isocratic system, and various authors have proposed a variety of mobile phases. The detection requires absorbance or fluorescence measurements carried out at 250\u2013254 nm and at l 5260\u2013285 nm, l 5375\u2013380 nm, respectively. The detection limit is about 0.3\u201310 ng/ml but the ex em most important aspect is related to the sample treatment, mainly when body fluids are under examination. The plasma samples obtained from human blood are pre-treated with an acid or acetonitrile deproteinization and the supernatant after centrifugation is successively extracted before RP-HPLC injection. Capillary Electrophoresis methods are also discussed. This new analytical approach might be the expected evolution, in fact the analyses are improved with regard to time and performance, in particular coated capillary as well as addition of stabilisers have been employed. The time of analysis is shortened arriving at less than half a minute. Furthermore by using an electrochemical detection, and having a calibration linearity in the range of 0.2\u201320.0 ng/ ml, the detection limit is 0.15 mg/ ml. The measurements of acyclovir and penciclovir have been presented but in the future other related drugs will probably be available using CE methods

Salivary free insulin-like growth factor-i levels: effects of an acute physical exercise in athletes

The offer of human saliva IGF-I (sIGF-I) measurement in athletes investigation is a new proposal. The aim was to investigate the physical exercise effect on sIGF-I and explore plasma free IGF-I relation

Growth hormone isoforms,segments/fragments:Does a link exist with multifunctionality?

Endocrine diagnostic tests are dependent on the molecular characteristics of protein hormones, a property that is also intimately tied to function. The structure-function relationship is of particular importance for multifunctional protein hormones such as growth hormone (GH). For clinical diagnosis, it is imperative to understand the relationship between GH structure (and its molecular fragments) and function and their potential interaction with single or multiple receptors. The existence of a single or aggregated intact GH 22 kDa form such as the 20 kDa GH isoform has been described, but GH fragments cannot be excluded a priori. Recent advances and probable similarity of GH with other protein hormones such as natriuretic peptides (ANP, BNP and their proANP and proBNP fragments) and POMC (ACTH, beta-endorphin, etc.) lend support to the hypothesis that GH fragments should also be present. This brief review focuses on GH heterogeneity and feasible post-synthesis events. The aim of the review is to describe which GH forms/fragments have already been recognized and/or are potentially present in the circulation. The impacts of GH isoforms (namely the intact 20 and 22 kDa isoforms) and fragments thereof on quantitative measurement are discussed with reference to traditional immunoassay technology and innovative immunofunctional laboratory assay