Anna Bertaso

Rapid and direct determination of creatinine in urine using capillary zone electrophoresis

BACKGROUND In clinical medicine creatinine determination is used for the diagnosis of renal diseases and muscular dysfunctions. Also, in forensic toxicology creatinine concentration is used as a standardization tool for the quantitative measurement of therapeutic or illicit drugs and xenobiotics in urine. The present work was aimed at developing a robust and reliable CE determination of creatinine in urine, meeting the needs of simplicity, rapidity and low cost required by routine toxicological screening. METHODS The optimized buffer electrolyte was composed of 200 mM phosphate and 200 mM acetic acid (pH 3.8). The separation capillary (50 microm x 10 cm of effective length) was made of naked fused silica. Separations were carried out under 25 kV potential. Urine samples were diluted 20 fold with water and directly injected. Direct UV absorption detection at 200 nm was employed. RESULTS Linearity was assessed in the range 0.2-32 mM. Precision tests resulted in CVs % below 0.56% for migration times and below 3.78% for peak area ratios (analyte/I.S.). CONCLUSIONS The described CE creatinine assay meets the strict requirements of forensic analysis and looks particularly useful to test the possible adulteration or dilution of urine samples undergoing toxicological screening.

Micellar electrokinetic chromatography: a new simple tool for the analysis of synthetic cannabinoids in herbal blends and for the rapid estimation of their logP values.

For the first time a capillary separation based on micellar electrokinetic chromatography (MEKC) with diode array detection (DAD) was developed and validated for the rapid determination of synthetic cannabinoids in herbal blends. Separations were carried out on a 30 μm(ID) × 40 cm uncoated fused silica capillaries. The optimized buffer electrolyte was composed of 25 mM sodium tetraborate pH 8.0, 30 mM SDS and n-propanol 20% (v/v). Separations were performed at 30 kV. Sample injection conditions were 0.5 psi, 10s. Diazepam and JWH-015 were used as internal standards. The determination of the analytes was based on the UV signal recorded at 220 nm, corresponding to the maximum wavelength of absorbance of the molecules, whereas peak identification and purity check were also performed on the basis of the acquisition of UV spectra between 200 and 400 nm wavelengths. Under the described conditions, the separation of the compounds was achieved in 25 min without any significant interference from the matrix. Linearity was assessed within a concentration range from 5 to 100 μg/mL. The intra-day and inter-day imprecision values were below 2.45% for relative migration times and below 10.75% for relative peak areas. The present method was successfully applied to the direct determination of synthetic cannabinoids in 15 different herbal blend samples requiring only sample dilution. In addition, the developed MEKC separation was also applied to estimate the octanol/water partition coefficients (logP) of these new and poorly known molecules.

Dextromethorphan/levomethorphan issues in a case of opiate overdose.

Dextromethorphan is the d-isomer of 3-methoxy-N-methylmorphinan (methorphan), a synthetic analogue of codeine. It interacts with the σ-opioid receptors with resulting antitussive activity, while it does not show any significant affinity for the μ and δ opioid receptors, which are responsible for analgesic and central nervous system depressant effects. On this basis, d-methorphan is widely used all over the world as an over-the-counter cough suppressant drug. Indeed, there are hundreds of cough and cold preparations containing d-methorphan alone or in combination with analgesic (acetaminophen, acetyl salicylic acid), decongestant (phenylephrine, pseudoephedrine), anthistaminic (chlorpheniramine, brompheniramine, pheniramine), and/or expectorant mucolytic agents. In large doses, d-methorphan and its metabolite dextrorphan antagonize the actions of excitatory amino acids on N-methyl-d-aspartate (NMDA) receptors as do phencyclidine and ketamine. This action is responsible for the hallucinogenic and dissociative effects experienced by the subjects who abuse d-methorphan. Moreover, both d-methorphan and its metabolite dextrorphan are reported to inhibit reuptake of serotonin, and to have competitive 5HT1 agonist activity. On this basis, d-methorphan abuse could lead also to serotonin syndrome. Actually, many papers report fatal and non-fatal cases of abuse of d-methorphan especially among teenagers. Levomethorphan is the l-isomer of 3-methoxy-N-methylmorphinan and, unlike d-methorphan, is a potent narcotic analgesic. For this reason it is not commercially available and in many countries, including Italy, it is listed as a controlled substance. Moreover, the hepatic O-demethylation of l-methorphan produces levorphanol, which is a pure opioid agonist used to treat severe pain. Levorphanol has the same properties as morphine with respect to the potential for habituation, tolerance, physical dependence and withdrawal syndrome, but it is 4–5 times more potent than morphine and has a longer half-life. Starting from 2010, the Italian National Early Warning System (NEWS, Department of Antidrug Policy, Presidency of the Council of Ministers) collected numerous warnings from national collaborating centres (forensic laboratories, law enforcement, health services, research centres) related to heroin containing methorphan. Moreover, the use of methorphan as adulterant is reported in recent scientific literature. Both notifications and scientific papers often report methorphan as an adulterant, since the analytical methods used for heroin analysis cannot distinguish between the two stereoisomers. On the other hand, some papers report the identification of dextromethorphan because the gas chromatography–mass

The use of finger-prick dried blood spots (fpdbs) and capillary electrophoresis for carbohydrate deficient transferrin (cdt) screening in forensic toxicology

Continued progress in chronic alcohol abuse investigation requires the development of less invasive procedures for screening purposes. The application of finger‐prick and related dried blood spots (fpDBS) for carbohydrate deficient transferrin (CDT) detection appears suitable for this aim. Therefore, the goal of this project was to develop a screening method for CDT using fpDBS with CZE analysis. Blood samples prepared by finger‐prick were placed on DBS cards and left to air dry; each dried fpDBS disc was shredded into small pieces and suspended in acid solution (60 μL of HCl 120 mmol/L). After centrifugation (10 min at 1500 × g), the collected sample was adjusted to pH 3.5. After an overnight incubation, the pH was neutralised and an iron rich solution was added. After 1 h, CZE analysis was carried out. A group of 47 individuals was studied. Parallel serum samples were collected from each investigated subject and the %CDT for each sample was measured using HPLC and CZE techniques. The fpDBS transferrin sialo isoform electropherograms were similar to those obtained with serum. Moreover, fpDBS CZE CDT percentage levels demonstrated significant statistical correlation with those obtained from serum for both HPLC and CZE %CDT (p < 0.01; r2 = 0.8913 and 0.8976, respectively), with %CDT from 0.8 to 13.7% for fpDBS and from 0.7 to 12.7% for serum. The newly developed fpDBS procedure for CDT analysis provides a simple and inexpensive tool for use in population screening.

Analytical and diagnostic aspects of carbohydrate deficient transferrin (CDT): A critical review over years 2007-2017.

The need for investigating alcohol abuse by means of objective tools is worldwide accepted. Among the currently available biomarkers of chronic alcohol abuse, carbohydrate-deficient transferrin (CDT) is one of the most used indicator, mainly because of its high specificity. However, some CDT analytical and interpretation aspects are still under discussion, as witnessed by numerous research papers and reviews. The present article presents a critical review of the literature on CDT appeared in the period from 2007 to 2017 (included). The article is organized in the following sections: (1) introduction, (2) pre-analytical aspects (3) analytical aspects (4) diagnostic aspects (5) concluding remarks. As many as 139 papers appeared in the international literature and retrieved by the search engines PubMed, Web of Science and Scopus are quoted.