Rapid analysis of caffeine in "smart drugs" and "energy drinks" by microemulsion electrokinetic chromatography (MEEKC).

A novel method based on microemulsion electrokinetic chromatography (MEEKC) with diode array detection (DAD) for rapid determination of caffeine in commercial and clandestine stimulants, known as "energy drinks" and "smart drugs", is described. Separations were carried out in 50 cm × 50 µm (ID) uncoated fused silica capillaries. The optimized buffer electrolyte was composed of 8.85 mM sodium tetraborate pH 9.5, SDS 3.3% (w/v), n-hexane 1.5% (v/v) and 1-butanol 6.6% (v/v). Separations were performed at a voltage of 20 kV. Sample injection conditions were 0.5 psi, 3 s. Diprofilline was used as internal standard. The determination of the analytes was based on the UV signal recorded at 275 nm, corresponding to the maximum wavelength of absorbance of caffeine, whereas peak identification and purity check was performed on the basis of the acquisition of UV radiation between 200 and 400 nm wavelengths. Under the described conditions, the separation of the compounds was achieved in 6 min without any interference from the matrix. Linearity was assessed within a caffeine concentration range from 5 to 100 µg/mL. The intra-day and inter-day precision values were below 0.37% for migration times and below 9.86% for peak areas. The present MEEKC method was successfully applied to the direct determination of caffeine in smart drugs and energy drinks.

Monitoring compliance to therapy during addiction treatments by means of hair analysis for drugs and drug metabolites using capillary zone electrophoresis coupled to time-of-flight mass spectrometry.

Capillary electrophoresis coupled to time-of-flight mass spectrometry was used in the present work for the determination of therapeutic and abused drugs and their metabolites in the hair of subjects undergoing addiction treatments, in order to monitor their compliance to therapy. For this purpose a rapid, qualitative drug screening method was adopted based on capillary electrophoresis hyphenated with time-of-flight mass spectrometry, which had earlier been developed and validated for the forensic-toxicological analysis of hair, limitedly to illicit/abused drugs [1]. Sampling of hair was carried out in order to refer to a time window of about two months from the date of sampling (i.e. 2cm ca. from cortex). A single extraction procedure was applied, allowing the determination in the hair matrix of "drugs of abuse" referred to the past abuses, and therapeutic drugs prescribed in the detoxification program as well as their metabolites. Analyte identification was based on accurate mass measurements and comparison of isotope patterns, providing the most likely matching between accurate mass value and elemental formula. Small molecules (<500Da) of forensic and toxicological interest could be identified unambiguously using mass spectrometric conditions tailored to meet a mass accuracy ≤5ppm. In the present study, the proposed approach proved suitable for the rapid broad spectrum screening of hair samples, although needing further confirmation of results by using fragmentation mass spectrometry.

[Assessment of fitness to drive in correlation with narcotic and psychotropic drug use. Epidemiologic study in Verona]. / La valutazione dell'idoneità alla guida in relazione all'uso di sostanze stupefacenti e psicotrope. Studio epidemiologico della casistica veronese.

Driving under the influence of drugs is a serious problem for road traffic safety. According to the Italian Road Traffic Code, the driving licence must not be issued to anyone who abuses, is addicted to, or suffers for dependence to illicit or psychotropic drugs. The diagnosis of such clinical conditions is performed by Provincial Medical Commissions of the Public Health Service also on the basis of drugs of abuse testing results on urine and/or hair samples. This study aimed at examining test results obtained by the Forensic Toxicology laboratory of the Department of Public Health & Community Medicine, University of Verona, upon request of the local Medical Commission, over the period 2003-2008 with the purposes of (i) defining trends in drug abuse in the examined population (ii) identifying specific risk factors for testing positive and for relapse, (iii) selecting the most effective and efficient analytical strategy to detect illicit drugs use. During the study period, cocaine was the most frequently detected illicit drug. The comparison of results from urine and hair testing confirmed the complementary features of these two biological substrates and the importance to have both data in order to increase the sensitivity in detecting illicit drug use. Moreover, this study showed that testing for driving fitness is an effective deterrent to illicit drug use, as only about one quarter of subjects testing positive at the first testing are still positive at the second testing.

Screening for pharmaco-toxicologically relevant compounds in biosamples using high-resolution mass spectrometry: a 'metabolomic' approach to the discrimination between isomers.

High-resolution mass spectrometry (HRMS) enables the identification of a chemical formula of small molecules through the accurate measurement of mass and isotopic pattern. However, the identification of an unknown compound starting from the chemical formula requires additional tools: (1) a database associating chemical formulas to compound names and (2) a way to discriminate between isomers. The aim of this present study is to evaluate the ability of a novel 'metabolomic' approach to reduce the list of candidates with identical chemical formula. Urine/blood/hair samples collected from real positive cases were submitted to a screening procedure using ESI-MS-TOF (positive-ion mode) combined with either capillary electrophoresis or reversed phase liquid chromatography (LC). Detected peaks were searched against a Pharmaco/Toxicologically Relevant Compounds database (ca 50,500 compounds and phase I and phase II metabolites) consisting of a subset of PubChem compounds and a list of candidates was retrieved. Then, starting from the mass of unknown, mass shifts corresponding to pre-defined biotransformations (e.g. demethylation, glucuronidation, etc.) were calculated and corresponding mass chromatograms were extracted from the total ion current (TIC) in order to search for metabolite peaks. For each candidate, the number of different functional groups in the molecule was automatically calculated using E-Dragon software (Talete srl, Milan, Italy). Then, the presence of metabolites in the TIC was matched with functional groups data in order to exclude candidates with structures not compatible with observed biotransformations (e.g. loss of methyl from a structure not bearing methyls). The procedure was tested on 108 pharmaco-toxicologically relevant compounds (PTRC) and their phase I metabolites were detected in real positive samples. The mean list length (MLL) of candidates retrieved from the database was 7.01 +/- 4.77 (median, 7; range, 1-28) before the application of the 'metabolomic' approach, and after the application it was reduced to 4.08 +/- 3.11 (median 3, range 1-17). HRMS allows a much broader screening for PTRC than other screening approaches (e.g. library search on mass spectra databases). The 'metabolomic' approach enables the reduction of the list of candidate isomers.

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 CV's % 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.

Rapid optimized separation of bromide in serum samples with capillary zone electrophoresis by using glycerol as additive to the background electrolyte.

After decades of neglect, bromide has recently been re-introduced in therapy as an effective anti-epileptic drug. The present paper describes the methodological optimization and validation of a method based on capillary zone electrophoresis for the rapid determination of bromide in serum using a high-viscosity buffer and a short capillary (10 cm). The optimized running buffer was composed of 90 mM sodium tetraborate, 10mM sodium chloride, pH 9.24 and 25% glycerol. The separation was carried out at 25 kV at a temperature of 20 degrees C. Detection was by direct UV absorption at 200 nm wavelength. The limit of detection (signal-to-noise ratio=5) in serum was 0.017 mM. The precision of the method was verified in blank serum samples spiked with bromide, obtaining intra-day and day-to-day tests, relative standard deviation values

Capillary zone electrophoresis (CZE) coupled to time-of-flight mass spectrometry (TOF-MS) applied to the analysis of illicit and controlled drugs in blood.

A new method for the determination of illicit and abused drugs in blood by capillary zone electrophoresis-electrospray ionization-time-of-flight mass spectrometry is proposed, in view of its application in clinical and forensic toxicology. The analytes (methamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, methadone, cocaine, morphine, codeine, 6-acethylmorphine, benzoylecgonine) were separated with capillary zone electrophoresis by applying 15 kV within 25 min, in an uncoated fused-silica capillary (75 microm x 100 cm) using a 25 mM ammonium formate electrolyte solution (pH 9.5). The capillary electropherograph was coupled to time-of-flight mass spectrometry through an orthogonal electrospray ionization source, with a coaxial sheath liquid interface. The sheath liquid was composed of isopropanol-water (1:1 v/v) containing 0.5% formic acid delivered at 4 microL/min. Forensic drugs were identified by exact mass determination (mass accuracy typically < or =5 ppm) and by matching of the isotopic pattern. Under optimized conditions, linearity was assessed in the range 10-2000 ng/mL, with correlation coefficients between 0.9744 and 0.9982 for all the analytes. LODs were in the range of 2-10 ng/mL (S/N > or =3) and LOQs of 10-30 ng/mL. The CVs (tested at 40 and 800 ng/mL in biological matrix) were below 2.97% for migration times and below 14.61% for peak area ratios (analyte/internal standard). Blood samples were extracted by using a liquid-liquid extraction procedure and injected under field-amplified sample stacking conditions. The method was successfully applied to real cases.