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The coupling of capillary electrophoresis-electrospray ionization and time-of-flight mass spectrometry, combining efficiency and speed of separation with high mass accuracy and fast scanning capability, was for the first time applied to the determination of drugs of abuse (amphetamine, methamphetamine, MDA, MDMA, ephedrine, cocaine, morphine, codeine) and their metabolites in hair (6-MAM, benzoylecgonine). Experimental conditions were as follows. Separation: voltage 15 kV, uncoated fused-silica capillary (75 microm ID, 100 cm total length), running electrolyte 25 mM ammonium formate, pH 9.5, field-amplified sample stacking injection. Forensic drugs could be identified by exact mass determination (mass accuracy typically < or = 5 ppm) and by match of the isotopic pattern. The method was fully validated, showing limit of detections (LODs) suitable for the determination of all the compounds below the cut-off usually adopted for hair analysis (0.1 ng/mg). Analytical precision in real matrices (tested at 0.1 and 1.0 ng/mg) was typically characterized by CV's < or = 24% in both intra-day and day-to-day experiments. Quantitative determination was also tested by using a single internal standard (folcodine). Results, although with a moderate accuracy, conceivably depending on the lack of deuterated internal standards, proved useful for diagnostic use of the results from hair analysis. A single liquid-liquid extraction procedure was applied for all analytes, allowing the detection of a broad spectrum of basic drugs and their major metabolites.

In forensic toxicology, hair analysis has become a well established analytical strategy to investigate retrospectively drug abuse histories. In this field, gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry are currently used, often after preliminary screening with immunoassays. However, on the basis of previous applications to pharmaceutical analysis, capillary zone electrophoresis coupled to ion trap mass spectrometry looks also highly promising. The purpose of the present work was the development of a simple and rapid CZE-MS method for sensitive and quantitative determination of the main drugs of abuse and their metabolites (namely, 6-monoacetylmorphine, morphine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethampthetamine (MDMA), benzoylecgonine, ephedrine and cocaine) in human hair. Hair samples (100 mg) were washed, cut and incubated overnight in 0.1 M HCl at 45 degrees C, then neutralized with NaOH and extracted by a liquid-liquid extraction method. CZE separations were carried out in a 100 cm x 75 microm (I.D.) uncoated fused silica capillary. The separation buffer was composed of 25 mM ammonium formate, pH 9.5; the separation voltage was 15 kV. Electrokinetic injections were performed at 7 kV for 30 s under field amplified sample stacking conditions. ESI-ion trap MS detection was performed in the ESI positive ionization mode using the following conditions: capillary voltage 4 kV, nebulizer gas (nitrogen) pressure 3psi, source temperature 150 degrees C and drying gas (nitrogen) flow rate 8l/min. A sheath liquid, composed of isopropanol-water (50:50, v/v) with 0.5% formic acid, was delivered at a flow rate of 4 microl/min. The ion trap MS operated in a selected ion monitoring mode (SIM) of positive molecular ions for each drug/metabolite. Collision induced fragmentation was also possible. Nalorphine was used as internal standard. Under the described conditions, the separation of all compounds, except amphetamine/methamphetamine, MDA/MDMA and morphine/6-MAM was achieved in 20 min, with limits of detection lower than the most severe cut-offs adopted in hair analysis (i.e. 0.1 ng/mg). Linearity was assessed within drug concentration ranges from 0.025 to 5 ng of each analyte/mg of hair. Analytical precision was fairly acceptable with RSD's < or = 3.06% for migration times and < or = 22.47% for areas in real samples, in both intra-day and day-to-day experiments. On these grounds, the described method can be proposed for rapid, selective and accurate toxicological hair analysis for both clinical and forensic purposes.

Background: The techniques universally believed as most reliable for determination of Carbohydrate-Deficient Transferrin (CDT) are HPLC and capillary electrophoresis (CE). Recently a reagent kit for CDT analysis to be used in a multicapillary electropherograph has been introduced. The present work was aimed at a validation of this new commercial system by application of the analytical chemistry parameters and by comparison with validated CE and HPLC methods., Methods: One hundred forty serum samples were analyzed with multicapillary CE (Capillarys, Sebia, France) using kit reagents (CAPILLARYS CDT assay), with single capillary electropherograph (P/ACE MDQ, Beckman Coulter, USA) using an original method developed by our group and with HPLC, performed on a gradient HPLC (Shimadzu Europe, Germany), using column and reagents provided in kit (ClinRep, Recipe, Germany)., Results: The separation efficiency of the multicapillary system was about 15,000 theoretical plates/column. The resolution of the transferrin isoforms ranged from 1.23 to 1.67. The variation coefficient was < 10% in both intra-run and between run tests. The correlation of results of multicapillary CE with those obtained with single CE and HPLC was statistically significant., Conclusions: The multicapillary system shows high productivity with good analytical performances; however, a confirmation of positive results with an alternative method is required.

Objective: There is a paucity of studies on quantitative determination of carbohydrate-deficient transferrin (CDT) in newborns. The aim of our study was therefore to determine CDT concentrations in newborns by using capillary zone electrophoresis (CZE)., Material and Methods: Capillary blood was collected from the heels of 28 at-term healthy newborns, simultaneously with the Guthrie card screening. Forty-seven adults were examined as controls. CZE separations were performed with a P/ACE MDQ capillary electropherograph in uncoated fused-silica capillaries using a commercial reagent kit. After iron saturation, the samples were loaded by application of 0.5 psi for 15 s, and separated under 28kV with UV detection. All relevant transferrin (Tf) glycoforms were separated within 7 min. CDT quantification (%CDT) was carried out by calculating the percentage ratio between the sum of the peak areas of CDT-related glycoforms and the sum of peak areas of all Tf glycoforms., Results: In most cases, good separations of Tf glycoforms were obtained. In the newborns the %CDT was 0.51 versus 0.66 in adults (difference not statistically significant). Trisialo-Tf concentration was significantly lower in newborns (3.20) than in adults (4.11). Furthermore, pentasialo-Tf appeared to be lower in newborns (7.30) than in adults (14.00), but because complete separation of the peaks of tetrasialo- and pentasialo-Tf was not always possible, this finding could not be confirmed statistically., Conclusions: CZE showed definite advantages in terms of volume of blood to be collected, simplicity and standardization of analysis and, because of the direct detection of the separated zones, accuracy of quantification. The present study provides the basic information in the search for glycosylation defects in newborns.

The present work was aimed at the development of a capillary electrophoretic analysis of gamma-hydroxybutyric acid (GHB) using electrospray ion trap mass spectrometry to achieve the direct and unequivocal detection of this analyte in human urine. Optimized capillary electrophoretic conditions were: injection, 20 s at 0.5 psi (1 psi = 6894.76 Pa); buffer electrolyte, 12.5 mM ammonium formate adjusted to pH 8.35 with diethylamine; fused silicacapillary: 100 cm x 50 microm i.d.; separation voltage, 25 kV (forward polarity) + 0.5 psi; room temperature. Electrospray and mass spectrometric conditions were: drying gas and nebulizing gas (nitrogen) at flow rate 3 l/min, temperature 250 degrees C, nebulizer pressure: 10 psi; sheath liquid solution: methanol-water (90:10) containing 0.1% ammonia delivered at 3 microl/min; spray voltage 3.5 kV. Mass spetrometric detection was carried out in the selected ion monitoring mode of negative molecular ions at 103 m/z for GHB and 115 m/z for maleic acid (I.S.). Under these conditions the baseline separation of GHB and the I.S. was obtained. The selectivity of the analysis allowed for direct injection of unextracted urine, previously diluted 1:4 with water. Linearity was assessed in the GHB concentration range from 80 to 1280 microg/ml in urine. Analytical sensitivity (as limit of detection) resulted about 5 microg/ml in water and 20 microg/ml in original urine. Analytical precision was fairly acceptable with R.S.D. values lower than 5% for migration times and 18% for quantitation in real samples, in both intra day and day-to-day experiments. On these grounds, the developed method can be adopted for rapid identification of acute intoxications from GHB in humans.