Federica Bortolotti

Recent advances in the applications of CE to forensic sciences (2001-2004)

The present article reviews the applications of CE in forensic science covering the period from 2001 until the first part of 2005. The overview includes the most relevant examples of analytical applications of capillary electrophoretic and electrokinetic techniques in the following fields: (i) Forensic drugs and poisons, (ii) explosive analysis and gunshot residues, (iii) small ions of forensic interest, (iv) forensic DNA and RNA analysis, (v) proteins of forensic interest, and (vi) ink analysis.

Evaluation and optimization of capillary zone electrophoresis with different dynamic capillary coatings for the determination of carbohydrate-deficient transferrin in human serum.

Serum transferrin (Tf) comprises several isoforms with up to two complex oligosaccharide chains containing zero to eight sialic acid residues and neutral sugars. The major glycoform, known as tetrasialo-Tf, contains four sialic acid residues and accounts for about 80% of whole Tf in human serum. Carbohydrate-deficient transferrin (CDT) encompasses isoforms that are deficient in carbohydrate chains and consequently in sialic acid residues (including asialo-, monosialo- and disialo-Tf) and is a well known marker for chronic alcohol abuse. Recently capillary zone electrophoresis (CZE) has been reported as a tool extremely effective for the simultaneous, individual, quantitative determination of CDT isoforms. Three CZE methods that feature different dynamic capillary coatings were evaluated and optimized for CDT determination in human serum of alcohol abusers and control subjects. CZE separation was performed in alkaline borate buffers after serum sample saturation with iron, electropherograms were detected at 200 nm, data were evaluated as % area of disialo-Tf in relation to tetrasialo-Tf and peak identification was accomplished via relative migration times to tetrasialo-Tf, immunosubtraction and enzymatic sequential cleavage of sialic acid residues. Dynamic capillary coatings with diaminobutane, spermine and a double coating produced by commercially available proprietary agents were investigated and found to be suitable for determination of CDT in human serum. For all three approaches, best results were obtained in 50 microm I.D. fused-silica capillaries of 50 cm effective length and a capillary cartridge temperature of 20-25 degrees C. Using 3 mM 1,4-diaminobutane or 0.02 mM spermine in a borate-based running buffer of pH 8.3 provided data of remarkable similarity with resolution of di-, tri-, tetra- and pentasialo-Tf within 15-18 min. With the double coating, asialo-Tf and Tf isoforms with two to six sialic acid residues were baseline separated. Compared to the two amine-based procedures, the run times were found to be somewhat shorter, the detector signals higher, the applied power level significantly lower and the reproducibility better.

Recent advances in the application of CE to forensic sciences, an update over years 2009-2011.

This article reviews and comments the applications of CE to forensic sciences covering the short period from 2007 until the first months of 2009, being the latest update of two previous review papers covering the years from 2001 to 2004 and from 2005 to 2007. The overview includes the most relevant examples of analytical applications of capillary electrophoretic and electrokinetic techniques in the following fields: (i) illicit and abused drugs, (ii) small ions of forensic interest (iii) proteins and peptides, (iv) forensic deoxyribonucleic acid, (v) dyes and inks. As many as 69 references are quoted.

Capillary zone electrophoresis and artificial neural networks for estimation of the post-mortem interval (PMI) using electrolytes measurements in human vitreous humour

Abstract Determination of electrolyte concentrations (mainly potassium) in vitreous humour has long been considered an important tool in human death investigations for the estimation of the post-mortem interval (PMI). On the basis of its well known potential in ion analysis, capillary zone electrophoresis (CZE) has recently been applied to achieve a rapid and simultaneous determination of inorganic ions in this extracellular fluid. In the present work, artificial neural networks (ANN) were applied for modelling of the relationship of multicomponent CZE analysis of K+, NH4+, Na+, and Ba2+ ions in vitreous humour with PMI. In a study based on 61 cases with different causes of death and a known PMI ranging from 3 to 144 h, the use of ANNs considering all inorganic ion data from the human vitreous humour, achieved a substantial improvement of post-mortem interval prediction. Good linear correlation was observed (r2 = 0.98) and in comparison to the traditional linear least squares (LLS) method applied only to K+ levels in the vitreous humour, the prediction of PMI with ANN was improved by a factor of 5 from ≈± 15 h to less than 3 h.

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 μm × 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 μL/min. Forensic drugs were identified by exact mass determination (mass accuracy typically ≤5 ppm) and by matching of the isotopic pattern.

Use of β-cyclodextrin in the capillary zone electrophoretic separation of the components of clandestine heroin preparations

The present paper describes the methodological optimization and validation of a capillary zone electrophoresis method for the rapid determination of heroin, secondary products and additives present in clandestine heroin samples, by using 20 mM beta-cyclodextrins in phosphate buffer, pH 3.23. Applied potential was 15 kV and separation temperature was 24 degrees C; detection was by UV absorption at 200 nm wavelength. Heroin samples were first dissolved in CHCl3-MeOH (96:4, v/v) and injected by pressure (0.5 p.s.i., 3 s; 1 p.s.i.=6894.76 Pa) after evaporation of the organic mixture and reconstitution in aqueous buffer. Under the described conditions, phenylethylamine (internal standard), morphine, monoacetylmorphine, heroin, acetylcodeine, papaverine, codeine and narcotine were baseline resolved in less than 10 min. The limit of detection was better than 1 microg/ml for each analyte. The study of the intra-day and day-to-day precision showed, in terms of migration times, RSDs < or = 0.71% and, in terms of peak areas, RSDs < or = 3.2%. Also, the evaluation of linearity and analytical accuracy of the method provided good results for all the analytes investigated, thus allowing its application to real cases of seized controlled drug preparations.

CEC-ESI ion trap MS of multiple drugs of abuse.

This article describes a method for the separation and determination of nine drugs of abuse in human urine, including amphetamines, cocaine, codeine, heroin and morphine. This method was based on SPE on a strong cation exchange cartridge followed by CEC‐MS. The CEC experiments were performed in fused silica capillaries (100 μm×30 cm) packed with a 3 μm cyano derivatized silica stationary phase. A laboratory‐made liquid junction interface was used for CEC‐MS coupling. The outlet capillary column was connected with an emitter tip that was positioned in front of the MS orifice. A stable electrospray was produced at nanoliter per minute flow rates applying a hydrostatic pressure (few kPa) to the interface. The coupling of packed CEC columns with mass spectrometer as detector, using a liquid junction interface, provided several advantages such as better sensitivity, low dead volume and independent control of the conditions used for CEC separation and ESI analysis. For this purpose, preliminary experiments were carried out in CEC‐UV to optimize the proper mobile phase for CEC analysis. Good separation efficiency was achieved for almost all compounds, using a mixture containing ACN and 25 mM ammonium formate buffer at pH 3 (30:70, v/v), as mobile phase and applying a voltage of 12 kV. ESI ion‐trap MS detection was performed in the positive ionization mode. A spray liquid, composed by methanol–water (80:20, v/v) and 1% formic acid, was delivered at a nano‐flow rate of ∼200 nL/min. Under optimized CEC‐ESI‐MS conditions, separation of the investigated drugs was performed within 13 min. CEC‐MS and CEC‐MS2 spectra were obtained by providing the unambiguous confirmation of these drugs in urine samples. Method precision was determined with RSDs values ≤3.3% for retention times and ≤16.3% for peak areas in both intra‐day and day‐to‐day experiments. LODs were established between 0.78 and 3.12 ng/mL for all compounds. Linearity was satisfactory in the concentration range of interest for all compounds (r2≥0.995). The developed CEC‐MS method was then applied to the analysis of drugs of abuse in spiked urine samples, obtaining recovery data in the range 80–95%.

Potassium concentration differences in the vitreous humour from the two eyes revisited by microanalysis with capillary electrophoresis

This paper presents a study of the variability of potassium concentrations in the vitreous humour of the two eyes of the same body at identical postmortem interval. The study was carried out by collecting microsample amounts (50 microl) of vitreous humour and by using an original method of capillary electrophoresis with indirect detection. The electrophoretic separations were carried out in a pH 4.5 running buffer composed of 5 mmol/l imidazole, 5 mmol/l 18-crown-6 ether and 6 mmol/l alpha-hydroxybutyric acid. Detection was by indirect UVabsorption at 214 nm. Vitreous humour samples were collected from 57 medico-legal autopsies or external examinations of cases of sudden natural or violent deaths. All samples prior to analysis were diluted 1:20 with a 40 microg/ml aqueous solution of barium, the used internal standard, and finally injected by nitrogen pressure. The mean concentrations of potassium measured in the two eyes of all the cases included in the present study ranged from 4.1 to 23.5 mmol/l with the postmortem interval values varying from 7 to 144 h. A highly significant (P<0.0001) linear correlation was found between these two parameters as described by the equation: y=0.1698x+2.3587, r=0.89. The intra-eye variability of potassium concentrations was low with an average RSD of 3.89% (+/- 1.83 SD) (48 eyes, five samples per eye). No statistically significant difference was found between the potassium concentrations in the two eyes of the same subject in a group of 24 cases, excepting a single case.

Capillary zone electrophoresis of potassium in human vitreous humour: validation of a new method

The analysis of potassium in the vitreous humour has long been regarded as an important tool in medicolegal and forensic toxicological investigation, particularly for the determination of the post-mortem interval. The present work was aimed at the optimisation and validation of a reliable, simple and fast capillary electrophoresis method for potassium analysis in the human vitreous humour with indirect UV detection at a wavelength of 214 nm. Electrophoretic separations were carried out in a running buffer comprising 5 mM imidazole, 5 mM 18-crown-6 ether and 6 mM D,L alpha-hydroxybutyric acid (HIBA), adjusted to pH 4.5. Constant voltage runs were carried out by applying a voltage of 500 V/cm at 25 degrees C. The samples were injected in the hydrodynamic mode at the anodic end of the capillary (0.5 p.s.i. for 10 s; 1 p.s.i. = 6894.76 Pa). The method showed good linearity in the concentration range from 6.5 mM to 16.25 microM, with an r2 value of 0.9994. The limit of detection, based on a signal-to-noise ratio of three, was 9.0 microM. Absolute intra-day RSDs of migration times were <0.40%, while the day-to-day values were < or =1.72%. Absolute peak area reproducibility was always better than 2.50%. A comparison of capillary electrophoresis with flame photometry on twelve real autopsy samples showed an excellent correlation with an r2 value of 0.9333. A preliminary application to real cases (20 subjects) was carried out plotting vitreous humour potassium vs. post-mortem interval with a resulting r2 of 0.904 and a Y-intercept of 4.75 mM, in agreement with the existing literature.

Dermal nitrate: an old marker of firearm discharge revisited with capillary electrophoresis

The present work describes a capillary electrophoretic method for nitrite and nitrate determination to be used as a screening tool for investigating the residues of firearm discharge. The use of capillary electrophoresis allowed the rapid determination of nitrite and nitrate, which are major inorganic components of gunshot residues, offering a quantitative and selective alternative to the traditional paraffin test (dermal nitrate test). The method is simpler, cheaper, and faster than the modern approaches to gunshot residue analysis based on the determination of barium, lead and antimony by using flameless absorption spectrometry, inductively coupled plasma‐mass spectrometry (ICP‐MS), or scanning electron microscopy. The analysis was carried out in a bare fused‐silica capillary (75 νm inner diameter) with a 100 mM borate buffer (pH 9.24). The detection was by UV absorption at 214 nm. Separation took place under reversed voltage of 15 kV. Bromide was used as the internal standard. Sensitivity was about 1 mM for both nitrite and nitrate. Reproducibility (intraday and day‐to‐day) was also good with relative standard deviations (RSDs) < 1.0% for relative migration times and < 4.5% for peak areas in both standard solutions and real matrix. Hair and skin samples from a victim shot in the head were successfully analyzed for the presence of nitrite and nitrate.