First detection of ethylphenidate in human fatalities after ethylphenidate intake.

Methylphenidate, a psychostimulant drug from the group of amphetamines is, among others, established in the treatment of attention deficit hyperactivity disorder and narcolepsy. It is also known to have a certain potential of abuse. In combination with alcohol, the metabolite ethylphenidate was detected in human plasma in small amounts. However, ethylphenidate is sold as "research chemical" via the Internet. It was put under German narcotics law in July 2013. In a recent case, where a deceased person was found in his apartment, the police seized a plastic bag with the inscription "ethylphenidate". An autopsy of the 32-year-old man yielded a mitral valve endocarditis, which must have persisted a while before death, in combination with a pneumonia. At the Forensic Toxicological Centre (FTC) in Munich femoral blood, liver, pericardium fluid, urine, stomach content and hair of the deceased were analyzed for ethylphenidate after sample preparation by an LC-Triple TOF 5600. Calibration curves were spiked with a methanolic 1mg/mL solution of ethylphenidate (substance provided by the State Office of Criminal Investigation in Munich) in whole blood in comparison to liver and femoral blood, in serum in comparison to pericardium fluid and in urine in comparison to urine and stomach content, respectively. Ethylphenidate was detected in all analyzed matrices. The spectrums of the human specimen were compared to those obtained from the calibration curves and identified as ethylphenidate. The measured concentrations were for femoral blood 110ng/mL, for liver 180ng/g, for pericardium fluid 131ng/mL, for urine 987ng/mL and for stomach content 20.7ng/mL, respectively. The stomach contained 200mL of a brownish-coloured liquid, resulting in a total amount of 4000ng ethylphenidate. The lowest calibrator for whole blood and serum was 1ng/mL and for urine 10ng/mL. As far as it is known to the authors, these are the first ethylphenidate levels measured in a case of ethylphenidate intake. Therefore these results can only be compared to methylphenidate concentrations with therapeutic levels ranging from 5 to 60ng/mL in serum. As the toxic levels for methylphenidate start from approximately 500ng/mL serum, we estimate that ethylphenidate in the concentrations mentioned above is not in a directly lethal range. But it has to be considered, that amphetamine-like drugs as methylphenidate are known for their cardiovascular side effects (like tachycardia and arrhythmia) and might therefore have contributed to death, which was attributed to endocarditis in combination with pneumonia.

Optimization strategies and modeling of separations of dansyl-amino acids by cyclodextrin-modified capillary electrophoresis.

Presented in this study is an approach to optimize conditions for capillary electrophoresis separations of multianalyte enantiomeric pairs (D- and L-dansyl (Dns)-amino acids) that involves the rational use of combinations of cyclodextrins (CDs) as enantioselective running buffer additives. Migration data is experimentally obtained for a range of concentrations for native CDs used individually and employed to determine inclusion constants for the Dns-amino acids of interest. An expression for the mobility of the amino acids when multiple (two in this work) CDs are present in the running buffer is used to simulate separations for more complex CD systems. A chromatographic response function involving predicted resolution is generated to gauge the quality of these separations. Simplex methods are then employed for the first time to optimize conditions for the separation of amino acid enantiomers. The validity of this approach is demonstrated for separations of five Dns-amino acid enantiomers using gamma- and beta-CDs at various concentrations. Extending the dual-CD approach to other CDs and increasing the number of CDs beyond two should be possible. To this end, preliminary experiments are performed by using several available single-isomer, derivatized CDs (individually) to determine if they have potential for further studies. Although results with these particular derivatized CDs are not encouraging, we did find that molecular mechanics modeling is useful in interpreting those cases in which low inclusion constants possibly contributed to the ineffectiveness of the CDs.

Parameters affecting reproducibility in capillary electrophoresis.

It is well known that poor quantitative reproducibility substantially limits the practical implementation of capillary electrophoresis (CE) separations in chemical analysis. The principal sources of variance in observed peak areas are irreproducible flow rate, which influences on-column detector response, and inconsistent injection volume or amount. An overview of studies by researchers to address the reproducibility issue will be presented. In addition, current efforts in our laboratory to assess sources of quantitative variance for separations of dansylated amino acids using an automated CE system are presented and related when appropriate to the body of existing knowledge on this important topic. A comparison of different injection methods (hydrostatic vs. electrokinetic) and approaches (e.g., high vs. low pressure), the effect of random changes in electroosmotic flow (EOF) due to air bubbles in the CE capillary, and choice of certain peak integration parameters in terms of peak area reproducibility are presented. Under optimum conditions relative standard deviation (RSD) values in raw peak area are typically 2.0%. With nonoptimum conditions (e.g., with air bubbles in capillary), RSD values can substantially degrade. However, normalizing with retention times, internal standards, or observed electrophoretic current produces RSD values in a range of 1.4-2.3%.

Capillary electrophoresis of phospholipids with indirect photometric detection.

The capillary electrophoresis (CE) separation of different anionic phospholipid classes including phosphatidic acids (PA), phosphatidylserine, phosphatidylinositol, phosphatidylglycerol (PG), and cardiolipin using indirect detection with adenosine monophosphate (AMP) is described. A standard mixture of PAs (C14, C16, and C18) can be separated in 10 min by CE using 5 mM AMP and 100 mM boric acid in 10% water--80% methanol--10% acetonitrile. Although nonionic surfactants such as Brij 35 can improve the CE resolution of PAs, the separation time and the baseline noise are both increased. Optimization of the organic solvent in the running electrolyte is important. Methanol provides faster electroosmotic flow than propanol, and 10% acetonitrile effectively reduces migration time further by a factor of 1.4-2.2, depending on the phospholipid. The concentration limit of detection ranges from approximately 2 to 6 mg/L, and the mass limit of detection is as low as 21 pg. Linearity from 19 to 100 mg/L is established for cardiolipin and C16-PG. Phospholipids in soybean and brain extract samples could be profiled.

Separation of phosphorylated sugars using capillary electrophoresis with indirect photometric detection.

Adenosine monophosphate (AMP) and naphthalene disulfonate (NDS) have been characterized as electrolytes for the indirect photometric detection of phosphorylated sugars and other organophosphorus compounds of biochemical interest. This work has resulted in the CE separation on an uncoated capillary using 5 mM AMP and 100 mM boric acid at pH 7.2 of six metabolites (glucose-6-phosphate [G6P], fructose-6-phosphate [F6P]), fructose-1,6-bisphosphate [F-1,6-P], dihydroxyacetone phosphate [DHAP], glyceraldehyde-3-phosphate [G3P], and 2-phosphoglycerate [2-PG] or 3-phosphoglycerate [3-PG]) found in the glycolytic pathway. The detection limits using a 5-sec injection time were between 0.5 and 1 mg/L for these compounds, with the exception of G3P. Resolution between 3-PG and 2-PG is possible by the addition of magnesium ion, although the separation time is longer. A successful separation of five monophosphorylated sugars (G6P, F6P, ribose-5-phosphate [R5P], sucrose-6-phosphate [S6P], and 2-PG) has been performed using the same conditions as for the glycolytic pathway separation. A separation of bisphosphorylated sugars (glucose-1,6-bisphosphate [G-1,6-P],F-1,6-P, ribulose-1,5-bisphosphate [Ru-1, 5P], and sedoheptulose-1,7-bisphosphate [S-1, 7P]) could not be performed with AMP unless magnesium chloride was added. With NDS, a separation of these bisphosphorylated sugars can be obtained without the addition of magnesium chloride.