Andrea N. Hagardorn

Application of Micellar Electrokinetic Capillary Chromatography to Forensic Analysis of Barbiturates in Biological Fluids

Micellar electrokinetic capillary chromatography (MECC) is a form of capillary zone electrophoresis. Addition of a surfactant produces micelles in an aqueous/organic buffer. Separation of drugs is obtained via differences in the electrophoretic mobilities of the analytes within the capillary, resulting from their electrophoretic velocity and the electroosmotic flow of the buffer in a given electric field. The migration order is determined by the differential partitioning of the drugs between the micelles and the aqueous/organic phase. Barbiturates were extracted from various biological fluids at pH 4.5 with TOXI-TUBES B. MECC analyses were performed using a Waters Quanta 4000 Capillary Electrophoretic System with a 745 Data Module with a 75 microns x 60 cm capillary and an aqueous/organic buffer of 85% 10 mM borate, 10 mM phosphate, 100 mM sodium dodecyl sulfate and 15% acetonitrile at a pH of 8.5 with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: phenobarbital, 7.78 min.; butalbital, 8.01 min.; butabarbital, 8.23 min.; mephobarbital (internal standard), 8.88 min.; amobarbital, 9.41 min.; pentobarbital, 10.03 min. and secobarbital, 10.79 min. Correlation coefficients (r) between peak areas and concentration ranges of 3 to 60 micrograms/mL were from 0.964 to 0.999. Coefficients of variation (CV) ranged from 2.6 to 8.6% between days and 2.3 to 9.8% within day. Application of this methodology to four forensic cases of butalbital intoxication detected concentrations of 0.7 to 12.7 micrograms/mL in blood; 0.8 to 1.9 micrograms/mL in vitreous humor and 1.5 to 7.6 micrograms/mL in urine. MECC is applicable to forensic analysis of barbiturates extracted from biological fluids.

Postmortem Determination of the Biological Distribution of Sufentanil and Midazolam after an Acute Intoxication

A case is presented of a death caused by self-injection of sufentanil and midazolam. Biological fluids and tissues were analyzed for midazolam by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) and for sufentanil by GC/MS. Midazolam was extracted from basified fluids or tissues homogenated with n-butyl chloride and analyzed by HPLC by using a phosphate buffer: acetonitrile (60:40) mobile phase on a mu-Bondapak C18 column at 240 nm. Sufentanil was extracted from basified fluids and tissue homogenates with hexane:ethanol (19:1). GC/MS methodology for both compounds consisted of chromatographic separation on a 15-m by 0.25-mm inside diameter (ID) DB-5 (1.0-micron-thick film) bonded phase fused silica capillary column with helium carrier (29 cm/s) splitless injection at 260 degrees C; column 200 degrees C (0.8 min) 10 degrees C/min to 270 degrees C; and electron ionization and multiple ion detection for midazolam (m/z 310), methaqualone (IS, m/z 235), sufentanil (m/z 289), and fentanyl (IS, m/z 245). Sufentanil concentrations were: blood 1.1 ng/mL, urine 1.3 ng/mL, vitreous humor 1.2 ng/mL, liver 1.75 ng/g, and kidney 5.5 ng/g. Midazolam concentrations were: blood 50 ng/mL, urine 300 ng/mL, liver 930 ng/g, and kidney 290 ng/g. Cause of death was attributed to an acute sufentanil/midazolam intoxication and manner of death a suicide.

Spectral differentiation and gas chromatographic/mass spectrometric analysis of the lacrimators 2-chloroacetophenone and o-chlorobenzylidene malononitrile.

2-Chloroacetophenone (CN) and o-chlorobenzylidene malononitrile (CS) are the most common chemical agents used as lacrimators in the United States. There is a lack of complete spectral data on these compounds in the literature. Spectral data (ultraviolet, fluorescence, proton nuclear magnetic resonance, and infrared) and a gas-liquid chromatographic/mass spectrometric method are presented that differentiate and identify CN and CS. These methods and data were used to identify a forensic science specimen from an accidental intoxication.

Plasma levels and kinetic disposition of 2,4-dinitrophenol and its metabolites 2-amino-4-nitrophenol and 4-amino-2-nitrophenol in the mouse

Eleven groups of six ICR mice were dosed orally with 22.5 mg/kg 2,4-dinitrophenol. Groups were sacrificed at 0, 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 96 h post-treatment and plasma was collected for analysis of dinitrophenol, 2-amino-4-nitrophenol, and 4-amino-2-nitrophenol content. Analyses were performed by capillary gas chromatography--mass spectrometry after liquid--liquid extraction of plasma specimens spiked with two internal standards. Quantification was based upon peak-area ratios of base peaks obtained from the three analytes and the trideuterated internal standards 2,4-dinitrophenol and 2-amino-4-nitrophenol. Plasma concentrations for each analyte versus their respective time periods were subjected to pharmacokinetic analysis. Of the two monoamine metabolites, 2-amino-4-nitrophenol was present in the greater amount and had an elimination half-life of 46 h from plasma while that of 4-amino-2-nitrophenol was 26 h.

Analysis and kinetics of 2,4-dinitrophenol tissues by capillary gas chromatography—mass spectrometry

Five groups of six ICR mice were orally dosed with 22.5 mg/kg 2,4-dinitrophenol. Groups were sacrificed at 1, 3, 6, 12, and 24 h post treatment, and serum, liver, and kidney tissues were collected for analysis of dinitrophenol content. Quantitation was performed via a capillary gas chromatography--mass spectrometry technique after liquid--liquid extraction of biological specimens spiked with a trideuterated dinitrophenol internal standard. Concentration versus time data for each tissue were subjected to pharmacokinetic analysis. Similar two-compartment open models were found to characterize most phases of the disposition of this compound. The kidney appears to maintain a more persistent low concentration of 2,4-dinitrophenol.

A Fatal Interaction of Methocarbamol and Ethanol in an Accidental Poisoning

A case is presented of a fatal drug interaction caused by ingestion of methocarbamol (Robaxin) and ethanol. Methocarbamol is a carbamate derivative used as a muscle relaxant with sedative effects. Therapeutic concentrations of methocarbamol are reported to be 24 to 41 micrograms/mL. Biological fluids were screened for ethanol using the Abbott TDx system and quantitated by gas-liquid chromatography (GLC). Determination of methocarbamol concentrations in biological tissue homogenates and fluids were obtained by colorimetric analysis of diazotized methocarbamol. Blood ethanol concentration was 135 mg/dL (0.135% w/v) and urine ethanol was 249 mg/dL (0.249% w/v). Methocarbamol concentrations were: blood, 257 micrograms/mL; bile, 927 micrograms/L; urine, 255 micrograms/L; gastric, 3.7 g; liver, 459 micrograms/g; and kidney, 83 micrograms/g. The combination of ethanol and carbamates is contraindicated since acute alcohol intoxication combined with carbamate usage can lead to combined central nervous system depression as a result of the interactive sedative-hypnotic properties of the compounds.