Timothy A. Robert

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.

Prevalence of heroin markers in urine for pain management patients.

Surveys of current trends indicate heroin abuse is associated with nonmedical use of pain relievers. Consequently, there is an interest in evaluating the presence of heroin-specific markers in chronic pain patients who are prescribed controlled substances. A total of 926,084 urine specimens from chronic pain patients were tested for heroin/diacetylmorphine (DAM), 6-acetylmorphine (6AM), 6-acetylcodeine (6AC), codeine (COD), and morphine (MOR). Heroin and markers were analyzed using liquid chromatography tandem mass spectrometry (LC-MS-MS). Opiates were analyzed following hydrolysis using LC-MS-MS. The prevalence of heroin use was 0.31%, as 2871 were positive for one or more heroin-specific markers including DAM, 6AM, or 6AC (a known contaminant of illicit heroin). Of these, 1884 were additionally tested for the following markers of illicit drug use: 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), methamphetamine (MAMP), 11-nor-9-carboxy-Δ(9)-tetracannabinol (THCCOOH), and benzoylecgonine (BZE); 654 (34.7%) had positive findings for one or more of these analytes. The overall prevalence of heroin markers were as follows: DAM 1203 (41.9%), 6AM 2570 (89.5%), 6AC 1082 (37.7%). MOR was present in 2194 (76.4%) and absent (<LOQ) in 677 (23.6%) of the heroin-positive specimens. COD was present in 1218 (42.4%) specimens. Prevalence of combinations for specimens containing MOR were as follows: DAM only 13 (0.59%), 6AM only 1140 (52.0%), 6AC only 24 (1.1%), DAM/6AM/6AC 710 (32.4%), 6AM/6AC 188 (8.6%), DAM/6AM 113 (5.2%), DAM/6AC 6 (0.27%). Importantly, the prevalence of combinations for specimens without MOR were as follows: DAM only 161 (23.8%), 6AM only 217 (32.1%), 6AC only 92 (13.6%), DAM/6AM/6AC 50 (7.4%), 6AM/6AC 7 (1.0%), DAM/6AM 145 (21.4%), DAM/6AC 5 (0.74%). Unexpected patterns of excretion were observed, such as the presence of DAM and 6AC in the absence of 6AM and MOR; therefore, multiple heroin markers may be useful to assess for heroin use.

Analysis of Nitrite in Adulterated Urine Samples by Capillary Electrophoresis

A simple method for analyzing nitrite in urine has been developed to confirm and quantify the amount of nitrite in potentially adulterated urine samples. The method involved separation of nitrite by capillary electrophoresis and direct UV detection at 214 nm. Separation was performed using a bare fused silica capillary and a 25 mM phosphate run buffer at a pH of 7.5. Sample preparation consisted of diluting the urine samples 1:20 with run buffer and internal standard, and centrifuging for 5 min at 2500 rpm. The sample was hydrodynamically injected, then separated using -25 kV with the column maintained at 35 degrees C. The method had upper and lower limits of linearity of 1500 and 80 microg/mL nitrite, respectively, and a limit of detection of 20 microg/mL. The method was evaluated using the National Committee for Clinical Laboratory Standards (NCCLS) protocol (Document EP10-A2), and validated using controls, standards, and authentic urine samples. Ten anions, ClO-, CrO4(-2), NO3-, HCO3-, I-, CH3COO-, F-, SO4-, S2O8(-2), and Cl-, were tested for potential interference with the assay. Interferences with quantitation were noted for only CrO4(-2) and S2O8(-2). High concentrations of Cl- interfered with the chromatography. The method had acceptable accuracy, precision, and specificity.

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.