Postmortem Urine Specimens Research Articles

Development of a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry for human urine (RaDPi-U).

Drug screening tests are mandatory in the search fordrugs in forensic biological samples, and immunological methods and mass spectrometry (e.g., gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry) are commonly used for that purpose. However, these methods have some drawbacks, and developing new screening methods is required. In this study, we develop a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry (PESI-MS/MS), which is an ambient ionization mass spectrometrymethod, for human urine, named RaDPi-U. RaDPi-U is carried out in three steps: (1) mixing urine with internal standard (IS) solution and ethanol, followed by vortexing; (2) pipetting the mixture onto a sample plate for PESI; and (3) rapid-fire analysis by PESI-MS/MS. RaDPi-U targets 40 forensically important drugs, which include illegal drugs, hypnotics, and psychoactive substances. The analytical results were obtained within 3min because of the above-mentioned simple workflow of RaDPi-U. The calibration curves of each analyte were constructed using the IS method, and they were quantitatively valid, resulting in good linearity (0.972-0.999) with a satisfactory lower limit of detection and lower limit of quantitation (0.01-7.1ng/mL and 0.02-21ng/mL, respectively). Further, both trueness and precisions were 28% or less, demonstrating the high reliability and repeatability of the method. Finally, we applied RaDPi-U to three postmortem urine specimens and successfully detected different drugs in each urine sample. The practicality of the method is proven, and RaDPi-U will be a strong tool as a rapid-fire drug screening method not only in forensic toxicology but also in clinical toxicology.

Usefulness of Heart-Type Fatty Acid-Binding Protein Measurement in Postmortem Urine Specimens.

Heart-type fatty acid-binding protein (HFABP) is a 15-kDa substance reported to pass through the renal tubules and be renally excreted. Therefore, it is possible that its concentration in the urine collected postmortem may reflect antemortem blood levels. We measured the postmortem urine concentration of HFABP in 94 forensic autopsy cases and compared it between acute myocardial infarction (AMI), sepsis, heat stroke cases, and asphyxia cases as control cases to examine its diagnostic validity. Kidney tissue collected at autopsy was immunostained with antibodies against HFABP to evaluate the correlation with the urinary measurements. Urinary HFABP was significantly higher in AMI, sepsis, and heat stroke cases than in asphyxia cases. Quantitative immunostaining results showed no significant differences between any 2 groups. The usefulness of kidney immunostaining for HFABP in elucidating the cause of death was low. Two reasons may explain the lack of significant differences in kidney immunostaining: nonspecific leakage of tubular epithelial HFABP into the tubules because of postmortem changes and oliguria due to dehydration caused by heat stroke. In conclusion, the measurement of urinary HFABP may be useful in elucidating the cause of death; however, the kidney HFABP immunostaining was not significantly different from AMI.

Forensic laboratory validation of immunochromatography and gas chromatography/mass spectrometry methods for the detection of methamphetamine and amphetamine in postmortem urine specimens

Identification of methamphetamine and its metabolite, amphetamine, both in biological and non-biological samples, is important in forensic toxicology practice. One of the key elements in forensic drug testing is the possibility to use inexpensive screening tests to obtain immediate presumptive results. In the present study the reliability of urine ABON® immunochromatography screening device for the detection of methamphetamine and amphetamine in postmortem urine samples was evaluated. The reliability of the test was assessed by comparing ABON® screening results with confirmatory gas chromatography/mass spectrometry (GC/MS). The analysed samples were 600 positive and 600 negative immunochromatographic results that were confirmed using GC/MS. GC/MS results were used as reference to determine sensitivity, specificity, false and true positives and negatives and positive and negative prediction values. Sensitivity of the ABON® device was 99% for methamphetamine in postmortem urine specimens. ABON® test was also specific enough (97%). Negative and positive predictive values were 99% and 97% respectively. In the GC/MS method, good chromatographic separation was obtained in the presence of licit and illicit drugs. The limit of detection and limit of quantification were equal to 5, 10 ng/mL respectively. The method was linear from 15–1500 ng/mL of methamphetamine and the coefficient of determination was 0.9915. Recovery efficiency was 94%. In conclusion, performance of the ABON® test device is adequate for presumptive determination of methamphetamine in forensic urine drug screening.

Determination of ethyl glucuronide in urine using reversed-phase HPLC and pulsed electrochemical detection (Part II)

A direct, versatile method for the determination of ethyl glucuronide (EtG), a biomarker of ethanol consumption, in urine has been developed using reversed-phase liquid chromatography with pulsed electrochemical detection (PED). EtG and methyl glucuronide (MetG), which serves as an internal standard, are readily separated using a mobile phase consisting of 1% acetic acid/acetonitrile (98/2, v/v). Post-column addition of NaOH allows for the detection of all glucuronides using PED at a gold working electrode. Upon optimization, EtG was found to have a limit of detection of 0.03 μg/mL (7 pmol; 50 μL injection volume) and repeatability at the limit of quantitation of 1.7%R.S.D. (relative standard deviation). Solid-phase extraction (SPE) using an aminopropyl phase was used to remove interferents in urine samples prior to their analysis. Compound recovery following SPE was approximately 50 ± 2%. The forensic utility of this method was further validated by the analysis of 29 post-mortem urine specimens, whose results agreed strongly with certified determinations.

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Identification of methamphetamine and its metabolite, amphetamine, both in biological and non-biological samples, is important in forensic toxicology practice. One of the key elements in forensic drug testing is the possibility to use inexpensive screening tests to obtain immediate presumptive results. In the present study the reliability of urine ABON® immunochromatography screening device for the detection of methamphetamine and amphetamine in postmortem urine samples was evaluated. The reliability of the test was assessed by comparing ABON® screening results with confirmatory gas chromatography/mass spectrometry (GC/MS). The analysed samples were 600 positive and 600 negative immunochromatographic results that were confirmed using GC/MS. GC/MS results were used as reference to determine sensitivity, specificity, false and true positives and negatives and positive and negative prediction values. Sensitivity of the ABON® device was 99% for methamphetamine in postmortem urine specimens. ABON® test was also specific enough (97%). Negative and positive predictive values were 99% and 97% respectively. In the GC/MS method, good chromatographic separation was obtained in the presence of licit and illicit drugs. The limit of detection and limit of quantification were equal to 5, 10 ng/mL respectively. The method was linear from 15–1500 ng/mL of methamphetamine and the coefficient of determination was 0.9915. Recovery efficiency was 94%. In conclusion, performance of the ABON® test device is adequate for presumptive determination of methamphetamine in forensic urine drug screening.

Stability of Ecgonine Methyl Ester in Postmortem Urine Specimens

In this study, 25 postmortem urine specimens testing positive, for cocaine and ecogonine methyl ester (EME) by full scan electron impact gas chromatography/mass spectrometry, were used to evaluate the stability of EME in refrigerated and frozen conditions. After an initial quantitation (t = 0), these specimens were split and stored at either 4 degrees C or -20 degrees C. At several intervals, over a six month period, the specimens were tested for cocaine and EME. Twenty-two of the frozen specimens were within 20% of their t = 0 EME concentration after 6 months; 19 of the 25 refrigerated specimens showed similar stability. At least 50% of the EME present was detected in all specimens under both storage conditions. In addition, there was no evidence to suggest that EME concentrations increased over time even though decreases in cocaine concentrations were observed over the same time period. This suggests that the presence of EME in urine specimens indicates in vivo conversion of cocaine and, therefore, use of cocaine.

Simultaneous determination of 6-monoacetylmorphine, morphine and codeine in urine using high-performance liquid chromatography with combined ultraviolet and electrochemical detection

A method is described for the simultaneous determination of 6-monoacetylmorphine (6-MAM), morphine and codeine in post-mortem urine specimens using reversed-phase high-performance liquid chromatography with dual ultraviolet spectrophotometric and electrochemical detection. The limits of detection for a 1-ml urine sample were 0.04 mg/l for 6-MAM and 0.05 mg/l for both morphine and codeine. The presence of 6-MAM in urine indicates prior use of heroin and enables differentiation between morphine- and heroin-related deaths.

Incidence of Cannabinoids in Medical Examiner Urine Specimens

Cannabinoid use was studied in a nonspecific population of postmortem urine specimens in the State of Maryland. Of 500 sequential specimens screened for cannabinoids by enzyme multiplied immunoassay EMIT, 63 (13%) were initially positive and 58 (12%) were confirmed positive (92%). It was observed that geographic location and race did not correlate with cannabinoid prevalence. Cannabinoid use was observed to be strongly age related, with peak use by the 21- to 25-year-old age group where 22% of the cases were positive. Use of cannabinoids was also closely linked to homicides, which represented nearly half of the positive cases but only 13% of the total cases. When comparing manner of death, the greatest percent of confirmed positives was seen in homicide (26%) and drug-related (17%) deaths. The incidence of cannabinoid use was found to be more than 3 times as great in drug-related (17%) as compared to natural deaths (5%). The percent of cannabinoid-positive cases from vehicle-related accidents was low (6%) and that from nonvehicle-related accidents somewhat higher (10%). Other drugs appeared in cannabinoid-positive cases. Most prevalent was ethanol N = 18, followed by morphine (from heroin, N = 11), quinine N = 11, and cocaine N = 11. Phencyclidine (PCP) occurred twice and several other drugs were reported only once. Of the 25 homicide cases screened for drugs, 64% were positive for some drug including ethyl alcohol. Thus it appears that a high percentage of homicide cases are drug related. Males greatly outnumbered females (56:2) in positive cases, but the number of female specimens received was small.

A method for the determination of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid in urine using high performance liquid chromatography with electrochemical detection.

11-Nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) was isolated using solid phase extraction and identified with high performance liquid chromatography (HPLC) with electrochemical detection (EC). HPLC/EC was compared to an established HPLC procedure using ultraviolet (UV) detection. Statistical analysis revealed good precision, linearity, and correlation between the HPLC/EC and HPLC/UV methods. Within-run and between-run precision for the HPLC/EC vs. HPLC/UV analyses yielded coefficients of variation of 4.1 vs. 6.7% and 8.3 vs. 10.9%, respectively. Standard curves over a range of 20 to 160 ng/mL of THC-COOH in fortified urine on four separate analytical runs yielded correlation coefficients of greater than 0.99 for each method. Quantitative results from 15 positive urine specimens also compared favorably between the two methods (r = 0.9923). Using an immunoassay screening technique with confirmation by HPLC/UV and high efficiency thin layer chromatography (HETLC), further evaluation of the HPLC/EC method was achieved by examining 50 cannabinoid positive and 50 cannabinoid negative clinical urine specimens and 500 sequentially collected postmortem urine specimens.