Simultaneous quantification of diazepam, flunitrazepam and metabolites in reinforced clostridial medium by liquid chromatography-tandem mass spectrometry.

A novel liquid chromatography-tandem mass spectrometry method was validated for identification and quantification of diazepam, flunitrazepam and metabolites in reinforced clostridial medium (RCM), a complex matrix used to provide the nutrients required for bacterial growth. The method was designed for subsequent use in the investigation of gastrointestinal bacteria as a potential source of postmortem alteration of drugs of abuse and respective metabolite concentrations. A literature review yielded no experimental means or model for the extraction and analysis of samples from RCM or similar bacterial medium. Development and validation of a new experimental method were therefore critical. In future work, this method could be adapted and extended to similar organic compounds of interest. The calibration curves extended from 0.100 to 500 ng/mL. Analyte recoveries ranged from 95 to 119% and matrix effects from 97 to 119%. Bias was ≤±17.6%, within-run precision ≤12.2%, and between-run precision ≤11.7% across all concentration levels. The limits of detection and quantitation ranged from 0.100 to 1 ng/mL. Dilution integrity was maintained for 1:2 and 1:5 dilutions. Analytes were stable through two freeze-thaw cycles and processed samples for 48 h. Method robustness was evaluated by changes in buffer composition and column temperature as well as samples prepared by an alternate analyst.

Production of human metabolites by gastrointestinal bacteria as a potential source of post-mortem alteration of antemortem drug/metabolite concentrations.

Previous studies have demonstrated that bacterial species are capable of transforming complex chemical substances. Several of these species, native to the human gastrointestinal tract, are active in postmortem decomposition. They have potential to cause biotransformations affecting compound-to-metabolite ratios within the human body, especially after death. Investigation of postmortem effects could supply valuable information, especially concerning compound identification and confirmation. The purpose of this research was to investigate the effects of Escherichia coli, Bacteroides fragilis, and Clostridium perfringens on diazepam and flunitrazepam in Reinforced Clostridial Medium, and to compare bacterial biotransformation products to those of human metabolism. A decrease in diazepam concentration between pre- and post-incubation was observed for samples inoculated with Escherichia coli (14.7-20.2%) as well as Bacteroides fragilis (13.9-25.7%); however there was no corresponding increase in concentration for the monitored human metabolites. Flunitrazepam demonstrated a greater concentration loss when incubated with individual bacterial species as well as mixed culture (79.2-100.0%). Samples incubated with Bacteroides fragilis, Clostridium perfringens, and mixed culture resulted in nearly complete conversion of flunitrazepam. Increased 7-aminoflunitrazepam concentrations accounted for the majority of the conversion; however discrepancies in the mass balance of the reaction suggested the possibility of a minor metabolite that was not monitored in the current analysis. These experiments served as a pilot study and proof of concept that can be adapted and applied to a realm of possibilities. Ultimately, this methodology would be ideal to study compounds that are too toxic or lethal for animal and human metabolic investigations.

Potential biomarkers of smoked fentanyl utilizing pyrolysis gas chromatography-mass spectrometry.

Fentanyl is a potent opioid analgesic that is increasingly becoming a choice drug of abuse. Fentanyl transdermal patches (FTPs) are easily obtained and consumed by smoking the reservoir gel and/or the whole patch. This allows for an increased bioavailability when inhaled. A method using analytical pyrolysis was developed to identify possible biomarkers associated with smoked fentanyl and FTPs. Pyrolysis was carried out under anaerobic and aerobic conditions using helium and air coupled to a gas chromatograph-mass spectrometer. The presence of a trap enhanced recovery and afforded a positive identification of pyrolytic products. Anaerobic and aerobic pyrolysis of fentanyl and FTPs consistently yielded propionanilide as the major pyrolytic product along with pyridine and previously reported metabolites (norfentanyl and despropionyl fentanyl). Analysis of fentanyl resulted in chlorine-containing compounds, presumably formed from the HCl salt of fentanyl. Analysis of FTPs showed significant polymeric and hydrocarbon compounds and products likely derived from the gel matrix. Fentanyl in the FTPs was in the citrate salt form; therefore, the chlorine-containing pyrolytic products obtained with the neat drug were not observed. Based on this application, it may be possible to identify what salt form of the drug was smoked based on pyrolytic products and to target distinguishing metabolic products for future research.

Bioassays for bomb-makers: proof of concept.

Clandestine bomb-makers are exposed to significant amounts of explosives and allied materials. As with any ingested xenobiotic substance, these compounds are subject to biotransformation. As such, the potential exists that characteristic suites of biomarkers may be produced and deposited in matrices that can be exploited for forensic and investigative purposes. However, before such assays can be developed, foundational data must be gathered regarding the toxicokinetics, fate, and transport of the resulting biomarkers within the body and in matrices such as urine, hair, nails, sweat, feces, and saliva. This report presents an in vitro method for simulation of human metabolic transformations using human liver microsomes and an assay applicable to representative nitro-explosives. Control and metabolized samples of TNT, RDX, HMX, and tetryl were analyzed using high-performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) and biomarkers identified for each. The challenges associated with this method arise from solubility issues and limitations imposed by instrumentation, specifically, modes of ionization.

Forensic discrimination of glass using cathodoluminescence and CIE LAB color coordinates: a feasibility study.

Cathodoluminescence (CL) spectroscopy has been shown to be useful for differentiating typical evidentiary glass samples. CL occurs when a surface is bombarded with an electron beam as in scanning electron microscopy and most of this luminescence is in the visible range. In effect, CL imparts color to colorless evidence and as a result, proven methods of forensic color analysis can be applied. In this work, spectral data dimensions were reduced to three and plotted in the CIE LAB color space. This approach allows for incorporation of uncertainties generated principally by intra-sample variation. NIST glass standards were used for method development and validation while potential case applications were studied with collections of window, consumer, and auto headlamp glasses. Using refractive index as the initial grouping variable, all of the window and consumer glasses were differentiated as were 6 of 10 automobile headlamp glasses. The potential advantages of CL include low cost instrumentation, its non-destructive nature, and ease of operation. The current limitations of CL in this context are the lack of databases and standards and the relatively low resolution of typical CL spectra.

A microfluidic device for presumptive testing of controlled substances.

A simple microfluidic device (MFD) has been developed to perform multiple color and crystal tests for controlled substance analysis. The MFD method uses less sample and reagents and generates less waste than traditional spot plate methods while performing several tests simultaneously. This methodology provides significantly more analytical information for a single sample analysis. The current generation device is the size of a microscope slide with four analytical channels: one for microcrystal tests and three for color tests. The optimized devices were subjected to a rigorous validation study using comparative replicate analyses and several operators. Target analytes were methamphetamine, amphetamine, cocaine, and oxycodone and color test reagents used were the Marquis, Simon, and cobalt thiocyanate. For the crystal tests, platinic chloride was used. The validation study showed the MFD's limits of detection to be in the picogram range. Positive tests results were observed in complex mixtures in which the controlled substance was present at concentrations of 5-10% (w/w). The microcrystal reagents showed greater sensitivity than color test reagents when used in the device. Reagent use and waste generation using the devices was 95% less that that used and generated using the traditional methods. The device performance was also shown to be operator independent.

Comparative analysis of gamma-hydroxybutyrate and gamma-hydroxyvalerate using GC/MS and HPLC.

This paper describes two analytical techniques used to separate and quantify gamma-hydroxybutyrate (GHB) and gamma-hydroxyvalerate (GHV). The first technique was a N,O-bis(trimethylsilyl)triflouro-acetimide-trimethylchlorosilane derivatization, followed by gas chromatography/mass spectrometry analysis using an HP-5 capillary column at a rate of 1.0 mL/min with a run time of 9.25 min. This technique was found to be sensitive (LOD 1 pg on column) and gave a low average error (5%) in a beverage study. When supplemented by a surrogate spike, the method yielded 97% analyte recovery from beverages. The second technique was high-performance liquid chromatography/UV (HPLC/UV) using a C-18 column with a (20:80% v/v) methanol:dibasic phosphoric buffer (10 mM, pH 3) at a rate of 1.00 mL/min with a run time of 7.5 min. UV detection occurred at 254 nm. This method was found to be less sensitive (LOD 0.05 microg on column) for direct analysis of aqueous samples. To remove interferences seen in the beverage study, a liquid-liquid extraction before HPLC analysis was tested. However, a decreased sensitivity (LOD 100 microg on column) and irreproducible peak profiles resulted.

Application of a pyroprobe to simulate smoking and metabolic degradation of abused drugs through analytical pyrolysis.

Smoking of illicit drugs can produce unique metabolic biomarkers. Smoking conditions can be partially modeled via pyrolysis, a process that decomposes a chemical compound by extreme heat. Pyrolytic decomposition was found to be useful as a limited metabolic mimic in that analytical pyrolysis can be used to generate some of the same compounds produced by metabolic degradation. This project focused on the pyrolysis of cocaine and methamphetamine using a pyroprobe coupled with a GC/MS and more generally, potential applications of pyrolysis to forensic toxicology. Common diluents including lidocaine, caffeine, and benzocaine were pyrolyzed in mixtures with cocaine and methamphetamine. Correlations between pyrolytic and metabolic degradations revealed that this method has the capability to produce some of the reported metabolites such as norcocaine and cocaethylene for cocaine, and amphetamine for methamphetamine. The results demonstrate that analytical pyrolysis has the potential to identify some metabolic products and to supplement in vivo and enzymatic studies.

Validation of a headspace GC method for the analysis of a pyrolytic product of methamphetamine in urine.

A method has been developed and validated using headspace GC-FID for the identification of 1-phenylpropene in urine. This compound is a pyrolytic product of methamphetamine that has been previously proposed as a marker for smoked methamphetamine. The instrumentation used is the same as employed for blood alcohol determination. The extraction-free procedure is rapid, simple, and quantitative using 2-phenylpropene as the internal standard. The method was validated for linearity over a range of 0.1-20 microg/mL with a limit of detection of 0.05 microg/mL, limit of quantification of 0.1 microg/mL, interday accuracy within 3.2 to -5.3%, intraday accuracy better than 7.5%, interday precision of 7.5 to 10.7%, intraday precision of 2 to 8.6%, and recovery above 80%. For the robustness determination in urine, the accuracy of four different sources of urine at the mid control level (1 microg/mL) ranged from 1.6 to 19% error. The % relative standard deviation of the different urine sources ranged from 3.1 to 11%. Urine samples from nine methamphetamine-positive cases investigated by the Office of the Chief Medical Examiner of West Virginia were included in the study. 1-Phenylpropene was found in two methamphetamine-positive cases (0.25 and 0.44 microg/mL).

Chemical composition and structure of the microcrystals formed between silver(I) and gamma-hydroxybutyric acid and gamma-hydroxyvaleric acid.

This study examined microcrystals formed by silver with gamma-hydroxybutyric acid (GHB) and gamma-hydroxyvaleric acid (GHV), the five-carbon analog of GHB, in the presence of silver, copper, and lanthanide nitrates. Distinct microcrystals formed with silver (+1) and lanthanum (+3) ions but not with the copper (+2) ions. The crystals formed with GHB were distinctly different than those formed with GHV and in all cases, the drug microcrystals were easily distinguishable from reagent crystals. X-ray diffraction analysis provided definitive structure for the microcrystals. The morphological differences between the silver-GHB and silver-GHV crystals were characterized using simple measurements such as size and angles provided by image recognition software. The utility of the test for casework was demonstrated using spiked beverage samples.