Tool In Forensic Toxicology Research Articles

Cross-reactivity of 24 cannabinoids and metabolites in blood using the Immunalysis Cannabinoids Direct enzyme-linked immunosorbent assay.

With wider availability of synthetic and semi-synthetic cannabinoids in the consumer space, there is a growing impact on public health and safety. Forensic toxicology laboratories should keep these compounds in mind as they attempt to remain effective in screening for potential sources of human performance impairment. Enzyme-linked immunosorbent assay (ELISA) is a commonly utilized tool in forensic toxicology, as its efficiency and sensitivity make it useful for rapid and easy screening for a large number of drugs. This screening technique has lower specificity, which allows for broad cross-reactivity among structurally similar compounds. In this study, the Cannabinoids Direct ELISA kit from Immunalysis was utilized to assess the cross-reactivities of 24 cannabinoids and metabolites in whole blood. The assay was calibrated with 5 ng/mL of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and the analytes of interest were evaluated at concentrations ranging from 5 to 500 ng/mL. Most parent compounds demonstrated cross-reactivity ≥20 ng/mL, with increasing alkyl side-chain length relative to Δ9-tetrahydrocannabinol resulting in decreased cross-reactivity. Of the 24 analytes, only the carboxylic acid metabolites, 11-nor-9-carboxy-Δ8-tetrahydrocannabinol, 11-nor-9(R)-carboxy-hexahydrocannabinol and 11-nor-9(S)-carboxy-hexahydrocannabinol, were cross-reactive at levels ≤10 ng/mL. Interestingly, 11-nor-9(R)-carboxy-hexahydrocannabinol demonstrated cross-reactivity at 5 ng/mL, where its stereoisomer 11-nor-9(S)-carboxy-hexahydrocannabinol, did not. As more information emerges about the prevalence of these analytes in blood specimens, it is important to understand and characterize their impact on current testing paradigms.

Effect of CYP2D6, 2C19, and 3A4 Phenoconversion in Drug-Related Deaths.

Molecular autopsy is a very important tool in forensic toxicology. However, many determinants, such as co-medication and physiological parameters, should be considered for optimal results. These determinants could cause phenoconversion (PC), a discrepancy between the real metabolic profile after phenoconversion and the phenotype determined by the genotype. This study's objective was to assess the PC of drug-metabolizing enzymes, namely CYP2D6, 2C19, and 3A4, in 45 post-mortem cases where medications that are substrates, inducers, or inhibitors of these enzymes were detected. It also intended to evaluate how PC affected the drug's metabolic ratio (MR) in four cases. Blood samples from 45 cases of drug-related deaths were analyzed to detect and determine drug and metabolite concentrations. Moreover, all the samples underwent genotyping utilizing the HaloPlex Target Enrichment System for CYP2D6, 2C19, and 3A4. The results of the present study revealed a statistically significant rate of PC for the three investigated enzymes, with a higher frequency of poor metabolizers after PC. A compatibility was seen between the results of the genomic evaluation after PC and the observed MRs of venlafaxine, citalopram, and fentanyl. This leads us to focus on the determinants causing PC that may be mainly induced by drug interactions. This complex phenomenon can have a significant impact on the analysis, interpretation of genotypes, and accurate conclusions in forensic toxicology. Nevertheless, more research with more cases in the future is needed to confirm these results.

Simultaneous Quantification of 25 Fentanyl Derivatives and Metabolites in Oral Fluid by Means of Microextraction on Packed Sorbent and LC-HRMS/MS Analysis.

Fentanyl and fentalogs’ intake as drugs of abuse is experiencing a great increase in recent years. For this reason, there are more and more cases in which it is important to recognize and quantify these molecules and related metabolites in biological matrices. Oral fluid (OF) is often used to find out if a subject has recently used a psychoactive substance and if, therefore, the person is still under the effect of psychotropics. Given its difficulty in handling, good sample preparation and the development of instrumental methods for analysis are essential. In this work, an analytical method is proposed for the simultaneous determination of 25 analytes, including fentanyl, several derivatives and metabolites. OF was collected by means of passive drool; sample pretreatment was developed in order to be fast, simple and possibly semi-automated by exploiting microextraction on packed sorbent (MEPS). The analysis was performed by means of LC–HRMS/MS obtaining good identification and quantification of all the analytes in less than 10 min. The proposed method was fully validated according to the Scientific Working Group for Forensic Toxicology (SWGTOX) international guidelines. Good results were obtained in terms of recoveries, matrix effect and sensitivity, showing that this method could represent a useful tool in forensic toxicology. The presented method was successfully applied to the analysis of proficiency test samples.

A possible new oxidation marker for hair adulteration: Detection of PTeCA (1H-pyrrole-2,3,4,5-tetracarboxylic acid) in bleached hair.

Hair analysis has become a valuable tool in forensic toxicology to assess drug or alcohol abstinence. Yet, hair adulteration by cosmetic products presents a major challenge for forensic hair analysis. Oxidative treatments, e.g. bleaching, may lead to analyte loss and thereby to false negative results. Currently, the eumelanin degradation product 1H-pyrrole-2,3,5-tricarboxylic acid (PTCA) serves as a marker for oxidative hair treatment, but requires the definition of cut-off values. To investigate further eumelanin degradation products as markers for oxidative hair treatment, hair samples with and without in vitro bleaching (hydrogen peroxide (H2 O2 ) concentrations 1.9% up to 12%; incubation times 15 min, 30 min, 60 min) were analyzed by liquid chromatography coupled to high-resolution time of flight mass spectrometry (HPLC-HRMS). The distribution of eumelanin degradation products along the hair shaft was investigated for routine applicability after segmentation of cosmetically untreated hair samples and authentically treated hair samples. The signals of the eumelanin degradation products PTCA, 1H-pyrrole-2,3,4-tricarboxylic acid (isoPTCA), and 1H-pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) were found to be significantly elevated after in vitro bleaching already with low H2 O2 concentrations and after short incubation times. In contrast to PTCA and isoPTCA, PTeCA was not detectable in cosmetically untreated segments up to 12 cm from hair root and was only formed through the oxidation process. The results of the study show that the detection of PTeCA within the proximal 3 to 6 cm segment can be applied to reliably detect hair adulteration attempts through hair bleaching.

Micellar liquid chromatographic method for the simultaneous determination of citalopram hydrobromide with its two demethylated metabolites. Utility as a diagnostic tool in forensic toxicology

A micellar liquid chromatographic method has been developed for the simultaneous determination of citalopram hydrobromide (CTA) with its two demethylated metabolites namely; desmethyl citalopram hydrochloride (DCTA) and didesmethyl citalopram tartrate (DDCTA). Separation was conducted on a C18 column using a mobile phase composed of 0.18 M sodium dodecyl sulphate (SDS), 15% 1-propanol, 0.3% tri-ethylamine, adjusted to pH 4 with 0.2 M o-phosphoric acid and adopting UV detection at 240 nm. Analysis was performed at 60 °C applying a flow rate of 2 mL/min. The proposed method was linear over the concentration ranges of 1.0–200.0, 0.6–200.0, and 0.5–200.0 μg/mL for CTA, DCTA, and DDCTA respectively, with corresponding limits of detection (LOD) of 0.5, 0.4, and 0.3 μg/mL and limits of quantification (LOQ) of 0.8, 0.5, and 0.4 μg/mL. The method was fully validated which allowed its application for the determination of CTA in its tablets. Moreover, the proposed method was extended to assay CTA with its metabolites in rat tissue organs samples which allowed the method to be used as a diagnostic tool in forensic toxicology.

Drugs of abuse extraction from whole blood using supported liquid extraction (SLE) and Extrahera automation prior to UPLC-MS/MS analysis

Whole blood continues to be a valuable tool in forensic toxicology for the immediate and near-term detection of illicit drugs and in cases where no other sample is available. Screening drugs of abuse can be complicated due to the wide variation of functional groups associated with different analyte classes. Most extraction techniques cannot extract all analytes using a single procedure without using non-optimal extraction protocols resulting in compromised extract cleanliness. Supported liquid extraction allows for the simultaneous analysis of cross functional analytes in a single extraction protocol without forfeiting extract cleanliness. The objective was to develop an automatable, common extraction procedure for multiple drug panels from whole blood matrix using supported liquid extraction (SLE) prior to UPLC-MS/MS analysis. The drug suites included amphetamines, benzodiazepines, cocaine, opiates, ketamine, buprenorphine, Z-drugs and THC-COOH. Blank whole blood was spiked with drugs from various panels at 13 ng/mL for the analytes and appropriate deuterated internal standards. Whole blood extraction was performed using ISOLUTE SLE+ 1 mL capacity columns evaluating pH control and optimum extraction solvent combinations. The resultant extracts were evaporated to dryness and reconstituted in mobile phase for subsequent UPLC-MS/MS analysis. Samples were analyzed using a Waters ACQUITY UPLC coupled to a Quattro Premier XE triple quadrupole mass spectrometer (Waters). Positive ions were acquired using electrospray ionisation in the MRM mode. Initial investigations focused on sample treatment. Extraction conditions were evaluated using spiked whole blood pre-treated 1:1 (v/v) with various ammonium hydroxide (aq) concentrations from 0.1 % up to 2 %. 0.1 % NH 4 OH (aq) was selected as the pre-treatment as many of the drugs are basic in nature, however an environment at this pH value also allowed concomitant extraction of THC-COOH. Loading 1 mL of pre-treated whole blood led to the extraction of matrix components in the form of red blood cells. Evaluation of various loading volumes from 500 to 900 μL resulted in selection of 750 μL (1:1, v:v) as the optimal loading volume for extract cleanliness. Dichoromethane, dichloromethane/isopropanol acid and methyl tert-butyl ether combinations with various modifiers were evaluated for extraction efficiency. Benzoylecgonine could only be extracted efficiently when using dichloromethane as the extraction solvent. The optimum extraction protocol provided recoveries greater than 70 % for nearly all drugs spiked into whole blood. Benzoylecgonine and buprenorphine recovered 47 % and 52 % respectively. RSD values for all drugs were under 10 %. Lower recoveries for benzoylecgonine and buprenorphine did not prevent quantitation at sub-ng/mL concentrations. Evaporative effects were eliminated using 0.05 M HCl in methanol prior to evaporation. Calibration curves constructed from 1–500 ng/mL demonstrated excellent linearity and r 2 > 0.99 for all analytes. LLOQs were determined to be sub-ng/mL for each analyte when extracting 375 μL of whole blood, with the exception of THC-COOH (5 ng/mL). MRM analysis for common phospholipid molecules demonstrated the optimum method yields extremely clean extracts. This method was transferred to the Extrahera sample automation platform for repeat investigations into recovery, reproducibility and linearity and in nearly all analytes, the automation approach improves upon the manual approach for acquired data while also saving operator time. This poster describes the suitability of ISOLUTE SLE+ for the rapid and reliable extraction of multiple analyte panels from whole blood in a single, fully-automatable assay on the Extrahera platform, prior to UPLC-MS/MS analysis.

A new software tool for confident identification and quantitation of drugs in urine and serum

Aim High-resolution time-of-flight mass spectrometry has become an excellent tool in forensic toxicology. The accurate mass based inherent characteristics like sensitive wide scope screening together with retrospective and general unknown analysis capabilities make it an ideal tool for this work. We describe the development and evaluation of a new software tool allowing screening and quantification using ultra-high-resolution LC-Q-ToF, accurate mass analysis. The new software allows applying the diagnostic ion concept, using enhanced confirmation criteria such as isotopes, adducts, fragments and qualifier ratios for reducing false positive detection rates. Methods Sixty-one compounds covering several compound classes based on their relevance in post-mortem and routine drug screening were used in this study. After acetonitrile precipitation, urine and serum samples were spiked with the toxicological compound mixes at four levels (between 10–500 ng/mL) and analyzed in ESI(+) mode by LC-Q-ToF-MS (Impact II, Bruker Daltonics, USA), in full scan and bbCID modes using a 14 minute gradient, reverse phase UHPLC separation. The data acquired in bbCID mode were processed with the new software package including a database with more than 2000 compounds of forensic relevance. Results In the full scan ToF-MS channel, all compounds were detected at all concentration levels, no false negatives were encountered. In urine, the high matrix load produced a high number of false positives (547 compared to the 276 expected positives). Only seven compounds were causing ∼ 75% of all -false positives. These false positives typically arose due to a very low detection threshold. In total 35 different compounds appeared as false positives. After applying the enhanced diagnostic ion detection criteria the false positives were completely removed. A challenge was tramadol in the presence of o-desmethylvenlafaxine as these compounds have the same retention time, same sum-formula and the same main fragment ion. However, two smaller characteristic fragments allowed the unambiguous identification of o-desmethylvenlafaxine. Conclusion The screening method with the new software is robust and has been shown to work reproducibly for forensic toxicological screening of blood and urine samples. The application of the ‘diagnostic ion’ concept has proven to be a very powerful tool to eliminate false positive findings.

Comparison of Tissue Homogenate Analytical Results With and Without Standard Addition

Standard addition (SA) has occasionally been utilized as an analytical tool in forensic toxicology. It is recommended for difficult matrices such as tissue or decomposed specimens in which the accurate quantitation of drug may be problematic. However, the additional preparation time and increased use of specimen may limit its general applicability. In this study, the authors compared SA with direct extraction (DE) and quantitation against a blood calibration curve to assess whether the quantitative results were significantly different. Twenty-two postmortem cases were assayed for drugs such as cocaine and metabolites, opioids, and antidepressants by solid-phase extraction followed by gas chromatography-mass spectrometry operated in the selected ion monitoring mode. A two-tailed, homoscedastic Student's t-test demonstrated that the two sets of data were not statistically different (p = 0.81). In addition, SAs were more likely to demonstrate nonlinearity (r(2) < 0.98).

A preliminary investigation into the use of biosensors to screen stomach contents for selected poisons and drugs

The bioluminescence response of two genetically modified ( lux-marked) bacteria to potentially toxic compounds (PTCs) in stomach contents was monitored using an in vitro assay. Cells of Escherichia coli HB101 and Salmonella typhimurium both carrying the lux light producing gene on a plasmid (pUDC607) were added to stomach contents containing various concentrations of organic and inorganic compounds. There was some variability in the response of the two biosensors, but both were sensitive to the herbicides glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T); pentachlorophenol (PCP), and inorganic poisons arsenic and mercury at a concentration range likely to be found in stomach contents samples submitted for toxicological analysis. This study demonstrates that biosensor bioassays could be a useful preliminary screening tool in forensic toxicology and that such a toxicological screening should include more than one test organism to maximise the number of PTC's detected. The probability of false positive results from samples containing compounds that may interfere with the assay such as over-the-counter (OTC) drugs and caffeine in tea and coffee was also investigated. Of the substances tested only coffee has the potential to cause false positive results.

Evaluation of the Cedia(R) Heroin Metabolite (6-AM) Immunoassay with Urine Specimens from a Criminal Justice Drug-Testing Program

The ability to differentiate illicit from legitimate drug use in a drug-testing program would decrease costs by reducing the number of screening specimens requiring confirmation and also reduce the stigma attached to positive preliminary test results. Because many screening tests for drug detection use immunoassays, increasing the specificity of these tests has been a goal of manufacturers. In this study we evaluated the utility of one such assay, the Cedia heroin metabolite (6-acetylmorphine, 6-AM) assay to reliably detect heroin use. Specimens (N = 525) from a criminal justice drug-testing program were screened with this assay (cutoff concentration = 10 ng/mL 6-AM) and any positive samples were confirmed by gas chromatographic-mass spectrometric analysis (lower reporting limit for 6-AM = 5 ng/mL). The confirmation rate for the enzyme immunoassay (EIA) was 98% (517/525). Specimens contained 6-AM at concentrations ranging from 5 to 16,923 ng/mL (mean = 1251; median = 317). All confirmed specimens also contained morphine (range: 8-222,427 ng/mL; mean = 11,203 ; median = 4134). When challenged with standard drug solutions, the EIA correctly identified drug-free urine and produced positive results (lowest concentration, in ng/mL, that produced a positive result) with morphine at 10,000; oxycodone at 61,000; codeine at 60,000; hydromorphone at 10,000; hydrocodone at 60,000, 6-AM at 10, and pentazocine at 35,000 ng/mL. The Cedia heroin metabolite (6-AM) assay produced a high confirmation rate when challenged with urine specimens and therefore should be a useful tool in forensic toxicology. Potential users should be aware that high concentrations of other opioids (e.g., morphine, oxycodone) and structurally related compounds (e.g., pentazocine) may produce positive results.

Rapid determination of carboxyhemoglobin in blood by Oximeter

Different methods to determine carboxyhemoglobin (COHb) in blood are described in the literature. In our laboratory three methods to analyze COHb in post-mortem blood samples were compared: the spectrophotometric method of Maehly, a gas chromatographic method with a thermal conductivity detector (GC-TCD) and the Oximeter. Several COHb containing blood samples of deceased persons were analyzed. Results of all three methods were comparable for low concentrations (ca. 10% COHb) as well as for high concentrations (ca. 80% COHb) regardless of the viscosity of the blood samples. The advantages of the Oximeter when compared to Maehly’s method and GC-TCD are extreme short time of analysis (<1 min), very small blood volume required (<0.1 ml) and easy handling. In our opinion application of the Oximeter is not limited to analyses of blood samples from living persons (e.g. in clinical toxicology); it can as well be used for the determination of COHb in post-mortem blood samples. Hence it is a useful and time saving tool in forensic toxicology.

Capillary electrophoresis: a new tool in forensic toxicology. Applications and prospects in hair analysis for illicit drugs

Capillary electrophoresis, the modern approach to instrumental electrophoresis, is probably the most rapidly expanding analytical technique that has appeared in recent years. In the hands of forensic toxicologists, capillary electrophoresis (CE) represents a powerful new analytical tool, which has proved suitable for the investigation of illicit drugs in seized preparations and also in complex biological matrices, among which is hair. CE can be applied according to different separation mechanisms, and among those that are toxicologically relevant are capillary zone electrophoresis and micellar electrokinetic capillary chromatography, which display different selectivities. For the investigation of hair for drugs of abuse, capillary electrophoresis proved effective, providing simultaneous determinations of different drugs without derivatization, with acceptable sensitivity (typically better than 1 ng of drug per mg of hair). The possibility of carrying out determinations of the same analytes, based on different separation mechanisms (capillary zone electrophoresis and micellar electrokinetic chromatography) with the same instrumentation, simply changing the buffer composition, provides an interesting possibility of ‘internal’ confirmation of the results.

The Gas Chromatograph-Mass Spectrometer as a New and Important Tool in Forensic Toxicology

Abstract The growing use of drugs in this country has caused the work of the toxicological chemist to become increasingly difficult. More and more, the courts, police departments, and coroners have had to rely on the chemist for evidence in respect to identification of drugs. In many cases testing procedures commonly used today do not, in fact, establish the identity of a substance of interest with a desirable degree of certainty and the presumptive identification needs confirmation by other techniques.