Urine toxicology screening by liquid chromatography time-of-flight mass spectrometry in a quaternary hospital setting.

OBJECTIVE: Validation of a non-targeted method for urine drug screening (UDS) by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF), and comparison to an established GC-MS method in a hospital setting. METHODS: 217 UDS specimens sent to a quaternary hospital pathology department, were analysed by a CEDIA® immunoassay screen (six drug panels; amphetamines, barbiturates, benzodiazepines, cocaine metabolites, cannabinoids and opiates) on an Abbott Architect instrument. Specimens were subsequently analysed by an established non-targeted qualitative GC-MS method and results compared with a general unknown screening method by LC-QTOF that was under evaluation as a replacement method. RESULTS: 42 selected drugs were evaluated; limits of identification ranged from 2 to 100 µg/L and most drugs (n = 39) were stabile for 24 h after preparation. Matrix effects greater than 25% were observed in seven of the selected drugs. 87% of the specimens tested positive to 1 or more drug panels in a CEDIA® screen. A total of 537 positive drug findings were identified by GC-MS compared to 1,267 positive findings by LC-QTOF. On average, each GC-MS screen identified 2.5 ± 1.8 drugs and the LC-QTOF screen identified 5.8 ± 3.2 drugs. No drugs were identified in 11.3% of the GC-MS screens, whereas drugs were detected in 99% of these by the LC-QTOF. In almost all instances, the LC-QTOF screen could provide mass spectrometric confirmatory results of positive immunoassay screens and was able to identify a wider range of additional drugs and drug metabolites. CONCLUSIONS: The described general unknown screening (non-targeted, qualitative) LC-QTOF method can detect a larger range of drugs encountered in a hospital setting. The method has been shown to be suitable for comprehensive toxicology screening in a clinical toxicology laboratory.

Evaluation of the diagnostic performance of an oral fluid screening test device for substance abuse at traffic controls.

INTRODUCTION: The aim of this study was to evaluate the analytical performance of the Kite Biotechnology Oral fluid (OF) screening test device, which is used for roadside screening of cannabis, opiates, amphetamines, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), cocaine and benzodiazepines by comparing samples with matched plasma samples, analysed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) for confirmation. METHODS: OF and plasma samples were obtained simultaneously from a total of 100 subjects. OF samples were analysed by OF screening test based on immunochromatography. The OF screening test cut-off values were 50 ng/mL for amphetamines (d-amphetamine) and methamphetamine/MDMA (d-methamphetamine), 30 ng/mL for cocaine (benzoylecgonine), 40 ng/mL for opiates (morphine), 20 ng/mL for benzodiazepines (nordazepam), and 25 ng/mL for cannabis (Δ9-tetrahydrocannabinol). LC-MS/MS method validation was performed according to the CLSI C62-A recommendations with the following parameters: matrix effect, lower limit of quantification (LLOQ), linearity, intra-day and inter-day precision and accuracy. RESULTS: The overall specificity, accuracy and negative predictive values (NPV) were acceptable and met the DRUID standard of >80%. The OF screening test device showed good sensitivity for cocaine, amphetamines and opiates, whereas it indicated poor sensitivity for methamphetamine/MDMA (66.7%) and failed to detect cannabis and benzodiazepines. CONCLUSION: The present study is the first report to evaluate the Kite Biotechnology OF screening test device. The diagnostic performance of the OF screening test device was acceptable for opiates, cocaine and amphetamines, but it was insufficient for methamphetamine/MDMA, benzodiazepines and cannabis because of sensitivity issues.

Evaluation and applicability of Alere iCup DX 14 for rapid postmortem urine drug screening at autopsy.

Performing point-of-care urine drug screen testing at autopsy by a forensic pathologist may provide an early indication of the presence of analytes of interest during autopsy. An evaluation for the screening of 14 classes of common drugs of abuse in postmortem urine by the point-of-care screening device, Alere iCup DX 14, is presented. One hundred ninety postmortem urine samples were screened with the iCup occurring at autopsy by the forensic pathologist. Positive and negative results obtained from the screening kit were evaluated against confirmatory test results obtained using routine forensic toxicology analyses that employed LC-MS/MS and GC-MS to detect a combination of over 85 common drugs of abuse and medications. Sensitivity for each respective iCup drug class ranged from 66% (buprenorphine) to 100% (methadone, tricyclic antidepressants). Specificity for each respective iCup drug class ranged from 89% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, 3,4-methylenedioxymethamphetamine, methadone). Positive predictive values ranged from 44% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, methylenedioxymethamphetamine, methadone), while negative predictive values ranged from 96% (methamphetamine) to 100% (barbiturates, methadone, tricyclic antidepressants). A high false-positive rate was yielded by the benzodiazepine class. The lack of fentanyl screening in the point-of-care device is a significant limitation considering its prolific prevalence in forensic casework. The results obtained in the study should be acknowledged when considering the use of the Alere iCup DX 14 in the context of postmortem casework to help indicate potential drug use contemporaneously with autopsy and when requiring such preliminary results prior to the release of a final forensic toxicology report.

Hyphenated high-resolution mass spectrometry-the "all-in-one" device in analytical toxicology?

This trend article reviews papers with hyphenated high-resolution mass spectrometry (HRMS) approaches applied in analytical toxicology, particularly in clinical and forensic toxicology published since 2016 and referenced in PubMed. The article focuses on the question of whether HRMS has or will become the all-in-one device in these fields as supposed by the increasing number of HRMS presentations at scientific meetings, corresponding original papers, and review articles. Typical examples for the different application fields are discussed such as targeted or untargeted drug screening, quantification, drug metabolism studies, and metabolomics approaches. Considering the reviewed papers, HRMS is currently the only technique that fulfills the criteria of an all-in-one device for the various applications needed in analytical toxicology.Graphical abstract.

Providing illicit drugs results in five seconds using ultra-portable NIR technology: An opportunity for forensic laboratories to cope with the trend toward the decentralization of forensic capabilities.

The analysis of illicit drugs faces many challenges, mainly regarding the production of timely and reliable results and the production of added value from the generated data. It is essential to rethink the way this analysis is operationalised, in order to cope with the trend toward the decentralization of forensic applications. This paper describes the deployment of an ultra-portable near-infrared detector connected to a mobile application. This allows analysis and display of results to end users within 5s. The development of prediction models and their validation, as well as strategies for deployment within law enforcement organizations and forensic laboratories are discussed.

A threshold LC-MS/MS method for 92 analytes in oral fluid collected with the Quantisal® device.

A study of impaired driving rates in the province of Québec is currently planned following the legalization of recreational cannabis in Canada. Oral fluid (OF) samples are to be collected with a Quantisal® device and sent to the laboratory for analysis. In order to prepare for this project, a qualitative decision point analysis method monitoring for the presence of 97 drugs and metabolites in OF was developed and validated. This high throughput method uses incubation with a precipitation solvent (acetone:acetonitrile 30:70 v:v) to boost drug recovery from the collecting device and improve stability of benzodiazepines (e.g., α-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam, nitrazepam). The Quantisal® device has polyglycol in its stabilizing buffer, but timed use of the mass spectrometer waste valve proved sufficient to avoid the glycol interferences for nearly all analytes. Interferences from OF matrices and 140 potentially interfering compounds, carryover, ion ratios, stability, recovery, reproducibility, robustness, false positive rate, false negative rate, selectivity, sensitivity and reliability rates were tested in the validation process. Five of the targeted analytes (olanzapine, oxazepam, 7-aminoclonazepam, flunitrazepam and nitrazepam) did not meet the set validation criteria but will be monitored for identification purposes (no comparison to a cut-off level). Blind internal proficiency testing was performed, where six OF samples were tested and analytes were classified as "negative", "likely positive" or "positive" with success. The final validated OF qualitative decision point method covers 92 analytes, and the presence of 5 additional analytes is screened in this high throughput analysis.

Development of a Pencil Drawn Paper-based Analytical Device to Detect Lysergic Acid Diethylamide (LSD)*, †.

The need for agile and proper identification of drugs of abuse has encouraged the scientific community to improve and to develop new methodologies. The drug lysergic acid diethylamide (LSD) is still widely used due to its hallucinogenic effects. The use of voltammetric methods to analyze narcotics has increased in recent years, and the possibility of miniaturizing the electrochemical equipment allows these methods to be applied outside the laboratory; for example, in crime scenes. In addition to portability, the search for affordable and sustainable materials for use in electroanalytical research has grown in recent decades. In this context, employing paper substrate, graphite pencil, and silver paint to construct paper-based electrodes is a great alternative. Here, a paper-based device comprising three electrodes was drawn on 300 g/m2 watercolor paper with 8B pencils, and its efficiency was compared to the efficiency of a commercially available screen-printed carbon electrode. Square wave voltammetry was used for LSD analysis in aqueous medium containing 0.05 mol/L LiClO4 . The limits of detection and quantification were 0.38 and 1.27 µmol/L, respectively. Both electrodes exhibited a similar voltammetric response, which was also confirmed during analysis of a seized LSD sample, with recovery of less than 10%. The seized samples were previously analyzed by GCMS technique, employing the full scan spectra against the software spectral library. The electrode selectivity was also tested against 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine. It was possible to differentiate these compounds from LSD, indicating that the developed paper-based device has potential application in forensic chemistry analyses.

LC-QTOF-MS Identification of Major Urinary Cyclopropylfentanyl Metabolites Using Synthesized Standards.

Cyclopropylfentanyl is a fentanyl analog implicated in 78 deaths in Europe and over 100 deaths in the United States, but toxicological information including metabolism data about this drug is scarce. The aim of this study was to provide the exact structure of abundant and unique metabolites of cyclopropylfentanyl along with synthesis routes. In this study, metabolites were identified in 13 post-mortem urine samples using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Samples were analyzed with and without enzymatic hydrolysis, and seven potential metabolites were synthesized in-house to provide the identity of major metabolites. Cyclopropylfentanyl was detected in all samples, and the most abundant metabolite was norcyclopropylfentanyl (M1) that was detected in 12 out of 13 samples. Reference materials were synthesized (synthesis routes provided) to identify the exact structure of the major metabolites 4-hydroxyphenethyl cyclopropylfentanyl (M8), 3,4-dihydroxyphenethyl cyclopropylfentanyl (M5) and 4-hydroxy-3-methoxyphenethyl cyclopropylfentanyl (M9). These metabolites are suitable urinary markers of cyclopropylfentanyl intake as they are unique and detected in a majority of hydrolyzed urine samples. Minor metabolites included two quinone metabolites (M6 and M7), not previously reported for fentanyl analogs. Interestingly, with the exception of norcyclopropylfentanyl (M1), the metabolites appeared to be between 40% and 90% conjugated in urine. In total, 11 metabolites of cyclopropylfentanyl were identified, including most metabolites previously reported after hepatocyte incubation.

The Trouble With Kratom: Analytical and Interpretative Issues Involving Mitragynine.

Mitragynine is the primary active alkaloid in the leaves of the tropical tree Mitragyna speciosa, and goes by the popular names "Kratom", biak-biak and maeng da. Mitragynine is increasingly seen in forensic toxicology casework including driving under the influence of drugs and medicolegal death investigation cases. The toxicity of mitragynine continues to be debated in the scientific community as advocates highlight its long history of use in Southeast Asia and testimonials to its benefits by present-day users, while opponents point to an increasing number of adverse events tied to mitragynine use in Western societies. Quantitative reports of mitragynine in biological specimens from forensic investigations in the literature are sparse and may be influenced by poor analyte stability and inadequate resolution of mitragynine from its diastereomers, which could lead to falsely elevated concentrations and subsequently render those reported concentrations inappropriate for comparison to a reference range. Over the course of 27 months, 1,001 blood specimens submitted to our laboratory tested positive for mitragynine using a sensitive and specific quantitative LC-MS/MS method; concentrations ranged from 5.6-29,000 ng/mL, with mean and median concentrations of 410 ± 1,124 and 130 ng/mL, respectively. Mitragynine presents an analytical challenge that requires a method that appropriately separates and identifies mitragynine itself from its isomers and other related natural products. We describe a validated analytical method and present a short series of case reports that provide examples of apparent adverse events, and the associated range of mitragynine concentrations. This type of analytical specificity is required to appropriately interpret mitragynine concentrations detected in biological specimens from forensic casework and assess its potential toxicity.

A Novel Liquid Chromatography Tandem Mass Spectrometry Technique using Multi-Period-Multi-Experiment of MRM-EPI-MRM3 with Library Matching for Simultaneous Determination of Amphetamine Type Stimulants Related Drugs in Whole Blood, Urine and Dried Blood Stain (DBS)-Application to Forensic Toxicology Cases in Malaysia.

A novel mass spectrometry detection technique based on a multi-period and multi- experiment (MRM-EPI-MRM3) with library matching in a single run for fast and rapid screening and identification of amphetamine type stimulants (ATS) related drugs in whole blood, urine and dried blood stain was developed and validated. The ATS-related drugs analyzed in this study include ephedrine, pseudoephedrine, amphetamine, methamphetamine, MDMA (3,4-Methylenedioxymethamphetamine), MDA (3,4-Methylenedioxyamphetamine), MDEA (3,4-Methylenedioxy-N-ethylamphetamine) and phentermine. The relative standard deviation for inter and intraday was less than 15% while recoveries ranged from 80% to 120% for all three matrices, i.e., whole blood, urine and dried blood stain. All compounds gave library matching percentage of more than 85% based on the purity. This method was proven to be simple and robust, and provide high confident results complemented with library matching confirmation.

A Tool for Automatic Correction of Endogenous Concentrations: Application to BHB Analysis by LC-MS-MS and GC-MS.

Several substances relevant for forensic toxicology purposes have an endogenous presence in biological matrices: beta-hydroxybutyric acid (BHB), gamma-hydroxybutyric acid (GHB), steroids and human insulin, to name only a few. The presence of significant amounts of these endogenous substances in the biological matrix used to prepare calibration standards and quality control samples (QCs) can compromise validation steps and quantitative analyses. Several approaches to overcome this problem have been suggested, including using an analog matrix or analyte, relying entirely on standard addition analyses for these analytes, or simply ignoring the endogenous contribution provided that it is small enough. Although these approaches side-step the issue of endogenous analyte presence in spiked matrix-matched samples, they create serious problems with regards to the accuracy of the analyses or production capacity. We present here a solution that addresses head-on the problem of endogenous concentrations in matrices used for calibration standards and quality control purposes. The endogenous analyte concentration is estimated via a standard-addition type process. This estimated concentration, plus the spiked concentration are then used as the de facto analyte concentration present in the sample. These de facto concentrations are then used in data analysis software (MultiQuant, Mass Hunter, etc.) as the sample's concentration. This yields an accurate quantification of the analyte, free from interference of the endogenous contribution. This de facto correction has been applied in a production setting on two BHB quantification methods (GC-MS and LC-MS-MS), allowing the rectification of BHB biases of up to 30 µg/mL. The additional error introduced by this correction procedure is minimal, although the exact amount will be highly method-dependent. The endogenous concentration correction process has been automated with an R script. The final procedure is therefore highly efficient, only adding four mouse clicks to the data analysis operations.

Qualitative identification of imidacloprid in postmortem animal tissue by gas chromatography-tandem mass spectrometry.

During an avian mass mortality event investigation at the National Fish and Wildlife Forensic Laboratory in Ashland, OR, imidacloprid became an insecticide of concern. A qualitative analytical toxicology screen of seeds, plucks (tongue, esophagus, and trachea), and ventricular contents was requested. A method for the extraction and qualitative analysis of the insecticide in animal tissues was therefore developed. The procedure relies on a combined Food Emergency Response Network (FERN) and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach to sample extraction followed by qualitative analysis by gas chromatography-tandem mass spectrometry. Since imidacloprid is not amenable to the conditions of gas chromatography, a trimethylsilyl derivative was created and characterized. Proposed mechanisms for the creation of this derivative and its mass spectrum are described. The imidacloprid-trimethylsilyl (TMS) derivative was detected in all samples submitted.

Validation of the Neogen ELISA Benzodiazepine Kit using Clonazepam as the Target Molecule for Blood and Urine.

This study demonstrates the validation of a semi-quantitative method for the rapid screening of whole blood and urine specimens using clonazepam as the target molecule for the Neogen® Benzodiazepine kit. Decision points were validated at 10.0 ng/mL for whole blood and 25.0 ng/mL for urine. The validation design was based on the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation and included the evaluation of sensitivity, precision, specificity, carryover, hook effect, drift, ruggedness/robustness and a case sample evaluation. The experimental limit of detection for clonazepam was determined to be at least 5.0 ng/mL in whole blood and at least 10.0 ng/mL in urine. Excellent precision was demonstrated when the assay was evaluated using the mean of three replicates from five separate runs (n = 15) at the decision point and at concentration levels ±50% and +100% of the decision point. Although the method was optimized and exceptional precision was demonstrated at each level, the current SWGTOX validation requirements for a valid decision point were not fulfilled. However, both the blood and urine matrix did meet the proposed revision of the SWGTOX requirements for determining a valid decision point promulgated by the American Academy of Forensic Sciences Standards Board and the assay was reliably able to detect benzodiazepines without interference from matrix components or other compounds routinely detected in authentic case samples. Case sample results were comparable with those obtained when the samples were initially screened using oxazepam as the target molecule. The Neogen® Benzodiazepine kit using clonazepam as the target molecule exhibited cross-reactivity for 29 different benzodiazepines and demonstrated excellent precision and sensitivity in both whole blood and urine, making it an efficient and reliable method to screen for benzodiazepines, even though the validation did not fulfill current SWGTOX requirements for a valid decision point.

Validation of a Fully Automated Immunoaffinity Workflow for the Detection and Quantification of Insulin Analogs by LC-MS-MS in Postmortem Vitreous Humor.

The analysis of biological specimens collected at autopsy for the presence of exogenous insulin(s) is of special interest in select death investigations as they may be suspected in the cause of a death. Technical challenges include the limited stability of insulin, and the forensic requirement of differentiating endogenous insulin from pharmaceutical analogs. A novel method was developed for the detection and quantification of human insulin, Glulisine, Lispro, Aspart, Glargine and Detemir in vitreous fluid. An immunoaffinity extraction procedure is performed followed by separation of the insulin α- and ß-chains. Liquid chromatography tandem mass spectrometry analysis of the ß-chain allows for the unequivocal identification of each insulin analog. The analytical measurement range for each insulin was 0.5-25 ng/mL. The method was evaluated for accuracy, precision, carryover, interferences and stability. Eight vitreous fluid samples collected from cases where untoward insulin use was suspected were subjected to analysis. Positive results were obtained from three samples, and a detailed case history is provided for one of these cases. Even though insulin instability in postmortem biological fluid remains a challenge, this method allows for a reliable forensic-level analysis in vitreous fluid.

Analysis of Acetyl Fentanyl in Postmortem Specimens by Gas Chromatography-Mass Spectrometry (GC-MS): Method Validation and Case Report.

In 2013, the Centers for Disease Control and Prevention released a warning regarding a new recreational drug, acetyl fentanyl. Acetyl fentanyl is a µ-opioid receptor agonist, and its pharmacological effects include euphoria, altered mood, miosis and central nervous system depression. The objective of this report was to develop a sensitive and specific method for the quantitation of acetyl fentanyl by gas chromatography-mass spectrometry in postmortem casework. Acetyl fentanyl was isolated from biological matrices using solid-phase extraction and acetyl fentanyl-13C6 was employed as an internal standard. The method was validated utilizing the Scientific Working Group for Forensic Toxicology's published method validation parameters, and the biological matrices used for analysis were postmortem blood and urine. In addition to the quantitation of acetyl fentanyl, a demographic study of cases obtained from the Rhode Island Office of State Medical Examiners and the University of Florida Health Pathology Laboratories-Forensic Toxicology Laboratory was performed to examine potential risk factors for acetyl fentanyl use. The results from this study found that the blood concentrations in these individuals ranged from 17 to 945 ng/mL. This suggests acetyl fentanyl is less potent than its prototype drug, fentanyl and requires an increased dose to achieve its desired effects. The demographic analysis indicated white males aged 21-40 years and individuals with a previous history of drug use have the highest risk for acetyl fentanyl abuse.

Application of High-Resolution UPLC-MSE/TOF Confirmation in Forensic Urine Drug Screening by UPLC-MS/MS.

The transition from presumptive (immunoassay) drug screening to definitive screening has continued in the practice of analytical toxicology. Development of a ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) screening method for over sixty drugs and metabolites (analytes) in urine has been reported by the authors and has been applied in probation, drug court, social services, chemical dependency, pain management and addiction medicine casework. Testing by the definitive screening method has increased both the rate and diversity of initial-positive drug findings, due to the lower positive thresholds and wider panel of analytes. Use of definitive screening in forensic casework, however, requires retesting of initial-positive analytes using a second method based upon a different analytical technique with at least equivalent sensitivity and selectivity. Consequently, a UPLC-MSE/TOF method for confirmation of the initial-positive analytes has been adapted; the method is for targeted confirmation and is based upon an alternate mass spectrometry technology and column separation. Both the initial screen and the confirmatory analysis employ threshold accurate calibration for normalization of matrix effects, without the use of stable isotopes. Validation and application of the complete workflow, in forensic urine drug testing casework, is reported.

Postmortem Brain-Blood Ratios of Amphetamine, Cocaine, Ephedrine, MDMA and Methylphenidate.

Brain tissue may serve as a useful supplement to blood in postmortem investigations. However, reference concentrations for central stimulant drugs are scarce in brain tissue. This study involves some frequently used stimulants: amphetamine, cocaine, ephedrine, MDMA and methylphenidate. We present concentrations from brain and blood and brain-blood ratios of the analytes from autopsies. The cases were grouped according to the cause of death: A: The compound solely caused a fatal intoxication. B: The compound contributed to a fatal outcome in combination with other drugs, alcohol or disease. C: The compound was not related to the cause of death. Analyses were carried out using solid-phase extraction and ultra high-performance liquid chromatography. Paired brain and femoral blood concentrations from 133 cases were analysed. Positive correlations were observed for all analytes with correlation coefficients ranging from 0.58 to 0.95. The following median brain-blood ratios were obtained: cocaine 2.0 (range 0.20-7.0), amphetamine 3.2 (range 1.5-4.5), ephedrine 2.3 (range 1.1-6.2), MDMA 3.9 (range 0.92-5.1) and methylphenidate 2.4 (0.92-4.6). The concentrations in femoral blood generally agreed with the literature for all compounds. The metabolite of cocaine, benzoylecgonine, was also quantified in brain and blood from 60 cases, and the median brain-blood ratio was 0.66 with 10-90 percentiles of 0.39-1.27. The results of this study can aid the toxicological investigation in determining the cause of death.

Application of TDA AAS to Direct Mercury Determination in Postmortem Material in Forensic Toxicology Examinations.

Mercury is a heavy metal with high toxicity, the level of which depends on the form of the metal. One of the newer techniques for determining trace amounts of total mercury in various materials, including biological samples, is thermal decomposition, amalgamation and atomic absorption spectrometry (TDA AAS). The TDA AAS method was optimized and validated using a mercury analyzer (DMA-80). The limits of detection for mercury were 0.10 and 0.20 µg/L (nickel and quartz boats, respectively). The working range of the calibration curve was at least from 0.6 to 200 ng Hg/mL; the intra-day precision in samples (RSD)-in the range of: 1.66-6.86% (blood), 0.82-1.47% (urine) and 2.01-3.44% (hair); the inter-day precision (over 8 days): 2.51%, and 2.5% (blood spiked with 2.5 and 10 ng Hg, respectively), 5.10% and 3.16% (urine spiked with 2.0 and 6.0 ng Hg, respectively). The accuracy (as relative error, mean value) determined on the basis of the study of reference materials of blood (Seronorm Trace Elements Whole Blood L-1, L-2, L-3), urine (Seronorm Trace Elements Urine, Urine L-2), and hair (Human Hair NIES CRM No. 13) was: 2.00% (blood), 0.50% (urine) and 0.86% (hair); recovery of 2.5 ng Hg (blood): 93-97%. The method was used for the determination of mercury in 76 samples of various biological matrices, including samples of whole blood, urine, hair, bile and vitreous humor. Mercury concentrations in postmortem blood (n = 24) were in the range: 0.61-12.4 µg/L (median 3.02 µg/L); urine (n = 12): 0.16-2.19 µg/L (median 0.81 µg/L); hair (n = 14): 0.08-0.53 µg/g (median 0.22 µg/g); bile (n = 12): 1.15-7.11 µg/L (median 2.41 µg/L and vitreous humor (n = 13): 0.22-1.01 µg/L (median 0.47 µg/L). The method is suitable for the purposes of forensic toxicology analysis.

Development and Validation of a Novel All-Inclusive LC-MS-MS Designer Drug Method.

Designer drugs including synthetic cannabinoids and synthetic cathinones are an increasing problem due to the ease of access to these compounds. They present analytical challenges inasmuch as the compound structures are numerous and growing within each class. Typically each class of designer compounds is analyzed separately due to differences in chemistry, desired cut-offs or other reasons. Physicians treating "high-risk" patients typically order tests for all "illicit" substances which can span several test classes. Despite that multiple classes of designer drugs are ordered together, there has not been a comprehensive confirmatory test developed to date. Presented here is a novel comprehensive designer drug LC-MS-MS method that combines synthetic cannabinoids and synthetic cathinones, etizolam, a designer benzodiazepine and mitragynine (kratom), a natural product analgesic. This method improves laboratory throughput with a cycle time of ~4.5 min which affords resolution of crucial isomers, such as ethylone and butylone. Development of this method also provided an opportunity to update the list of compounds within the method. Analytes with fewer than five positive specimens in a year of testing with previous separate methods were removed as old and not current. New analytes were added based on reports from NMS Laboratories and the US Drug Enforcement Administration testing and drug seizures, which included etizolam, its major metabolite α-hydroxyetizolam as well as newer synthetic cannabinoids (5-fluoro ADB metabolite 7, AB-FUBINACA metabolite 3, AB-FUBINACA metabolite 4 and MDMB-FUBINACA metabolite M1) and synthetic cathinones (N-ethyl pentylone). Finally, the impact of the new analytes and cut-off changes are discussed in context with patient results from the first 4 months of testing after implementation of the method in the lab.

A Gas Chromatography-Triple Quadrupole Mass Spectrometry Assay for the Quantification of Opiates in Human Blood Samples.

A simplified protein precipitation method in combination with gas chromatography-triple quadrupole mass spectrometry (GC-MS-MS) analysis was developed and validated for the simultaneous determination of codeine, morphine, 6-acetylmorphine (6-MAM), hydrocodone and hydromorphone in human blood samples. A protein precipitation with 10% trichloroacetic acid followed by solid-phase extraction using a mixed-mode cartridge was used to separate the analyte from the blood samples. A BSTFA + 1% TMCS was used for derivatization of opiates prior to the analysis. Codeine-D3, morphine-D3, 6-acetylmorphine-D3 and hydrocodone-D6 were used as internal standards. The GC-MS-MS was operated under multiple-reaction monitoring mode using electron ionization technique. The transition ions used for quantitation were 371 → 234 for codeine, 429 → 146 for morphine, 399 → 287 for 6-MAM, 299 → 228 for hydrocodone and 357 → 314 for hydromorphone. The method was linear over the concentration range 2.5-1000 ng/mL for all analytes, except hydrocodone which was linear over 5-1000 ng/mL with a correlation coefficient (r2) = 0.99. The limit of detection was 1.0 ng/mL for all compounds except hydrocodone which was 2.5 ng/mL. The limit of quantitation was 2.5 ng/mL for all compounds except hydrocodone which was 5.0 ng/mL. The precision (% RSD) was within 1.26-14.81 and the accuracy (% Bias) was within -6.29-10.93% for all compounds. The method successfully analyzed morphine (305 ng/mL) and 6-acetylmorphine (6-MAM) (2.3 ng/mL) in a human blood sample received from an opiate user.