Novel and Nonroutine Benzodiazepines and Suvorexant by LC-MS-MS.

Benzodiazepines are a commonly prescribed class of drugs that have the potential for abuse. The Palm Beach County Sheriff's Office received drug seizure submissions that included novel and/or nonroutine benzodiazepines of increasing prevalence from 2017 to 2019. This prompted the development of a method of analysis for these compounds in biological specimens. The method tests for 16 novel and nonroutine benzodiazepines and suvorexant in whole blood by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The target analytes included bromazepam, clobazam, clonazolam, clotiazepam, diclazepam, estazolam, etizolam, flualprazolam, flubromazepam, flubromazolam, loprazolam, lormetazepam, phenazepam, prazepam, suvorexant, tetrazepam and triazolam. The method uses 200 µL of sample, protein precipitation and an instrument run-time of 8 min. The limit of detection was either 1 or 5 ng/mL and the limit of quantitation was either 5 or 25 ng/mL depending on the analyte. The method was validated for quantitative analysis for 15 out of the 17 analytes. Flubromazepam and prazepam were validated for qualitative identification only. A quadratic calibration model (r2 > 0.990) with 1/x weighting was used for all analytes for quantitative analysis. The calibration range was either 5-100 or 25-500 ng/mL depending on the analyte. The coefficient of variation of replicate analyses was within 14% and bias was within ±14%. The method provides a sensitive, efficient and robust procedure for the quantitation and/or qualitative identification of select novel and nonroutine benzodiazepines and suvorexant using LC-MS-MS and a sample volume of 200 µL.

1,1-Difluoroethane Forensic Aspects for the Toxicologist and Pathologist.

1,1-Difluoroethane (DFE) is a halogenated hydrocarbon that is commonly used as a propellant in air duster products. Herein, the pharmacology of DFE was reviewed, and questions relevant to medicolegal investigations were addressed. Particular emphasis was given to detection time in biological specimens and the range, onset and duration of effects. DFE may be abused as an inhalant and is rapidly absorbed through the lungs. Onset of central nervous system (CNS) depressant effects is within seconds and the duration may only last minutes. The effects may lead to impairment of human performance, including confusion, lethargy, impaired judgment, loss of motor coordination and loss of consciousness. Death may result even after the first use. With heavy use or in combination with other CNS depressants, extended periods of drowsiness or loss of consciousness may be observed with an increased risk of a fatal event. A majority of impaired driving investigations where DFE was identified included a collision demonstrating the significant impact its use may have on traffic safety. When DFE is identified alone, without other drugs that cause CNS impairment, the effects may not be observable minutes after the crash, making identification of its use difficult. Although concentrations dissipate rapidly, DFE has been detected in blood specimens collected up to 3 hours after the driving incident. Two studies on passive exposure presented herein demonstrated that it is unlikely to detect DFE above concentrations of ∼2.6 µg/mL in blood or urine due to even extreme unintentional exposure. Alternative specimens such as brain, lung and tracheal air should be considered in some postmortem investigations. DFE has been identified in blood specimens from postmortem cases at concentrations from 0.14 to 460 µg/mL and in impaired driving cases from 0.16 to 140 µg/mL.

Inferences and Legal Considerations Following a Blood Collection Tube Recall.

In mid-2019, medical, forensic and legal communities were notified that a certain shipment of evacuated blood sampling tubes were recalled by the manufacturer. This recall order described that the preservative sodium fluoride (100 mg) and anticoagulant potassium oxalate (20 mg) were missing from a small batch of 10-mL evacuated tubes. This gave cause for concern for possible implications in criminal justice (e.g., in drink-driving offenses) when blood-alcohol concentrations are interpreted. In reality, the lack of an anticoagulant would have been immediately obvious during sample preparation, owing to the formation of a large clot in the tube when received. Certain impairing drugs (e.g., cocaine and 6-acetylmorphine) are unstable in blood and tend to degrade without an enzyme inhibitor, such as sodium fluoride, present. In reviewing available literature related to current practices and the stability of ethanol in stored blood samples, there does not appear to be a clear consensus regarding the amount of sodium fluoride preservative necessary, if any at all, when blood is taken from living subjects under sterile conditions for typical forensic ethanol analysis.

Fentanyl and Driving Impairment.

The incidence of fentanyl in forensic toxicology analyses in the USA has dramatically increased over the past several years. The increase in death cases has been well studied; however, little has been reported on the impact to drug impaired driving. Fentanyl driving while under the influence of drugs (DUID) case data from 2014 to 2019 is presented. The data were obtained from three toxicology laboratories in the Northeast, Southeast, and Midwest regions of the USA. Fentanyl whole blood concentrations ranged from 0.1 to 157 ng/mL in living drivers with a 466% to 524% increase in fentanyl-positive DUID cases from 2014 to 2019, depending on the US region. The vast majority of fentanyl cases involved poly-drug use. Twenty case histories are presented where fentanyl was the only drug identified. The mean (standard deviation) fentanyl concentration for these cases was 5.2 ± 3.8 ng/mL with a median of 3.7 ng/mL, and the concentrations ranged from 2.0 to 16 ng/mL. Naloxone administration was documented in exactly half of these cases similar to another study involving carfentanil-impaired driving. The case histories also demonstrate that some recreational opioid users may display limited signs of impairment either due to tolerance or naloxone administration. The top three observations in common among the cases were the driver was found unresponsive behind the wheel, the vehicle left the travel lane or roadway, and the driver was involved in a crash. The increase in fentanyl use not only poses a risk for overdose and death, but is also a significant concern for traffic safety. This study supports the movement of fentanyl from a Tier II drug to Tier I due to its significant potential for impairment and increase in prevalence in impaired driving cases.

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.

Driving Under the Influence of Drugs: When the Law Misses the Mark.

According to Florida law, an individual is not guilty of driving under the influence of drugs unless impairment is observed and is due to one or more controlled drugs listed in the Florida Statutes. Many prescription drugs, over-the-counter drugs and novel psychoactive compounds that can cause significant impairment are not included in this list. Five other states within the USA including Alaska, Hawaii, Massachusetts, New York and Oregon have similar or other restrictive language in their impaired driving statutes. From January of 2007 to February of 2018, 1,344 blood specimens and 1,796 urine specimens were analyzed for drugs in impaired driving cases in Palm Beach County, Florida. Over the past 11 years, 21% (212 out of 1,028) of all drug-positive blood specimens and 47% (711 out of 1,527) of all drug-positive urine specimens contained at least one non-controlled drug, often mixed with controlled drugs. Despite documentation of observed impairment with the concurrent identification of impairing drugs, an impaired driving charge could not be supported due to the phrasing of the law in Florida. If the intent of drug-impaired driving laws is to improve safety by removing impaired drivers from the road, a more all-encompassing "any impairing drug" law would be more appropriate. Linking the charge to a drug possession law framework or using other restrictive language is not the most effective means to improve road safety.

Carfentanil in Impaired Driving Cases and the Importance of Drug Seizure Data.

Drug seizures containing carfentanil continue to increase in Palm Beach County, FL, USA despite international efforts to control the distribution of the drug. The analysis of drug seizures from the county in 2016 and 2017 demonstrated that carfentanil was the most commonly identified fentanyl analog and was most often detected in combination with heroin, fentanyl, furanyl fentanyl and/or other fentanyl analogs. Carfentanil is an ultra-potent opioid requiring a method with adequate sensitivity for detection in blood specimens from impairment cases. Previous research indicated that carfentanil could not be identified in biological specimens by routine drug testing protocols and that detection requires targeted analysis with greater sensitivity. Due to the prevalence of carfentanil in drug seizures, a sensitive targeted qualitative method by liquid chromatography tandem mass spectrometry in antemortem blood samples was evaluated, validated and implemented. The method included identification of carfentanil, acetyl fentanyl, beta-hydroxythiofentanyl, butyryl fentanyl, fentanyl, furanyl fentanyl, kavain, mitragynine, MT-45 and U-47700. In 2017 carfentanil was the second most frequently detected drug, after ethanol, in driving under the influence blood cases. Of all blood cases in which drug testing was performed (n = 145), carfentanil was detected in 38% followed by alprazolam (29%), fentanyl (27%), delta-9-tetrahydrocannabinol (24%) and morphine (23%). In toxicology cases carfentanil was rarely identified alone (only four cases) and was most commonly identified with other opioids (73% of cases), benzodiazepines (43%) and stimulants (29%). The high incidence of carfentanil-positive cases detected by this method underscores the importance of continually monitoring regional drug seizure trends, and evaluating the adequacy of toxicology testing panels based on these trends.

Cost/Benefit Analysis of Case Management Policies in a DUI Lab.

A study was previously conducted and published describing the magnitude of the under-reporting of drugs in driving under the influence (DUI) cases by using a blood drug screen (BDS) case management protocol and to determine whether not reporting those drugs would have a meaningful impact on the DUI cases. A follow-up study presented herein was conducted to generate a larger dataset for evaluation and to compare the results to the original study. For this follow-up study of 576 cases, the laboratory BDS protocol was modified so that a BDS was performed for all felony cases and all misdemeanor cases with a BAC < 0.15 g/dL, regardless of the officer's request. A cost analysis estimate was also conducted using purchasing and statistical data for calendar year 2014. It was estimated that on average a BDS had a materials cost 30 times greater than a BAC and required over six times as much analyst time. To perform a BDS on every case as has been recommended, the estimated analysis materials cost and analyst time were 218 and 193% of the old protocol, respectively. The results of this follow-up study futher support the insufficiency of presenting drug positivity as a justification for completing drug analysis on every DUI case. For the vast majority of cases with a BAC > 0.08 g/dL, the drugs detected are not significant for supporting a DUI and do not warrant the substantial increase in analysis cost and time required.

Validation of the Neogen® Fentanyl ELISA Kit for Blood and Urine.

The Neogen® Fentanyl ready-to-use enzyme-linked immunosorbent assay kit was validated following the Scientific Working Group for Forensic Toxicology Standard Practices for Method Validation in Forensic Toxicology Laboratory Guidelines. Two decision points, 0.5 and 1 ng/mL, were successfully validated for whole blood. For urine, two decision points, 1 and 5 ng/mL, were also successfully validated. The validation included the evaluation of sensitivity, precision, specificity, carryover, plate drift, ruggedness/robustness and a case sample evaluation. The empirically determined limit of detection was 0.25 ng/mL for blood and 0.5 ng/mL for urine. Precision was determined at five different concentrations ranging from 0.25 to 1.5 ng/mL with 15 replicates at each level for whole blood and demonstrated a <2.4% coefficient of variation (CV). In urine, the CV was <5.6% at six different concentrations from 0.5 to 7.5 ng/mL with 15 replicates at each level. Cross-reactivity was evaluated for norfentanyl, acetyl fentanyl, 4-anilino-N-phenethylpiperidine, beta-hydroxythiofentanyl, butyryl fentanyl and furanyl fentanyl.

An Efficient, Robust Method for the Determination of Cannabinoids in Whole Blood by LC-MS-MS.

Due to the high prevalence of cannabinoids in forensic toxicology casework, it is desirable to have an efficient method that uses a small volume of blood and requires a minimal sample preparation. Although many methods have been reported, they are often labor intensive, require special sample preparation materials, use 1 mL or more of specimen or are difficult to replicate. The liquid chromatography with tandem mass spectrometry (LC-MS-MS) method presented herein employs a rapid and simple liquid-liquid extraction, has been successfully applied in two different laboratories, uses 0.5 mL of specimen and was extensively validated. The validated limit of detection and limit of quantitation were 1 ng/mL for delta-9-tetrahydrocannabinol (THC) and 11-hydroxy-delta-9-tetrahydrocannabinol (OH-THC) and 5 ng/mL for 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCA). Each analyte demonstrated a zero-order linear range (r2 > 0.990) with 1/x weighting of 1-40 ng/mL for THC and OH-THC and 5-200 ng/mL for THCA. The coefficient of variation of replicate analyses was within 14%. Bias was within ±13% of the prepared concentration. The validated method provides a sensitive, efficient and robust procedure for the quantitation of cannabinoids in blood using LC-MS-MS and a sample volume of 0.5 mL.

Long-Term Blood Alcohol Stability in Forensic Antemortem Whole Blood Samples.

The effect of long-term room temperature storage on the stability of ethanol in whole blood specimens was investigated. One hundred and seventeen preserved whole blood case samples (110 of 117 with two tubes of blood in each case) were used for this study. One tube from each case was initially tested for blood alcohol concentration (BAC) for criminal driving under the influence proceedings. Cases positive for ethanol ranged in BAC from 0.023 to 0.281 g/dL. The second tube, if present, remained sealed. All blood samples were then stored at room temperature. After 5.4-10.3 years, the opened tubes were reanalyzed for BAC by the same laboratory that performed the initial testing using the same method and same instrumentation. After the same storage period, the unopened tubes were sent to a different laboratory, using a different method and different instrumentation, and reanalyzed for BAC after a total of 5.6-10.5 years of room temperature storage. Seven samples initially negative for alcohol remained negative. All samples initially positive for ethanol demonstrated a decrease in BAC over time with a statistically significant difference in loss observed based on blood sample volume and whether or not the tube had been previously opened. The decrease in BAC ranged from 0.005 to 0.234 g/dL. Tubes that were not previously opened and were more than half full demonstrated better BAC stability with 89% of these tubes demonstrating a loss of BAC between 0.01 and 0.05 g/dL.

Case management in a DUI lab: effect on drugs reported.

An evaluation of an internal laboratory decision to implement a protocol for limiting drug testing based on ethanol concentration in laboratory analysis for driving under the influence (DUI) cases is presented. The described case management strategy is supported by known impairment of ethanol at relatively high concentrations, difficulty assigning a level of contributing impairment from drugs in the presence of high ethanol levels and the likelihood that the drug results may be suppressed at trial. Although the results of this study reinforce the assertion that such protocols lead to the under reporting of drugs in DUI cases, for the majority of cases, 95% in this study, the drug analysis results were not significant and did not warrant the time and resources needed for the additional blood drug testing. Furthermore, the study demonstrated that a high drug positivity rate does not necessarily mean that those drug results are legally or pharmacologically meaningful. Additional research should be conducted with quantitative drug results and casework impact of blood drug screen protocols as previous studies only report drug positivity rates and not whether the drug results would be meaningful to the case.

Identification of volatiles by headspace gas chromatography with simultaneous flame ionization and mass spectrometric detection.

Volatiles are frequently abused as inhalants. The methods used for identification are generally nonspecific if analyzed concurrently with ethanol or require an additional analytical procedure that employs mass spectrometry. A previously published technique utilizing a capillary flow technology splitter to simultaneously quantitate and confirm ethyl alcohol by flame ionization and mass spectrometric detection after headspace sampling and gas chromatographic separation was evaluated for the detection of inhalants. Methanol, isopropanol, acetone, acetaldehyde, toluene, methyl ethyl ketone, isoamyl alcohol, isobutyl alcohol, n-butyl alcohol, 1,1-difluoroethane, 1,1,1-trifluoroethane, 1,1,1,2-tetrafluoroethane (Norflurane, HFC-134a), chloroethane, trichlorofluoromethane (Freon®-11), dichlorodifluoromethane (Freon®-12), dichlorofluoromethane (Freon®-21), chlorodifluoromethane (Freon®-22) and 1,2-dichlorotetrafluoroethane (Freon®-114) were validated for qualitative identification by this method. The validation for qualitative identification included evaluation of matrix effects, sensitivity, carryover, specificity, repeatability and ruggedness/robustness.

Ethanol analysis by headspace gas chromatography with simultaneous flame-ionization and mass spectrometry detection.

Ethanol is the most frequently identified compound in forensic toxicology. Although confirmation involving mass spectrometry is desirable, relatively few methods have been published to date. A novel technique utilizing a Dean's Switch to simultaneously quantitate and confirm ethyl alcohol by flame-ionization (FID) and mass spectrometric (MS) detection after headspace sampling and gas chromatographic separation is presented. Using 100 µL of sample, the limits of detection and quantitation were 0.005 and 0.010 g/dL, respectively. The zero-order linear range (r(2) > 0.990) was determined to span the concentrations of 0.010 to 1.000 g/dL. The coefficient of variation of replicate analyses was less than 3.1%. Quantitative accuracy was within ±8%, ±6%, ±3%, and ±1.5% at concentrations of 0.010, 0.025, 0.080, and 0.300 g/dL, respectively. In addition, 1,1-difluoroethane was validated for qualitative identification by this method. The validated FID-MS method provides a procedure for the quantitation of ethyl alcohol in blood by FID with simultaneous confirmation by MS and can also be utilized as an identification method for inhalants such as 1,1-difluoroethane.

A study of blood alcohol stability in forensic antemortem blood samples.

The effect of long-term storage on alcohol stability in preserved forensic antemortem blood samples was investigated. Thirty-two whole blood case samples (each with two tubes of blood) were used for this study. One tube from each case was analyzed for blood alcohol concentration (BAC) for court proceedings of driving under the influence (DUI), and all blood samples were then stored under refrigeration. After the storage time (ranging from 13 to 39 months) both tubes of blood for each case were reanalyzed for BAC and the results were compared to the original analysis. Seven samples originally negative for alcohol analysis remained negative. The comparative data for 25 samples demonstrated various losses in BAC in both tubes. A significant loss with a mean of 0.015g/dL, was observed in previously opened tubes compared to a mean loss of 0.010g/dL in unopened tubes. In order to determine the effect of other storage conditions, the same blood samples were then stored at room temperature for 6 months followed by 38°C for 7 and 28 days and analyzed for BAC at the end of each storage time period. The seven alcohol negative cases remained negative when stored at room temperature or at 38°C. Six months of storage at room temperature decreased BAC further for both tubes of the alcohol positive cases with a mean loss of 0.014g/dL. Further storage at 38°C for 7 days did not cause any significant change in BAC. Storage at 38°C for 28 days caused some loss in BAC which was determined to be significant by statistical analysis.

Quantitation of opioids in whole blood by electron impact-gas chromatography-mass spectrometry.

Opioids are frequently encountered in Forensic Toxicology casework. A PubMed literature search was conducted to find a method using electron impact-gas chromatography-mass spectrometry to examine whole blood specimens. A previously published method was identified, and an updated version was provided by the State of North Carolina Office of the Chief Medical Examiner. This procedure was used as a starting point for development and validation of a refined procedure to be used in the Palm Beach County Sheriff's Office Forensic Toxicology laboratory for routine analysis of antemortem forensic toxicology case samples. Materials and instrumentation common to most forensic toxicology laboratories were utilized while obtaining detection limits from 1 to 10 ng/mL and quantitation limits of 2.5 to 10 ng/mL using 1 mL of whole blood. Target compounds were chosen based on applicability to the method as well as availability and common use in the United States and include dihydrocodeine, codeine, morphine, hydrocodone, 6-monoacetylmorphine, hydromorphone, oxycodone, and oxymorphone. Each analyte demonstrated two zero-order linear ranges (r(2) > 0.990) over the concentrations evaluated (from 2.5 to 500 ng/mL). The coefficient of variation of replicate analyses was less than 12%. Quantitative accuracy was within ± 27% at 2.5 ng/mL, ± 11% at 10 ng/mL, and ± 8% at 50 ng/mL. The validated method provides a more sensitive procedure for the quantitation of common opioids in blood using standard laboratory equipment and a small amount of sample.

Quantitation of benzodiazepines in whole blood by electron impact-gas chromatography-mass spectrometry.

Benzodiazepines are frequently encountered in forensic toxicology. A literature search was conducted to find a simple method using electron impact-gas chromatography-mass spectrometry (EI-GC-MS) to examine whole blood specimens for the most commonly encountered benzodiazepines in the United States. A recently published method was identified in the literature search and used as a starting point for development of a new procedure to be used for routine analysis of forensic toxicology case samples. The procedure was then developed and validated as a rapid and efficient method for the screening and quantitation of benzodiazepines in blood using liquid-liquid extraction and EI-GC-MS in selective ion monitoring mode. Materials and instrumentation common to most forensic toxicology laboratories were utilized while obtaining LODs from 5 to 50 ng/mL and LOQs of 50 ng/mL or less using 1 mL of sample. Target compounds were chosen based on availability and common use in the United States and include diazepam, desalkylflurazepam, nordiazepam, midazolam, oxazepam, temazepam, lorazepam, clonazepam, and alprazolam (relative elution order). The linear range (r2 > 0.990) was validated from 50 to 1000 ng/mL for all analytes. The CV of replicate analyses at both 50 and 200 ng/mL was less than 4%. Quantitative accuracy was within +/- 16% at 50 ng/mL and within +/- 7% at 200 ng/mL. The validated method provides an efficient procedure for the quantitation of a broad range of the most common benzodiazepines in blood at meaningful limits of detection and quantitation using standard laboratory equipment and a small amount of sample.

Psilocin identified in a DUID investigation.

Psilocin was identified in a urine specimen collected during a routine driving under the influence of drugs investigation, the first for this laboratory. The subject did not exhibit any response to an automobile crash, indicating the he may have been unaware of the severity of the situation or his immediate surroundings. The urine specimen gave a positive result on a fluorescence polarization immunoassay amphetamine/methamphetamine assay, and psilocin was determined to have 1.3% cross-reactivity at 50 mg/L. Psilocin was confirmed by gas chromatography-mass spectrometry following an extraction for acidic, neutral, and basic drugs. Hydrolysis and derivatization techniques were not employed. The urine concentration of psilocin decreased rapidly, although the specimen was maintained at 4 degrees C.