Measurement of Buprenorphine and Norbuprenorphine in Urine.

Buprenorphine, a synthetic opioid possessing both analgesic and opioid receptor antagonist properties, has proven to be an effective therapeutic aid for opioid dependency and chronic pain management. The downside, as with all opioids, natural or synthetic, is its potential for misuse and abuse. The euphoria induced by buprenorphine leads to abuse. Additionally, individuals with an active addiction to short-acting opioids such as heroin may use buprenorphine between doses of their drug of choice to stave off withdrawal symptoms. As such, buprenorphine monitoring is utilized in medication-assisted therapy programs for opioid dependency, as well as chronic pain management settings. Buprenorphine may also be included in drug testing programs for law enforcement purposes. The assay described here was designed to detect and quantify both buprenorphine and its metabolite norbuprenorphine.

Identification of novel third-generation synthetic cannabinoids in products by ultra-performance liquid chromatography and time-of-flight mass spectrometry.

Synthetic cannabinoids are a group of compounds that are structurally diverse and are commonly found in various herbal incense and potpourri blends, which are sold in convenience stores, smoke shops and over the Internet. During the past few years, multiple state and federal legislations have been enacted controlling various subsets of these compounds that have been detected in compound categories generally considered the first and second product generations. As shown in previous studies, as compounds become controlled, new compounds emerge and become prevalent. We report on the emergence and prevalence of five different compounds (A796,260, MAM-2201, UR-144, URB597 and XLR-11) in the state of Indiana through their qualitative detection in solid-dosage herbal products via rapid solvent extraction and ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/ToF). We demonstrate the use of UPLC/ToF to be a suitable tool in the identification of these substances in a crime laboratory or forensic laboratory setting, which ultimately enables a laboratory to design assays for the detection of specific analytes in biological specimens in regard to regional trends and prevalence.

Analysis of first and second generation legal highs for synthetic cannabinoids and synthetic stimulants by ultra-performance liquid chromatography and time of flight mass spectrometry.

Various "legal high" products were tested for synthetic cannabinoids and synthetic stimulants to qualitatively determine the active ingredient(s). Ultra-performance liquid chromatography with accurate mass time-of-flight mass spectrometry (UPLC-TOF) was used to monitor the non-biological specimens utilizing a customized panel of 65+ compounds comprised of synthetic cannabinoids, synthetic stimulants and other related drugs. Over the past year, the United States Drug Enforcement Agency has controlled five synthetic cannabinoid compounds (JWH-018, JWH-073, JWH-200, CP-47,497 and CP-47,497-C8) and three synthetic stimulant compounds (3,4-methylenedioxypyrovalerone, mephedrone and methylone) that were previously reported to be detected in these legal high products. Through our analyses of first and second generation products, it was shown that many of these banned substances are no longer used and have been replaced by other derivatives that are federally legal. Since enactment of the federal bans on synthetic cannabinoids and synthetic stimulants, 4.9% of the products analyzed at our facility contained at least one controlled substance. The remaining 95.1% of products contained only uncontrolled drugs. We demonstrate the UPLC-TOF methodology to be a powerful tool in the qualitative identification of these designer drugs, thus enabling a laboratory to keep current with the drugs that are being sold as these designer products.

Detection of JWH-018 and JWH-073 by UPLC-MS-MS in postmortem whole blood casework.

Synthetic cannabinoids have been detected in various herbal blends sold legally in convenience stores, smoke shops, and on the Internet. Many of these compounds have extreme forensic significance. We developed and validated a rapid ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of trace concentrations of two of these compounds, JWH-018 and JWH-073, in human blood. Samples underwent liquid-liquid extraction at pH 10.2 into ethyl ether. Tandem mass spectrometry was performed in positive electrospray ionization mode with multiple reaction monitoring using two transitions and one calculated ion transition ratio for each analyte. Deuterated analogs were used as internal standards. Total run time was 2.6 min. The linear dynamic range was 0.05-50 ng/mL with a limit of detection of 0.01 ng/mL for each analyte. Intra-run imprecision (at two different concentration levels, 2 and 8 ng/mL) was 3.9-10.3% for JWH-018 and 3.5-6.2% for JWH-073. Inter-run imprecision was 6.5-7.2% for JWH-018 and 4.8-5.5% for JWH-073. Intra-run accuracy was 95.9-112.7% for JWH-018 and 92.6-104.7% for JWH-073. Inter-run accuracy was 99.1-107.0% for JWH-018 and 97.7-102.0% for JWH-073. Carryover, exogenous drug interferences, ion suppression and matrix selectivity were also assessed. The method has been applied to postmortem forensic casework received by the laboratory and has proven to be robust and reliable. Concentrations of authentic samples have ranged from 0.1-199 ng/mL for JWH-018 and 0.1-68.3 ng/mL for JWH-073.

Identification and quantitation of amphetamine, methamphetamine, MDMA, pseudoephedrine, and ephedrine in blood, plasma, and serum using gas chromatography-mass spectrometry (GC/MS).

Amphetamine, methamphetamine, MDMA, pseudoephedrine, and ephedrine are measured in blood, serum, and plasma using gas chromatography coupled to mass spectrometry (GC/MS). Following a simple liquid-liquid extraction, analytes are derivatized with heptafluorobutyric anhydride (HFBA) and 1 microL injected onto a HP-5MS 15-meter capillary column. Quantitation of each analyte is accomplished using a multi-point calibration curve and deuterated internal standards. The method provides a simple, robust, and reliable means to identify and measure these analytes.

Simultaneous determination and quantification of 12 benzodiazepines in serum or whole blood using UPLC/MS/MS.

The benzodiazepines are a large, commonly prescribed family of psychoactive drugs. We describe a method permitting the simultaneous detection and quantification of 12 benzodiazepines in serum using ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS). Analytes included alprazolam, temazepam, oxazepam, nordiazepam, clonazepam, lorazepam, diazepam, chlordiazepoxide, midazolam, flunitrazepam, 7-aminoclonazepam, and 7-aminoflunitrazepam. Sample pretreatment is simple consisting of protein precipitation using cold acetonitrile (ACN) mixed with the deuterated internal standards. Samples were capped and vortexed for 5 min to ensure maximum precipitation. Following a 5-min centrifugation period, 400 microL of the supernatant was transferred to a clean tube and evaporated down under nitrogen. Samples were reconstituted in 200 microL of a deionized water:ACN (80:20) mixture and transferred to appropriate vials for analysis. Chromatographic run time was 7.5 min, and the 12 analytes were quantified using multiple reaction monitoring (MRM) and 6-point calibration curves constructed for each analyte at concentrations covering a clinically significant range.

Identification and quantitation of cocaine, benzoylecgonine, and cocaethylene in blood, serum, and plasma using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS).

Cocaine is a widely abused stimulant. Numerous methods exist for the identification of the drug, or more commonly, one of its metabolites in urine. Urine testing is useful for most cases, but it is necessary to use other matrices in forensic situations and when subjects are anuric. We describe a novel method for the analysis of cocaine, benzoylecgonine, and cocaethylene in blood, serum, and plasma utilizing ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Sample preparation has been minimized to a simple deproteinization step in which each specimen is mixed with an acetonitrile-internal standard mixture. The method has excellent precision across the linear range of 25-2,000 ng/mL for each analyte. With a run-time of 4 min, this method provides a significant improvement over traditional GC/MS methods.

Detection and quantification of cocaine and benzoylecgonine in meconium using solid phase extraction and UPLC/MS/MS.

The simultaneous determination and quantification of cocaine and its major metabolite, benzoylecgonine, in meconium using UPLC-MS/MS is described. Ultra-performance liquid chromatography (UPLC) is an emerging analytical technique which draws upon the principles of chromatography to run separations at higher flow rates for increased speed, while simultaneously achieving superior resolution and sensitivity. Extraction of cocaine and benzoylecgonine from the homogenized meconium matrix was achieved with a preliminary protein precipitation or protein 'crash' employing cold acetonitrile, followed by a mixed mode solid phase extraction (SPE). Following elution from the SPE cartridge, eluents were dried down under nitrogen, reconstituted in 200 microL of DI water:acetonitrile (ACN) (75:25), and injected onto the UPLC/MS/MS for analysis. The increased speed and separation efficiency afforded by UPLC, allowed for the separation and subsequent quantification of both analytes in less than 2 min. Analytes were quantified using multiple reaction monitoring (MRM) and six-point calibration curves constructed in negative blood. Limits of detection for both analytes were 3 ng/g and the lower limit of quantitation (LLOQ) was 30 ng/g.

Quantitation of morphine, codeine, hydrocodone, hydromorphone, oxycodone, oxymorphone, and 6-monoacetylmorphine (6-MAM) in urine, blood, serum, or plasma using liquid chromatography with tandem mass spectrometry detection.

Opiates and opioids currently rank among the most commonly prescribed pain medications. We describe two liquid chromatography tandem mass spectrometry (LC-MS-MS) methods for the quantification of morphine, codeine, hydrocodone, hydromorphone, oxycodone, oxymorphone, and 6-monoacetylmorphine (6-MAM). In the first, urine samples are pretreated by acidifying with sodium acetate containing appropriate deuterated internal standards and hydrolyzed with beta-glucuronidase. Samples are cooled, diluted with water, vortexed, centrifuged, and a portion is transferred to an autosampler vial for analysis. The second method allows for the measurement of the compounds in blood, serum, or plasma specimens. Analysis of these samples involves pretreatment with acetonitrile containing deuterated internal standards to deproteinize the sample, which is subsequently vortexed and centrifuged. A portion of the organic layer is transferred to a clean test tube, dried under nitrogen, and reconstituted with water for analysis. Quantitation of analytes is accomplished using a commercially available single-point calibrator (urine samples) or an in-house prepared six-point standard curve (blood samples).

Simultaneous quantification of amphetamine and methamphetamine in meconium using ISOLUTE HM-N-supported liquid extraction columns and GC-MS.

A procedure is described for the rapid extraction and quantification of amphetamine and methamphetamine from meconium using ISOLUTE HM-N-supported liquid extraction columns and gas chromatography-mass spectrometry (GC-MS). Because of the matrix complexity of meconium samples, extraction and sample preparation prior to instrumental analysis can prove difficult and time-consuming. The present study introduces a novel sample preparation technique for the simultaneous quantification of amphetamine and methamphetamine in meconium using GC-MS. Extraction of both analytes was achieved using ISOLUTE HM-N-supported liquid extraction columns containing a modified form of diatomaceous earth. Limits of detection for both analytes were 30 ng/g and the lower limit of quantitation was 75 ng/g. Linearity was achieved over the range 75-3000 ng/g. The methodology showed excellent intrarun precision with %CV values ranging from 2 to 8% for both analytes. Interrun precision experiments produced %CV values between 7 and 10% for both analytes. The reported methodology proved suitable for the accurate quantification of amphetamine and methamphetamine in meconium samples and greatly reduced the sample preparation time normally required for traditional solid-phase extraction. The development and validation of such analytical methodologies will prove beneficial for the identification of prenatal substance abuse, which is an ongoing concern across socioeconomic lines.

Diesel fumes do kill: a case of fatal carbon monoxide poisoning directly attributed to diesel fuel exhaust with a 10-year retrospective case and literature review*.

While it is known that diesel fuel combustion engines produce much lower concentrations of carbon monoxide (CO) than gasoline engines, these emissions could certainly generate lethal ambient concentrations given a sufficient amount of time in an enclosed space and under suitable environmental conditions. The authors report a case of CO poisoning which was initially referred for autopsy as a presumed natural death of a truck driver found in the secure cab of a running diesel tractor trailer truck. Completion of the preliminary investigation ascribed death to complications of ischemic heart disease (IHD), pending toxicological analysis that included quantification of CO. When the toxicology results showed lethal blood COHbg, the cause of death was re-certified as CO intoxication secondary to inhalation of (diesel) vehicular exhaust fumes. Because of the unique source of fatal CO intoxication in this case, the contributory IHD and the possible contaminants in the putrefied blood, a 10-year retrospective review was conducted on all nonfire related CO deaths autopsied (n = 94) at the Office of the Chief Medical Examiner in Louisville, KY from 1994 to 2003. For validation of the COHbg detection method used by the Kentucky Office of Forensic Toxicology (KYOFT), blood samples from these cases along with controls were submitted to three laboratories using various analytical methods yielding no statistically significant differences. Lastly, an extensive literature review produced no scientifically reported cases of fatal CO poisoning attributed to diesel fuel exhaust.

Osmol gap as a surrogate marker for serum propylene glycol concentrations in patients receiving lorazepam for sedation.

STUDY OBJECTIVES: To correlate serum propylene glycol concentration with osmol gap, serum lactate concentration, and amount of propylene glycol administered to mechanically ventilated patients receiving continuous infusions of lorazepam (80% propylene glycol by weight), and to characterize the prevalence of hyperosmolality and range of serum propylene glycol concentrations in this patient population. DESIGN: Prospective, controlled, observational study. SETTING: Adult surgical and cardiothoracic intensive care units (ICUs) of a 1200-bed, urban, tertiary care, teaching hospital. PATIENTS: Sixty-four consecutively enrolled intensive care patients requiring mechanical ventilation and pharmacologic sedation. INTERVENTION: Thirteen patients received continuous infusions of high-dose lorazepam (> or = 6 mg/hr) for a minimum of 36 hours, and 26 received continuous infusions of low-dose lorazepam (2-5.99 mg/hr) for 36 hours. Twenty-five control patients received sedatives that did not contain propylene glycol. MEASUREMENTS AND MAIN RESULTS: Serum propylene glycol and lactate concentrations, osmolality, and basic metabolic profiles were obtained 72-108 hours after ICU admission. Clinical data, drug administration, and severity of illness scores were recorded. Osmol gap and the amount of propylene glycol administered before serum sampling predicted propylene glycol concentrations (r(2)=0.692, p<0.05). Osmol gap alone also predicted serum propylene glycol concentrations (r(2)=0.532, p<0.05). Serum lactate concentrations did not correlate with serum propylene glycol concentrations. Unlike the low-dose and control patients, eight (62%) of 13 high-dose patients had osmol gaps above 10. All 13 high-dose patients had serum propylene glycol concentrations previously associated with toxicity. CONCLUSION: Osmol gap can be used as a surrogate marker for serum propylene glycol concentration. In critically ill patients receiving lorazepam for sedation, an osmol gap above 10 was associated with concentrations previously reported to cause toxicity.

Reconstitution of nucleosome positioning, remodeling, histone acetylation, and transcriptional activation on the PHO5 promoter.

The PHO5 gene promoter is an important model for the study of gene regulation in the context of chromatin. Upon PHO5 activation the chromatin structure is reconfigured, but the mechanism of this transition remains unclear. Using templates reconstituted into chromatin with purified recombinant yeast core histones, we have investigated the mechanism of chromatin structure reconfiguration on the PHO5 promoter, a prerequisite for transcriptional activation. Footprinting analyses show that intrinsic properties of the promoter DNA are sufficient for translational nucleosome positioning, which approximates that seen in vivo. We have found that both Pho4p and Pho2p can bind their cognate sites on chromatin-assembled templates without the aid of histone-modifying or nucleosome-remodeling factors. However, nucleosome remodeling by these transcriptional activators requires an ATP-dependent activity in a yeast nuclear extract fraction. Finally, transcriptional activation on chromatin templates requires acetyl-CoA in addition to these other activities and cofactors. The addition of acetyl-CoA results in significant core histone acetylation. These findings indicate that transcriptional activation requires Pho4p, Pho2p, nucleosome remodeling, and nucleosome acetylation. Furthermore, we find that DNA binding, nucleosome remodeling, and transcriptional activation are separable steps, facilitating biochemical analysis of the PHO5 regulatory mechanism.

Evaluation of a no-pretreatment cyclosporin A assay on the Dade Behring dimension RxL clinical chemistry analyzer.

BACKGROUND: Monitoring whole-blood concentrations of cyclosporin A (CsA) is common practice in the management of solid organ and bone marrow transplant recipients. In a multicenter study we evaluated a new, direct (no pretreatment) CsA assay on the Dade Behring Dimension RxL system and compared results with those from the Abbott TDx CsA immunoassay and a HPLC method. METHODS: Whole-blood samples from heart (n = 111; 35 patients), liver (n = 201; 44 patients), kidney (n = 279; 65 patients), and miscellaneous organ (n = 77; 12 lung, 12 bone marrow, 5 kidney/pancreas, and 1 pancreas patient) recipients were obtained from patient populations of the participating institutions. Routine clinical monitoring of CsA was performed using either the TDx method or HPLC. RESULTS: The minimum detectable concentration of CsA averaged 9.4 microg/L, and the lower limit of quantification was 30 microg/L. The method was linear from 30 to 500 microg/L. Cross-reactivity with seven different CsA metabolites ranged from 0.0% to 5.7% for the Dimension RxL assay compared with 0.4-15.9% for the TDx assay. Total imprecision (CV) averaged 6.2%, and within-run imprecision averaged 4.9%. Passing-Bablok linear regression analyses of all samples from two sites yielded the following: RxL = 0.81 x TDx - 16.8; and RxL = 1.12 x HPLC - 1.7. CONCLUSIONS: The Dade Behring CsA assay for the random-access Dimension platform offers adequate performance characteristics for routine clinical use, does not require a manual pretreatment step, and demonstrates less cross-reactivity with CsA metabolites than another commonly used immunoassay.