Choosing the right mouse model: comparison of humanized NSG and NBSGW mice for in vivo HSC gene therapy.

ABSTRACT: In vivo hematopoietic stem cell (HSC) gene therapy is an emerging and promising area of focus in the gene therapy field. Humanized mouse models are frequently used to evaluate novel HSC gene therapy approaches. Here, we comprehensively evaluated 2 mouse strains, NSG and NBSGW. We studied human HSC engraftment in the bone marrow (BM), mobilization of BM-engrafted HSCs into circulation, in vivo transduction using vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vectors (VSV-G LVs), and the expression levels of surface receptors needed for transduction of viral vectors. Our findings reveal that the NBSGW strain exhibits superior engraftment of human long-term HSCs compared with the NSG strain. However, neither model resulted in a significant increase in circulating human HSCs after mobilization. We show that time after humanization as well as human chimerism levels and platelet counts in the peripheral blood can be used as surrogates for human HSC engraftment in the BM. Furthermore, we observed low expression of the low-density lipoprotein receptor, a requirement for VSV-G LV transduction, in the human HSCs present in the murine BM. Our comprehensive characterization of humanized mouse models highlights the necessity of proper validation of the model and methods to study in vivo HSC gene therapy strategies.

The preanalytical stability of emerging cannabinoid analogs (delta-8 THC and its metabolites, delta-10 THC and carboxy-HHC) in urine.

There has been a surge in the presence and use of cannabinoids since the federal legalization of hemp (Agricultural Improvement Act of 2018). This increase is attributed not only to the use of ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol, the most abundant phytocannabinoid components of cannabis and hemp, respectively, but also to the use of many other emerging THC analogs. Structurally, these analogs are similar to ∆9-THC. Urine specimens for drug analysis are often collected offsite and transported to a laboratory for analysis. Screening assays are usually the first step in urine drug testing. These assays are usually qualitative and automated, which for negative specimens, reduce cost and reporting time. The stability of ∆9-THC and its metabolites has been known for some time; however, the stability of emerging analogs has not been elucidated, and therefore, assuming equivalent storage stability can be erroneous. Previous work assessed the cross-reactivity of ∆8-THC and its major metabolites, the ∆10-THC chiral analogs and the chiral 11-COOH-hexahydrocannabinol analogs. Stability was assessed for each analyte at a concentration two times greater than the analytes' determined decision point. Samples were prepared in drug-free urine at three different pHs (4.5, 7 and 9) and stored at three different temperatures (4°C, 20°C and 45°C) in triplicate. Samples were analyzed utilizing the Lin-Zhi International Cannabinoids Enzyme Immunoassay cannabinoid screening kit calibrated at the 25 ng/mL cut-off. Overall, the cannabinoid analogs produced diminishing instrument responses depending on pH and temperature. The parent analogs were not detected after a single day at 45°C regardless of pH. In general, carboxylic acid analogs at the acidic pH (4.5) produced diminished instrument responses when compared to their counterparts stored at neutral (7) and basic (9) pH. The time, storage temperature and pH of urine specimens may affect the screening results of specimens collected for cannabinoid drug screening.

The cross-reactivity of cannabinoid analogs (delta-8-THC, delta-10-THC and CBD), their metabolites and chiral carboxy HHC metabolites in urine of six commercially available homogeneous immunoassays.

There has been an exponential surge in the presence and use of cannabinoids since the federal legalization of hemp (Agricultural Improvement Act of 2018). This growth is attributed to delta-9-tetrahydrocannabinol (delta-9-THC) and cannabidiol (CBD), the most abundant phytocannabinoid components of cannabis and hemp, respectively, but with many other emerging THC analogs. Structurally, these analogs are similar to delta-9-THC, yet very little information is available about their potency and even less information is available regarding their detectability using commercially available cannabinoid screening kits. Due to their structural similarity, current cannabinoid homogeneous immunoassay screening methods may be able to detect these emerging cannabinoid analogs and their metabolites. Six urine immunoassay kits (Abbott Cannabinoids-Abbott Diagnostics, LZI Cannabinoids (cTHC) Enzyme Immunoassay-Lin-Zhi International, DRI® Cannabinoid Assay and CEDIA™ THC-Thermo Fisher Scientific, ONLINE DAT Cannabinoid II-Roche Diagnostics and Syva EMIT®II Plus-Siemens Healthineers) were evaluated at two different cutoff concentrations: 50 ng/mL and 20 or 25 ng/mL, assay dependent. The analysis was performed on an Abbott Architect Plus c4000 (Abbott Diagnostics). Delta-8-THC, CBD, olivetol and their major metabolites, and delta-10-THC and HHC carboxylic acid chiral analogs were evaluated. The cross-reactivity was evaluated by preparing each analyte at 20, 50, 100 and 1,000 ng/mL in urine. Analytes that did not cross-react at 1,000 ng/mL for a cutoff were considered not detectable. If detected, the lowest concentration was used as the decision point to determine the precision at the immunoassay's cutoff. The six commercially available urine cannabinoid homogeneous immunoassay screening kits cross reacted with the following analogs: delta-8-THC, 11-OH-delta-8-THC, 11-COOH-delta-8-THC, 6-OH-CBD, 7-OH-CBD, all delta-10-THC and HHC carboxylic acid chiral analogs and olivetol with varying selectivity depending on the screening kit and cutoff concentration. The kits did not cross-react with the following analogs: CBD, 7-COOH-CBD, Abnormal CBD, CBDA-A and olivetolic acid.

Evaluation of Strontium Interference in Calcium Measurement Procedures and Content in Supplements as Measured by ICP-MS.

BACKGROUND: Bone health supplements containing strontium are available without prescription, however, the effects of strontium interference on clinical laboratory calcium measurement procedures are unknown. METHODS: To evaluate strontium interference on total calcium measurements, plasma pools with exogenously added strontium were measured by 3 total calcium measurement procedures. For ionized calcium measurements, whole blood pools prepared with exogenously added strontium were measured by 2 ionized calcium measurement procedures. An inductively coupled plasma mass spectrometry assay (ICP-MS) was validated for research measurements of strontium content in commercially available supplements. RESULTS: Exogenous strontium addition to plasma caused positive bias for total calcium measurements. Strontium concentrations of 1.0 mg/dL (0.114 mmol/L), 2.5 mg/dL (0.284 mmol/L), and 5.0 mg/Dl (0.568 mmol/L) resulted in mean biases of 1.9% to 3.5%, 4.9% to 9.0%, and 10.8% to 19.2%, respectively, for total calcium measurement procedures. Biases for ionized calcium measurements were less than 4.5% for a strontium concentration of 5.0 mg/dL (0.568 mmol/L). An in-house-developed ICP-MS assay for strontium in commercially available supplements exhibited within-laboratory and within-run coefficients of variation of less than 3%, and a linear response was obtained over the assay analytical measurement range of 10 to 100 000 ng/mL (0.0001 to 1.141 mmol/L). Strontium recovery for the ICP-MS assay was 97.1% to 105.3%. The largest amount of strontium measured in dietary supplements was 395 mg in a 1054 mg tablet. CONCLUSIONS: Some dietary supplements contain larger amounts of strontium than indicated on the product label. High concentrations of strontium may cause significant interference for total calcium measurement procedures, but ionized calcium measurement procedures are not significantly affected.

Detection of 58 fentanyl analogs using ARK fentanyl II and Immunalysis fentanyl immunoassays.

OBJECTIVES: The ability to detect fentanyl analogs in urine aids in patient management. Little is published about the new ARK™ Fentanyl II Assay formulation's ability to detect fentanyl analogs. Norfentanyl (fentanyl metabolite) cross-reactivity with the ARK II assays is 7%, while the Immunalysis SEFRIA assay norfentanyl cross-reactivity is approximately 0.005%. The purpose of this study was to determine the new ARK II and SEFRIA fentanyl assays' detection of 58 fentanyl analogs. DESIGN & METHODS: Drug-free urine was fortified with 0-100 ng/mL (0-0.297 µmol/L) of the fentanyl analog and analyzed using the previously evaluated immunoassays. Results were compared to molecular structure. Of the 58 analogs tested at ≤ 100 ng/mL (0-0.297 µmol/L), the ARK II and SEFRIA assays produced 51 and 57 positive results respectively. The cross-reactivity of the assay was predominantly determined by the location of the modification. Most modifications to the aniline ring and/or amide group did not affect the ARK II or SEFRIA assay. Modifications to the piperidine ring decreased detection by ARK II assay. Of the 7 compounds which were undetected by the ARK II assay, all had modifications to the N-alkyl chain. Norsufentanil was not detected by either assay and was the only analog not detected by the SEFRIA assay. CONCLUSIONS: The ARK II and Immunalysis fentanyl immunoassays can detect a range of fentanyl analogs with acryl, butyryl, or furanyl modifications to the amide group or aniline ring of the molecule. N-alkyl chain and piperidine ring modifications significantly affect the ARK II assay's ability to detect the analogs, while the SEFRIA assay appeared less affected and detected all analogs tested except for norsufentanil, which was also not detected by the ARK II assay.

Ivabradine toxicity: a case report.

BACKGROUND: We describe a case of symptomatic bradycardia resulting from ivabradine toxicity by measurement of ivabradine levels, of which there are limited reports in the literature. CASE PRESENTATION: A 43-year-old White female presented with several days of near syncope and dizziness accompanied by a drop in her heart rate to 50 beats per minute. She was taking ivabradine for inappropriate sinus tachycardia. After excluding several other causes of bradycardia, we made the diagnosis of ivabradine toxicity by measurement of serum ivabradine levels, an approach that is currently not clinically available. CONCLUSIONS: Measurement of serum ivabradine levels and knowledge of the pharmacokinetic properties of the drug can be utilized to confirm the diagnosis of ivabradine toxicity.

Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells.

Clinical applications of hematopoietic stem cell (HSC) gene editing are limited due to their complex and expensive logistics. HSC editing is commonly performed ex vivo using electroporation and requires good manufacturing practice (GMP) facilities, similar to bone marrow transplant centers. In vivo gene editing could overcome this limitation; however, electroporation is unsuitable for systemic in vivo applications to HSCs. Here we evaluated polymer-based nanoparticles (NPs), which could also be used for in vivo administration, for the delivery of mRNA and nucleases to human granulocyte colony-stimulating factor (GCSF)-mobilized CD34+ cells. NP-mediated ex vivo delivery showed no toxicity, and the efficiency was directly correlated with the charge of the NPs. In a side-by-side comparison with electroporation, NP-mediated gene editing allowed for a 3-fold reduction in the amount of reagents, with similar efficiency. Furthermore, we observed enhanced engraftment potential of human HSCs in the NSG mouse xenograft model using NPs. Finally, mRNA- and nuclease-loaded NPs were successfully lyophilized for storage, maintaining their transfection potential after rehydration. In conclusion, we show that polymer-based NP delivery of mRNA and nucleases has the potential to overcome current limitations of HSC gene editing. The predictable transfection efficiency, low toxicity, and ability to lyophilize NPs will greatly enhance the portability and provide a highly promising platform for HSC gene therapy.

Pressure Necrosis Requiring Fasciotomy After Kratom Overdose.

INTRODUCTION: Kratom (Mitragyna speciosa) is a popular plant-based extract that has dose-dependent stimulatory and sedative effects. It has been used for self-treatment of opioid withdrawal and can result in seizures, hepatotoxicity, and infectious complications from bacterial contamination. Reports of morbidity and mortality associated with Kratom may be confounded by coingestants. We report a case of severe rhabdomyolysis and pressure necrosis leading to fasciotomy in a patient who was using Kratom. CASE REPORT: A 31-year-old male with substance use presented to the emergency department after loss of consciousness for 6 hours after smoking Kratom. He was found to have rhabdomyolysis, acute renal and hepatic injury, and electrolyte disturbances. No ethanol was detected, and urine drug screen was negative. Over the next 3 hours, the patient developed signs of compartment syndrome and he was transferred to the operating room for fasciotomy. He required continuous renal replacement therapy for 48 hours and his labs and clinical status improved. He was discharged 18 days later. A serum and urine sample from the first day of presentation were analyzed for mitragynine and 7-hydroxymitragynine using an Ultra Performance Liquid Chromatography-Tandem Mass Spectrometer (UPLC-MSMS) method. The serum mitragynine was 5 ng/mL and the urine mitragynine 6 ng/mL. CONCLUSIONS: Although there are numerous reports of opioids resulting in prolonged periods of immobilization and rhabdomyolysis, this is not commonly reported in Kratom overdoses.This case report highlights the profound sedative effect of Kratom and the potential of pressure necrosis injury resulting in rhabdomyolysis and compartment syndrome requiring fasciotomy.

Negligible Nux Vomica: Homeopathic Nux Vomica remedies don't contain strychnine?

INTRODUCTION: The St. Ignatius bean of the Strychnos ignatii tree and Nux Vomica homeopathic products presumably could contain the toxic alkaloids strychnine and brucine. This study aimed to determine the amount of these toxic alkaloids in some commercially available Nux Vomica products and the St. Ignatius bean and to determine if overdose of these products could result in clinically significant toxicity. METHODS: Using ultra-performance liquid chromatography-tandem mass spectrometry, various formulations of Nux Vomica products and St. Ignatius beans were analyzed for strychnine, and brucine with detection limits set at 0.1 ng/g. RESULTS: None of the analyzed Nux Vomica products contained any detectable strychnine or brucine, while the expected strychnine dose from a St. Ignatius bean would be < 0.001 mg. CONCLUSIONS: Overall, our study reveals that the amount of strychnine in homeopathic Nux Vomica products or St. Ignatius beans are not likely to result in clinically significant strychnine toxicity.

Neonatal Exposure to Tramadol through Mother's Breast Milk.

Tramadol is an opioid used in the treatment of moderate to moderately severe pain. Tramadol's use during pregnancy is generally avoided and may cause some reversible withdrawal effects in neonates, and its use during lactation is not licensed by the manufacturer. A small clinical trial reported infants were exposed to <3% of a mother's tramadol dose through breast milk with no evidence of harmful effects. Presented is a case study of breast milk, neonatal urine, and neonatal oral fluid for the analysis of tramadol and its metabolites, along with the validation of a method for the analysis of tramadol, O-desmethyltramadol, and N-desmethyltramadol in breast milk. Tramadol and its metabolites were extracted by solid-phase extraction after saponification of breast milk to remove lipids. Samples were analyzed by ultra-pressure liquid chromatography-tandem mass spectrometry. To the author's knowledge, this is the first report of tramadol and its metabolites in neonatal oral fluid. The breast milk concentrations were 63, 22, and 76 ng/mL for the analysis of tramadol, O-desmethyltramadol, and N-desmethyltramadol, respectively, on day of life 12. On day of life 20, the breast milk concentrations were 1,254, 388, and 937 ng/mL for the analysis of tramadol, O-desmethyltramadol, and N-desmethyltramadol, respectively. Oral fluid concentrations were 1,011, 1,499, and 406 ng/mL for the analysis of tramadol, O-desmethyltramadol, and N-desmethyltramadol, respectively, on day of life 20. Oral fluid concentrations were similar to breast milk for tramadol, almost four times higher for O-desmethyltramadol, and less than half for N-desmethyltramadol. The absolute infant dose was calculated to be 10 µg/kg/day and 294 µg/kg/day for tramadol on day of life 12 and 20, respectively.

Determining the Damage and Failure Behaviour of Textile Reinforced Composites under Combined In-Plane and Out-of-Plane Loading.

The absence of sufficient knowledge of the heterogeneous damage behaviour of textile reinforced composites, especially under combined in-plane and out-of-plane loadings, requires the development of multi-scale experimental and numerical methods. In the scope of this paper, three different types of plain weave fabrics with increasing areal weight were considered to characterise the influence of ondulation and nesting effects on the damage behaviour. Therefore an advanced new biaxial testing method has been elaborated to experimentally determine the fracture resistance at the combined biaxial loads. Methods in image processing of the acquired in-situ CT data and micrographs have been utilised to obtain profound knowledge of the textile geometry and the distribution of the fibre volume content of each type. Combining the derived data of the idealised geometry with a numerical multi-scale approach was sufficient to determine the fracture resistances of predefined uniaxial and biaxial load paths. Thereby, CUNTZEmathsizesmall's three-dimensional failure mode concept was incorporated to predict damage and failure. The embedded element method was used to obtain a structured mesh of the complex textile geometries. The usage of statistical and visualisation methods contributed to a profound comprehension of the ondulation and nesting effects.

A Case Study Evaluating the Efficacy of an Ad Hoc Hospital Collection Device for Fentanyl in Infant Oral Fluid.

Neonatal drug exposure is currently assessed using meconium, urine, blood, hair, or umbilical cord tissue/blood. Due to the invasiveness, challenges, and limitations of collection, and/or analytical difficulties of these matrices, oral fluid may be a more desirable matrix in diagnosing opioid exposure and risk for opioid withdrawal in neonatal abstinence syndrome. Traditional oral fluid collection devices are not viable options as they are too large for neonates' mouths and may contain chemicals on the collection pad. Unstimulated and stimulated infant oral fluid samples have been used for therapeutic drug monitoring as an alternative matrix to blood. The objective of this study was to assess the viability of a simple oral fluid collection system using a sterile foam-tipped swab rinsed in phosphate-buffered saline. Two infants were administered fentanyl for post-operative pain relief while hospitalized in the Neonatal Intensive Care Units at the Children's Hospital of Richmond of Virginia Commonwealth University. Oral fluid samples were collected at 16 h, 2 days, and/or 7 days following the start of intravenous infusion of fentanyl. Samples were analyzed by ultra-high-pressure liquid chromatography-tandem mass spectrometry for fentanyl and norfentanyl after solid-phase extraction. In one of the three samples tested, fentanyl and norfentanyl were detected at concentrations of 28 and 78 ng/mL, respectively. Based on the infusion rate, the theoretical oral fluid fentanyl concentration at steady state was calculated to be 33 ng/mL.

Are Urine Propylene Glycol or Vegetable Glycerin Markers of E-cigarette Use or Abstinence?

Objectives: Urine propylene glycol (PG) and vegetable glycerin (VG) were evaluated as potential markers for discriminating ECIG users from non-users and verifying ECIG abstinence. Methods: Urine samples from 51 ECIG users (collected pre/post 12-hours ECIG abstinence), and 50 controls (who do not use nicotine/tobacco) were analyzed for urine cotinine, PG, and VG concentration. Results: Of 42 ECIG users with pre-abstinence urine cotinine indicating nicotine use, mean (SD) urine cotinine concentration was 1053.7 ng/ml (874.5) and for controls was 1.93 ng/ml (0.4); after abstinence, ECIG users' mean cotinine decreased to 615.4 ng/ml (753.0). For ECIG users, mean urine PG pre-abstinence was 25.6 mcg/ml (20.0) and was 9.8 mcg/ml (13.5) for controls; after abstinence, ECIG users' mean urine PG decreased to 9.7 mcg/ml (15.0; ps < .05). For ECIG users, mean urine VG pre-abstinence was 7.5 mcg/ml (7.1) and was 13.2 mcg/ml (25.0) for controls; after abstinence, ECIG users' mean VG decreased to 5.0 mcg/ml (4.4; ps < .05). Conclusions: ECIG users' mean urine PG was greater than controls and decreased after 12-hours ECIG abstinence suggesting urine PG may be useful for discriminating ECIG users from non-users and verifying short-term abstinence.

Determination of Cannabinoids in Breast Milk Using QuEChERS and Ultra-Performance Liquid Chromatography and Tandem Mass Spectrometry.

Use of marijuana and cannabinoids has been on the rise in recent years, including in childbearing women. This has resulted in cannabinoids being more frequently identified in breast milk as a result of its high lipid content and cannabinoids having a high lipophilicity, thereby exposing the breastfeeding infant to cannabinoids and other marijuana constituents. Presented is a method for the analysis of Δ9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD) in breast milk. THC, CBN, CBD and their isotopically labeled standards were extracted from breast milk using a modified QuEChERS method and analyzed using ultra-performance liquid chromatography and tandem mass spectrometry. As a result of the high lipid content of breast milk, saponification of the lipids was necessary to improve overall extraction efficiency. The process efficiency percentage for THC, CBD and CBN are 55%, 80% and 25%, respectively. The recovery percentage for THC, CBD and CBN are 95%, 118% and 85%, respectively. The matrix effect percentage for THC, CBD and CBN are 53%, 66% and 26%, respectively. Linearity was assessed from 1 to 100 ng/mL for THC, CBN and CBD and had r2 > 0.996. Validation controls were prepared at 1, 3, 20, 80 and 300 ng/mL (dilution control), and the bias was determined to be less than ±20% with %CVs <15% for all controls. Due to the limited access of genuine breast milk for routinely preparing matrix matched calibration and control materials, Enfamil® Premium™ Newborn Infant Formula (0-3 months) was evaluated as a breast milk substitute. No significant differences were observed for THC, CBN and CBD using either breast milk or formula as the matrix; thus, it was determined to be an acceptable breast milk matrix substitute. The modified QuEChERS method was determined to be a robust, reliable method for the determination of THC, CBN and CBD in breast milk.

Using Papaverine and Its Metabolites, 6-Desmethyl Papaverine and 4',6-Didesmethyl Papaverine as Biomarkers to Improve the Detection Time of Heroin Use.

Opioid usage in the USA has increased over the past decade, with prescriptions increasing from 76 million in 1991 to 207 million in 2013. New regulations have curbed the number of prescriptions, leading to an increase in heroin use. Heroin-related overdoses have quadrupled between 2000 and 2015. The traditional urinary biomarkers for indicating heroin use are a combination of morphine and 6-acetyl morphine (6-AM). Morphine is detectable in urine for several days. 6-AM is detected in urine for 2-8 hours. Papaverine has been proposed as an alternative heroin biomarker. It has been reported to have a 1-2 day detection window. Papaverine metabolites have been reported to have up to a 3-day detection window. Presented is a method for the detection of papaverine and its metabolites, 6-desmethyl papaverine (6-DMP) and 4', 6-didesmethyl papaverine (4,6-DDMP), in urine using a modified Waters® MCX™ microelution method. An ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS-MS), with a Waters' BEH C18 column, and 20 mM ammonium formate water: 20 mM ammonium formate methanol mobile phase was employed. Calibration curves were linear from 0.1 to 50 ng/mL. No interferences were observed from the analysis of multicomponent therapeutic drug or drugs of abuse control materials; intra- and inter-run precision tests were acceptable. A total of 428 genuine urine specimens where heroin use was suspected were analyzed. These included 101 6-AM and 179 morphine only positive samples as well as 6 morphine-negative samples where papaverine and/or metabolites were detected. The determined concentrations in these samples for papaverine, 6-DMP and 4,6-DDMP ranged from 0.10 to 994, 0.10 to 462 and 0.12 to 218 ng/mL, respectively. The method was rugged and robust for the analysis of papaverine and metabolites, 6-DMP and 4,6-DDMP. The use papaverine and metabolites, 6-DMP and 4,6-DDMP has the potential to increase the detection window of heroin use.

An Ultra-High-Pressure Liquid Chromatographic Tandem Mass Spectrometry Method for the Analysis of Benzoyl Ester Derivatized Glycols and Glycerol.

Presented is an ultra-high-pressure liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method developed for the detection of propylene glycol, glycerol, ethylene glycol and diethylene glycol using isotopically labeled standards in urine as part of ongoing studies to evaluate whether urinary propylene glycol and/or vegetable glycerin concentration are indicators of recent use. Propylene glycol and vegetable glycerol are found in many products that are consumed and used including electronic cigarettes (e-cigarettes). E-cigarettes are battery-powered devices used as an alternative to traditional cigarettes. The liquid formulations aerosolized in these devices largely consist of propylene glycol and/or vegetable glycerol. Published reports regarding the ratio of propylene glycol to glycerol content in these formulations ranged from 50:50 to 100 percent of either. For the analysis of urine specimens from both users and non-users of e-cigarettes, calibrators, controls and specimens were derivatized using benzoyl chloride prior to analysis. They were analyzed using a Waters AcQuity Xevo TQ-S Micro UPLC-MS/MS. Chromatographic separation was performed on an AcQuity UPLC BEH C18 1.7 um, 2.1 × 50 mm, column using a 20 mM ammonium formate in water and 20 mM ammonium formate in methanol as the mobile phase. The method was validated using SWGTOX guidelines for linearity, precision and accuracy, stability, carryover and limit of detection. The linear range was determined using a seven-point calibration curve ranging between 0.5 and 100 mcg/mL. The bias for all validation controls was determined to be ±20% of the expected concentrations with CVs of <15%. A total of 124 urine specimens analyzed collected with 50 specimens collected from self-reported non-smokers (cigarettes/e-cigarettes) confirmed cotinine free using the DRI® Cotinine Assay (Thermo Scientific, Waltham, MA) and 74 specimens collected before and after 12 hours self-reported e-cigarettes abstinence e-cigarette users. Propylene glycol and glycerol were determined to have concentration ranges of "none detected" to 1470 and "none detected" to 2950 mcg/mL, respectively.

Analysis of carbenoxolone by ultra-high-performance liquid chromatography tandem mass spectrometry in mouse brain and blood after systemic administration.

Carbenoxolone is a derivative of glycyrrhetinic acid found in the root of Glycyrrhiza glabra, colloquially known as licorice. It has been used as a treatment for peptic and oral ulcers. In recent years, carbenoxolone has been utilized in basic research for its ability to block gap junctional communication. Better understanding the distribution of carbenoxolone after systemic administration can lead to a better understanding of its potential sites of action. Presented is an ultra high-performance liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) method for the identification and quantification of carbenoxolone in mouse blood and brain tissue. Twenty mice were injected intraperitoneally with 25 mg/kg carbenoxolone and brain tissue and blood were collected for analysis. Blood concentrations (mean ± SD) at 15, 30, 60 and 120 min were determined to be (n = 5) 5394 ± 778, 2636 ± 836, 1564 ± 541 and 846 ± 252 ng/mL, respectively. Brain concentrations (mean ± SD) at 15, 30, 60 and 120 mins were determined to be (n = 5) 171 ± 62, 102 ± 35, 55 ± 10 and 27 ± 9 ng/g, respectively. The analysis of these specimens at the four different time points resulted in blood and brain half-lives in mice of ~43 and 41 min, respectively. The UHPLC-MS/MS method was determined to be sensitive and robust for quantification of carbenoxolone.

Acute Toxicity From Intravenous Use of the Tricyclic Antidepressant Tianeptine.

Tianeptine (7-[([3-chloro-6,11]-dihydro-6-methyldibenzo[c,f][1,2]thiazepin-11-yl) amino] heptanoic acid S, S dioxide) is a tricyclic compound prescribed as an antidepressant in European countries, but is not currently approved for use in the United States. There are few published case reports of tianeptine intoxication. Presented is the first case of acute toxicity associated with the intravenous use of tianeptine. A 36-year-old male intentionally injected tianeptine powder intravenously to "help him see into the future". He became unresponsive and a bystander called emergency medical services. Upon arrival to the Emergency Department, excessive constriction of the pupils, sedation, and a respiratory rate of 6 respirations per minute (rpm) were noted. Blood and urine were collected ~2 h post admission. The patient's serum ethanol concentration was 133 mg/dL. His toxicity was successfully reversed with two doses of naloxone 0.4 mg IV. He was started on a naloxone infusion at 0.2 mg/h and discharged 13 h after admittance awake, alert and oriented. The patient's urine sample screened negative for common drugs of abuse and total tricyclic antidepressants. A high performance liquid chromatography tandem mass spectrometry method was developed and validated to quantify tianeptine in urine. The calibration range was 1-100 ng/mL with linear regression correlation (r2) of 0.9996 or greater. The limit of quantitation was administratively set at 1 ng/mL. The bias of the assay was determined to be within ±20% of the target value for each quality control specimen. The intra-day and inter-day precision did not exceed 15% coefficient of variation for each quality control specimen. Matrix effects, absolute recovery, carryover and specificity were also evaluated. The patient's tianeptine urine concentration was determined to be 2 ng/mL.