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Interest has increased in the role of N-acyl amino acids in a variety of disease states and as potential pharmacotherapies. Recently, N-oleoyl glycine and N-oleoyl alanine have shown promise in reducing the rewarding effects of drugs of abuse and alleviating withdrawal signs in rodent models. Previously published methods for the quantitation of these analytes by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in tissue were part of extensive lipidomic panels which may result in limited sensitivity and selectivity and also reported low recovery. Presented is a method for the extraction and HPLC-MS/MS analysis of N-oleoyl glycine and N-oleoyl alanine. The bias and precision of the assay were determined to be within ± 20%. The method was shown to be reliable and robust, with over 90% recovery for the low-level analytes. Increasing concentrations of N-oleoyl glycine and N-oleoyl alanine were quantitated in mouse brain and plasma following exogenous administration. This method was developed to serve to support studies investigating the pharmacokinetics and involvement of N-oleoyl glycine and N-oleoyl alanine in drug dependence and other diseases., (© 2023 The Authors. Journal of Separation Science published by Wiley-VCH GmbH.)

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., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The kidneys play an important role in the long-term regulation of blood pressure by control of salt and water balance in the body through various systems including the endocannabinoid system. The endocannabinoid system consists of the two major cannabinoid receptor agonists, anandamide (AEA) and 2-arachidonylglycerol (2-AG), their hydrolyzing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and the cannabinoid receptors, CB 1 and CB 2 . AEA can be converted into 12- and 15(S)-hydroperoxyeicosatetraenoic acid ethanolamides by 12-LOX and 15-LOX, respectively and can form epoxyeicosatrienoic acid- (EET-EAs) (5,6-, 8,9-, 11,12-, 14,15-) and hydroxyeicosatetraenoic acid- (HETE) ethanolamides. Furthermore, the EET-EAs produce a secondary metabolism by microsomal epoxide hydrolase to form the corresponding dihydroxyeicosatetraenoic acid-EAs (DiHETE-EA). Reference material was not available for DiHETE-EA. These metabolites were synthesized by incubation of the corresponding EET-EAs with mouse liver cytosol containing epoxide hydrolases. Presented is a solid phase extraction and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for the extraction and quantitation of AEA, 2-AG, their metabolites, oleoylethanolamide (OEA), and palmitoylethanolamide (PEA), and the in vivo formation of the DiHETE-EAs in kidney after a single intravenous bolus administration of 20 mg/kg of anandamide in C57BL/6 J and FAAH KO mice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (© 2018 John Wiley & Sons, Ltd.)

A high-performance liquid chromatography tandem mass spectrometry method was developed for the detection and quantification of 6-methyl-3-(2-nitro-1-(thiophen-2-yl)propyl)-2-phenyl-1H-indole (ZCZ-011) using 2-phenylindole as the internal standard (ISTD). ZCZ-011 was synthesized as a possible positive allosteric modulator with the CB1 cannabinoid receptor. The analytical method employs a rapid extraction technique using Clean Screen FASt™ columns with a Positive Pressure Manifold. FASt™ columns were originally developed for urine drug analysis but we have successfully adapted them to the extraction of brain tissue. Chromatographic separation was performed on a Restek Allure Biphenyl 5 µ, 100 × 3.2 mm column (Bellefonte, PA). The mobile phase consisted of 1:9 deionized water with 10 mmol ammonium acetate and 0.1% formic acid-methanol. The following transition ions (m/z) were monitored for ZCZ-011: 363 > 207 and 363 > 110 and for the ISTD: 194 > 165 and 194 > 89. The FASt™ columns lowered and stabilized the ion suppression over the linear range of the assay (40-4,000 ng/g). The method was evaluated for recovery, ion suppression, accuracy/bias, intraday and interday precision, bench-top stability, freeze-thaw and post-preparative stability. The method was successfully applied to brain tissue from C57BL/6J mice that received intraperitoneal (i.p.) injections with 40 mg/kg of ZCZ-011 or vehicle., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

While Marijuana continues to be the most widely used illicit drug, abuse of synthetic cannabinoid (SCB) compounds in 'Spice' or 'K2' herbal incense products has emerged as a significant public health concern in many European countries and in the USA. Several of these SCBs have been declared Schedule I controlled substances but detection and quantification in biological samples remain a challenge. Therefore, we present a liquid chromatography-tandem mass spectrometry method after liquid-liquid extraction for the quantitation of CP-47,497, CP-47,497-C8 and JWH-250 in mouse brain. We report data for linearity, limit of quantification, accuracy/bias, precision, recovery, selectivity, carryover, matrix effects and stability experiments which were developed and fully validated based on Scientific Working Group for Forensic Toxicology guidelines for forensic toxicology method validation. Acceptable coefficients of variation for accuracy/bias, within- and between-run precision and selectivity were determined, with all values within ±15% of the target concentration. Validation experiments revealed degradation of CP-47, 497 and CP-47,497-C8 at different temperatures, and significant ion suppression was produced in brain for all compounds tested. The method was successfully applied to detect and quantify CP-47,497 in brains from mice demonstrating significant cannabimimetic behavioral effects as assessed by the classical tetrad paradigm., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We present a high-performance liquid chromatography triple quadrupole mass spectrometry (HPLC-MS-MS) method for the identification and quantification of nine serotonin 5-HT2A receptor agonist hallucinogenic substances from a new class of N-methoxybenzyl derivatives of methoxyphenylethylamine (NBOMe) designer drugs in human urine: 25H-NBOMe, 2CC-NBOMe, 25I-NBF, 25D-NBOMe, 25B-NBOMe, 2CT-NBOMe, 25I-NBMD, 25G-NBOMe and 25I-NBOMe. This assay was developed for the Virginia Commonwealth University Clinical and Forensic Toxicology laboratory to screen emergency department specimens in response to an outbreak of N-benzyl-phenethylamine derivative abuse and overdose cases in Virginia. The NBOMe derivatives were rapidly extracted from the urine specimens by use of FASt™ solid-phase extraction columns. Assay performance was determined as recommended for validation by the Scientific Working Group for Forensic Toxicology (SWGTOX) for linearity, lower limit of quantification, lower limit of detection, accuracy/bias, precision, dilution integrity, carryover, selectivity, absolute recovery, ion suppression and stability. Linearity was verified to be from 1 to 100 ng/mL for each of the nine analytes. The bias determined for the NBOMe derivatives was 86-116% with a <14% coefficient of variation over the linear range of the assay. Four different NBOMe derivatives were detected using the presented method in patient urine specimens.

2CC-NBOMe {4-chloro-2,5-dimethoxyphenethyl-N-[(2-methoxyphenyl) methyl] ethanamine} and 25I-NBOMe {2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl) methyl] ethanamine} are of a class of N-benzyl phenethylamine derivatives whose synthesis was first reported in the scientific literature in 2011. Recent reports from 'personal drug experience websites' and in the popular press indicate these drugs are the latest in a series of designer 'Bath Salt' drugs of abuse. The presented high-performance liquid chromatography triple quadrupole mass spectrometry (HPLC/MS/MS) method was developed for the detection and quantification of 2CC-NBOMe and 25I-NBOMe in serum of intoxicated emergency department patients. The assay applies 2-​(2,​5-​dimethoxyphenyl)-​N-​(2-​methoxybenzyl) ethanamine (25H-NBOMe) as the internal standard. Samples were extracted using solid-phase extraction columns. The chromatographic separation was performed on a Luna 3 µ C8(2) 100 Å, 100 × 2.0 mm, column. Detection was accomplished by multiple-reaction monitoring via an electrospray ionization source operating in the positive ionization mode. The calibration curves were linear over the investigated concentration range, 30-2000 pg/mL, with a lower limit of detection of 10 pg/mL for both 2CC-NBOMe and 25I-NBOMe. The method proved suitable for serum clinical toxicology testing. Two severely intoxicated emergency department patients were determined to have serum concentrations of 250 and 2780 pg/mL of 25I-NBOMe using the presented method., (Copyright © 2013 John Wiley & Sons, Ltd.)

The disposition of the cannabimimetic naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in mice following inhalation of the smoke of the herbal incense product (HIP) 'Buzz' is presented. A high-pressure liquid chromatography with electrospray ionization triple quadrupole mass spectrometer (HPLC/MS/MS) method was validated for the analysis of JWH-018 in the specimens using deuterated Δ(9) -tetrahydrocannabinol (d(3) -THC) as the internal standard. JWH-018 was isolated by cold acetonitrile liquid-liquid extraction. Chromatographic separation was performed on a Zorbaz eclipse XDB-C(18) column. The assay was linear from 1 to 1000 ng/mL. Six C57BL6 mice were sacrificed 20 min after exposure to the smoke of 200 mg 'Buzz' containing 5.4% JWH-018. Specimen concentrations of JWH-018 were: blood, 54-166 ng/mL (mean 82 ± 42 ng/mL); brain, 316-708 ng/g (mean 510 ± 166 ng/g); and liver, 1370-3220 ng/mL (mean 1990 ± 752 ng/mL). The mean blood to brain ratio for JWH-018 was 6.8 and ranged from 4.2 to 10.9. After exposure, the responses of the mice were consistent with cannabinoid receptor type 1 activity: body temperatures dropped 7.3 ± 1.1 °C, and catalepsy, hyperreflexia, straub tail and ptosis were observed. The brain concentrations and physiological responses are consistent with the hypothesis that the behavioral effects of 'Buzz' are attributable to JWH-018., (Copyright © 2012 John Wiley & Sons, Ltd.)

A sensitive, specific, and rapid high-pressure liquid chromatography/mass spectrometry/mass spectrometry method was developed for the quantitation of 11 tricyclic antidepressants and/or their metabolites; fluoxetine and norfluoxetine; cyclobenzaprine; and trazodone in urine. Samples were alkalinized with 0.2 N NaOH and extracted into 2 ml of hexane: ethyl acetate (1:1), evaporated to dryness, and reconstituted with 100 μl of 20 mM ammonium formate: methanol (20:80). The chromatographic separation was performed using an Allure Biphenyl 100 × 3.2 mm, 5-μ column with a mobile phase consisting of 20 mM ammonium formate: methanol (20:80 v/v) at a flow rate of 0.5 ml/min. The detection was accomplished by multiple-reaction monitoring via electrospray ionization source operating in the positive ionization mode. The calibration curve was linear over the investigated concentration range, 25-2,000 ng/ml, for each analyte using 1.0 ml of urine. The lower limit of quantitation for each analyte was 25 ng/ml. The intra- and inter-day precisions had coefficient of variation less than 15% and the accuracy was within the range from 88% to 109%. The method proved adequate for the tricyclic antidepressants analysis of urine for emergency clinical toxicology and pain management compliance testing., (© 2012 Wiley Periodicals, Inc.)

We present an evaluation of a new phencyclidine (PCP) enzyme immunoassay (PCPI) for detection in urine. The PCPI was evaluated by testing 523 urine specimens. Controls containing 0 ng/mL of PCP and -25% (negative control) and +25% (positive control) of the 25 ng/mL cutoff calibrator were analyzed with each batch. All urines were analyzed by high-performance liquid chromatography- tandem mass spectrometry (HPLC-MS-MS) for PCP. Of the 523 specimens tested, 218 yielded positive results by the PCP assay. HPLC-MS-MS confirmed the presence of at least 25 ng/mL in 214 specimens, indicating four false-positive results containing 11, 13, 16, and 20 ng/mL PCP. Three specimens yielded a negative result; however, PCP concentrations were within 20% of the 25 ng/mL cutoff value. The overall agreement of PCPI and HPLC-MS-MS results was 98.7%. The sensitivity of the PCPI was 0.982 and the specificity 0.987. Testing at 100 ng/mL of other drugs or their metabolites demonstrated no cross-reactivity. The within-run precision was CV = 2% (n = 12); the between-run precision was CV = < 6% (n = 4). The assay was found linear from -50% to 150% (12.5-37.5 ng/mL) of the cutoff concentration. The Lin-Zhi PCPI provides a precise, reliable method for the routine detection of phencyclidine in urine specimens.

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. [ABSTRACT FROM AUTHOR]

The use of electronic cigarettes (e-cigs) has expanded from a nicotine delivery system to a general drug delivery system. The internet is rife with websites, blogs and forums informing users how to modify e-cigs to deliver illicit drugs while maintaining optimal drug delivery of their device. The goal of this study was to qualitatively identify the presence of methamphetamine in the aerosol produced by an e-cig and to quantitatively assess the effect voltage on the concentration of aerosolized methamphetamine. A KangerTech AeroTank electronic cigarette containing a 30, 60 or 120 mg/mL of methamphetamine in 50:50 propylene glycol: vegetable glycerin formulation was used to produce the aerosol. To qualitatively identify aerosolized methamphetamine, the aerosol was generated at 4.3 V, trapped in a simple glass trapping system, extracted using solid-phase microextraction (SPME), and analyzed by high-resolution Direct Analysis in Real Time AccuTOF™ Mass Spectrometry (DART-MS). To assess the effect of voltage on the concentration of aerosolized methamphetamine, the aerosol was generated at 3.9, 4.3 and 4.7 V, trapped and quantified using gas chromatography mass spectrometry (GC/MS). SPME-DART-MS and SPME-GC-MS demonstrated the aerosolization of methamphetamine. The concentration of aerosolized methamphetamine at 3.9, 4.3 and 4.7 V was not statistically different at 800 ± 600 ng/mL, 800 ± 600 ng/mL and 1,000 ± 800 ng/mL, respectively. The characterization of the vapors produced from e-liquids containing methamphetamine provides an understanding of the dose delivery dynamics of e-cigarettes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We present a traumatic fatality of a 19-year-old man who had ingested blotter paper containing 25INBOMe [2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine]. Postmortem specimens were analyzed by high performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS). Toxicology findings for fluids based upon blood or urine calibrators were as follows: peripheral blood, 405 pg/mL; heart blood, 410 pg/mL; urine, 2.86 ng/mL; and vitreous humor, 99 pg/mL. While findings based upon the method of standard additions were: gastric contents, 7.1 μg total; bile, 10.9 ng/g; brain, 2.54 ng/g and liver, 7.2 ng/g. To our knowledge the presented case is the first postmortem case of 25I-NBOMe intoxication documented by toxicological analysis of tissues and body fluids. [ABSTRACT FROM AUTHOR]

The article presents a study which investigates the effect of the marijuana cannabinoids in mouse brain tissue with the use of an Applied Biosystems 3200 Q trap. The study developed a liquid chromatography- tandem mass spectrometry (LC-MS-MS) method for the examination, along with the of a turbo V source for TurbolonSpay to a Shimadzu SCL HPLC system. Results show that the proportional mean brain cannabinoid concentrations are linked to the amounts of the cannabinoids in the inhaled marijuana.