PurposeDexmedetomidine (DEX) and esketamine (ESK) are often used together during anesthesia. This study aimed to establish a sensitive and reliable HPLC-MS/MS method for simultaneous quantification of ESK and its active metabolite norketamine (NORK) in beagle dog plasma and to investigate the pharmacokinetic drug-drug interactions (DDIs) between DEX and ESK/NORK.MethodsA simple protein precipitation method using acetonitrile was applied for plasma sample preparation. After chromatographic separation, analytes were detected by HPLC-MS/MS in positive ion mode using multiple reaction monitoring (MRM). The mass transitions were m/z 238.10→125.10 for ESK, m/z 224.10→125.10 for NORK, and m/z 354.20→209.00 for the internal standard (proadifen). Six beagle dogs were intramuscularly administered 1 mg/kg ESK alone in the first period (ESK group). After a washout, the same dogs received intravenous DEX (2 µg/kg) for 7 consecutive days, followed by co-administration of ESK (DEX+ESK group). The pharmacokinetic parameters of ESK and NORK were calculated using DAS software. Independent-sample t test was used to compare the differences of pharmacokinetic parameters between ESK group and DEX+ESK group, and P < 0.05 indicated a statistically significant difference.ResultsBoth ESK and NORK exhibited good linearity within the concentration range of 1–400 ng/mL, and the methodological validation met the requirements. When ESK was used in combination with DEX, the main pharmacokinetic parameters of ESK and NORK changed, the Cmax, AUC(0–24) and AUC(0-∞) of ESK increased, the Cmax, of NORK decreased, and the AUC(0–12) and AUC(0-∞) of NORK increased too.ConclusionA novel HPLC-MS/MS method was developed and validated and successfully applied to simultaneously quantify ESK and NORK in beagle dog plasma. The pharmacokinetic DDI results indicate that DEX could inhibit the metabolism of ESK, alter pharmacokinetic characteristics of ESK and its metabolite NORK, and significantly increase the systemic exposure of both ESK and NORK.

To construct an aptamer-functionalized carboxylated graphene oxide (CGO) fluorescent sensor to achieve highly sensitive and specific detection of ketamine (KET) and its metabolite norketamine (NK) using an aptamer capable of simultaneously recognizing KET and NK. A specific aptamer for simultaneous recognition of KET and NK was screened using graphene oxide-systematic evolution of ligand by exponential enrichment (GO-SELEX) and molecular docking techniques. The aptamer, labeled with Cy5 fluorescence, was chemically conjugated to CGO to construct an aptamer-functionalized CGO fluorescent sensor. By optimizing detection conditions, including the mass concentration of CGO, aptamer concentration, reaction temperature, and incubation time, quantitative analysis of the target analytes was achieved using the ratio of fluorescence intensity changes before and after target addition. The stability of the sensor in biological matrices was evaluated by monitoring fluorescence intensity changes over incubation time in blank blood and urine, in comparison with the traditional physical adsorption-based CGO fluorescent sensor. Spiked recovery experiments in blank blood and urine were conducted to compare performance with that of HPLC-MS/MS. A specific aptamer A5 was selected and chemically conjugated with CGO to construct the aptamer-functionalized CGO fluorescent sensor. Under optimized conditions, the proposed fluorescent sensor exhibited a linear detection range of 1.0-5.0 ng/mL for KET, with a limit of detection (LOD) of 0.86 ng/mL; while for NK, the linear detection range was 1.0-5.0 ng/mL, with an LOD of 0.70 ng/mL. Compared with the CGO fluorescent sensor constructed via physical adsorption, this sensor demonstrated greater stability in blood and urine. The spiked recovery rates of KET and NK in blank blood and urine ranged from 81.50% to 110.03%, exhibiting detection performance comparable to that of HPLC-MS/MS. The aptamer screening method offers a novel approach for selecting aptamers targeting drugs and their metabolites. The constructed aptamer-functionalized CGO fluorescent sensor provides an efficient and reliable strategy for the high-performance detection of KET and NK.

Background: Human nails are primarily composed of keratin, a fibrous protein. As nails grow, drugs that have been misused can become embedded in the keratin fibers, allowing detection even months after use. This prolonged retention has led to increased interest in utilizing keratinized matrices for both forensic and therapeutic purposes. Unlike traditional biological samples such as blood and urine, which have shorter detection windows, keratinized matrices can preserve evidence of repeated drug exposure for extended periods. Materials and Methods: This study analyzes nail clippings from five individuals with documented ketamine (KET) misuse. Before liquid chromatography-mass spectrometric analysis, the samples underwent a series of preparatory steps, including decontamination, hydrolysis in 1 M sodium hydroxide at 90°C for one hour with sonication, extraction using ethyl acetate, and reconstitution in methanol. Results: Calibration curves were created using reference standards, covering KET concentrations from 0.20 ng/mg to 16 ng/mg and norketamine (NKT) concentrations from 0.30 ng/mg to 16 ng/mg. The limits of quantification and detection for KET in spiked nail clippings were found to be 0.2 ng/mg and 0.05 ng/mg, respectively. For NKT, the respective values were 0.07 ng/mg and 0.15 ng/mg. Conclusion: This study presents a targeted analytical method for detecting KET and its metabolite, NKT, in human nails. The proposed approach could be valuable for applications such as monitoring drug misuse, investigating drug-facilitated crimes, and assessing cases of medical negligence.

Rapid on-site detection of illicit drugs in urine using C18 pipette-tip based solid-phase extraction coupled with a miniaturized mass spectrometer

Herein, we report the synthesis of aryl derivatives of ketamine and of ketamine's major metabolites hydroxynorketamine (HNK), norketamine (NK), and dehydronorketamine (DHNK) via a microwave-assisted Diels-Alder reaction to form the substituted cyclohexane core structure. Starting with aryl acrylic esters as dienophiles and siloxybutadienes as diene counterparts, a wide range of substituted arylcyclohexylamines was obtained after several modification steps of the initial Diels-Alder products [El Sheikh, S.; Weber, H.; Kortenbrede, L.; Drouvé, N. A broadly applicable Diels-Alder based Synthesis of Ketamine related Arylcyclohexylamines. ChemRxiv 2022, 10.26434/chemrxiv- 2022-xf1l9].

IntroductionKetamine is a phenylcyclohexylamine derivative comprising a racemic mixture of S- and R-ketamine that possesses anesthetic, analgesic, anti-inflammatory, and antidepressant activity. Oral (including extended release [PO]), intravenous (IV) sublingual (SL), transmucosal (TM), intranasal (IN), intramuscular (IM), rectal (PR), and subcutaneous (SC) formulations have been developed since its commercialization in 1970.ObjectivesTo review and understand the impact of different forms and formulations on the pharmacokinetics of ketamine.MethodsThe extant literature on ketamine metabolism and formulations was reviewed and discussed.ResultsIV (racemic) ketamine (KET) has been shown to improve depressed mood within 4 hours with maximal effect at 24 hours. KET is a chiral molecule with two optimal isomers, R- and S-KET. KET is stereoselectively metabolized by CYP2B6 and CYP3A4 initially via nitrogen demethylation to active metabolite, norketamine (NK); there is no interconversion between R- and S-KET. NK is further metabolized to hydroxynorketamine (HNK) by CYP3A4 and CYP3A5; and dehydronorketamine (DHNK) by CYP2B6. Additional metabolic pathways exist including a direct enantioselective hydroxylation of KET to 6-hydroxyketamine (HK). Bioavailablity is greatest (100%) with the IV racemic KET formulation, but as low as 8% for oral S-KET due to extensive first-pass metabolism; the KET: NK ratio is a measure of first pass metabolism. NK plasma levels are higher with oral S-KET than KET as a result of local intestinal metabolism effects. Additionally, greater plasma concentrations are noted with IV bolus doses of S-KET vs. racemic KET or R-KET. S-KET possesses a longer elimination half-life than racemic KET due to inhibition by R-KET. KET is primarily renally eliminated and twice as fast in children vs. adults.ConclusionsComplex interactions are reported between ketamine form (racemic/enantiomer), formulation, dose, and route of administration that impact on clinical variables and thus, outcome.Disclosure of InterestNone Declared

Chiral LC-MS/MS method for the simultaneous determination of (R,S)-ketamine, (R,S)-norketamine, and (2R,6R;2S,6S)-hydroxynorketamine in mouse plasma and brain

Ketamine-associated cystitis is characterized by suburothelial inflammation and urothelial cell death. Norketamine (NK), the main metabolite of ketamine, is abundant in urine following ketamine exposure. NK has been speculated to exert toxic effects in urothelial cells, similarly to ketamine. However, the molecular mechanisms contributing to NK-induced urothelial cytotoxicity are almost unclear. Here, we aimed to investigate the toxic effects of NK and the potential mechanisms underlying NK-induced urothelial cell injury. In this study, NK exposure significantly reduced cell viability and induced apoptosis in human urinary bladder epithelial-derived RT4 cells that NK (0.01–0.5 mM) exhibited greater cytotoxicity than ketamine (0.1–3 mM). Signals of mitochondrial dysfunction, including mitochondrial membrane potential (MMP) loss and cytosolic cytochrome c release, were found to be involved in NK-induced cell apoptosis and death. NK exposure of cells also triggered the expression of endoplasmic reticulum (ER) stress-related proteins including GRP78, CHOP, XBP-1, ATF-4 and -6, caspase-12, PERK, eIF-2α, and IRE-1. Pretreatment with 4-phenylbutyric acid (an ER stress inhibitor) markedly prevented the expression of ER stress-related proteins and apoptotic events in NK-exposed cells. Additionally, NK exposure significantly activated JNK, ERK1/2, and p38 signaling and increased intracellular calcium concentrations ([Ca2+]i). Pretreatment of cells with both PD98059 (an ERK1/2 inhibitor) and BAPTA/AM (a cell-permeable Ca2+ chelator), but not SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), effectively suppressed NK-induced mitochondrial dysfunction, ER stress-related signals, and apoptotic events. The elevation of [Ca2+]i in NK-exposed cells could be obviously inhibited by BAPTA/AM, but not PD98059. Taken together, these findings suggest that NK exposure exerts urothelial cytotoxicity via a [Ca2+]i-regulated ERK1/2 activation, which is involved in downstream mediation of the mitochondria-dependent and ER stress-triggered apoptotic pathway, consequently resulting in urothelial cell death. Our findings suggest that regulating [Ca2+]i/ERK signaling pathways may be a promising strategy for treatment of NK-induced urothelial cystitis.

Wastewater-based monitoring of 2-fluoro-deschloroketamine abuse from 2019 to 2021 in a southern Chinese province

Development of a rapid and sensitive LC-MS/MS assay for the quantification of commonly abused drugs in Asia in a micro-segment of a single hair using microwave-assisted extraction and dansyl chloride derivatization

Synthesis of a novel polydopamine and C18 dual-functionalized magnetic core-shell mesoporous nanocomposite for enrichment and analysis of widely abused illegal drugs in urine samples on site and in the laboratory

Ketamine (K) and its main metabolite, norketamine (NK), are chiral compounds that have been found in effluents from wastewater treatment plants (WWTPs) and aquatic environments. Little is known about their enantioselective biodegradation during sewage treatment; however, this information is pivotal for risk assessment, the evaluation of WWTP performance and wastewater epidemiological studies. The aim of this study was to investigate the biodegradation pattern of the enantiomers of K by activated sludge (AS) from a WWTP. For that, an enantioselective liquid chromatography with diode array detection (LC-DAD) method was developed and validated to quantify the enantiomers of K and NK. Both K and NK enantiomers were separated, in the same chromatographic run, using a Lux® 3 µm cellulose-4 analytical column under isocratic elution mode. The method was demonstrated to be linear (r2 &gt; 0.99) and precise (&lt;11.3%). Accuracy ranged between 85.9 and 113.6% and recovery ranged between 50.1 and 86.9%. The limit of quantification was 1.25 µg/mL for the enantiomers of NK and 2.5 µg/mL for K. The method was applied to monitor the biodegradation assay of the enantiomers of K by AS for 14 days. K was poorly biodegraded, less than 15% for both enantiomers, and enantioselectivity in the biodegradation was not observed. The metabolite NK and other possible degradation products were not detected. This work reports, for the first time, the behavior of both enantiomers of K in biodegradation studies.

Tissue-specific accumulation, elimination, and toxicokinetics of illicit drugs in adult zebrafish (Danio rerio)

Estimation of the psychoactive substances consumption within 12 wastewater treatment plants service areas in a certain city of Guangxi, China applying wastewater-based epidemiology.

Simultaneous enantioselective analysis of illicit drugs in wastewater and surface water by chiral LC-MS/MS: A pilot study on a wastewater treatment plant and its receiving river.

A fast GC-MS screening procedure for ketamine and its metabolites in urine samples

Targeted mass spectrometry of ketamine and its metabolites cis-6-hydroxynorketamine and norketamine in human blood serum

This study presents a retrospective analysis of the prevalence of drug abuse in Shanghai by hair analysis. Files and toxicology analysis results of a total of 5,610 cases requesting for hair analysis of abused drugs at the Academy of Forensic Science (AFS) in Shanghai over 12months between August 2018 and July 2019 were reviewed. All cases of drug abuse identified by hair analysis were from the public security organs in Shanghai, China. Hair samples were analyzed for drugs of abuse and related metabolites, mainly including amphetamine (AMP), methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), tetrahydrocannabinol (THC), ketamine (K), norketamine (NK), cocaine (COC), benzoylecgonine, morphine, 6-acetylmorphine, flunitrazepam, and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Among the 5,610 cases, 1,713 (30.5%) were positive for drugs of abuse, with amphetamine-type stimulants (ATS) (57%), including amphetamines (AMP and MA) (48%), MDMA and MDA (9%), being the most frequently detected drugs, followed by THC (14%), COC (8%), 5-MeO-DIPT (8%), and K (7%). The majority (75%) of positive hair samples were from male subjects. Overall, 77% of abusers were younger than 44years old. The proportion of female subjects (22.3%) under 24years was larger than that of male subjects (7.8%). There were 132 cases (7.7%) in which more than one type of drug was detected among 1,713 drug-positive cases. The most common combination was MDMA and K. The present study characterizes the current toxicological profile of drug abuse cases and provides a scientific basis for drug abuse prevention. Moreover, the hair concentration distributions of the commonly abused drugs in positive cases have been reported.

Environmental behavior of methamphetamine and ketamine in aquatic ecosystem: Degradation, bioaccumulation, distribution, and associated shift in toxicity and bacterial community

Male Sprague-Dawley rats (n = 18) were randomly divided into three groups: a saline group (20 mL/kg by gavage), a ketamine (KET) group (100 mg/kg by gavage), and a KET (the same routes and doses) combined with levo-tetrahydropalmatine (l-THP; 40 mg/kg by gavage) group (n = 6). Blood samples were acquired at different time points after drug administration. A simple and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to determine the concentrations of KET and its metabolite, norketamine (NK), in rat plasma. Chromatographic separation was achieved using a BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with chlorpheniramine maleate (Chlor-Trimeton) as an internal standard (IS). The initial mobile phase consisted of acetonitrile–water with 0.1% methanoic acid (80 : 20, v/v). The multiple reaction monitoring (MRM) modes of m/z 238.1→m/z 179.1 for KET, m/z 224.1→m/z 207.1 for NK, and m/z 275→m/z 230 for Chlor-Trimeton (IS) were utilized to conduct a quantitative analysis. Calibration curves of KET and NK in rat plasma demonstrated good linearity in the range of 2.5–500 ng/mL (r > 0.9994), and the lower limit of quantification (LLOQ) was 2.5 ng/mL for both. Moreover, the intra- and interday precision relative standard deviation (RSD) of KET and NK were less than 4.31% and 6.53%, respectively. The accuracies (relative error) of KET and NK were below -1.41% and -6.07%, respectively. The extraction recoveries of KET and NK were more than 81.23 ± 3.45% and 80.42 ± 4.57%, respectively. This sensitive, rapid, and selective UPLC-MS/MS method was successfully applied to study the pharmacokinetic effects of l-THP on KET after gastric gavage. The results demonstrated that l-THP could increase the bioavailability of KET and promote the metabolism of KET. The results showed that l-THP has pharmacokinetics effects on KET in rat plasma.