Effects of gender on ketamine-induced conditioned placed preference and urine metabonomics.

OBJECTIVE: The aim of this study was to examine whether or not there was a gender difference in CPP (conditioned placed preference) induced by ketamine and to further explore the effect of sex on metabolic responses to ketamine inducing in SD rats. METHODS: We measured ketamine-induced conditioned place preference and ketamine-induced metabolic changes in urine by using (1)H nuclear magnetic resonance (NMR) coupled with principal component analysis (PCA), partial least squares (PLS) and orthogonal signal correction (OSC) analysis. RESULTS: In the CPP experiment, ketamine served as a positive reinforcing agent in both male and female rats, but, in particularly, the preference score of female rats was significantly higher than that of male rats. Compared with male rats, the metabolic trajectory fluctuation of the female rats was relatively larger. At the same time, different metabolites (1, 3-dimethyluric acid, cysteine-S-sulfate, glyceraldehydes, glycine, ribitol, acetoacetic acid, creatine, 3-methyladenine, hypotaurine, taurine, dimethylglycine and theobromine) between male and female rats were found. CONCLUSIONS: Female Sprague-Dawley rats were more sensitive to the ketamine-induced CPP than male rats. The fluctuation ranges of metabolic trajectory and metabolite contents in urine were both different between female and male rats. This would provide targeted suggestions for ketamine abuser, for example, men and women should take different drug withdrawal therapeutic methods.

Determination of ketamine, norketamine and dehydronorketamine in urine by hollow-fiber liquid-phase microextraction using an essential oil as supported liquid membrane.

Here, we present a method for the determination of ketamine (KT) and its main metabolites, norketamine (NK) and dehydronorketamine (DHNK) in urine samples by using hollow-fiber liquid-phase microextraction (HF-LPME) in the three-phase mode. The fiber pores were filled with eucalyptus essential oil and a solution of 1.0mol/L of HCl was introduced into the lumen of the fiber (acceptor phase). The fiber was submersed in the alkalinized urine containing 10% NaCl, and the system was submitted to lateral shaking (2400rpm) during 30min. Acceptor phase was withdrawn from the fiber, dried and the residue was then derivatized with trifluoroacetic anhydride (TFAA) for further determination by gas chromatography-mass spectrometry (GC-MS). The calibration curves were linear over the specified range and limits of detection (LoDs) obtained for KT, NK and DHNK were below the cut-off value (1.0ng/mL) recommended by the United Nations Office on Drugs and Crime (UNODC). A totally "green chemistry" approach of the sample extraction was obtained by using essential oil as a supported liquid membrane in HF-LPME. The developed method was successfully validated and applied to urine samples collected from two clinical cases in which KT was suspected to be involved.

Drug testing physicians for substances of abuse: case report of a false-positive result.

The risk of a false-positive urine drug screen is one of the major impediments to widespread implementation of drug testing programs in anesthesiology. A case of a false-positive urine screen for ketamine in an anesthesia provider is presented, with recommendations for methods of managing such an event.

A novel derivatization approach for determination of ketamine in urine and plasma by gas chromatography-mass spectrometry.

A new method was developed for determination of ketamine (KT) in urine and plasma samples by derivatization and gas chromatography-mass spectrometry. In this article, KT was first derivatized with sodium nitrite to volatile N-nitrosamines under acidic condition. Then the derivative had been identified by the mass spectra. The derivatization conditions including the amount of hydrochloric acid, the amount of sodium nitrite, reaction temperature, reaction time and the extraction reagents were optimized. Calibration curves were linear in the range of 0.04-20 µg mL(-1) for KT, and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.01 µg mL(-1) and 0.04 µg mL(-1), respectively. The results of recovery indicated that the method had good precision and reproducibility. Compared with existing derivatization methods, this method provided a rapid, convenient, effective and low-cost way for gas chromatography method of KT quantification.

Validation of an enzyme-linked immunosorbent assay screening method and a liquid chromatography-tandem mass spectrometry confirmation method for the identification and quantification of ketamine and norketamine in urine samples from Malaysia.

An ELISA and a liquid chromatography-tandem mass spectrometry (LC-MS-MS) confirmation method were developed and validated for the identification and quantitation of ketamine and its major metabolite norketamine in urine samples. The Neogen ketamine microplate ELISA was optimized with respect to sample and enzyme conjugate volumes and the sample preincubation time before addition of the enzyme conjugate. The ELISA kit was validated to include an assessment of the dose-response curve, intra- and interday precision, limit of detection (LOD), and cross-reactivity. The sensitivity and specificity were calculated by comparison to the results from the validated LC-MS-MS confirmation method. An LC-MS-MS method was developed and validated with respect to LOD, lower limit of quantitation (LLOQ), linearity, recovery, intra- and interday precision, and matrix effects. The ELISA dose-response curve was a typical S-shaped binding curve, with a linear portion of the graph observed between 25 and 500 ng/mL for ketamine. The cross-reactivity of 200 ng/mL norketamine to ketamine was 2.1%, and no cross-reactivity was detected with 13 common drugs tested at 10,000 ng/mL. The ELISA LOD was calculated to be 5 ng/mL. Both intra- (n = 10) and interday (n = 50) precisions were below 5.0% at 25 ng/mL. The LOD for ketamine and norketamine was calculated statistically to be 0.6 ng/mL. The LLOQ values were also calculated statistically and were 1.9 ng/mL and 2.1 ng/mL for ketamine and norketamine, respectively. The test linearity was 0-1200 ng/mL with correlation coefficient (R(2)) > 0.99 for both analytes. Recoveries at 50, 500, and 1000 ng/mL range from 97.9% to 113.3%. Intra- (n = 5) and interday (n = 25) precisions between extracts for ketamine and norketamine were excellent (< 10%). Matrix effects analysis showed an average ion suppression of 5.7% for ketamine and an average ion enhancement of 13.0% for norketamine for urine samples collected from six individuals. A comparison of ELISA and LC-MS-MS results demonstrated a sensitivity, specificity, and efficiency of 100%. These results indicated that a cutoff value of 25 ng/mL ketamine in the ELISA screen is particularly suitable and reliable for urine testing in a forensic toxicology setting. Furthermore, both ketamine and norketamine were detected in all 34 urine samples collected from individuals socializing in pubs by the Royal Malaysian Police. Ketamine concentrations detected by LC-MS-MS ranged from 22 to 31,670 ng/mL, and norketamine concentrations ranged from 25 to 10,990 ng/mL. The concentrations of ketamine and norketamine detected in the samples are most ikely indicative of ketamine abuse.

Use of human microsomes and deuterated substrates: an alternative approach for the identification of novel metabolites of ketamine by mass spectrometry.

In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by liquid chromatography (LC)-tandem mass spectrometry. Microsomal synthesis produced dehydronorketamine, seven structural isomers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the metabolites were also produced from tetra-deuterated isotopes of ketamine or norketamine as substrates. Five metabolites (three hydroxynorketamine and two hydroxyketamine isomers) gave chromatographically resolved components with product ion spectra indicating the presence of a phenolic group, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave unique masses in accordance with the predicted elemental composition. The metabolites, including the phenols, were subsequently confirmed to be present in urine of subjects after oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine, including an intact glucuronide conjugate, are here reported for the first time. The use of biologically synthesized deuterated material as an internal chromatographic and mass spectrometric marker is a viable approach to aid in the identification of metabolites. Metabolites that have particular diagnostic value can be selected as candidates for chemical synthesis of standards.

Urinary excretion rates of ketamine and norketamine following therapeutic ketamine administration: method and detection window considerations.

Ketamine is widely used in veterinary medicine. Its medical application in humans is limited to children because in adults it induces severe psychedelic episodes. In recent years, teenagers have abused ketamine as a recreational and "club drug" because of its hallucinogenic and stimulant effects. Ketamine is also misused as a "date-rape" drug (to induce amnesia in unsuspecting victims). Sensitive gas chromatography-mass spectrometry-negative chemical ionization (GC-MS-NCI) and liquid chromatography-mass spectrometry-atmospheric pressure chemical ionization (LC-MS-APCI) methods were applied for the simultaneous quantification of ketamine and its major metabolite, norketamine, in urine. Urine samples were collected from hospitalized children who had received ketamine as an anesthetic. Individual urine samples were collected up to 16 days after drug administration. Using the GC-MS-NCI method, ketamine was detected in the urine of the children from only the day of drug administration up to 2 days after drug administration. Its concentrations ranged from 29 to 1410 ng/mL. Norketamine (measured in concentrations of 0.1-1442 ng/mL) was detected up to 14 days. Using the LC-MS-APCI method, norketamine was detected up to 6 days after drug administration, ranging in concentrations of 2-1559 ng/mL, while ketamine was detected up to 11 days (2-1204 ng/mL). In the urine taken from one child, ketamine was not detected through the entire 16-day period using both methods. The detection window for the analytes is highly dependent on the method used for determination and varies between individuals.

Quantitative detection of ketamine, norketamine, and dehydronorketamine in urine using chemical derivatization followed by gas chromatography-mass spectrometry.

A repeatable and highly sensitive analytical method using gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode (SIM) is developed for the simultaneous detection of ketamine (KT), norketamine (NK), and newly introduced dehydronorketamine (DHNK) in urine. The test specimen along with the deuterium analogues as internal standards (IS): d4-KT for KT and d4-NK for NK/DHNK, was extracted on an automatic solid-phase extraction (SPE) apparatus. The extracted eluate then was dried and derivatized with N-methyl-bis(trifluoroacetamide) (CF3CONCH3COCF3, MBTFA). Finally, the cooled derivatized solution was directly injected into the GC-MS system for analysis. The proposed process achieves high sensitivity for the detection of KT, NK, and DHNK. Correlation coefficients derived from typical calibration curves in the range of 20-2000 ng/mL are 1.000 for KT and NK, 0.999 for DHNK. The limits of detection (LODs) and limits of quantitation (LOQs) are 0.5-1.0 and 1.5-3.0, respectively. The overall method recoveries of KT, NK, and DHNK are 82.2-93.4. The intra- and inter-day run deviations are smaller than 5.0%. The analytical scheme was also applied to the determination of KT, NK, and DHNK in 20 KT suspected urine specimens, and the results reconfirm that DHNK is a main metabolite of KT.

Use of SPE and LC/TIS/MS/MS for rapid detection and quantitation of ketamine and its metabolite, norketamine, in urine.

Ketamine (K) has become more and more popular for drug abuse in recent years. A lot of pre-treatment work such as extraction and derivatizing increase difficulties in the tests for ketamine in biological specimens. A rapid method to detect and quantitate ketamine and its metabolite norketamine in urine used deuterated dilution followed by solid phase extraction and liquid chromatography/TurboIonSpray/tandem mass spectrometry (LC/TIS/MS/MS) is described. Control recovery for both low and high concentrations can reach to 90%. Ten ketamine positive urines were examinated by this method. Concentrations ranged from 114 to 2925 ng/mL and from 453 to 9805 ng/mL for norketamine. The method was sensitive, specific, accurate and provided easy operation to detect and quantitate ketamine and its metabolites in urine.

A rapid and sensitive ESI-MS screening procedure for ketamine and norketamine in urine samples.

Traditionally, ketamine was analyzed with gas chromatography (GC) equipped with nitrogen-phosphorus detection, flame-ionization detection, and mass selective detection (MSD) or with liquid chromatography-mass spectrometry (LC-MS). These procedures are sensitive but tedious and slow. There is no commercial immunoassay for ketamine. We have developed a simple and rapid electrospray ionization MS (ESI-MS) procedure to screen ketamine and norketamine (NK) in urine samples. Samples were spiked with ketamine-d(4) (K-d(4)) as internal standard and extracted with 0.2 mL of hexane. An experienced technician can prepare a batch of 60 samples in 1 h. An Agilent LC-MSD trap system with autosampler was employed to inject 10-microL extracted samples directly for mass analysis without chromatographic separation. Total analysis time was 1.3 min per sample. The ESI-MS was operated in scan mode. The ion pairs (m/z 238/242 for K/K-d(4) and m/z 224/242 for NK/K-d(4)) extracted from the full scan mass spectrum were used for quantification. Because of the nature of the ion trap mass detector employed, the presence of other compounds at high concentration could cause the suppression of target analyte ion intensity determined. Limits of detection were 3 ng/mL for ketamine and 15 ng/mL for NK. Carryover was 0.28% for ketamine and 0.39% for norketamine. Within-run precision (%CV) for K and NK at 3 different concentrations (80, 200, and 600 ng/mL) was 4.0% to 14.7%. A group of 168 urine samples collected from disco-dancing club participants were screened with ESI-MS and confirmed with GC-MS. The sensitivity was 97.1% and specificity was 85.7%. These results indicated that the ESI-MS screening procedure is rapid, sensitive, accurate, and reliable.

Gas chromatography-isotope dilution mass spectrometry preceded by liquid-liquid extraction and chemical derivatization for the determination of ketamine and norketamine in urine.

An analytical scheme using gas chromatography (GC)-isotope dilution mass spectrometry (MS) assisted by precedent liquid-liquid extraction (LLE) and chemical derivatization (ChD) is described for the simultaneous determination of ketamine (KT) and its major metabolite, norketamine (NK), in urine. The simultaneous ChD of the two analytes, one primary amine and one secondary amine, with pentafluorobenzoyl chloride (PFBC) has not only enhanced their instrumental responses and mass-spectrum uniqueness but also afforded more proper yet easier selection of qualifier and quantifier ions and hence achieved more evidential identification and quantitation. Thus, the regression calibration curves for KT and NK in urine are linear within 100-5000 ng/ml, with correlation coefficients typically exceeding 0.99 and NK curves exclusively showing larger slopes than KT curves. The method limits of detection (LODs) determined by two definitions for KT and NK range from 3 to 75 ng/ml, and limits of quantitation (LOQs) from 9 to 100 ng/ml. The mean recoveries (N = 3) calculated for the LLE/ChD of KT and NK from 50 and 100 microl, respectively, of a 100 microg/ml urinary spike vary from 71.0 to 97.8%, with NK consistently giving better recoveries than KT. The precisions (N = 3) calculated for the total analyses of four real-case samples are typically below 12.3%. GC-MS operated in the positive ion chemical ionization (PCI) mode can offer both qualitative and quantitative information complementary to those given by the EI mode. The proposed scheme is simple, effective, reliable, and robust. It may serve as a confirmatory protocol for forensic urine drug testing.

Urine concentrations of ketamine and norketamine following illegal consumption.

Ketamine, an anesthetic agent primarily used in veterinary medicine and pediatrics, continues to gain in popularity in the drug abuse scene or 'Rave Wave' of all-night dance clubs. The Division of Forensic Toxicology Laboratory (Office of the Armed Forces Medical Examiner) at the Armed Forces Institute of Pathology, as the primary analytical laboratory for criminal investigative agencies in the Department of Defense (DOD), has seen requests for ketamine analysis rise from 1 in 1997 to 116 in 2000. This increasing abuse has led the DOD Urine Drug Testing Laboratories to consider adding ketamine screening to their random urinalysis program. However, before ketamine testing can be implemented as standard policy, concentrations of ketamine and metabolites in urine need to be evaluated after actual drug use. There is very little information regarding the pharmacokinetics of ketamine, especially concentrations of the drug or its two major metabolites, norketamine and dehydronorketamine, that can be expected in urine. In fact, dehydronorketamine has been believed to be an analytical artifact caused by the high temperatures of gas chromatography. In this paper, we attempt to resolve this issue with the development of a liquid chromatography-mass spectrometry (LC-MS) method. The urine concentrations of ketamine, norketamine and dehydronorketamine (presumptive) detected in 33 "positive" cases received in our laboratory since 1998 are reported. Quantitations were accomplished with LC-MS. Ketamine concentrations ranged from 6 to 7744 ng/mL. Norketamine concentrations ranged from 7 to 7986 ng/mL and dehydronorketamine (presumptive) concentrations ranged from 37 to 23,239 ng/mL.