Ketamine is a NMDA receptor antagonist used since 1970 for the induction of anesthesia. It has a rapid onset and short duration of action, so it is a preferred agent for short-term surgical procedures. Ketamine possesses sedative and analgesic properties and maintains hemodynamic stability; it therefore is used in patients during the neuro-reanimation period (1). Ketamine produces posthypnotic emergence reactions, such as prolonged hallucination and delirium. The frequency of ketamine abuse is increasing (2), and fatal ketamine poisoning cases have been reported (3)(4).
Ketamine is metabolized by the hepatic microsomal cytochrome P450 enzymes to two major metabolites, norketamine (NK) and dehydronorketamine (DHNK), which are further biotransformed to glucuronide conjugates and then excreted in the urine (5). The activities of these metabolites have not been well studied in humans, but in animals, it was reported that they possess an anesthetic effect and that they might be responsible for the emergent reactions to ketamine anesthesia (5). In view of the growing importance of ketamine, both as a therapeutic agent and more recently as a drug of abuse, we studied the stability of ketamine and its two major metabolites, NK and DHNK, in biological samples under various conditions to optimize the conditions for transport and storage of these biological samples. Little has been reported about the stability of ketamine and its metabolites in human blood samples. In a recent study, ketamine was found to be stable in plasma samples stored at −20 °C for up to 3 months (6). In this study, the stability of ketamine metabolites was not investigated. Idvall et al. (7) reported that ketamine and its metabolites were stable in plasma samples stored at −20 °C for up to 2 months.
To date, no data on the stability of ketamine and its metabolites in human blood are available, so we proposed to investigate the stability of these compounds in human blood samples under different storage conditions. We studied the stability of ketamine and its metabolites NK and DHNK in human serum stored at −20 °C for 6 months and at 4 °C for 2 days. The stability of these compounds in human blood stored at 4 and 20 °C for 2 h was also examined.
Ketamine, NK, DHNK, and nortilidine (internal standard) were generously supplied by Parke-Davis Laboratories. Lyotrol® drug-free human serum was obtained from Biomerieux. Solvents for the extraction procedure and for HPLC (Uvasol grade), potassium dihydrogen phosphate, and boric acid were purchased from Merck. Ketamine, NK, and DHNK were analyzed according to the chromatographic method described by Bolze and Boulieu (8). The chromatographic system consisted of a Waters 715 Ultrawisp injector connected to a 510 series pump and coupled to a PDA 996 photodiode array detector and a COMPAQ Pentium III computer. The column used was a reversed-phase silica gel end-capped Purospher® RP-18e (125 × 4 mm; 5 μm bead size) purchased from Merck. The samples were extracted according to a published procedure (8). Briefly, 0.5 mL of the biological sample was alkalinized with boric acid (pH 13) and extracted twice with a mixture of dichloromethane-ethyl acetate (80:20 by volume) followed by a back-extraction with 2 mol/L HCl. After evaporation of the acid layer and reconstitution of the residue with mobile phase, 60 μL was injected into the HPLC column.
To study the stability of ketamine and its metabolites in stock solutions, we prepared aqueous solutions of ketamine, NK, DHNK, and nortilidine at a concentration of 500 μg/L. The solutions were stored at −80 °C and analyzed in five replicates at time zero (t0) and every 2 weeks of storage up to 6 months.
To study the stability of ketamine and its metabolites in human serum stored at −20 and 4 °C, we added ketamine, NK, and DHNK at a concentration of 500 μg/L to a pool of human serum (Lyotrol). For the stability study at −20 °C, we divided a serum sample into six 3-mL aliquots and stored them at −20 °C. Aliquots were analyzed in five replicates at t0 and every 2 weeks of storage up to 10 weeks. For the stability study at 4 °C, 3-mL aliquots were drawn at t0 and at 2, 4, 6, 8, 10, 24, 30, and 48 h and immediately stored at −20 °C until analysis. The assay was performed within 15 days of storage, and five replicates were used in each case.
The stability of the compounds of interest was also assessed in human blood left at room temperature or at 4 °C. Ketamine, NK, and DHNK at a concentration of 500 μg/L were added to blood samples from volunteers. The samples were divided into two aliquots: one was stored at room temperature (∼20 °C) and the other at 4 °C. At t0 and at 30, 60, and 120 min, 8 mL of each aliquot was removed and immediately centrifuged at 2000g for 10 min at 20 °C. The plasma was decanted and stored immediately at −20 °C until analysis. The assay was performed within 15 days of storage, and five replicates were used in each case. Results were compared using the nonparametric Mann-Whitney test. Two-tailed values <0.05 were considered significant. Statistical analysis was performed using Graphpad Instat software.
Ketamine, NK, DHNK, and nortilidine were stable in aqueous solutions at −80 °C for at least 6 months. These solutions were used for the addition experiments in biological samples. No significant difference was observed in the concentrations of ketamine and its metabolites in human serum left at 4 °C for 2 days. Storage of ketamine, NK, and DHNK in human serum at −20 °C did not produce significant changes in concentrations over a period of 10 weeks. These data are in accordance with those reported previously (7). As shown in Table 1⇓ , plasma concentrations of ketamine and NK remained constant when the centrifugation of blood was delayed for 2 h, and the stability of the two compounds was not affected by the change in storage temperature from 4 to 20 °C. On the other hand, a significant decrease in the plasma DHNK concentration was observed when blood samples were kept at 4 °C, whereas surprisingly, the DHNK concentration did not change significantly when blood samples were left for 2 h at room temperature.
After 30 min of storage at 4 °C, the plasma concentration of DHNK was, on average, 68% of the initial concentration. Furthermore, we also observed that the plasma concentrations of DHNK measured at t0 represented, on average, only 75% of the concentration added to blood and stored at 4 °C or at room temperature. These results could suggest that DHNK is unequally distributed between plasma and blood cells and that rapid permeation of this compound into blood cells may occur. Chemical degradation is not likely because the increase in temperature from 4 to 20 °C slowed the decrease in DHNK concentrations. Further investigations should be done to study the effect of various temperatures on the plasma concentration of DHNK. Moreover, the determination of DHNK concentrations in blood cells to estimate the partitioning behavior of this compound may be warranted.
In conclusion, the present study shows the necessity of observing rigorous conditions for the accurate estimation of ketamine metabolite concentrations in blood samples. The collected blood should be centrifuged without delay at ambient temperature to avoid the decrease in DHNK concentrations, which is most likely attributable to the permeation of this compound into the blood cells. The plasma samples can be transported at 4 °C within 2 days and can be stored at −20 °C for 10 weeks without any change in the concentrations of ketamine, NK, and DHNK.
Stability of ketamine and its metabolites in human blood.
- © 2001 The American Association for Clinical Chemistry
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