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Regula Theurillat1 M. Paula Larenza2 Karsten Feige3 Regula BettschartWolfensberger2 Wolfgang Thormann1 1 Clinical

Pharmacology Laboratory, University of Bern, Bern, Switzerland 2 Equine Department, Vetsuisse ¨ Faculty, University of Zurich, ¨ Zurich, Switzerland 3 Clinic for Horses, University of Veterinary Medicine, Hannover, Germany

Received February 21, 2014 Revised April 7, 2014 Accepted April 21, 2014

Research Article

Development of a method for analysis of ketamine and norketamine enantiomers in equine brain and cerebrospinal fluid by capillary electrophoresis Ketamine and norketamine are being transported across the blood brain barrier and are also entering from blood into cerebrospinal fluid (CSF). Enantioselective distributions of these compounds in brain and CSF have never been determined. The enantioselective CE based assay previously developed for equine plasma was adapted to the analysis of these compounds in equine brain via use of an acidic pre-extraction of interferences prior to liquid/liquid extraction at alkaline pH. CSF can be treated as plasma. With 100 mg of brain tissue and 0.5 mL of CSF or plasma, assay conditions for up to 30 nmol/g and 6 ␮M, respectively, of each enantiomer with LOQs of 0.5 nmol/g and 0.1 ␮M, respectively, were established and the assays were applied to equine samples. CSF and plasma samples analyzed stemmed from anesthetized patient horses and brain, CSF and plasma were obtained from anesthetized horses that were euthanized with an overdose of pentobarbital. Data obtained indicate that ketamine and norketamine enantiomers are penetrating into brain and CSF with those of ketamine being more favorably transported than norketamine, whereas metabolites of norketamine are hindered. More work is required to properly investigate possible stereoselectivities of the ketamine metabolism and transport of metabolites from blood into brain tissue and CSF. Keywords: Brain / CSF / Enantiomer / Ketamine / Norketamine DOI 10.1002/elps.201400093

1 Introduction Ketamine ((R,S-2-(2-chlorophenyl)-2-methylamino)cyclohexanone; for chemical structure of S-ketamine see insert in Fig. 1) is an intravenous analgesic and anesthetic drug widely used in clinical practice of man and animals. Ketamine interacts with opioid receptors, muscarinic acetylcholine receptors, and different voltage-gated channels. Its neurophysiologic effect is mainly based on the noncompetitive antagonism on the N-methyl-D-aspartate receptor. Ketamine is administered as racemate (in most countries) or as S-ketamine (e.g. in Germany to humans and Switzerland to cats). The S-enantiomer is more potent than the R-enantiomer and exhibits a greater clearance and faster anesthetic recovery compared to the racemate. The pKa value of ketamine is 7.5 (it is thus partially positively charged at physiological pH) and the partition coefficient expressed as

Correspondence: Professor W. Thormann, Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland E-mail: [email protected] Fax: +41-31-632-4997

Abbreviation: CSF, cerebrospinal fluid

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the log P (octanol/water) value is 3.1. Metabolites formed include norketamine (main active metabolite with a calculated log P value of 1.7), various hydroxynorketamine compounds and 5,6-dehydronorketamine [1–4]. Although used extensively in horses, the metabolism, pharmacokinetics and clinical effects of S- and R-ketamine were incompletely documented for this species [5]. This prompted us to focus on the use of racemic ketamine versus S-ketamine for anesthesia and pain therapy in horses and the characterization of the stereoselectivity of equine ketamine metabolism in vivo and in vitro [6–12]. Ketamine and norketamine enantiomers in human and animal plasma can be determined by HPLC using expensive, enantioselective columns that are based upon immobilized ␣1 -acid glycoprotein [5,13–15] or cellulose tris(3,5-dimethylphenylcarbamate) coated silica-gel (Chiralcel OD) [16]. An enantioselective GC method that features a ␤-CD based chiral stationary phase has also been reported [17]. As an alternative to these methods, enantioselective CE assays with sulfated CDs as chiral selector for analysis of ketamine and norketamine enantiomers in equine plasma, urine and in vitro preparations were developed in our laboratory [18–20]. Ketamine and norketamine are small hydrophobic compounds that are transported across the blood brain barrier. They are also expected to enter from blood into cerebrospinal

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gether with first analytical results obtained with samples of patient horses that were anesthetized with medetomidine and racemic ketamine and from horses that received xylazine and racemic ketamine or S-ketamine prior to euthanizing with an overdose of pentobarbital.

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TIME (min) Figure 1. Enantioselective electropherograms of equine brain extracts obtained with a cerebellum brain sample collected after i.v. administration of 1.1 mg/kg S-ketamine and using (A) the liquid/liquid extraction at alkaline pH and (B) an acidic preextraction prior to the liquid/liquid extraction at alkaline pH. (C) presents data obtained with 500 ␮L of arterial plasma that was collected 4 min after administration of S-ketamine. The data presented in (D) represent the temporal behavior of the current for the analysis of the sample shown as (B). The insert in (A) depicts the chemical structure of S-ketamine (R = CH3 ) and S-norketamine (R = H). The insert in (B) depicts data obtained after extraction of 200 ␮L of CSF that was collected 4 min after S-ketamine administration. Electropherograms are presented with a 0.01 AU y-axis shift. Key: S-K, S-ketamine; SNK, S-norketamine; S-HNK, S-hydroxynorketamine; S-DHNK, S5,6-Dehydronorketamine; IST, (+)-pseudoephedrine added as internal standard.

fluid (CSF), the fluid that surrounds the brain and spinal cord. A penetration from brain into CSF is also possible. This transport, however, occurs most likely to a much smaller extent [21, 22]. Very little is known about the amounts of ketamine and norketamine present in brain [23–27] and CSF [28–30]. No information could be found for equines and no paper reported the stereoselective distributions of the compounds of interest after application of racemic ketamine. Thus, the distribution of the enantiomers of ketamine and norketamine in equine brain and CSF in comparison to that of plasma as well as the occurrence of norketamine metabolites in these compartments attracted our attention. The enantioselective CE-based assay previously developed for equine plasma was adapted to the analysis of these compounds in brain and CSF. In this paper, the development of the method is presented to C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

All chemicals used were of analytical grade. Racemic ketamine hydrochloride was obtained from the pharmacy of the Inselspital (Bern, Switzerland) and racemic norketamine hydrochloride was purchased as methanolic solution (1 mg/mL of the free base) from Cerilliant (Round Rock, USA). Tris and acetic acid (100%) were from Merck (Darmstadt, Germany), (+)-pseudoephedrine hydrochloride, H3 PO4 (85%), ethylacetate and diammonium hydrogenphosphate were from Fluka (Buchs, Switzerland), and dichloromethane was purchased from Biosolve (Valkenswaard, the Netherlands). Sulfated ␤-CD (7–11 mol sulfate/mol ␤-CD) was obtained from SigmaAldrich Chemie (Schnelldorf, Germany) as batches 13307MA and 13112JD. Bovine plasma was received from cattle at the local slaughter house. An aqueous solution comprising 100 ␮M of each enantiomer of ketamine and norketamine was used for calibration, whereas a solution of (+)-pseudoephedrine (149 ␮M) was employed as internal standard solution. A pH 2.5 buffer comprising 50 mM Tris and phosphoric acid was used to prepare the running buffer. 2.2 Horse samples The study was performed with the permission of the Committee for Animal Experimentation, Canton Z¨urich, Switzerland and the State Office for Consumer Protection and Food Safety in accordance with the German Animal Welfare Law. Brain tissue, CSF, arterial plasma of blood drawn from the left transverse facial artery (a branch of the external carotid artery) and venous plasma of blood collected from the left jugular vein stemmed from two horses that were anesthetized with boluses of xylazine (1.1 mg/kg) and racemic ketamine (2.2 mg/kg) or S-ketamine (1.1 mg/kg) and were euthanized with an overdose of pentobarbital, all i.v. Blood and CSF samples were collected simultaneously 4 min after ketamine injection and immediately before administration of pentobarbital. Brain samples of different brain regions (cerebellum, corpus callosum, and frontal cortex) were collected within 30 min after euthanizing the animals. Blank brain tissue was from a horse that was euthanized with an overdose of pentobarbital. CSF and arterial plasma were taken from three patient horses that were anesthetized with acepromazine (30 ␮g/kg, i.m.), medetomidine (7 ␮g/kg i.v. loading dose followed by a constant rate infusion of 3.5 ␮g/kg/h), racemic ketamine (2.2 mg/kg, i.v.), and inhaled isoflurane in oxygen (end-tidal concentration 1.0–1.4%). Samples were collected 7 to 15 min after ketamine injection and one horse received an additional www.electrophoresis-journal.com

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topup of ketamine (0.2 mg/kg) 5 min before sampling at 15 min. All samples were frozen at −20°C until analysis. 2.3 Sample preparation for CSF and plasma The conditions used were similar to those reported previously for analysis of equine plasma [6–11, 18–20]. Extraction of 0.5 mL CSF, plasma, or calibrator solution (bovine plasma fortified with the enantiomers of ketamine and norketamine) was effected with addition of 30 ␮L internal standard ((+)pseudoephedrine) solution, 500 ␮L of 0.2 M NaOH and 5 mL of a dichloromethane/ethylacetate (75/25% v/v) solvent mixture using a 10 mL glass tube. After shaking (10 min) and centrifugation at 3500 rpm (5 min), the organic (lower) phase was recovered, acidified with 30 ␮L 50 mM phosphoric acid, and evaporated to dryness at 40°C under a gentle stream of air. The residue was reconstituted in 200 ␮L methanol, transferred to a 300 ␮L Eppendorf tube, and again evaporated. The final residue was dissolved in 30 ␮L of 5 mM Tris/phosphate buffer (pH 2.5; tenfold diluted running buffer without chiral selector) and analyzed.

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lary of 45 cm total length (Polymicro Technologies, Phoenix, AZ, USA). The applied voltage was −17 kV (current: about −30 ␮A), the cartridge temperature was 20°C and the sample tray temperature was 20°C. In order to decrease the running time to 18 min, a constant coflow of 0.1 psi (1 psi = 6894.76 Pa) toward the anode was applied throughout the run. The detection wavelength of the PDA detector was set to 195 nm. Sample injection was effected with pressure of 1.5 psi for 8 s. A 50 mM Tris/phosphate buffer, pH 2.5, containing 10 mg/mL sulfated ␤-CD as a mixture of two lots (30% 13307MA and 70% 13112JD as described in Theurillat et al. [19]) was employed as separation medium. Fresh buffer was prepared every day. In the morning, the capillary was rinsed with 1 M NaOH, 0.1 M NaOH, and bidistilled water (10 min each) followed by 0.1 M HCl and running buffer (5 min each). Before each experiment the capillary was sequentially rinsed with 0.1 M HCl and running buffer (2 min each).

3. Results and discussion 3.1 Analysis of brain samples

2.4 Sample preparation for brain An aliquot of brain tissue (about 100 mg) or calibrator tissue (blank brain tissue fortified with the enantiomers of ketamine and norketamine) is weighed in a 2 mL Eppendorf vial. A stainless steel bead of 5-mm diameter and 0.5 mL water are added prior to homogenization on a Mixer Mill 300 (Retsch, Haan, Germany) set to 20 Hz for 3 min. After addition of 30 ␮L internal standard ((+)-pseudoephedrine) solution, the sample was vortexted and the homogenate transferred to a 10 mL glass tube. The Eppendorf vial was rinsed with 200 ␮L water and this solution was also transferred to the 10 mL glass tube. Then 200 ␮L of acetic acid (20%) and 5 mL of a dichloromethane/ethylacetate (75/25% v/v) solvent mixture were added to the glass tube. After shaking (10 min) and centrifugation at 3500 rpm (5 min), the aqueous (upper) phase was recovered and put into a new 10 mL glass vial before addition of 200 ␮L of 2 M NaOH and 5 mL of dichloromethane/ethylacetate (75/25% v/v). After shaking for 10 min and centrifugation at 3500 rmp for 5 min, the organic (lower) phase was recovered, acidified with 30 ␮L 50 mM phosphoric acid and evaporated to dryness at 40°C under a gentle stream of air. The residue was reconstituted in 200 ␮L methanol, transferred to a 300 ␮L Eppendorf tube, and again evaporated. The final residue was dissolved in 30 ␮L of 5 mM Tris/phosphate buffer (pH 2.5; tenfold diluted running buffer without chiral selector) and analyzed.

2.5 CE instrumentation and running conditions Analyses were performed on the ProteomeLab PA 800 Capillary Electrophoresis Analyzer (Beckman Coulter, Fullerton, CA, USA) using a 50 ␮m id uncoated fused-silica capil C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

As discussed previously [18–20], ketamine and norketamine enantiomers can be separated and analyzed using a 50 mM Tris-phosphate buffer (pH 2.5) containing 10 mg/mL of sulfated ␤-CD. Experiments are performed in an untreated fused-silica capillary and having reversed polarity, indicating that the migrating drug complexes have a negative charge (strong complexation). Sulfated ␤-CD from Aldrich comprises a mixture of ␤-CD molecules with 7–11 mol sulfate/mol ␤-CD [31], mixtures that vary from batch to batch to an extent which led to large differences in the separation of ketamine enantiomers [19]. Excellent resolution for ketamine and many of its metabolites was obtained with a blend of two batches [19, 20] (cf. Section 2.5) and this mixture was employed for the data presented in this paper. Typical electropherograms obtained with extracts of an equine brain sample and plasma of a horse that received S-ketamine are presented in Fig. 1. The electrophoretic configuration is characterized with a stable current of about −30 ␮A (Fig. 1D). Using the liquid/liquid extraction procedure at alkaline pH (which was used for plasma, urine, and in vitro samples [18–20] as described in Section 2.3) for extraction of ketamine and norketamine enantiomers in brain revealed electropherograms with a large interference (Fig. 1A). Furthermore, the sample comprised tiny particles (aggregates) that produced sharp spikes. The interference and the spikes prevented quantitation of the compounds of interest. With a liquid/liquid extraction of interferences at acidic pH, similar to what was described for other brain assays [25], followed by the liquid/liquid extraction of the compounds of interest at alkaline pH, however, revealed extracts that provided clean electropherograms (Fig. 1B). The electropherograms presented as graphs A and B in Fig. 1 were from cerebellum of the horse that received S-ketamine after sedation www.electrophoresis-journal.com

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3.2 Analysis of plasma and CSF The conditions used for plasma and CSF were similar to those reported previously for analysis of equine plasma [6–11, 18–20] employing liquid/liquid extraction of the enantiomers at alkaline pH according to Section 2.3. Bovine plasma served as calibrator matrix. The calibration range was between 0.2 and 6.0 ␮M for each enantiomer. All calibration graphs were found to be linear (r ⬎ 0.999; F ⬎ 1590) and the  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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with xylazine. These data indicate that mainly S-ketamine (13.65 nmol/g of brain) and a small amount of S-norketamine (2.10 nmol/g) are found in this brain sample. No R-ketamine, no R-norketamine and no metabolites of S-norketamine were detected. Similar data were obtained with the samples from the other two brain regions (data not shown). Based on the data presented in Fig. 1B, an assay based on the two-step liquid/liquid extraction scheme (Section 2.4) was developed. Recoveries of all compounds were assessed in triplicates by comparing the responses of samples prepared in tenfold diluted buffer with those obtained after extraction from the brain matrix. The recoveries for the enantiomers of ketamine and norketamine were around 50% (between 49.9 and 51.8%; internal standard 65.7%), values that are lower compared to the recoveries of 70 to 80% reported for the alkaline liquid/liquid extraction from plasma [18] and microsomal preparations [32]. For assay calibration, blank brain tissue was fortified with aliquots of the solution containing the four compounds (four-point calibration between 1 and 30 nmol/g for each enantiomer) and sample preparation was identical to that used for brain samples. All calibration graphs were found to be linear (r ⬎ 0.998; F ⬎ 402). The quantitation limit for all enantiomers was 0.5 nmol/g (half the concentration of the lowest calibrator) and interday precision values (n = 3) expressed as RSDs were ⬍ 10%. Typical electropherograms obtained with the extracts of calibrators containing 1 and 15 nmol/g of each enantiomer are presented in Fig. 2A. Analysis of the cerebellum brain sample collected after administration of racemic ketamine to one horse revealed the data presented as Fig. 2B. S-ketamine, R-ketamine, S-norketamine, and R-norketamine enantiomer levels of this sample were determined to be 22.61, 21.86, 3.07, and 3.01 nmol/g of brain, respectively. Similar electropherograms were obtained with samples from the other brain regions. Ketamine and norketamine enantiomers were observed in all samples, whereas metabolites of norketamine were not detected. Mean (RSD) enantiomer levels of S-ketamine, Rketamine, S-norketamine, and R-norketamine in the three samples were 24.68 nmol/g of brain (13.12%), 23.65 nmol/g of brain (12.51%), 3.90 nmol/g of brain (19.86%), and 3.87 nmol/g of brain (20.86%), respectively. Mean S/R ratios (RSD) of ketamine and norketamine enantiomers in the three samples are 1.04 (0.78%) and 1.01 (1.08%), respectively. These data suggest that there is no stereoselectivity for the case with ketamine administration after xylazine sedation.

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TIME (min) Figure 2. Enantioselective electropherograms of equine brain extracts obtained with (A) a brain calibrator comprising 15 nmol/g of each ketamine and norketamine enantiomer and (B) a cerebellum brain sample of a horse that received 2.2 mg/kg racemic ketamine prior to euthanasia. The graph under the calibrator graph A was obtained with a brain calibrator containing 1 nmol/g of each compound. (C) and (D) present data obtained with 500 ␮L of arterial and venous plasma, respectively, samples from the same horse that were collected 4 min after administration of racemic ketamine. The insert in (B) depicts data obtained after extraction of 500 ␮L of CSF collected 4 min after ketamine administration. Electropherograms are presented with a 0.01 AU y-axis shift. Key: S-K, S-ketamine; R-K, R-ketamine; S-NK, S-norketamine; RNK, R-norketamine; S-HNK, S-hydroxynorketamine; R-HNK, Rhydroxynorketamine; S-DHNK, S-5,6-Dehydronorketamine; IST, (+)-pseudoephedrine added as internal standard.

LOQ for all enantiomers was taken as 0.1 ␮M. For all enantiomers, typical interday precision values expressed as RSD were ⬍ 10% [9,18]. Four min after intake of S-ketamine, arterial plasma drug levels of S-ketamine and S-norketamine were 1.55 and 1.37 ␮M, respectively (Fig. 1C, Table 1). Corresponding values in venous plasma were 1.72 and 0.23 ␮M, respectively (Table 1). No R-ketamine and R-norketamine was detected in these samples. However, S-5,6-dehydronorketamine and one S-hydroxynorketamine metabolite were detected (Fig. 1C), which is comparable to previous results [20]. For the case of racemic ketamine administration, the enantiomers of ketamine, norketamine, and one hydroxynorketamine together with a small peak of S-5,6-dehydronorketamine were detected in arterial plasma (Fig. 2C). S-ketamine, R-ketamine, S-norketamine, and R-norketamine were determined to www.electrophoresis-journal.com

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Electrophoresis 2014, 35, 2863–2869 Table 1. Ketamine and norketamine enantiomer levels under xylazine anesthesiaa),

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2.18 2.84 0.35 1.55 1.72 1.08

2.13 2.70 0.34 ND ND ND

2.07 0.49 ND 1.37 0.23 0.08

1.89 0.47 ND ND ND ND

a) Samples collected 4 min after ketamine injection and immediately prior to euthanasia. b) ND refers to not detected.

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be 2.18, 2.13, 2.07, and 1.89 ␮M, respectively (Table 1). Corresponding values in venous plasma were 2.84, 2.70, 0.49, and 0.47 ␮M, respectively (Fig. 2D, Table 1). The values for arterial plasma compare well with those obtained in a controlled study with xylazine sedated Shetland ponies [8] and the differences in arterial versus venous plasma data are comparable to those observed in ponies under isoflurane anesthesia [7, 33]. Pronounced differences in arterial and venous ketamine drug levels was also observed in humans [34] and is assumed to be the result of differences in drug distribution and elimination and indicates that the choice of sampling site can be important. Furthermore, these data suggest that there might be no stereoselectivity in the ketamine to norketamine Ndemethylation when racemic ketamine is administered in presence of xylazine, which would be in agreement with the observations reported for ponies [8]. Comparison of brain and plasma data revealed some interesting differences. Blood is supplied to the brain of horses by five pairs of arteries: the rostral cerebral, middle cerebral, caudal cerebral, rostral cerebellar, and caudal cerebellar [35]. All these paired vessels originate from the basilar and internal carotid artery, which in turn are distal and direct branches of the common carotid artery, respectively. Thus, ketamine and norketamine distributions found in arterial plasma and brain should be similar if both compounds are transported to the same extent across the blood brain barrier. Based on our data (compare Fig. 1B and C, as well as Fig. 2B and C), however, this is not the case. It appears that ketamine is easier penetrating into the brain compared to norketamine as ketamine is more hydrophobic than norketamine. More ketamine than norketamine is found in brain whereas, after 4 min, the opposite is true for arterial plasma. The distribution in brain (Fig. 2B) is more similar to the pattern found in venous plasma (Fig. 2D). Analysis of the CSF samples of the two horses under xylazine sedation collected 4 min after administration of ketamine revealed smaller quantities of ketamine enantiomers than in plasma and norketamine levels that were below or close to LOQ (Table 1, for electropherograms see inserts in Figs. 1B and 2B). These data indicate that both, ketamine and norketamine penetrate into CSF. No apparent stereoselectivity was observed. No norketamine metabolites were detected in these samples and amounts of norketamine enantiomers are again smaller compared to those of ketamine. The latter aspect may again indicate that

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norketamine is penetrating to a smaller extent into CSF compared to ketamine. In order to be able to better compare plasma and CSF drug levels of ketamine and norketamine enantiomers, arterial plasma and CSF were taken from three patient horses that received 2.2 mg/kg racemic ketamine after anesthesia with acepromazine, medetomidine, and isoflurane (cf. Section 2.2). Electropherograms monitored with the extracts of plasma and CSF of one horse collected 7 min after ketamine administration are presented in Fig. 3. In addition to the peaks originating from the enantiomers of ketamine and norketamine, both enantiomers of 5,6-dehydronorketamine and one hydroxynorketamine metabolite were detected in plasma (Fig. 3A), which is comparable to the metabolite pattern monitored previously in other equine plasma samples [19, 20]. The enantiomers of medetomidine, which were part of the sedation employed and would interfere with R-5,6-dehydronorketamine and S-hydroxynorketamine under the given CE conditions, were not detected. This was assured by analyzing the extract with another system in which

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the medetomidine enantiomers migrated ahead of all enantiomers of ketamine and its metabolites (data not shown). The monitored plasma levels of S- and R-ketamine were 0.93 and 0.88 ␮M, respectively. Corresponding values for S- and R-norketamine were 2.60 and 1.58 ␮M, respectively. Analysis of CSF comprised all enantiomers of ketamine and norketamine, but hardly any 5,6-dehydronorketamine and only small peaks for the hydroxynorketamine metabolite (Fig. 3B). The monitored levels of S- and R-ketamine were 1.70 and 1.65 ␮M, respectively, whereas those of S- and R-norketamine 0.75 and 0.50 ␮M, respectively. It was interesting to find that the level of norketamine found in CSF was smaller than the amount of ketamine and this was the case for all three samples. This was also found to be true for arterial plasma of two horses but not with the one whose data are shown in Fig. 3A. Mean S/R ratios (RSD) of ketamine and norketamine enantiomers in the plasma samples of the three horses are 1.03 (3.11%) and 2.30 (39.9%), respectively. These data suggest that there is a stereoselective N-demethylation of ketamine under the given conditions with medetomidine comedication which is similar to data obtained with ponies that were anesthetized with isoflurane [6, 7]. Mean S/R ratios (RSD) of ketamine and norketamine enantiomers in the three CSF samples were found to be 1.02 (1.50%) and 1.50 (0.30%), respectively. These data suggest that there is a somewhat lower amount of R-norketamine compared to S-norketamine present in these samples. However, insufficient data are available thus far to make a clear claim for that inequality.

4 Concluding remarks The assays reported represent the first approaches for the determination of ketamine and norketamine enantiomers in brain and CSF. They are reproducible and require small mounts of tissues and body fluids only. The data obtained indicate that analysis of ketamine and norketamine enantiomers in brain by CE is feasible but requires an acidic preextraction of interferences prior to liquid/liquid extraction of the compounds of interest at alkaline pH whereas CSF can be treated as plasma. Following ketamine infusion, ketamine and norketamine enantiomers penetrate into brain and CSF. Metabolites of norketamine that are observed in plasma, including 5,6-dehydronorketamine, could either not be or only as tiny peaks detected in these samples. After i.v. administration of racemic ketamine, all enantiomers of ketamine and norketamine are detected in CSF and brain, whereas with infusion of S-ketamine, S-ketamine and S-norketamine are detected only. With racemic ketamine, amounts of norketamine enantiomers found in blood are different. First data indicate that the same could be true for these enantiomers in CSF for the case of sedation with medetomidine. Under anesthesia with xylazine, however, this might not be the case. More brain and CSF samples will have to be analyzed to properly elucidate the stereoselectivity of the N-demethylation pathway of ketamine, including possible metabolism in brain [36], and the presence of metabolites of norketamine in these tissues.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Furthermore, stereoselectivities in the ketamine and norketamine enantiomer transport from blood into brain tissue and CSF should be studied as well. This work was sponsored by Vetsuisse Z¨urich and the Swiss National Science Foundation. S-ketamine was kindly supplied by Dr. E. Graeub AG, Bern, Switzerland. The authors have declared no conflict of interest.

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Development of a method for analysis of ketamine and norketamine enantiomers in equine brain and cerebrospinal fluid by capillary electrophoresis.

Ketamine and norketamine are being transported across the blood brain barrier and are also entering from blood into cerebrospinal fluid (CSF). Enantio...
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