Separation of hydroxynorketamine stereoisomers using capillary electrophoresis with sulfated β‐cyclodextrin and highly sulfated γ‐cyclodextrin

Abstract

The racemic N-methyl-d-aspartate receptor antagonist ketamine is used in anesthesia, analgesia and the treatment of depressive disorders. It is known that interactions of hydroxylated norketamine metabolites and 5,6-dehydronorketamine (DHNK) with the α -nicotinic acetylcholine receptor and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor are responsible for the antidepressive effects. Ketamine and its first metabolite norketamine are not active on these receptors. As stereoselectivity plays a role in ketamine metabolism, a cationic capillary electrophoresis based method capable of resolving and analyzing the stereoisomers of four hydroxylated norketamine metabolites, norketamine and DHNK was developed. The assay is based on liquid/liquid extraction of the analytes from the biological matrix, electrokinetic sample injection across a buffer plug and analysis of the stereoisomers in a phosphate background electrolyte (BGE) at pH 3 comprising a mixture of sulfated β-cyclodextrin (5 mg/mL) and highly sulfated γ-cyclodextrin (0.1%). The method was used to analyze samples of an in vitro study in which ketamine was incubated with equine liver microsomes and in plasma samples of dogs and horses that were collected after an i.v. bolus injection of racemic ketamine.

Enantioselective capillary electrophoresis provides insight into the phase II metabolism of ketamine and its metabolites in vivo and in vitro

“…and Lin and Lua described acid‐labile conjugates of ketamine, norketamine and DHNK which are most likely N‐glucuronides. In this work, insight into the phase II metabolism of ketamine and its metabolites was investigated by enantioselective capillary electrophoresis (CE), an approach that was previously employed for the assessment of the stereoselectivity of phase I metabolic pathways in vivo and in vitro . Urine samples of equines that received an i.v.…”

“…Equine urine collected 120 min after racemic ketamine (2.2 mg/kg) injection analyzed (A,C,E) in its native form and (B,D,F) after enzymatic hydrolysis with β‐glucuronidase/arylsulfatase from Helix pomatia using (A,B) method I with lamotrigine as internal standard , (C,D) method II with D‐(+)‐norephedrine as internal standard and (E,F) method III without internal standard . Key: 1: (2R,6R)‐6‐hydroxynorketamine (R‐I), 2: R‐II, 3: (2S,6S)‐6‐hydroxynorketamine (S‐I), 4: R‐IV, 5: S‐II, 6: R‐III, 7: S‐IV, 8: R‐norketamine, 10: S‐dehydronorketamine, 11: S‐norketamine, 12: R‐dehydronorketamine, 13: R‐ketamine, 14: S‐ketamine, IST: internal standard.…”

“…Key: 1: (2R,6R)‐6‐hydroxynorketamine (R‐I), 2: R‐II, 3: (2S,6S)‐6‐hydroxynorketamine (S‐I), 4: R‐IV, 5: S‐II, 6: R‐III, 7: S‐IV, 8: R‐norketamine, 10: S‐dehydronorketamine, 11: S‐norketamine, 12: R‐dehydronorketamine, 13: R‐ketamine, 14: S‐ketamine, IST: internal standard. Metabolites I to IV refer to HNK stereoisomers of and peak numbers are the same as in .…”

“…fused‐silica capillary (Polymicro Technologies, Phoenix, AZ, USA) of 45 cm total length (effective length 35 cm). If not stated otherwise, the chemicals and drug standards used were the same as reported before . Method I comprised the reversed polarity approach of Sandbaumhüter et al.…”

“…It is based on electrokinetic analyte injection using a BGE with 50 mM sodium dihydrogenphosphate solution adjusted with phosphoric acid to pH 3, 5 mg/mL sulfated β‐cyclodextrin (lot 04426HJ, Sigma‐Aldrich, St. Louis, MO, USA) and 0.1% highly sulfated γ‐cyclodextrin . Sample preparation in the three assays was effected by liquid/liquid extraction of the analytes at alkaline pH and internal standards were used for quantification as described previously . All three methods were employed in this phase II metabolism study in order to be able to exclude interferences.…”