Microassay for ketamine and metabolites in plasma and serum based on enantioselective capillary electrophoresis with highly sulfated γ-cyclodextrin and electrokinetic analyte injection

Abstract

For the assessment of stereoselective aspects of the metabolism of ketamine, an enantioselective CE-based microassay for determination of the stereoisomers of ketamine and three of its major metabolites in plasma and serum was developed. The assay is based on liquid/liquid extraction of the analytes of interest at alkaline pH from 0.05 mL plasma or serum followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3.0 phosphate buffer containing 0.66% of highly sulfated γ-cyclodextrin (HS-γ-CD). Key parameters for optimization are identified as being the amount of HS-γ-CD in the BGE, the length of the buffer plug and its concentration, the duration of electrokinetic injection, and the extraction medium. Diluted buffer in the plug is employed to ascertain sufficient analyte stacking due to a combination of field amplification and complexation. The newly developed microassay is robust (intraday and interday RSD < 5% and <9%, respectively) and well suited to determine enantiomer levels of ketamine and its metabolites down to 10 ng/mL. It is more sensitive, uses less plasma or serum, organic solvent, and analysis time compared to previous CE-based assays and was successfully applied to monitor ketamine, norketamine, 5,6-dehydronorketamine (DHNK), and 6-hydroxynorketamine (6HNK) stereoisomer levels in plasma of a Beagle dog that received a bolus of racemic ketamine or S-ketamine after sevoflurane anesthesia. The data suggest that the formation of DHNK and 6HNK occur stereoselectively.

Chiral Separations using Miniaturized Techniques: State of the Art and Perspectives

“…The chiral selector, positively charged at that pH, was moving in the opposite direction of the analytes (negatively charged), avoiding, in such a way, the presence of vancomycin in the interface . Other studies employing these techniques include the analysis of: mephenytoin and some of its metabolites;ecstasy and two of its metabolites in urine using MEKC with CDs; and ketamine and its metabolites in equine plasma and urine and in dog plasma, applying CE with charged CDs. Other applications related to the determination of drug enantiomers have also been reported …”

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.…”

“…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 was employed for analysis of ketamine, norketamine and DHNK. Method II represents an assay with electrokinetic analyte injection across a pressure injected buffer plug followed by cationic analysis in a BGE composed of 100 mM phosphate buffer (pH 3.0) and 0.66% highly sulfated γ‐cyclodextrin . This assay was applied to analyze the stereoisomers of ketamine, norketamine, DHNK and selected HNKs.…”