Ketamine, a well-known dissociative anesthetic, has inspired the synthesis of numerous structural analogues that are increasingly encountered on the illicit drug market as designer drugs.
These analogues often involve minor chemical modifications to the parent molecule, designed to evade existing narcotic regulations while retaining similar psychoactive effects. Many ketamine derivatives, like ketamine itself, possess a chiral center and thus exist as two enantiomers, whose pharmacological properties can differ markedly. Although the distinct activities of S- and R-ketamine are well established, little is known about the enantioselective behavior of emerging ketamine analogues.
In this work, enantioseparation strategies using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) were systematically developed and optimized for 11 novel ketamine derivatives, several of which were resolved into enantiomers for the first time.
HPLC separations employed four polysaccharide-based chiral stationary phases, while coupling to an optical rotation detector (ORD) enabled determination of the absolute enantiomer elution order. CE separations were performed using 2% (w/v) acetyl-β-cyclodextrin or carboxymethyl-β-cyclodextrin as chiral selectors in 10 mM disodium hydrogen phosphate buffer. All investigated compounds achieved at least partial resolution in both systems.
These findings provide new analytical tools for chiral profiling of ketamine analogues, supporting forensic and toxicological investigations in the context of the rapidly evolving designer drug landscape.
Eleven ketamine derivatives and a solution of enantiopure S-(+)-ketamine, all present as hydrochloride salts, were analyzed by both capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC).
Ketamine was purchased from Fagron GmbH, Glinde, Germany, and S-(+)-ketamine was obtained as Ketanest® S (5 mg/mL ampoules, Pfizer Manufacturing Belgium NV, Puurs, Belgium) supplied via Kwizda Pharma Distribution GmbH, Vienna, Austria.
The ketamine derivatives were acquired from various online NPS (new psychoactive substance) vendors or provided by the Austrian Federal Police for research purposes. All compounds were verified by gas chromatography–mass spectrometry (GC–MS) and, where necessary, by nuclear magnetic resonance (NMR) spectroscopy prior to analysis.
All chemicals were of analytical reagent grade or higher.
Approximately 1 mg of each ketamine derivative was dissolved in 1 mL of analytical-grade water.
The Ketanest® S solution (5 mg/mL S-(+)-ketamine in saline) was diluted to 1 mg/mL with analytical-grade water (Fisher Scientific, UK).
For some derivatives, an ultrasonic bath was used for complete dissolution.
CE analyses were performed using an Agilent 7100 Capillary Electrophoresis System (Agilent Technologies Inc., Santa Clara, CA, USA), equipped with single-wavelength UV detection at 210 nm.
The applied voltage was +25 kV, and the cassette temperature was maintained at 25 °C.
A fused silica capillary (50 μm i.d., 68.5 cm total length, 60 cm effective length) from MicroQuartz GmbH, Munich, Germany was used.
Sample introduction was carried out by dynamic injection (10 mbar for 5 s).
The background electrolyte (BGE) consisted of 10 mM Na₂HPO₄ (pH 2.5, adjusted with diluted H₃PO₄) containing 2% (w/v) of either acetyl-β-cyclodextrin or carboxymethyl-β-cyclodextrin. Cyclodextrins were fully dissolved by sonication for a few minutes.
All BGE solutions were filtered through 0.45 μm single-use syringe filters (Carl Roth, Germany) before use.
HPLC analyses were conducted using an Agilent 1260 Infinity II LC System (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a variable-wavelength UV detector and a Jasco OR-4090 Optical Rotation Detector (ORD) (Jasco Corporation, Tokyo, Japan).
UV detection was performed at 254 nm. All measurements were conducted under isocratic conditions at 25 ± 1 °C, with a flow rate of 1.0 mL/min and injection volume of 1 μL.
Two different mobile phases were used:
Four polysaccharide-based chiral stationary phases (CSPs) from Phenomenex Inc., Torrance, CA, USA were evaluated (see Table 2 for details).
Approximately 1 mg of each hydrochloride salt was dissolved in 1 mL of methanol.
For certain samples, 2-propanol was used as solvent, with sonication applied where necessary to ensure complete dissolution.
The Ketanest® S solution (5 mg/mL) was diluted with methanol to a final concentration of 1 mg/mL.