A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome

Abstract

A parallel chiral/achiral LC-MS/MS assay has been developed and validated to measure the plasma and urine concentrations of the enantiomers of ketamine, (R)- and (S)-Ket, in complex regional pain syndrome (CRPS) patients receiving a 5-day continuous infusion of a sub-anesthetic dose of (R,S)-Ket. The method was also validated for the determination of the enantiomers of the Ket metabolites norketamine, (R)- and (S)-norKet and dehydronorketamine, (R)- and (S)-DHNK, as well as the diastereomeric metabolites hydroxynorketamine, (2S,6S)-/(2R,6R)-HNK and two hydroxyketamines, (2S,6S)-HKet and (2S,6R)-Hket. In this method, (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK and the diastereomeric hydroxyl-metabolites were separated and quantified using a C(18) stationary phase and the relative enantiomeric concentrations of (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK were determined using an AGP-CSP. The analysis of the results of microsomal incubations of (R)- and (S)-Ket and a plasma and urine sample from a CRPS patient indicated the presence of 10 additional compounds and glucuronides. The data from the analysis of the patient sample also demonstrated that a series of HNK metabolites were the primary metabolites in plasma and (R)- and (S)-DHNK were the major metabolites found in urine. The results suggest that norKet is the initial, but not the primary metabolite and that downstream norKet metabolites play a role in (R,S)-Ket-related pain relief in CRPS patients.

Published by Elsevier B.V.

Figures

Figure 1

The key metabolic pathways of ketamine (Ket), where: A. The direct conversion of Ket to hydroxy-ketamine (HKet) metabolites; B. The conversion of Ket to norKet to dehydronorketamine (DHNK) and hydroxy-norketamine (HNK) metabolites.

Figure 1

The key metabolic pathways of ketamine (Ket), where: A. The direct conversion of Ket to hydroxy-ketamine (HKet) metabolites; B. The conversion of Ket to norKet to dehydronorketamine (DHNK) and hydroxy-norketamine (HNK) metabolites.

Figure 2

The enantioselective separations of (R,S)-Ket (peak 1R and 1S), (R,S)-norKet (2R and 2S) and (R,S)-DHNK (3R and 3S) on the AGP-CSP, where: A. Standard solutions; B. An extracted plasma sample obtained on Day 3 from a CRPS patient receiving (R,S)-Ket; C. An extracted urine sample obtained on Day 3 from a CRPS patient receiving (R,S)-Ket. See text for experimental details.

Figure 2

The enantioselective separations of (R,S)-Ket (peak 1R and 1S), (R,S)-norKet (2R and 2S) and (R,S)-DHNK (3R and 3S) on the AGP-CSP, where: A. Standard solutions; B. An extracted plasma sample obtained on Day 3 from a CRPS patient receiving (R,S)-Ket; C. An extracted urine sample obtained on Day 3 from a CRPS patient receiving (R,S)-Ket. See text for experimental details.

Figure 2

The enantioselective separations of (R,S)-Ket (peak 1R and 1S), (R,S)-norKet (2R and 2S) and (R,S)-DHNK (3R and 3S) on the AGP-CSP, where: A. Standard solutions; B. An extracted plasma sample obtained on Day 3 from a CRPS patient receiving (R,S)-Ket; C. An extracted urine sample obtained on Day 3 from a CRPS patient receiving (R,S)-Ket. See text for experimental details.

Figure 3

The achiral separation of (R,S)-Ket (peak 1), (R,S)-norKet (2), (R,S)-DHNK (3), (2S,6S;2R,6R)-HNK (4a), (2S,6R;2R,6S)-HNK (4b), (2S,6S)-HKet (5a) and (2S,6R)-HKet (5b) on a C18 column, where: A. Standard solution; B. Extracted sample obtained from the incubation of (R)-Ket with human microsomal preparation; C. Extracted sample obtained from the incubation of (S)-Ket with human microsomal preparations. See Table 2 for the assignment of the structures for peaks 4c–f, 5c–d and 6a–e and the text for experimental details.

Figure 3

The achiral separation of (R,S)-Ket (peak 1), (R,S)-norKet (2), (R,S)-DHNK (3), (2S,6S;2R,6R)-HNK (4a), (2S,6R;2R,6S)-HNK (4b), (2S,6S)-HKet (5a) and (2S,6R)-HKet (5b) on a C18 column, where: A. Standard solution; B. Extracted sample obtained from the incubation of (R)-Ket with human microsomal preparation; C. Extracted sample obtained from the incubation of (S)-Ket with human microsomal preparations. See Table 2 for the assignment of the structures for peaks 4c–f, 5c–d and 6a–e and the text for experimental details.

Figure 3

The achiral separation of (R,S)-Ket (peak 1), (R,S)-norKet (2), (R,S)-DHNK (3), (2S,6S;2R,6R)-HNK (4a), (2S,6R;2R,6S)-HNK (4b), (2S,6S)-HKet (5a) and (2S,6R)-HKet (5b) on a C18 column, where: A. Standard solution; B. Extracted sample obtained from the incubation of (R)-Ket with human microsomal preparation; C. Extracted sample obtained from the incubation of (S)-Ket with human microsomal preparations. See Table 2 for the assignment of the structures for peaks 4c–f, 5c–d and 6a–e and the text for experimental details.

Figure 4

The analysis of the concentrations of (R,S)-Ket and its metabolites in clinical samples obtained from a CRPS patient on Day 3 of a 5-Day infusion of (R,S)-Ket obtained using an achiral chromatographic method, where: A. An extracted plasma sample, in which the peak corresponding to (R,S)-Ket (peak 1) has been deleted from the chromatogram; B. An extracted urine sample in which the peaks corresponding to the glucuronides of the HNKs metabolites (6a–e) have been deleted from the chromatogram; C. An extracted urine sample in which only the peaks corresponding to the glucuronides of the HNKs metabolites (6a–e) are presented. See Table 2 for the assignment of the structures for peaks 4c–f, 5c–d and 6a–e and the text for experimental details.

Figure 4

The analysis of the concentrations of (R,S)-Ket and its metabolites in clinical samples obtained from a CRPS patient on Day 3 of a 5-Day infusion of (R,S)-Ket obtained using an achiral chromatographic method, where: A. An extracted plasma sample, in which the peak corresponding to (R,S)-Ket (peak 1) has been deleted from the chromatogram; B. An extracted urine sample in which the peaks corresponding to the glucuronides of the HNKs metabolites (6a–e) have been deleted from the chromatogram; C. An extracted urine sample in which only the peaks corresponding to the glucuronides of the HNKs metabolites (6a–e) are presented. See Table 2 for the assignment of the structures for peaks 4c–f, 5c–d and 6a–e and the text for experimental details.

Figure 4

The analysis of the concentrations of (R,S)-Ket and its metabolites in clinical samples obtained from a CRPS patient on Day 3 of a 5-Day infusion of (R,S)-Ket obtained using an achiral chromatographic method, where: A. An extracted plasma sample, in which the peak corresponding to (R,S)-Ket (peak 1) has been deleted from the chromatogram; B. An extracted urine sample in which the peaks corresponding to the glucuronides of the HNKs metabolites (6a–e) have been deleted from the chromatogram; C. An extracted urine sample in which only the peaks corresponding to the glucuronides of the HNKs metabolites (6a–e) are presented. See Table 2 for the assignment of the structures for peaks 4c–f, 5c–d and 6a–e and the text for experimental details.