On the Surprising Retention Order of Ketamine Analogs Using a Biphenyl Stationary Phase

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An unexpected retention order for the ketamine analogs methoxpropamine (MXPr) and methoxisopropamine (MXiPr) was observed when using a biphenyl stationary phase for liquid chromatography-mass spectrometry (LC–MS). For separations of isomeric pairs of drugs (ketamine analogs, tryptamines, fentanyls, and nitazenes), branched-chain compounds are eluted before straight-chain compounds when using acetonitrile or methanol in mobile phases with C18 or biphenyl stationary phases. However, when a biphenyl stationary phase was paired with methanol, the opposite retention order for MXPr and MXiPr occurred, which was not observed when using acetonitrile in the mobile phase. The phenomenon persisted in biphenyl columns from two vendors. This study suggests additional work may be needed to understand the retention mechanisms when using biphenyl stationary phases. It is an example that we can still expect the unexpected when investigating alternative stationary phases of reversed-phase liquid chromatography (RPLC).

Ketamine is categorized as a dissociative with a number of medical uses. It is employed in human and veterinary medicine for its anesthetic properties, and is also used for treating pain and (treatment-resistant) depression (1,2). However, ketamine and ketamine analogs are also used recreationally, posing significant health risks and legal consequences, and are therefore focus analytes in forensic or toxicological analysis. Two illicitly used ketamine analogs are the structural isomers methoxpropamine and methoxisopropamine (MXPr and MXiPr, Figures 1a and 1b). MXPr and MXiPr affect the activity of N-methyl-D-aspartate (NMDA) receptors in the central nervous system, and are sometimes referred to as designer drugs (3,4). Establishing a dedicated methodology for ketamine analogs and other emerging drugs is crucial to identifying these compounds in samples. In addition, it is necessary to have a sound method to study the metabolism, pharmacology, and toxicity of these potentially dangerous compounds.

A key tool for detecting drugs is mass spectrometry (MS), which allows measurements to be performed based on the mass-to-charge ratio (m/z) of substances. The MS instrument also enables us to fragment molecules of interest for subsequent measurements of the fragments. This two-step MS process (tandem mass spectrometry, or MS/MS) provides highly sensitive and selective measurements in forensics and toxicology, as well as a plethora of other applications. However, when two drugs are isomeric and additionally produce identical mass spectrometric data, the MS instrument will not provide sufficient information to distinguish the compounds; Figure 1 shows four examples of such isomeric pairs of drugs (featuring straight-chains or branched-chains). Nevertheless, separating the isomeric pairs prior to MS analysis can allow for the drugs to be confidently identified. For bioanalysis, liquid chromatography (LC) is often applied, as done in this study. Most often, LC separates compounds according to hydrophobicity (reversed-phase LC, or RPLC), pumping the sample through a separation column filled with particles featuring a hydrophobic C18 stationary phase (SP) (Figure 2a). However, at our laboratory, a biphenyl SP (Figure 2b) is also commonly used in, for example, forensic analysis, providing RPLC separations with excellent capabilities for separating isomers of several important drug classes (5,6). Regarding the mapping of biphenyl phase selectivity, factors such as polarity, shape selectivity, and polarizability have been discussed (7), but the driving forces of biphenyl selectivity are not yet completely understood.

Figure 1: Chemical structure of isomeric pairs of four drug classes. Ketamine analogs: (a) MXPr and (b) MXiPr, tryptamines; (c) 5-MeO-DPT and (d) 5-MeO-DiPT, fentanyls; (e) 4-FBF and (f) 4-FiBF; and nitazenes: (g) protonitazene and (h) isotonitazene. These compound pairs are termed isomeric, and as both their molecular mass and main mass fragments are identical, they represent challenges for analysis by mass spectrometry alone.

Figure 1: Chemical structure of isomeric pairs of four drug classes. Ketamine analogs: (a) MXPr and (b) MXiPr, tryptamines; (c) 5-MeO-DPT and (d) 5-MeO-DiPT, fentanyls; (e) 4-FBF and (f) 4-FiBF; and nitazenes: (g) protonitazene and (h) isotonitazene. These compound pairs are termed isomeric, and as both their molecular mass and main mass fragments are identical, they represent challenges for analysis by mass spectrometry alone.

Figure 2: Chemical structure of two RPLC SPs: (a) biphenyl; and (b) C18.

Figure 2: Chemical structure of two RPLC SPs: (a) biphenyl; and (b) C18.

In this article, we illustrate LC–MS/MS analysis of various isomeric pairs of drugs (straight-chain vs. branched-chain), with a particular emphasis on the retention time order of MXPr and MXiPr, which retention behavior deviated noteworthy from the other straight or branched pairs as a function of SP and membrane phase (MP), underlining that unexpected retention behaviors can be observed, depending on the combination of compound and separation conditions.

aterials and Methods

Chemicals

Methoxpropamine (≥ 98%, MXPr), methoxisopropamine (≥ 98%, MXiPr), 5-methoxy-N,N-dipropyltryptamine (≥ 98%, 5-MeO-DPT) , 5-methoxy-N,N-diisopropyltryptamine (≥ 98%, 5-MeO-DiPT), 4-fluorobutyrylfentanyl (≥ 98%, 4-FBF), and protonitazene (≥ 98%) were obtained from Cayman Chemical Company; 4-fluoroisobutyrylfentanyl (99.3%, 4-FiBF) and isotonitazene (99.5%) were obtained from Chiron.

Type 1 water (18.2 MΩ, H2O) was purified by a Milli-Q Advantage A10 purification system equipped with a Q-POD remote dispenser with a 0.22 μm filter from Merck KgaA. Acetonitrile (≥ 99.9%, ACN) was purchased from Avantor Performance Materials. Methanol (≥ 99.9%, MeOH) was purchased from Merck. Ammonium formate (≥ 98.5%) was acquired from VWR in Leuven, Belgium, while formic acid (98%) was obtained from VWR in Briare, France.

Equipment

Four different columns were used: a Kinetex Biphenyl column (2.1 × 100 mm, 1.7 μm) from Phenomenex; a Raptor Biphenyl column (2.1 × 100 mm, 1.8 μm) from Restek ; an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm); and an Acquity UPLC T3 HSS C18 column (2.1 × 100 mm, 1.8 μm), both from Waters.

The LC–MS used was manufactured by Agilent Technologies, comprising a 1290 Infinity II LC System with a high-speed pump, multisampler, and multicolumn thermostat, and a 6495D Triple Quadrupole MS.

Solutions

A mixture of the straight-chain isomers (MXPr, 5-MeO-DPT, 4-FBF, protonitazene) was prepared at a concentration of 1 μM of each in MeOH/H2O (1/1, v/v). A mixture of the branched-chain isomers (MXiPr, 5-MeO-DiPT, 4-FiBF, isotonitazene) was prepared at a concentration of 1 μM of each in MeOH/H2O (1/1, v/v).

Analysis

Each mixture was analyzed by LC–MS using the four columns and 10 mM ammonium formate buffer (adjusted to pH 3.1 by adding formic acid) as MP A and MeOH or ACN as MP B. For each analysis, 3 μL of the mixture was injected into the system with a flow rate of 0.600 mL/min. Separation was performed at 60 °C using the following gradient: 0–14.5 min; 5–50% MP B, 14.5–15 min; 50–100% MP B, 15–15.5 min; 100% MP B, 15.5–15.6; 100–5% MP B, 15.6–17.00; 5 % MP B. Mass spectrometric conditions for the compounds were optimized and key parameters are summarized in Table I.

Table I: Chemical formula, theoretical mass, and MRM transitions for the isomeric pairs.

Table I: Chemical formula, theoretical mass, and MRM transitions for the isomeric pairs.

Results and Discussion

Retention Time Stability

A single assay was conducted for each of the four columns, with nine injections for each of the isomer mixtures for both MeOH and ACN (36 injections in total per column). There was minimal drift in the retention times observed using the Kinetex Biphenyl column (RSD < 0.17%), the Raptor Biphenyl column (RSD < 0.10%), and the UPLC T3 HSS C18 column (RSD < 0.08%). Slightly more drift was noted in the series employing the UPLCBEH C18 column (RSD < 2.3%). Nevertheless, the retention order of all isomeric pairs could be confidently determined.

Chromatographic Retention Orders of Isomeric Pairs Using C18 Stationary Phase with MeOH and ACN

When employing a C18 SP (Figure 3), the branched-chain isomers were eluted prior to the straight-chain isomers for all four drug classes included in this study. This was the case with MeOH (Figure 3a) and ACN (Figure 3b) as organic modifiers in the MP. However, modifications in the retention order of the tryptamines and ketamine analogs were observed when changing the MP; with MeOH, tryptamines were eluted before ketamine analogs, but with ACN, the ketamine analogs were sandwiched between the tryptamines. This is in accordance with selectivity differences that can arise when switching the MP in RPLC.

Figure 3: Overlaid chromatograms (straight-chain isomers in red, and branched-chain isomers in blue) obtained using the UPLC T3 HSS C18 column and (a) MeOH or (b) ACN in MP. The retention orders were the same when using the UPLC BEH C18 column.

Figure 3: Overlaid chromatograms (straight-chain isomers in red, and branched-chain isomers in blue) obtained using the UPLC T3 HSS C18 column and (a) MeOH or (b) ACN in MP. The retention orders were the same when using the UPLC BEH C18 column.

Chromatographic Retention Orders of Isomeric Pairs Using Biphenyl with ACN

Using a biphenyl SP and ACN as an organic modifier in the MP (Figure 4b) also resulted in the branched-chain isomers eluting prior to the straight-chain isomers for all four drug classes. Compared to using the C18 SP with ACN, changes in retention orders of the drug classes were again seen related to the tryptamines and ketamine analogs. With biphenyl, both ketamine analogs were eluted before both tryptamines instead of being sandwiched between them as with C18. However, it is difficult to make strong claims regarding the superiority of one SP over the other regarding these particular compounds.

Figure 4: Overlaid chromatograms (straight-chain isomers in red, and branched-chain isomers in blue) obtained using the Raptor Biphenyl column and (a) MeOH or (b) ACN in MP. The retention orders were the same when using the Kinetex Biphenyl column.

Figure 4: Overlaid chromatograms (straight-chain isomers in red, and branched-chain isomers in blue) obtained using the Raptor Biphenyl column and (a) MeOH or (b) ACN in MP. The retention orders were the same when using the Kinetex Biphenyl column.

hromatographic Retention Orders of Isomeric Pairs Using Biphenyl with MeOH

Using a biphenyl SP and MeOH as an organic modifier in the MP (Figure 4a) resulted in a surprising retention order of the ketamine analogs. The branched-chain isomers were eluted prior to the straight-chain isomers for the fentanyls, nitazenes, and tryptamines (5,6,8), as described previously. This was not the case for the ketamine analogs. Instead, the branched-chain MXiPr were eluted after the straight-chain MXPr. This unexpected result was confirmed with new solutions, runs, and other columns. Apart from this “deviation," we could not make strong conclusions on which column or MP was the best choice. The retention orders across the different conditions are summarized in Table II.

Table II: Retention order of isomeric pairs of tryptamines, ketamine analogs, fentanyls, and nitazenes when using either biphenyl or C18 as SP and either methanol (MeOH) or acetonitrile ( ACN) as organic modifier in the mobile phase (MP).

Table II: Retention order of isomeric pairs of tryptamines, ketamine analogs, fentanyls, and nitazenes when using either biphenyl or C18 as SP and either methanol (MeOH) or acetonitrile ( ACN) as organic modifier in the mobile phase (MP).

Conclusions

Differences in the retention order of drugs of forensic interest confirm that the selectivity of C18 SPs changes when using different MPs. Even though reversed-phase separations are often simplistically described as being based on hydrophobicity, the traits of the chemicals involved may alter retention orders. For instance, the protic nature of MeOH and non-protic nature of ACN can lead to variations in retention. The selectivity of biphenyl SPs can be even more challenging to predict, as these are not yet fully understood (7). Chemical traits that may influence the chromatography include the analyte’s hydrophobicity, shape, and dipole moment, in addition to the MP, as exemplified in this study. Exploring these SPs and MPs may allow for improved separations. Still, as shown with the case study of ketamine analogs, the prediction of elution order must be done cautiously, especially when using chromatographic traits to predict the structure of unknown compounds like new designer drugs.

Acknowledgments

Financial support was obtained from the Research Council of Norway through its Centres of Excellence funding scheme, project number 262613, and partly from the UiO:Life Science convergence environment funding scheme. S.R.W. is a member of the National Network of Advanced Proteomics Infrastructure (NAPI), which is funded by the Research Council of Norway INFRASTRUKTUR-program (project number: 295910).

About the Authors

Line Noreng is a master's student in bioanalytical chemistry at the Department of Chemistry, University of Oslo (Norway). She conducted her master's project on ketamine analogs at Oslo University Hospital.

Steven Ray Wilson is a professor at the Department of Chemistry at the University of Oslo. His work focuses on LC-MS of biosamples, including organoids, organ-on-chip systems, and a number of other clinical applications in collaboration with Oslo University Hospital. Direct correspondence to: stevenw@kjemi.uio.no

Elisabeth Leere Øiestad is Senior Scientist at Oslo University Hospital and Associate Professor at the Department of Pharmacy, University of Oslo. Her research interests are analytical forensic toxicology, micro sampling, membrane extraction, and method development, especially for new psychoactive substances or alternative biological matrices.

Åse Marit Leere Øiestad is a senior scientist at Oslo University Hospital. Her research interests include analytical forensic toxicology, development of analytical methods with mass spectrometric techniques, new psychoactive substances, and post-mortem toxicology.

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