Ionisation of hydrophobic small molecules with poor ionisation potential in a complex mixture is challenging. These ions are suppressed in favour of ions corresponding to species with higher abundance or propensity towards ionisation.106,251 Ion suppression can be avoided by the separation of components in a mixture such that each analyte within the sample is introduced individually into the mass spectrometer. The separation process is carried out by reversed phase LC on an Acquity UPLC (Waters Corp., UK). Reversed phase LC separation can be challenging for hydrophobic analytes due to high column affinity and poor solubility in the polar mobile phase. As a result analyses can be time consuming and exhibit issues with poor peak shape and separation of components. These issues can be avoided by developing optimised chromatography for the system of interest, in this case small molecule intrinsic lipidation.
Development of chromatography for the study of small molecule intrinsic lipidation requires selection of a suitable stationary phase. A BEH C18 column (Waters Corp., UK) provides an excellent option due to availability, understanding of the column chemistry, and previous success in the separation of small molecules including hydrophobic analytes.11,39
A binary mixture of H2O (0.1 % formic acid) and MeCN (0.1 % formic acid) was selected as the mobile phase for analysis, given the compatibility with reversed phase LC, solubility of analytes in the solvent, and previous success in small molecule analysis. Building upon previous LC carried out for small molecules, the two solvents ran in a linear gradient at a flow rate of 0.400 mL/ min over a nine minute period.77 Ensuring parity with ionisation optimisation, procaine 2 was selected as the small molecule utilised to develop chromatography for small molecule intrinsic lipidation. Inclusion of N -acylated procaine and lysolipid standards provide a full picture of the predicted contents of a small molecule intrinsic lipidation reaction mixture. As such, 0.5 µg mL−1 solutions in 1:1 H2O:MeCN were prepared for procaine 2, N-palmitoyl procaine 35, and the lysolipid OPC.
Fig. 5.10 combines the the individual chromatograms for each of the predicted components of procaine lipidation under the chromatographic conditions described. Symmetrical and sharp peaks are observed, suggesting success in this aspect of chromatography. However, poor chromatographic resolution is observed for the peaks corresponding to N -palmitoyl procaine 35, and lysolipid OPC. Given the low abundance of acylated small molecule expected to form through small molecule intrinsic lipidation, and the poor ionisation potential of these
112 5.5. Optimisation of Chromatography
products, such poor resolution was predicted to result in ion suppression issues.
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Figure 5.10 Combined traces of procaine 2 (red), N -palmitoyl procaine 35 (green), and36 lysolipid OPC (blue). Analysis was conducted on a BEH C18 column (Waters Corp., UK) with the nine minute linear gradient of mobile phase A (H2O containing 0.1 % formic acid) and mobile phase B (MeCN containing 0.1 % formic acid) shown in grey.
Modification to the linear gradient was attempted in order to improve chromatographic resolution between N -palmitoyl procaine 35 and lysolipid by-products, thus avoiding ion suppression. Two modified gradients were developed, each reducing the gradient increase prior to acylated small molecule and lysolipid elution, thus facilitating improved resolution.
Fig. 5.11 presents chromatographic traces for species of interest under the two modified gradients highlighted in grey. Improved chromatographic resolution between N -palmitoyl procaine 35 and OPC is observed for both new gradients, however the second provides better separation attributed to its extended shallow gradient. Given sharp and symmetrical peak shape is also maintained, modified gradient two was determined to provide optimum chromatographic conditions for study of procaine 2 intrinsic lipidation.
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35 Figure 5.11 Chromatographic traces of procaine 2 (red), N -palmitoyl procaine 35 (green), and lysolipid OPC (blue) run on a BEH C18 column (Waters Corp., UK): (a) modified gradient one; (b) modified gradient two. Gradients both use mobile phase A (H2O containing 0.1 % formic acid), and mobile phase B (MeCN containing 0.1 % formic acid) shown in grey.
Validation of the chromatographic conditions developed through the study of procaine 2 is required in order to confirm their suitability for the more general study of small molecule
Chapter 5. Small Molecule Intrinsic Lipidation 113
intrinsic lipidation. One validation mechanism is the analysis of synthetically prepared acylated standards N -palmitoyl propranolol 12, N -palmitoyl tetracaine 37, and N -oleoyl procaine 36.
Comparing chromatograms of these acylated small molecules with those of lysolipid OPC, Fig. 5.12, allows for success of the optimised chromatography conditions to be determined.
Analysis revealed defined peak shapes and separation between reaction mixture components for all small molecule systems studied, validating the developed chromatography conditions.
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Figure 5.12 Chromatographic traces of lysolipid OPC (blue) synthetically acylated small molecules (green): (a) N -palmitoyl propranolol 12; (b) N -palmitoyl tetracaine 37; (c) N -oleoyl procaine 36. Analysis was conducted using modified gradient two on a BEH C18 column (Waters Corp., UK). Mobile phase A (H2O containing 0.1 % formic acid) and mobile phase B (MeCN containing 0.1 % formic acid) are shown in grey.
Application of optimised chromatography conditions of real small molecule intrinsic lipidation reaction mixtures is necessary to fully validate them. Samples containing DOPC liposomes and small molecule were prepared and incubated under physiological conditions for 72 hours.
Fig. 5.13 presents the small molecules selected for this validation process. Propranolol 1, procaine 2 and tetracaine 3 are included due to their pharmaceutical relevance and key role within the optimisation process. Small molecules 4 and 8 from the original screen of small molecule intrinsic lipidation of the QToF Premier (Waters Corp., UK) are also included. Otherwise, changes have been made to small molecules included within the set due to availability of small molecules, reassessment of potential reactivity, and the necessity of structural and functional diversity to ensure chromatographic validation.
114 5.5. Optimisation of Chromatography
Figure 5.13 Cationic amphiphilic small molecules studied during preliminary screening of small molecule intrinsic lipidation, in an attempt to validate optimised chromatographic conditions.
Figure 5.14 Chromatographic traces highlighting key issues associated with analysis of reaction mixtures under optimised chromatographic conditions on a BEH C18 column: (a) broad phospholipid peak (red) coelutes with acylated small molecule (*) resulting in ion suppression; (b) retention time fluctuation of second oleoylated propranolol peak (*) between analyses; (c) phospholipid eluting in blank run between analysis indicating column retention.
Validation of the optimised chromatographic conditions proved unsuccessful when faced with this more diverse selection of small molecules. Under the optimised conditions ion suppression of some acylated small molecules is likely, given their observed co-elution with phopholipid, shown in Fig. 5.14 (a). Furthermore, hydrophobic phospholipid present at high concentrations within the samples is retained by the BEH C18 column, Fig. 5.14 (c), causing poor peak shape and crossover contamination between samples. Propranolol 1 reaction mixtures exhibit peaks corresponding to the m/z of protonated oleoyl propranolol at two separate retention times. The
Chapter 5. Small Molecule Intrinsic Lipidation 115
later eluting of these peaks coelutes with phospholipid and undergoes significant fluctuations in retention time, Fig. 5.14 (b), making examination challenging under the chromatographic conditions.
Development of chromatography for the study of small molecule intrinsic lipidation proved impossible using a BEH C18 column (Waters Corp., UK). Improved chromatography was therefore developed by considering alternative stationary phases with proven records in the separation of hydrophobic or aromatic small molecules. Table 5.2 presents four available columns which fit this criteria, and describes the differences in their column chemistry.99
Column Name Column Chemistry
Acquity CSH C18
Trifunctional C18 ligand bonded to a Charged Surface Hybrid (CSH) particle substrate
Acquity BEH Phenyl Trifunctional C6 Phenyl, bonded to Ethylene Bridged Hybrid (BEH) substrate
Acquity BEH Shield RP18 Monofunctional embedded polar C18, bonded to Ethylene Bridged Hybrid (BEH) substrate
Acquity BEH C8 Trifunctional C8, bonded to Ethylene Bridged Hybrid (BEH) substrate
Table 5.2 Stationary phase column chemistries known to excel in the separation of hy-drophobic or aromatic small molecules.99
Considering the previous challenges associated with developing chromatography for small molecule intrinsic lipidation utilising acylated standards, a different approach was adopted on this occasion. In an analogous fashion to previously attempted validation experiments, reaction mixtures containing liposomes with either propranolol 1 or compound 46 were prepared. Propranolol 1 was selected due the interesting observation of multiple peaks of oleoylated propranolol m/z, one of which elutes at a late retention time. Compound 46 was selected due to the co-elution of acylated product and lysolipid, and its significant structural and functional differences to propranolol 1. Liposomes were prepared from phospholipid POPC, increasing the diversity of acyl chains available for transfer, and providing a wider variety of acylated small molecule and lysolipid products.
After 72 hours samples were analysed on each of the four stationary phases using first a linear gradient of H2O:MeCN (both containing 0.1 % formic acid) at a flow rate of 0.400 mL/ min over nine minutes, and then a linear gradient of H2O:MeOH (H2O containing 0.1 % formic acid) at a flow rate of 0.400 mL/ min over nine minutes. MeOH was included within the mobile phase in this optimisation step due to the increased solubility of lysolipids and phospholipids in MeOH
116 5.5. Optimisation of Chromatography
compared to MeCN, and the lack of success in previous optimisation attempts. However, MeOH was immediately ruled out as a mobile phase component based on chromatograms resulting from the linear nine minute gradient, Fig. 5.15. Delays were observed in the elution of reaction mixture components across all column types when MeOH was employed as a mobile phase. Delayed elution proved particularly problematic for hydrophobic analytes, including acylated small molecules, resulting in an undesirable increase in run time, coelution, and reduced peak quality.
Figure 5.15 Examples of propranolol 1 reaction mixtures with POPC analysed on five stationary phase types: (a) Acquity BEH C18; (b) Acquity CSH C18; (c) Acquity BEH Phenyl;
(d) Acquity BEH Shield RP18 ; (e) Acquity BEH C8. Mobile phase conditions shown in grey employed a 9 minuted linear gradient of H2O(0.1 % formic acid):MeOH. * indicates the peak corresponding to propranolol 1, the earliest eluting species in the mixture.
Fig. 5.16 and Fig. 5.17 show chromatograms of propranolol 1 and compound 46 reaction mixtures respectively, using a nine minute linear H2O:MeCN gradient. The Acquity CSH C18
(Waters Corp., UK) stationary phase provides little improvement compared to its Acquity BEH C18 (Waters Corp., UK) counterpart, exhibiting similar issues in separating mixture components and retention of phospholipids. Both the Acquity BEH C8 (Waters Corp., UK) and Acquity BEH Shield RP18 (Waters Corp., UK) stationary phases give improved separation
Chapter 5. Small Molecule Intrinsic Lipidation 117
of components compared to the Acquity BEH C18 (Waters Corp., UK) column, however issues remain with lipid retention and peak broadening. By contrast, the Acquity BEH Phenyl (Waters Corp., UK) stationary phase designed for aromatic small molecules achieves excellent component separation. Further, the Acquity BEH Phenyl (Waters Corp., UK) column consistently elutes phospholipid in a distinct peak even at high concentrations, minimising co-elution and preventing contamination between samples.
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Figure 5.16 TIC (black) of propranolol 1 reaction mixture with POPC, analysed on four stationary phase types: (a) Acquity CSH C18; (b) Acquity BEH Phenyl; (c) Acquity BEH Shield RP18 ; (d) Acquity BEH C8. Mobile phase conditions used a 9 minuted linear gradient of H2O:MeCN (both containing 0.1 % formic acid). Key species highlighted: (i) propranolol 1;
(ii) lysolipids PPC and OPC; (iii) POPC. Insets show EIC (red) for m/z 524.4, corresponding to protonated oleoyl propranolol 1 intrinsic lipidation products.
The Acquity BEH Phenyl (Waters Corp., UK) with a nine minute linear H2O:MeCN gradient clearly provided superior chromatographic results compared to other stationary and mobile phases. No further modifications to the linear gradient were deemed necessary, due to the elution and separation of all reaction components. As such, these conditions were considered suitable for the study of small molecule intrinsic lipidation, and were taken forward for this purpose.