Large-Scale Structural Characterization of Drug and Drug-Like Compounds by High-Throughput Ion Mobility-Mass Spectrometry

Abstract

Ion mobility-mass spectrometry (IM-MS) can provide orthogonal information, i.e., m/z and collision cross section (CCS), for the identification of drugs and drug metabolites. However, only a small number of CCS values are available for drugs, which limits the use of CCS as an identification parameter and the assessment of structure-function relationships of drugs using IM-MS. Here, we report the development of a rapid workflow for the measurement of CCS values of a large number of drug or drug-like molecules in nitrogen on the widely available traveling wave IM-MS (TWIM-MS) platform. Using a combination of small molecule and polypeptide CCS calibrants, we successfully determined the nitrogen CCS values of 1425 drug or drug-like molecules in the MicroSource Discovery Systems' Spectrum Collection using flow injection analysis of 384-well plates. Software was developed to streamline data extraction, processing, and calibration. We found that the overall drug collection covers a wide CCS range for the same mass, suggesting a large structural diversity of these drugs. However, individual drug classes appear to occupy a narrow and unique space in the CCS-mass 2D spectrum, suggesting a tight structure-function relationship for each class of drugs with a specific target. We observed bimodal distributions for several antibiotic species due to multiple protomers, including the known fluoroquinolone protomers and the new finding of cephalosporin protomers. Lastly, we demonstrated the utility of the high-throughput method and drug CCS database by quickly and confidently confirming the active component in a pharmaceutical product.

Figure 1

Summary of the bioactivities of the 1425 drug and drug-like compounds in the MicroSource Discovery Spectrum Collection, for which CCS values were measured in this study. The top 10 bioactivities and the number of compounds in each group are identified in the legend.

Figure 2

(A) IM-MS conformational space plot showing the trends in CCS–mass for PolyAla and drug-like calibrants, which were obtained on a drift tube (DT) IM-MS with nitrogen as the drift gas. (B) Errors of calibrated CCS values for PolyAla and Drug-like CCS calibrants as a function of drift time. Calibration errors were determined as the percent error of the calibrated CCS relative to the DT CCS value. (C) IM-MS conformational space plot showing the 1440 nitrogen CCS values of the 1425 drugs from the Spectrum Collection. Data points represent the average of three measurements. The solid line represents a power curve fit to the CCS–mass trendline of drugs, which excluded compound that deviated significantly from the main band and multiply charged species. The dashed lines represent the ±10% bands from the power curve. (D) Histogram of the relative standard deviations (RSDs) of the triplicate CCS measurements of the Spectrum Collection.

Figure 3

IM-MS conformational space plots showing the regions occupied by (A) lipids and peptides; (B) subclasses of antibiotics; (C) compounds of various densities; and (D) corticosteroid and nonsteroidal anti-inflammatory drugs (NSAIDs). Structures shown: cephalexin is a cephalosporin antibiotic, benzalkonium C12 is an amphiphilic ammonium, clioquinol is an antifungal drug, ibuprofen is a common NSAID, and cortisone is a common corticosteroid.

Figure 4

(A) Conformation of cefpodoxime proxetil, obtained through molecular modeling, that had a theoretical CCS value 0.65% different than that of lower experimental CCS value; (B) bimodal ATD of cefpodoxime proxetil annotated with the experimental CCS values; (C) conformation of cefpodoxime proxetil, obtained through molecular modeling, that had a theoretical CCS value 0.97% different than that of the higher experiment CCS value.

Figure 5

Comparison of the arrival time distribution (ATD) of the clindamycin standard in the Spectrum Collection (solid black line) and that of clindamycin from a 300 mg capsule prepared as a 1 ng/mL (approximately 0.5 ng/mL clindamycin) solution (dotted red line). The CCS values of clindamycin from the Spectrum Collection and the capsule are different by 0.2%.