HILIC chromatography is now a common method of sample introduction for mass spectrometry. In the analysis of small polar molecules, HILIC has proved to be a robust and selective tool that allows the application of Ultra-High-Throughput-LC-MS/MS methodologies, capable of >2000 samples/instrument/day. When designing chromatographic applications for such throughput, selectivity from isobaric interferences and phospholipid-based ion suppression becomes of paramount concern. Analytes containing certain functional groups have been found to be amenable to HILIC, and, during bioanalysis, isobars and phospholipids have shown to play a deleterious role in accurate quantitation of data. This paper describes strategies wherein functional groups, sorbent-bed selection, and non-intuitive mobile phase chemistries can be utilized to provide selective, suppression-free mass spectrometric detection of small polar analytes.
Nornicotine, Succinic Acid, and Orotic Acid, representing polar molecules <200 amu containing carboxylic acids and/or amines amenable to HILIC retention, were spiked into DI water and human plasma at 500 ng/mL. 300µL sample was precipitated with 1200µL acetonitrile; samples were vortexed, centrifuged, and 10µL supernatant was injected onto the chromatographic systems. Nornicotine and phospholipids were monitored via positive electrospray MS/MS detection; succinic acid and orotic acid were monitored via negative electrospray MS/MS detection. 28 mobile phase modifications on 7 HILIC-style columns were screened to demonstrate selective retention of the analytes. Comparison of retention times for analytes and phospholipids were used to determine the effect of mobile phase modifiers and the selective shifting of phospholipids/isobaric interferences from the analytes of interest.
Retention of the analytes was achieved on all analytical columns using a loading solvent of acetonitrile; a consistent gradient profile was used over the course of experimentation on all columns. Elution profiles based on changes in acid, buffer, and protic solvent concentrations were built by monitoring the retention times of the analytes, retention times of the 2 signal suppressing phospholipid classes (phosphatidyl- and lysophosphatidyl-cholines), and comparing the retention time differences between the two as the eluents were modified. Results demonstrate that elution using addition of protic organic solvents on silica and HILIC sorbent beds contributes to the early elution of phospholipids and analytes lacking amines (succinic acid), whereas the retention of aminated (nornicotine and orotic acid) molecules is relatively unchanged if that protic solvent is in the presence of buffering materials. Selectively shifting isobaric interferences and phospholipids from carboxylic acids, regardless of amine content, is achieved by utilizing ion exchange mechanisms derived from volatile buffers, adjustments to the charge states of phospholipids in solution, and aqueous content in gradient chromatography. Detailed examples will be shown to enable classification of the effects of solvents/functionalities on speed of analysis (including pressure considerations, reconditioning time, etc), and selectivity for analytes versus phospholipids/isobaric interferences. |
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