Liquid chromatography-mass spectrometry (LC-MS) is taking over as an analytical technique of choice in diverse fields, including research, pharmaceutical development, food and beverage testing and environmental monitoring. While a LC-MS analyst may be tempted to rely on the most commonly used sample prep techniques, columns and mobile phases in his/her field, we show successful LC-MS demands these workflow elements be chosen with the physico-chemical properties of the analyte in mind.
First, the analytes to be detected using LC-MS should be determined. Most relatively polar analytes in aqueous samples are best analyzed using electrospray ionization MS, whereas alternative ionization techniques like atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI) can provide better results for non-polar analytes. Analyte molecular properties, such as hydrophobicity, chemical structure, functional groups, pKa values and non-analyte sample components, should determine experimental design.
By identifying the analyte’s general chemical structure (aromatic, dipolar, amino acid) and then key functional groups (aromatics, hydroxyl groups, organic acids, ketones), the stationary phase can be chosen (for example, C18, C8, HILIC).
At this point, the sample matrix should be considered, and a sample preparation strategy drawn out. Sample preparation needs to be selected so it brings the sample into a solution and is free of particles for LC-MS analysis. Additional points of consideration include increasing analyte concentration and reducing sample complexity. For example, a plasma sample might benefit from solid phase extraction, which removes contaminants (proteins, lipids) and also concentrates the sample, whereas fruit and vegetable juices, with their high particle load, might benefit simply from dilution and filtration.
Following sample preparation, the actual separation strategy can be finalized using literature or separation methods for similar types of analytes as a starting point for LC-MS. This starting chromatography method can be fine-tuned to obtain desired resolution, sensitivity and peak characteristics (asymmetry, tailing) as needed for the application.
What parameters determine LC-MS success?
Sensitivity, resolution and reproducibility are key parameters of success and failure of LC-MS, accompanied by additional parameters such as efficiency, selectivity, speed and column lifetime. But each parameter is affected by multiple factors, derived from various aspects of the setup. Successful LC-MS requires systematic optimization of key components of the experiment. Usually, resolution, sensitivity and reproducibility are the most important parameters which need to be optimized prior to focusing on other parameters.
Parameter 1: Sensitivity
Sensitivity is the lowest level of analyte detectable above background. Though the type and setup of the mass spectrometer makes a difference, so do chromatographic factors, such as type of column, degree of column bleeding and effectiveness of mobile phase and sample preparation. The smaller the internal diameter of the column and the smaller the particle size of the stationary phase, the more sensitive the analysis. Column bleeding can be mitigated by column washing. Interfering molecules from the sample or mobile phase that may interfere with sensitivity, such as endogenous phospholipids, dosing media, leached extractable impurities, formulation agents and coeluting molecules (metabolites, degradation products) can be removed by extraction and microporous filtration of the sample and mobile phase. Finally, choosing high-quality, LC-MS-grade solvents can minimize organic impurities that compromise sensitivity (Figure 1).
Parameter 2: Resolution
Resolution, or how easily two peaks can be distinguished in a spectrum, is a function of the difference in retention times and the width of the peaks. It’s affected by capabilities of the mass spectrometer, such as mass resolution and peak resolution, but also chromatographic factors. Specifically, resolution can be affected by stationary phase characteristics (type, particle size, chemical modification, end capping, carbon loading, porosity, surface area).
Other factors affecting resolution are the composition and pH of the mobile phase. Most separations are reverse-phase, meaning the mobile phase is more polar than the stationary phase. To minimize unwanted salt precipitation inside the column, volatile solvents (methanol, acetonitrile and tetrahydrofuran) and freshly delivered ultrapure water are commonly used mobile phase components. These components vary in their polarity and therefore their impact on resolution. To achieve finer separations, mobile phases may contain buffers and sources of protons (such as formic acid), which can change the polarity of the analyte(s). Similarly, temperature, which can affect pH, can be varied to affect resolution.
Parameter 3: Reproducibility
Whether referring to reproducibility between columns or between runs, it depends on how well columns are manufactured and tested by the manufacturer, and how much user-to-user technical variation exists. LC-MS reproducibility depends on removal of sample components that interfere with separation, ionization and fragmentation. Reproducibility is also affected by the precision of the sample preparation method, and whether the method is introducing nonspecific analyte binding, extractable impurities or variable analyte recovery due to hold-up volume.
Parameter 4: Efficiency
Efficiency is usually expressed as plate count/number (N)—the higher the plate count, the better the separation. Efficiency can be assessed by measuring the dispersion of a peak; the narrower the peaks, the better the resolution, since narrow peaks take up less space in the chromatogram, allowing more peaks to be separated. Smaller stationary phase particles also contribute to higher efficiency, but result in higher backpressure. For a given particle size, increasing column length increases separation efficiency, but will require longer times for separation.
Parameter 5: Selectivity
Selectivity is the ratio of retention factors of two analytes, and therefore reflects the capacity of the column to retain certain analytes to a significantly greater extent than others. Besides column chemistry and type of packing, other factors that affect selectivity include the solvent/mobile phase composition and temperature.
Parameter 6: Speed
Separation speed is affected by packing material; monolithic columns allow fast separation using standard HPLC instruments. Small particles allow faster analyses, but produce high backpressure.
For all columns, the longer the column, the longer the separation time. In LC-MS, total analysis speed is also determined by the time required for mass spectrum acquisition, which is a function of the mass spectrometer.
Parameter 7: Column Lifetime
The column lifetime is the number of injections on a column without change in selectivity and efficiency. Column robustness and matrix tolerance can extend lifetime; for example, monolithic columns have high tolerance for “dirty” samples. Column lifetime is compromised by sample contaminants that adsorb strongly. Use guard columns and proper sample preparation to remove particulates (such as centrifugation, filtration and extraction) to extend column lifetime (Figure 2). If mobile phase pH isn’t within the specified range where the column is stable, column lifetime will decrease. Generally, for silica-based columns, acceptable column lifetimes can be achieved in the pH 2 to 8 range, whereas polymer-based or hybrid (silica-polymer) columns offer broader pH compatibility and hence suitable column life.