Application of LCMS in small-molecule drug development

As a technology, mass spectrometry (MS) has evolved to the point where it is used throughout the drug development process. In particular, when MS is coupled with high-performance liquid chromatography (HPLC) it adds an orthogonal detection function for sample analysis and provides unique capabilities for pharmaceutical analysis, such as sensitivity, selectivity, speed of analysis and rich mass information. This article describes the fundamentals and general applications of hyphenated LC-MS in supporting small-molecule drug development, such as impurity and degradant structural characterisation, and qualitative and quantitative analysis. In addition, some newly-developed technologies in LC and MS are discussed in terms of their future application within pharmaceutical development.

Introduction to LC-MS

LC is a separation technique that relies on analytes’ differences in partitioning behaviour between a flowing mobile phase and a stationary phase to separate the components in a mixture. For most small-molecule drug development, LC is now one of the most powerful tools in analytical chemistry1,2. MS uses the difference in mass-to-charge ratio (m/z) of ionised compounds to separate them from each other. It is often considered as the most sensitive detector and is typically coupled with other technologies, most commonly LC. This type of orthogonal-mass spectrometric methodology has facilitated drug development enormously, primarily due to the superior speed, sensitivity and selectivity of such ‘hyphenated’ techniques. At present there are four major stages of drug development: drug discovery, preclinical development, clinical development and manufacturing3 . The small-molecule drug’s early development is also known as chemistry, manufacturing and control (CMC).

Ionisation and LC-MS general applications

The primary difficulties of combining LC and MS have been the interface, the ionisation of analytes in a stream of condensed liquids and transfer of the ions into the high vacuum inside the mass spectrometer. The most common forms of ionisation modes coupled with LC in small-molecule research are electrospray ionisation (ESI); atmospheric pressure chemical ionisation (APCI); and atmospheric pressure photoionisation (APPI). As illustrated in Figure 1 ESI is better suited to higher-molecular-weight and polar compounds, while APCI is best suited for low- to medium-polarity compounds with a limited upper mass range (< m/z of 1,000).

Recent studies have shown that APPI expands the range of compounds that can be ionised and has become the third option of choice for less polar compounds, such as steroids and PAHs4,5.

Electron ionisation (EI) is typically used in gas chromatography-MS for small and volatile molecules. Aviv A. et al. have described an approach to bring EI back to LC-MS based on sample ionisation as vibrational cold molecules in a supersonic molecular beam (Cold EI)6 . Cold EI results in enhanced molecular ions that are often weak or missing in standard EI, however the technique is fully compatible with library identification. This renovated EI-LC-MS technique has great potential to identify unknown samples that cannot be ionised by ‘soft ionisation’ sources.

LC-MS for drug impurity identification and profiling

Impurity identification and profiling is critical to the assurance of patient safety and drug efficacy in a drug development and active pharmaceutical ingredient (API) manufacturing unit. Regulatory authorities have established clear and rigorous guidelines which dictate the identification of impurities at lower levels, depending upon dosage7,8. For most small-molecule drug-like compounds, LC is by far the most reliable and efficient separation technique, and serves as the main workhorse for impurity profiling.

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