Liquid chromatography-mass spectrometry (LC-MS) is the preferred tool in bioanalysis today. But what is LC-MS? LC-mass spectrometry combines the resolving power of liquid chromatography and the specificity of mass spectrometry detection. The LC component separates the analytes while the MS unit detects the analytes based on their mass-to-charge ratio.
Also called HPLC-MS analysis, LC-MS can provide crucial analyte data during the drug development process. Developing LC-MS analysis is more complex than HPLC method development. This intricacy becomes even more vital during method transfer. However, the current article focuses on the complexity revolving around the ionization process.
Researchers have long focused on the efficient interfacing of the LC and MS components. LC sample analysis has a broad mass range for samples, but the MS analyzer has limitations. Liquid chromatography unit employs high pressure to separate individual components, which in turn, generates a high gas load. On the other hand, the mass spectrometry unit requires a vacuum and a finite gas load. Besides, the LC unit needs near ambient temperature compared to elevated temperature for MS components. Finally, MS prefers volatile buffers, while LC uses inorganic buffers.
Hence, developing a robust interface was always necessary for LC-MS analysis. Ionization techniques were the answer to this interface crisis. The atmospheric pressure ionization source has overcome the limitation of traditional LC-MS techniques. The following section discusses the ionization process of LC-MS analysis.
Atmospheric Pressure Lonization(API)
Atmospheric pressure ionization (API) processes are suitable for analyzing polar and nonpolar, small, and large compounds. API can rapidly identify a spectrum of volatile and nonvolatile compounds and provide accurate and sensitive fragmentation and molecular weight data. Today, API is employed largely in metabolite confirmation studies in pharmaceutical and biomedical applications.
The API process has three basic steps, nebulization and charging, desolvation, and ion evaporation. Let us understand each of these steps in detail.
The HPLC unit has a nebulizing needle at the ground potential through which the effluent is pumped, whereas the spray passes through a high-potential semi-cylindrical electrode. The potential difference between the electrode and the needle is, it generates a strong electrical field, which charges the liquid surface and produces charged droplets. In the desolvation process, the capillary sampling orifice attracts the charged droplets. A counterflow of heated nitrogen drying gas shrinks the droplets and removes the uncharged material. Finally, in the ionization step, the shrunk droplets approach a threshold where the electrostatic forces are more than the cohesive forces. The ionization process continues until all analyte ions are desorbed into the gaseous phase. These ionized analytes then enter the MS unit for detection.
Atmospheric Pressure Chemical Ionization (APCI)
Atmospheric Pressure Chemical Ionization (APCI) is suitable for moderate molecular weight polar and nonpolar analytes. APCI is complementary to API. However, APCI employs a hot vaporizer chamber. This chamber evaporates the spray droplets and produces analyte molecules in a gas-phase solvent.
APCI has a charged corona needle that ionizes the analytes in the gas-phase solvent. In APCI analysis, the charge from ionized solvent molecules is transferred to the analyte molecules. These analyte molecules are then transported to the filter and detector.