Liquid chromatography mass spectrometry (LC-MS) is a powerful analytical chemistry tool used in the pharmaceutical, biotechnological, and environmental industries. This equipment combines the separation capabilities of high-pressure liquid chromatography (HPLC) and the mass analysis capabilities of mass spectrometry to provide very sensitive and specific detection of a wide range of molecules.
Samples containing complex mixtures of compounds are pumped through a chromatography column and separated into its components, mainly based on polarity of the molecules. As each component appears at the end of the column, they are channeled into the ion source, where the solvent is removed, and compounds become ionized before entering the mass analyzer and mass detector. The mass analyzer sorts ions by their masses (expressed as mass to charge ratio, or m/z), and the mass detector determines the abundance of each ion. There are different ion sources that use slightly different mechanisms for creating ions.
Tandem LC-MS (LC-MS/MS) is used as an analytical tool to detect and quantify molecules in a variety of samples. Our triple quadrupole LC-MS instrumentation allows us to focus on the mass of a specific target compound, to allow accurate and sensitive detection.
In triple quad mass spectrometers, compounds enter the ion source where they become ionized. The ions then pass through a quadrupole mass filter, which only allows an ion of a specified mass-to-charge ratio (m/z) to pass into the collision chamber. The selected ion is called the precursor ion. In the collision chamber, energy is applied to the precursor ion, to cause it to fragment. The ion fragments are then sent through a second quadrupole mass filter, where a specific fragment ion, known as the product ion, is selected and passed to the mass analyzer for sorting and detection. The specific pair of m/z values associated to the precursor and product ions selected is referred to as a “transition” and can be written as parent m/z → product m/z (for example 542→ 315).
The filtering and fragmentation of ions utilized in a triple quad LC-MS instrument has several advantages. Firstly, it allows for better signal-to-noise ratios, enabling very low limits of detection for quantitation of molecules. With a well-optimized method, molecules can be detected down to the picogram per milliliter (sub-ppb) level.
Secondly, the ability to select specific ions for detection means that components in a sample do not have to be completely separated from each other before detection. In quantitative analysis using HPLC with other detectors such as UV or RI, accurate analysis often depends on baseline separation of components. While chromatographic separation still plays a role in minimizing ion suppression, triple quad mass spectrometers allow for shorter run-times, which allows for higher-throughput analysis.
Finally, analysis by triple quad mass spectrometers allows for accurate and specific detection of compounds. The application of collision energy at different levels in the mass spectrometer collision chamber yields characteristic fragments that can be used for quantitation and confirmation of identification. Multiple parent ion → product ion transitions can be tracked for the same compound by rapidly switching between the different transition ion pairs. This is called multiple reaction monitoring (MRM) and allows for selectivity by monitoring multiple transitions for a given analyte. Usually, the product of one of the transitions is used for quantitation, while one or more additional transitions is used as identity confirmation.
For some applications, detectors other than mass spectrometric detectors are more suitable for detecting compounds of interest. At Emery Pharma we have several alternative detectors, which allows us to analyze a broad range of large and small molecules after separation with HPLC.
HPLC with UV detection is widely applied in quantitative methods, including many regulatory methods (e.g. USP). UV detection, using diode array (DAD) detectors, measures the absorption of light by molecules. Absorption can be measured at single or multiple wavelengths. Ultraviolet and visible (UV-vis) absorption spectra of molecules can also be measured, which can aid in identification of unknown compounds, or for confirmation of the purity of a sample.
Charged Aerosol Detector (CAD)
For compounds that lack UV chromophores, universal detectors are often necessary for analysis by HPLC. Charged Aerosol Detectors (CAD) are universal-type detectors that nebulize HPLC eluent using nitrogen gas to form droplets, and dry the droplets to form particles. This stream of particles is met by a positively charged gas stream, and the charge is transferred to the analyte particles. The charged analyte particles charge then transfers the charge to an electrometer, generating a signal that is directly proportional to the quantity of analyte present. Charged Aerosol Detectors can be used to analyze a wide range of analytes, small and large, including polymers, surfactants and other compounds that are not amenable to detection by HPLC-UV or LC-MS.
Evaporative Light Scattering Detector (ELSD)
Evaporative Light Scattering Detectors are another kind of universal detector. Similar to charged aerosol detectors, the HPLC effluent is nebulized to form aerosolized droplets. The droplets then pass through an inert drift tube, where the solvent is evaporated. The remaining particles are passed through a light, which scatters in the presence of particles, and is measured by a photomultiplier tube. These detectors are very versatile, and able to detect a range of compounds without the need for a chromophore.