Diab Elmashni outlines the advantages of liquid chromatography coupled with mass spectrometry detection.
Liquid chromatography coupled with mass spectrometry detection (LC/MS) is one of the most powerful analytical tools for organic compound analysis.
The key advantages of using LC/MS methods over HPLC methods include:
* Selectivity. Co-eluting peaks can be isolated by mass selectivity and are not constrained by chromatographic resolution.
* Peak assignment. A chemical fingerprint for the compound of interest is generated, ensuring correct peak assignment in the presence of complex matrices.
* Molecular weight information. Confirmation and identification of known and unknown compounds.
* Structural information. Controlled fragmentation enables structural elucidation.
* Rapid method development. Provides easy identification of eluted analytes without retention time validation.
* Sample matrix adaptability. Decreases sample preparation time.
* Quantitation Quantitative and qualitative data can be obtained easily with limited instrument optimisation.
How LC/MS detection works
An LC/MS detector consists of three major components: an ion source that generates ions at atmospheric pressure, a mass analyser which filters ions, and a detector that detects ions. As chromatographic peaks elute from the LC column and transfer to the ion source, two main processes occur. First, the ion source produces charged molecules or ions, and second the mobile phase is removed. Once the ions are created, they are extracted from the ion source and transferred to the mass analyser. Sample ions are then filtered by the quadrupole mass analyser according to their mass-to-charge ratio (m/z) prior to detection.
Atmospheric pressure ionisation is regarded as a asoft' ionisation process. This results in a mass spectrum typically dominated by a single ion that corresponds to the molecular weight of the compound, which is protonated in the positive ion mode (M+H)+ or deprotonated in the negative ion mode (M-H)-.
For example, a compound with a molecular weight of 200Da in the positive ion mode will result in a spectrum with a abase peak' at 201m/z. In negative ion mode (dependent on the chemistry of the compound in question), the spectrum will be dominated by an ion at 199m/z. Source fragmentation can be induced to form diagnostic fragment ions for structural confirmation. However, in some cases, the molecular ion is relatively unstable, which creates fragment ions. Using an LC/MS detector, it is possible to make structural predictions from the mass difference of the molecular ion and fragment ions seen in a mass spectrum. The degree of fragmentation can be precisely controlled by adjusting the Finnigan Surveyor MSQ Plus source voltage.
Obtaining data
The Finnigan Surveyor MSQ Plus has intuitive instrument software, with simple interactive method set up and point-and-click sequences available from standard templates. It features a built-in auto mass scale calibration, a full system Autotune, and automated instrument set up that does not require user input. It also allows the user to mix and overlap scan events, enabling both confirmation and quantitation in one single method. This software works on Microsoft Windows operating environments and utilises the Microsoft Office software package.
Chromatographic integrity in LC/MS
Regardless of flow rate and mobile phase composition, the peak shape is consistent between conventional LC detection and LC/MS detection.
The ESI FastLoc Probe for the Finnigan Surveyor MSQ Plus can be used with HPLC flow rates from capillary LC to conventional analytical LC, with flow rates ranging from 10µL/min to 2mL/min, without splitting. With the APCI FastLoc Probe, all conventional HPLC columns with flow rates from
0.2 to 2mL/min can be used without splitting.
The Finnigan Surveyor MSQ Plus has changed the scope of LC/MS applications. Constraints imposed by nonvolatile buffers and sample matrices, which are common with HPLC methods, are no longer applicable. The patented self cleaning source has proven to be effective in routinely analysing compounds in phosphate buffers, and working with limited sample preparation protocols such as protein precipitated plasma samples. Unlike other instruments, the Finnigan Surveyor MSQ Plus features a unique patented cone wash facility, which maintains instrument performance under the most rigorous conditions. The new titanium entrance cone provides increased durability and greater chemical resistance. In addition, the new square quadrupole RF/DC pre-filter lens has been designed for high efficiency ion transmission and protection from contamination for the analysing quadrupole.
The benefits of LC/MS quantitation
LC/MS adds additional confidence to your quantitation enabling you to obtain limits of detection which can be up to 1000 times greater than UV detection. The Finnigan Surveyor MSQ Plus is designed to provide simple mass information about specific analytes at an extremely cost effective outlay without the need to be an expert in the details of multi-stage MS/MS. Sensitivity and specificity both prevail in high abundance with the Finnigan Surveyor MSQ Plus LC/MS detector, which decrease costs and increases confidence.
Diab Elmashni is with Thermo Electron Corporation,
San Jose, CA, USA. www.thermoelectron.com
