HPLC-MS/MS consists of two different techniques. An analytic HPLC (High performance liquid chromatography) is connected with a mass spectrometer (MS). By the mass spectrometer thus a further separation process is coupled to the LC. The mass spectrometer also is used for the identification of components and the structure elucidation, see also FT-ICR-MS.
Mass spectrometry is a procedure for measuring the mass by measuring the m/z ratio of ions. In the HPLC coupling the compounds of interest are ionized with different inonization techniques e.g. like ESI (electrospray ionization) or APCI (atmospheric pressure chemical ionization). In the analyzer the ions are separated according to their mass/charge ratio, m/z. For gaining structural information, usually fragmenting is needed (MS/MS). With the equipment used by us fragmentation is possible in the second quadropole and with that the 3rd quadrupole with alternatively also can be used as linear ion trap these fragment-ions can be collected and analyzsed. The fragmentation is performed by collision with inert atoms in a so called collision cell between the first and the third quadrupole. By ESI or APCI a pseudo molecular ion is produced, which is isolated in the first analyzer quadrupole (Q1). In the second quadrupole (Q2) by impacts with an inert gas such as N2 or Ar energy is supplied to the ions (CID, Collision Induced Dissociation). Thus the ions fragement very specifically to lighter ions. In the third quadrupole (Q3) one can determine all products of the fragment ions, which allows conclusions on the structure of the original molecule. If only a known specific fragmention is monitored in a so-called MRM-mode (Multiple Reaction Monitoring), it is possible to quantify very sensitively and selectively.
LC-MS/MS is a highly sensitive method with for quantification in strongly matrix-loaded samples (plant extracts, ground excerpts, plasma among other things) also the structural elucidation of organic substances (synthesis products, natural substances) is a strength of LC-MS/MS.
- HPLC-part: binary pumping system
- MS-part: triple-quadrupol e.g. with linear ion trap which means that the third quadropole can be used as an ion trap alternatively ionization techniques: ESI, APCI, nano-ESI
Applications of LC-MS/MS
- structural elucidation of organic substances (synthesis products, natural substances) as pure substances or mixtures
- Quantification e.g. pesticides, antibiotics and their metabolites
characterization of proteins