LC-MS instruments are basically HPLC units with a mass spectrometry detector attached to it whereas LC-MS/MS is HPLC with two mass spectrometry detectors. The LC in LC-MS stands for liquid chromatography. The liquid chromatography part of LC-MS separates compounds within a sample and the mass spectrometer provides mass to charge ratio data which can help provide structural identity of the compound. Applications of LC-MS range from food analysis, environmental testing, drug development work and medical device testing.
The History of LC-MS
Combining chromatography with mass spec technology dates back to 1952 when A.T. James and A.J.P Martin were developing a gas chromatography mass spec unit but LC-MS development did not start until the early 1970s. Currently, many manufacturers produce LC-MS instrument, such as Agilent, Bruker, PerkinElmer, SCIEX, Shimadzu Scientific, Thermo Fisher Scientific, and Waters Corporation.
LC-MS/MS – How Does It Work?
Please see our HPLC page for information on how liquid chromatography (the LC in LC-MS) is able to separate compounds. There are minor differences between normal HPLC runs and runs using LC-MS. One of these differences is that the flow rate for LC-MS is slow than the standard HPLC flow rate of 1 mL/min. In addition, conventional HPLC columns are about 100-300 mm long while the columns used for LC-MS applications are substantially smaller, typically 30-50 mm in length. LC-MS columns also have a small internal diameter which provides better separation and can function at flow rates of less than 1 mL/min.
When the molecules are eluted from the chromatography column they are under pressure and the continuous flow cannot be directly detected by the mass spectrometer because mass spec units operate in a vacuum and requires the liquid to be passed through an interface. The interface removes the mobile phase used in the chromatography step and transfers the analyte to the mass spec unit. May different types of interfaces exist but the most commonly used interfaces are electrospray ionization (ESI) systems, atmospheric pressure chemical ionization (APCI) systems, or atmospheric pressure photo-ionization (APPI) systems. In all these interfaces, the liquid is nebulized into a fine spray, ionized and then transferred to the mass spec detector.
The mass spec detector measures the mass-to-charge ratio of ions by exposing the ions to a magnetic or electric fields which can alter the movement of the ions allowing the ions to be sorted based on their mass. The detector can then measure and amplify the ion current to quantitate the amount of sorted ions. If two mass spec detectors are used, then molecules with a particular mass-to-charge ratio can be chosen to undergo further analysis by fragmenting the ion by collision-induced dissociation or other fragmentation processes. These fragmented ions can then be detected by the second mass spec unit.
The mass spec detector transfers the mass-to-charge data to a computer which graphical presents the information as a mass spectrum. The mass spectrum of the sample can be used to determine the concentration of known or unknown compounds, find the mass of impurities and give insight into chemical structures.
LC-MS and LC-MS/MS Drug Development and Medical Device Applications
For small molecule PK studies, the bioanalysis is often performed using an LC-MS or LC-MS/MS instrument because they provide much higher sensitivity and specify than UV detectors that are common on HPLC units.
Peptide Mapping –
LC-MS/MS can be used to verify the amino acid sequence of a protein therapeutic. Typically this involves digesting the sample with a series of peptidases and the fragments can be ran on the LC-MS/MS to verify the expected mass from each fragment.
Identification of Post-Translational Modification –
Glycosylation and other post-translational modifications can be analyzed by LC-MS.
Quality Control –
LC-MS is a convenient method for identity testing using a reference standard and to determine purity of drug product or drug substance.
Extractable Leachable Studies –
Plastics from pharmaceutical packaging or exposed to the drug substance or drug product during the manufacturing process could leach into the product and potentially cause toxic side effects. Likewise materials used in the production of medical device could also cause patient harm. LC-MS/MS is a good instrument for analyzing compounds in extractable leachable studies.