Mass Spectrometry (MS) combined with the separation power of chromatography has revolutionised the way we do chemical analysis today. One of the core instruments of the School MS Facility H&LS, KCL is a Thermo Finnigan Surveyor liquid chromatograph interfaced directly to a Thermo Finnigan LCQ DECA XP facilitating the analysis of polar thermolabile compounds.

This instrument was purchased as a result of a Joint Research Initiative Fund grant award from the BBSRC in 2001 (Biotechnology and Biological Research Council grant number 18/JE514264). The principal applicant was Professor Catherine Rice-Evans from the School of Biomedical Sciences, who is an active member of the MS Steering Committee. Professor Rice-Evans shares an interest in applying this powerful technique with those on the Steering Committee from the School of Health and Life Sciences, and it is their inter-School collaboration that led to the successful grant application.

The LCQ DECA XP is one of the most sensitive instruments currently on the market. Detection limits are at the low-femtomole levels for full-scan mass spectra and in the low-femtomole to high-attomole levels for the more sensitive but less informative selective-ion monitoring (SIM) mode of operation. Excellent sensitivity is also possible for selective-reaction monitoring (SRM), which also enables additional selectivity compared with SIM operation, and MS/MS analyses that may provide additional structural information. A unique feature of ion trap instruments, the ability to perform MSn experiments, also achieves a high level of selectivity. The upper mass limit of this instrument is 4,000 and proportionately greater for multiply charged ions since mass/charge is measured.

The syringe pump can be used toinfuse samples directly into the mass spectrometer or to infuse samples into LC flow by means of a tee piece. The syringe pump is a device that delivers a solution (up to 500 mL) at a specified rate (0.05 to 100 mL/min). LCQ Deca XP employs an electronically-controlled, integrated dual syringe pump that delivers sample solution and/or sheath liquid from syringes into the API source.

The use of Atmospheric Pressure Ionisation (API) technique allows positive or negative ions to be detected. API offers soft ionisation resulting in little or no fragmentation. A typical API spectrum contains only the protonated (positive ion mode) or deprotonated (negative ion mode) molecular ion. The detected ion peaks are (M+z)/z and (M-z)/z in positive and negative ion mode, respectively, where M represents the molecular weight of the compound and z the charge (number of protons). MS spectra provide valuable molecular weight information of single and multiply charged ions and identification of the charge state of each peak in the charge-state envelope of a given compound. A computer routine is used to analyse multiply charged mass spectral data of mixtures of proteins and other biopolymers.
Detailed structural information may be obtained by fragmentation spectra from MS/MS experiments. These spectra provide a characteristic molecular fingerprint. The LCQ DECA XP is capable of sequential MS/MS experiments, MSn, where n is number of MS/MS experiments. In the first MS/MS (MS²) experiment, an ion selected from the MS spectrum is fragmented to give a fragment ion spectrum. Ions can be selected from that spectrum for further fragmentation (MS³) and so on. These spectra can be used for identification purposes, metabolic profiling, structure elucidation and peptide sequencing.

The API source can operate using electrospray (ESI), nanospray (NSI) or atmospheric pressure chemical ionisation (APCI) techniques.
In ESI, the sample solution is sprayed in a fine mist of charged droplets containing sample ions by application of a large negative or positive voltage (typically ±4.5 to ±5 kV). A flow of nitrogen drying gas is directed at droplets and individual positive or negative ions are produced. ESI accommodates a liquid flow of 1 mL/min to 1 mL/min. This ionisation technique is very suitable for the analysis of polar, thermally labile molecules such as drugs, DNA, RNA, sugars, peptides, and proteins.
NSI is essentially ESI operating at very low liquid flow rates of 100 nL/ml to several microlitres per minute in static or dynamic modes. Static NSI is a self-sustaining direct infusion of a low volume of sample over an extended period of time (1 to 5 mL can be sprayed over 30 to 60 min) and allows a thorough investigation of a sample in MS and. MSn modes. There is no LC attachment and instigation and maintenance of the spray conditions is assisted with a constant gas backpressure. Dynamic NSI allows connectivity to micro and nano LC columns adding the advantage of a chromatographic separation. The technique provides a tool for the most sensitive analytical challenges.

APCI vaporizes the sample solution at temperature up to 600 °C. Application of a high potential (typically ±3 to ±5 kV) produces reagent ion plasma, mainly from the solvent vapour. The sample vapour is ionised by ion-molecule reactions with the reagent ions in the plasma. APCI accommodates liquid flows of 100 mL/min to 2 mL/min. This type of ionisation technique is recommended for analysis of less polar, thermally stable molecules such as steroids.

The LCQ DECA XP can also be used successfully for quantitative work. Both ESI and APCI ionisation techniques can be employed and selective-reaction monitoring (SRM) mode is typically applied in this type of analysis. However, achieving a good precision data at very low concentration levels (less than 1 ng/mL) can be challenging.
The extremely useful MS/MS library and supporting NIST software is available with the instrument. There is also a possibility to create and use own custom MS/MS or MSn libraries. Mass Frontier and Metabolite ID software are used for the management, evaluation and interpretation of mass spectra in identification, structure elucidation and metabolic profiling work. Biomass software is used to analyse multiply charged mass spectra of biopolymers and Sequest software is essential for peptide sequencing analysis.

Examples of compounds suitable for LC/MS analysis

Document compiled by Anna Przyborowska - January 2002.