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Instruments and information

Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC/MS/MS)

Mass Spectrometry (MS) combined with the separation power of chromatography has revolutionised the way we do chemical analysis today. The MS Facility can offer several powerful mass spectrometers interfaced directly to the ultra performance liquid chromatography (UPLC)/high-performance liquid chromatography(HPLC) systems facilitating the analysis of polar and thermolabile compounds. The range of instruments include ion trap, single and triple quadrupole and high resolution (HR) mass spectrometers.

Mass spectrometry is one of the most sensitive analytical techniques currently available. 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 calibrated upper mass limit is 2,000 and proportionately greater for multiply charged ions since mass to charge ratio is measured.

The syringe pump can be used to infuse samples directly into the mass spectrometer or to infuse samples into a LC flow by means of a tee piece. The syringe pump is a device that delivers a solution (up to 500 µL) at a specified rate (0.05 to 100 µL/min). The majority of mass spectrometers employ 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.

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. A flow of nitrogen drying gas is directed at droplets and individual positive or negative ions are produced. ESI accommodates a liquid flow of 0.1 mL/min to 1 mL/min, but for the most instruments the most optimal is 0.2mL/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 µL can be sprayed over 30 to 60 min) and allows a thorough investigation of a sample in MS, MS/MS and, in the case of ion trap instruments, 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  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 0.5 mL/min to 2 mL/min. This type of ionisation technique is recommended for analysis of less polar, thermally stable molecules such as steroids.

Detailed structural information may be obtained by fragmentation spectra from MS/MS experiments. These spectra provide a characteristic molecular fingerprint. The ion trap instruments are 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 ion trap instruments are instruments of choice for qualitative work.

The extremely useful MS/MS library and supporting NIST software is available with the instrument. There is also a possibility to create and use one’s 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.

Although the ion trap instruments can also be used successfully for quantitative work, achieving a good precision data at very low concentration levels (less than 1 ng/mL) can be challenging. The triple quadrupole mass spectrometers are extremely sensitive and robust at very low levels (100pg/ml or less) in SRM mode. They provide extremely good precision and accuracy data. The triple quadrupole instruments are instruments of choice 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.

High resolution mass spectrometers are designed for compound identification, high-throughput and high-performance screening and for qualitative and quantitative analysis. They deliver high resolution accurate mass (HRAM) to provide fast, precise and reproducible results. High Resolving Power (up to 100,000) provides precise mass accuracy for complex sample analysis.

Accurate mass data is the current mass spectrometry standard that is being increasingly employed for rapid compound identification of biological markers and chemical products, providing in many cases, unambiguous chemical formula for small molecules and also peptides in complex biological extracts.  Such accurate mass measurement is particularly important in work involving the characterisation of drug candidates (pharmaceuticals), endogenous and xenobiotic metabolites, and environmental pollutants. 

Mass accuracy is key to facilitating metabolomics, which is a particularly fast growing area; the MS Facility is developing methodology and expertise to meet the increased demands in this area. In addition, high mass resolution can be used for very specific quantification of trace analytes. 

Examples of compounds suitable for LC/MS analysis:

Small Molecular weight < 1 kDa - drugs, endogenous compounds,vitamins, pesticides, toxins,conjugates (glucuronides, SO4) of compounds with m/z > 50

Middle Molecular weight 1 -10 kDa - synthetic polypeptides and polysaccharides

Large Molecular weight > 10 kDa - polypeptides, proteins, oligonucleotides, polysaccharides

LCMS systems available in the MS Facility:

 

Ion Trap

Triple quadrupole

High Resolution

Thermo Surveyor LC interfaced directly to Thermo LCQ DECA XP

Thermo Accela LC system interfaced directly to Thermo TSQ Access

Thermo Accela LC pump and CTC autosampler interfaced directly to Thermo Exactive

Thermo Accela LC system interfaced directly to Thermo LTQ XL with Electron Transfer Dissociation source

Waters Acquity  LC (nano) system  interfaced directly to Waters Quattro Premier  

 
 

Agilent HP 1100 LC system interfaced directly to Waters Quattro LC

 
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