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Hinrichs Lab - Liquid chromatography coupled to mass spectrometry (LC-MS)

Introduction

Liquid chromatography coupled to mass spectrometry (LC-MS) is used for the identification of polar, high molecular weight organic compounds and enables semi-quantitative determination of concentrations. This separation technique is based on the differential retention of compounds on a stationary phase. The analyte is injected into a stream of liquid mobile phase which is pumped at high pressure through a column packed with the selected stationary phase. Depending on the type and degree of interaction between compound and stationary phase, the compounds are retained for a certain time before elution from the column, thus separating the complex analyte mixture into individual compounds. Reversed phase (RP) separation is a widely used technique and is based on the combination of a gradient from polar to apolar mobile phase and RP columns. Lipid analysis, however, has traditionally used normal phase separation with a gradient from apolar to polar eluents due to the advantage of maintaining a separation pattern according to prominent lipid characteristics of polar head groups.

The separated compounds can either be collected in a fraction collector for purification and further processing (preparative LC) or be directed to a detector (analytical LC). A large variety of detectors can be coupled to LC systems. Some of these detectors are well suited for quantification of compounds with specific characteristics, e.g., fluorescence, absorbance at given wavelengths, or conductivity. Mass spectrometers are the detector of choice if structural identification is necessary. Such equipment is capable to determine the molecular mass of the compounds and, if MS2 mode is available, can obtain additional molecular and structural information by fragmentation of the parent ions.

In the Hinrichs Lab, LC-MS techniques are mainly employed for the characterization of intact polar lipids and core lipids. For this purpose, several specialized mass spectrometers are available (see below).

Thermo Surveyor LC

This equipment is mainly used for preparative HPLC in order to concentrate and purify compound classes. In this case, the goal of the chromatographic separation is not the characterization or quantification of a compound, but its isolation. Therefore, after being eluted from the column, the flow is directed to a fraction collector where fractions are collected according to a previously defined timetable. In order to purify large sample amounts, sample loading, flow rate and column size are considerably increased compared to analytical HPLC. If not used in preparative mode, this HPLC system can be connected to the Thermo Finnigan Ion Trap or to an evaporative light scattering detector (ELSD).

Agilent 6130 Single Quad MS

The Agilent 6130 MSD system is equipped with an Agilent 1200 series HPLC including cooled fraction collector and active splitter. The mass spectrometer is well suited for routine analysis of known compounds at low detection limits, especially in selected ion monitoring (SIM) mode. The ionization capabilities are widened by the multimode ion source which allows simultaneous electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). As this equipment is based on single quadrupole technology, fragmentation of parent ions is not possible and structural information cannot be obtained. The connection to a fraction collector via active splitter allows peak-based collection of individual compounds with simultaneous acquisition of mass spectra.

Thermo Finnigan LCQ Deca XP Plus Ion Trap MS

This system is based on ion trap technology and equipped with an Agilent 1200 series HPLC. The main advantage of the ion trap is the possibility of performing data-dependent multiple stage mass spectrometry (MSn) where several consecutive fragmentation steps on a selected ion are performed. This provides a wealth of structural information about the compounds of interest. The analytical capabilities can be extended by rapid switching between positive and negative ionization mode, where even more information regarding the chemical structure can be gained. ESI and APCI ion sources are available and additional possibilities and a dramatically improved sensitivity are offered by the camera-assisted nano electrospray ion source.

Bruker maXis UHR-qToF MS

The Bruker maXis ultra-high resolution quadrupole time-of-flight mass spectrometer (UHR-qToF) offers high mass accuracy (<2 ppm) and mass resolution (>40.000 for m/z = 922 Da), as well as high scan rates. This instrument is coupled to a Dionex Ultimate 3000 RSLC ultra high pressure LC (UHPLC) including a variable wave-length detector and is able to work with pressures up to 1034 bar. This allows the use of sub-two micron particle size columns and to achieve improved chromatographic separation, high chromatographic peak capacity, and shorter chromatographic runs. Due to the possibility to obtain exact mass data and high mass resolution in both MS1 full scan and MS2 fragment spectra, this system is ideal to characterize and identify unknown compounds. Also, separation of near-isobaric compounds and detection of compounds in very low concentration (1 pg on column) is improved. Four different ion sources are available: ESI, APCI, nano-ESI (liquid analysis) and direct probe-APCI (solid sample analysis without previous sample treatment).

ABSciEX QTRAP4500 Triple Quadrupole/Ion Trap MS

For the quantitative and highly sensitive analysis of targeted analytes, we use a dedicated triple quadrupole mass spectrometer. The ABSciEX QTRAP4500 Triple Quadrupole/Ion Trap MS is a hybrid of a classical triple quadrupole and a linear ion trap and offers superior sensitivity (lower limit of quantification of ~100 fg lipid on column) and excellent linearity over several orders of magnitude for targeted screening of compounds. The mass spectrometer is coupled to a Dionex Ultimate 3000 RSLC ultra high pressure LC (UHPLC), allowing the use of state-of-the-art chromatography methods and easy method transfer with the Bruker maXis QTOF instrument. The combination of typical triple quadrupole scanning modes such as multiple reaction monitoring (MRM), precursor ion scanning (PI), or neutral loss scanning (NL) with enhanced product ion (MSMS) scanning in the ion trap provides unique options for simultaneous quantification at highest sensitivity and identification of structures. The mass spectrometer is equipped with an ion source that can easily be switched between heated electrospray ionization (ESI) used for quantification of polar compounds (e.g. intact polar lipids, IPL) and atmospheric pressure chemical ionization (APCI) for compounds of intermediate polarity (e.g. core lipids). The ion source is very robust and supplies excellent ionization efficiency with minimal ion suppression, even in highly concentrated and complex matrices typical for marine sediment extracts.