Bruker Daltonics, Inc.

Separation Science & Metabolomics Laboratory, Metabolomics Australia, Murdoch University, Murdoch, Western Australia 	

The emerging field of metabolomics is defined as the nontargeted detection and quantification of small molecule metabolites that are found in biological materials. With the advantage of noninvasive techniques, metabolomics is finding a growing number of applications in: chronic disease, environmental exposure, functional genomics, neonatal screening, nutrition, sports science, toxicology, and transplantation to monitor the health of the transplant organ. 

To identify and quantify the vast range of chemically diverse primary and secondary metabolites, any analytical approach must encompass reliable sampling, precise detection methods, and advanced data handling and interpretation capabilities. With two recognized approaches to metabolomics, the first is referred to as "footprinting" (1) and is used when profiling metabolites within the environmental medium into which they were secreted by cells. Alternatively, a more untargeted method to metabolomics would be to analyze all metabolites present in a more blanketed approach much simpler and required less frequently, resulting in a more robust system for long operation. 

Transitioning Single Quadrupole (SQ) to Triple Quadrupole (TQ) GC–MS

While metabolomics studies can be carried out using single quadrupole (SQ) GC–MS instruments, these rely heavily on mass spectra deconvolution and some derivatization for polar compounds such as amino acids, organic acids, and simple sugars (2). The application of single quadrupole GC–MS requires chromatographic temperature ramps and spectral scan rate to be matched in order to maximize the number of compounds that can be routinely identified for metabolite profiling. The scan rate becomes a limiting factor in metabolite profiling and the ability to effectively deconvolute data sets is critical. 

To lessen the load of quantitative metabolomics, triple quadrupole (TQ) GC–MS has been utilized to excellent effect. The SCION™ TQ hardware (Figure 1) provides higher throughput and faster scan speeds for more efficient sampling runs, greater sensitivity, and zero cross talk and improved signal-to-noise ratio (S/N). Final outcomes include the ability to process more samples, resolve more compounds, and improve identification of compounds.  

Figure 1: Bruker’s SCION GCâMS TQ platform with PC illustrating the compound-based scanning (CBS) software.			

The transition from single quad to triple quad can be illustrated with the use of GC–MS-MS with a standard 43 component mixture used for a QA/QC mix with derivatization (2).  

Figure 2: Full scan mass spectrometry of 43 components QA/QC mix (TMS).			

Figure 2 illustrates a typical full scan with a significant peak (3). Following close examination of the peak, total ion chromatogram coelution of proline and leucine was identified. The need for deconvolution to resolve these compounds was necessary (see Figure 3).  

Figure 3: Upper: Full scan mass spectrometry of 43 components QC mix split (TIC filtered) with coelution at one peak.			

When investigating metabolomic samples there are several hundreds of compounds and several may coelute. These compounds can be very similar in structure, such as sugars and deconvolution may not resolve them. Expanding the capabilities of the GC–MS-MS system is ideal with an instrument that undertakes data dependent MS and combines it with a series of scheduled multiple reaction monitoring (MRM) transitions. This extended system can be set-up to reference known coelution and accurate retention times of compounds.  

Figure 4: Deconvolution of proline and leucine by compound-based scanning (CBS) software.			

In Figure 4 the mass spectra of the left and right markers are quite similar and dependent on the speed at which data can be collected. Deconvolution may not resolve the two compounds in the peak, but if analysis by MS-MS is undertaken (Figure 5), the baseline is cleaned up significantly improving S/N, and the limits of detection (LOD) and quantification are greatly improved. It then becomes obvious that two peaks exist at this retention time (RT), whereas in full scan it was only a single peak with coelution. Using the triple quad system, it only takes one transition to resolve the compounds in this instance.  

Figure 5: GCâMS-MS TQ of 43 components QA/QC mix illustrating two peaks for coeluted compounds proline and leucine.			

A triple quadrupole operating in MRM mode with compound-based scanning (CBS) software provides enhanced duty-cycle coverage so that resolution of co-eluted compounds is possible in the SCION GC–MS TQ. It also provides a faster scan rate with lower LOD and this will lead to improved metabolome coverage with routine metabolite profiling using built-in libraries.   

The TQ can also deliver precursor ion scans, product ion scans, neutral loss scans, and MRM to assist in the identification of unknowns as well as quantitatively detect metabolites in really complex mixtures at very low levels. 

Chemical ionization (CI) in combination with MRM mode provides high throughput metabolomics capability, where as full scan struggles to resolve compounds. In addition, when using CI, an improved transmission is possible. With the right combination of reagent gases, compound identification is further improved, more compounds are resolved, and S/N is substantially better. CI also provides higher mass to charge (

) species of precursor ions for MS-MS with far less interference from the matrix. TQ MS that combines CI and MRM offers the potential to increase metabolome coverage and identification and provides both the LODs and improved precision required to do so. 

Bruce Pebbles, Katherine Rousetty, Catherine Rawlinson, Garth Maker, Joel Gummer, Christian Krill, Hayley Abbis, and Bong Sze, Murdoch University; Australian Research Council, West Australian Government, Grain Research and Development Corporation, Grape and Wine Research and Development Corporation, Bioplatforms Australia and Murdoch University for funding; and Bruker Chemical and Applied Markets division. 

(3) "Transitioning from Single Quadrupole to Triple Quadrupole GC/MS-based Metabolomics" live webcast, 

tel. +1 (510) 683-4300, Fax. +1 (510) 490-6586  

Articles

Comparing Single Quadrupole with Triple Quadrupole GC–MS-Based Metabolomics

There are currently more than 1000 pesticides in use worldwide in the production of foodstuffs. There is a significant risk to human health and the environment due to increased pesticide use, poor agricultural practices and illegal use. Most countries have adopted strict regulations governing pesticide usage. Pesticide control laboratories are now under increased pressure to maximize both the range of pesticides detected in a single run and in complex matrices. 

In this study, we describe the development of a multi-residue pesticide testing method utilizing a Bruker 320-MS triple quadrupole mass spectrometer that provides a fast, sensitive, and robust procedure for the comprehensive analysis of a large number of pesticides at 0.1 to 0.5 µg/kg levels in a single analytical run in food samples. At the completion of the study, the results obtained were analyzed to determine if they met the important SANCO

quality control requirements for analysis of pesticides.

 Figure 1: Listing of the 212 pesticides measured in this study. 			

A dispersive extraction procedure known as QuEChERS was used to prepare samples for analysis. QuEChERS is a simple and rapid technique, which is suitable for multi-residue pesticide analysis in a wide variety of food products. The homogenized samples were extracted with an organic solvent, agitated, and centrifuged for 1 min. The supernatant was transferred into a sample tube containing MgSO

 and Bondesil™ PSA, agitated for 20 s and then centrifuged. This extract was then transferred to an autosampler vial for direct injection into the Bruker 320-MS GC–MS-MS system.

Figure 2: GCâMS-MS chromatogram of 212 pesticides at concentrations ranging from 0.1 to 5 Âµg/kg. 			

Injector Temp.  70 °C ramped to 300 °C 

Carrier Gas Helium at 1.2 mL/min, Electronic Flow Control (EFC), constant flow mode			

Column  FactorFour™ VF-5ms, 30 m × 0.25 mm × 0.25 µm  (Bruker Part Number CP8944)			

Column Temp. 60 °C, 2.0 min hold  20 °C/min to 150 °C 3 °C/min to 260 °C 30 °C/min to 290 °C, 6.0 min hold 

Detector Voltage  Extended Dynamic Range (EDR)			

Bruker 320-MS triple quadrupole mass spectrometer			

Samples containing a mixture of 212 pesticides were analyzed. List of the pesticides tested is shown in Figure 1. The rapid determination of all 212 pesticides was achieved with a single injection, monitoring at least two MRM transitions for each analyte to satisfy the SANCO quality control procedures. Figure 2 shows the full target ion chromatogram (TIC) for the 212 pesticides. Figure 3 shows a typical calibration curve for one the pesticides measured (tetraconozole). 

Figure 3: Calibration curve for tetraconazole. 			

To further satisfy the SANCO quality control procedures, the Bruker MS Workstation software has the ability to measure and confirm ion ratios of the primary and secondary transitions automatically. Retention times of analytes are checked and confirmed at the same time. Figure 4 shows an example of a variety of user-selectable views of the data that enable the analyst to view primary and confirmatory transitions, calibration curves, a sample report, and spectrum comparison in a single window. This customizability has a significant effect on the speed at which samples can be processed, and helps to improve throughput in a busy analytical laboratory.

Figure 4: Data for tomato matrix matched solution at 0.05  Âµg/kg. 			

The highly sensitive Bruker 320-MS GC–MS-MS triple quadrupole mass spectrometer, controlled with MS Workstation software, can carry out the simultaneous analysis of 212 pesticides, including isomers and metabolites in a food matix. The results of this analysis were fully compliant with the SANCO quality control procedures. 

There are currently more than 1000 pesticides in use worldwide in the production of foodstuffs. There is a significant risk to human health and the environment due to increased pesticide use, poor agricultural practices and illegal use.

Multi-Residue Pesticide Analysis Using a GC Triple Quadrupole Mass Spectrometer

The amaZon series is the next step in Bruker Daltonics' family of ion trap mass spectrometers. Incorporating a number of new innovations to Bruker's highly successful ion trap technology platforms, the amaZon series is a true leap forward in performance and reliability. 

Figure 1: Direct injection of 50â750 fg Reserpine. Even at 50 fg in MS-MS mode, the amaZon is able to achieve at 50:1 S/N. Note also the excellent reproducibility in these 5 replicate injections.			

Building upon Bruker's vast foundation of expertise with ion traps, the amaZon series of instruments features a number of key performance enhancements including: 					

Greater than 10× sensitivity improvement over previous generation systems

Outstanding mass resolution of up to 20,000

Extraordinarily broad mass range from 50 to 3,000 

Rapid scan speeds of up to 52,000 μs allowing full compatibility with fast separation techniques

The very latest in ETD/PTR technology for analysis of post translational modifications of peptides or proteins

High speed ion polarity switching for handling difficult or complex samples

Comprehensive MSn spectral libraries for easy molecular formula determinations

Figure 2: Dynamic range of amaZon. In this example 1â10,000 ppb of melamine on column was measured by the amaZon. This correlates to at least 4 orders of magnitude dynamic range with outstanding linearity.			

The amaZon series is available in 3 versions: 

for all protein and small molecule applications. 

for small molecule and protein applications at a value price. 

Designed for key applications in chemical reaction yield and purity determinations, proteomics, protein and glycan analysis, metabolomics, compound screening, and compound identification, the amaZon series is the top performing ion trap available today. 

Figure 3: Speed of amaZon. In this example, a compound in plasma separated by UPLC with a 1 s peak width is measured at 20 Hz in positive and negative ion mode with "zero delay" alternating ionization switching.			

The performance of the amaZon X Ion Trap System has been tested in terms of sensitivity, speed, and dynamic range for small molecule analysis. It has been shown that the amaZon is easily capable of detecting low femtagrams of a small molecule on column. The amaZon also displays at least four orders of magnitude dynamic range when quantitating small molecules in the ppb to ppm range. The instrument also possesses the capability to do "zero delay" polarity switching, even at UPLC separation speeds. All of these attributes demonstrate the high performance capabilities of the amaZon and demonstrate it's suitability for deployment in a number of small molecule analysis areas. 

The amaZon series is the next step in Bruker Daltonics' family of ion trap mass spectrometers.

Bruker Daltonics, Inc.

Articles

Comparing Single Quadrupole with Triple Quadrupole GC–MS-Based Metabolomics

Multi-Residue Pesticide Analysis Using a GC Triple Quadrupole Mass Spectrometer

Multi-Residue Pesticide Analysis Using a GC Triple Quadrupole Mass Spectrometer

Analysis of Small Molecules with amaZon: Enhanced Sensitivity, Speed, and Dynamic Range