Finding the perfect GC-MS

Environmental analysis laboratories face many challenges relating to sample preparation, analysis and efficient data reporting – but how do you choose the most appropriate GC-MS technology to suit individual analysis needs?

Environmental laboratories face unique challenges with delivering accurate, consistent, reproducible and traceable results while complying with stringent legislation. In the current regulatory environment, it is more important than ever to implement breakthrough technologies that facilitate regulatory compliance, ensure public safety and monitor quality throughout the entire sampling, testing and reporting process. GC-MS is a powerful technology, perfectly suitable to address the needs of environmental laboratories.

[caption id="attachment_27062" align="alignright" width="300" caption="Table 1: Nine criteria for selecting a GC-MS technology"][/caption]

Starting with the sample injection, features of an autosampler can have a beneficial effect on the performance of the entire system in the lab. There are currently a wide range of GC-MS technologies to choose from, including single quadrupole, ion trap and triple quadrupole. Often, it is very clear which one of these technologies is the most appropriate for a particular laboratory or application. However, there are times when the choice is a difficult one requiring the scientist to assess a number of different parameters to reach a final decision. Table 1 lists nine key selection criteria – the number of check marks indicates that a specific technology is well positioned to fulfil a given criterion. By ranking the criteria that are most important for a laboratory’s current analytical requirements and future plans, scientists can select the technology that best fits their overall performance needs.

Current state-of-the-art autosamplers offer a number of powerful capabilities, achieving accurate and reproducible injections of nano-volume samples at high speeds, approaching 100ms. Advanced systems, such as the Thermo Scientific TriPlus RSH autosampler, can handle the routine tasks of environmental laboratories, eliminating the associated errors and challenges. There are certain requirements that need to be taken into careful consideration when selecting an autosampler. In cases when a small sample volume is available, it is necessary to use a system that is able to get to the bottom of the sample without damaging the needle and still provide accurate injections. When a large number of samples must be analysed, it is preferable to implement an autosampler with a high throughput capability that will keep injecting samples, enabling unattended, continuous operation.

[caption id="attachment_27063" align="alignleft" width="300" caption="Figure 1: Chromatogram and chromatographic separations of semi-volatile compounds"][/caption]

Regardless of the sample quantity and the complexity of a particular application, it is always beneficial to choose an autosampler that provides the flexibility to easily and quickly change between liquid, headspace and solid phase micro extraction (SPME) injection modes. This new capability enables a laboratory to meet current and future possible needs with one autosampler. This superior capability ensures uninterrupted operation, thereby accelerating laboratory productivity. Additionally, choosing a system that can perform routine, prone-to-error tasks prior to the injection can help to substantially reduce the time spent on sample preparation, since the need for re-extraction due to errors is eliminated. The chosen autosampler will also need to be flexible enough to offer new expanded capabilities in line with changing laboratory needs. A basic liquid injection autosampler can be initially implemented, which can be subsequently enhanced with the addition of different sample handling trays, such as trays for keeping samples cool. Vortexing sample preparation capabilities could be a further advancement, while a barcode reader can help keep track of samples and ensure tighter control of chain of custody.

Single quadrupole GC-MS technology has been established as the gold standard in environmental laboratories offering many significant advantages, including superior robustness, accelerated sample throughput, high sensitivity, optimal precision and excellent reproducibility. Modern single quadrupole GC-MS systems are capable of continuous operation, analysing large numbers of samples without the need for cleaning and maintenance intervals. In addition, they offer the flexibility to easily and quickly switch between full scan and selected ion monitoring (SIM) modes for reliable screening of unknown library searchable spectra and low level analysis respectively. Being equipped with a highly inert, non-coated, high temperature ion source and powerful ion optics, single quadrupole GC-MS systems maintain optimal performance for long periods of time.

[caption id="attachment_27064" align="alignright" width="300" caption="Figure 2: Illustration of the fast scanning and separation capabilities of single quadrupole GC-MS technology"][/caption]

An operation that provides added flexibility to meet long term needs of the lab is pulsed positive ion negative ion chemical ionisation (PPINICI). This capability allows scientists to collect both positive and negative ions in the same analytical run. This is of particular benefit when analysing pesticides and polychlorinated biphenyls (PCBs). All of the data is collected into one data file that can be easily worked with through the software and makes archiving simple. One of the issues that analysts face in the laboratory is cleaning the source of the GC-MS system. With current powerful single quadrupole GC-MS systems, the source is removed and replaced with a clean one while the system remains under vacuum. This allows the system to maintain optimum, uninterrupted performance. Users can also quickly and easily switch between dedicated electron ionisation (EI) and chemical ionisation (CI) sources to address different application requirements.

Owing to all of the above capabilities, single quadrupole GC-MS technology is ideal for analysing semi-volatile compounds in contaminated samples, without the need to dilute and re-inject the samples. Figure 1 shows an example chromatogram and the respective chromatographic separations of semi-volatile compounds analysed using a single quadrupole GC-MS system, using a 1 µL injection at a 200 ng/µL per compound amount. Figure 2 demonstrates the instrument’s fast scanning and separation capabilities for the analysis of semi-volatile compounds. In the top mass chromatogram, the separations that are required by many methods globally are shown, whereas the bottom chromatograms show how three peaks are separated without tailing, as would typically happen when analysing these challenging compounds.

[caption id="attachment_27066" align="alignleft" width="300" caption="Figure 3: Full scan/SIM methodology on a single quadrupole GC-MS system for drinking water analysis"][/caption]

Three semi-volatile compounds were analysed in a drinking water sample using a single quadrupole GC-MS system to demonstrate the superior capabilities of the technology. The system was operated in alternating full scan/SIM mode (Figure 3). The full scan portion of the data file provides the quantitation at relatively higher levels and unknown screening. The SIM portion of the data file provides targeted low level analysis for three of the compounds. Two of the three targeted compounds showed method detection limits (MDLs) of 2 ppt. The third compound showed an MDL of 4 ppt. In order to meet the QC requirements and the low detection limits imposed in certain states of the USA, such as California, and in the European Union, SIM must be used, while switching between full scan and SIM modes allows analyses to be completed in one rather than two injections.

Ion trap GC-MS technology offers greater specificity and selectivity than single quadrupole GC-MS, thus being more suitable for analysing low analyte concentrations in very high matrix interferences. Just like single quadrupole GC-MS systems, ion trap GC-MS instruments can be operated in full scan mode. However, the most important advantage associated with ion trap GC-MS technology is its capability to perform tandem mass spectrometry (MS/MS) analyses, thereby allowing for targeted capture and subsequent fragmentation of specific compounds. As a result, matrix interferences are minimised and specificity is increased. In addition, by using the generated fragmentation pathways, it becomes possible to achieve reliable structural characterisation of the compounds under investigation. This capability is further enhanced when coupling the ion trap GC/MS-MS system with a CI source. Due to the time it takes to perform MS/MS in an ion trap, the practical number of different MS/MS functions should be kept to less than 60 per sample. However, it does give the possibility of MS/MS/MS/MS/MS.

[caption id="attachment_27069" align="alignright" width="300" caption="Figure 4: GC/MS-MS product ion spectrum of chlordane"][/caption]

An ion trap GC/MS-MS instrument was used to analyse chlordane in a sediment sample. When performing MS/MS analyses, the precursor ion is isolated. In this application example, mass to charge (m/z) 373 precursor ions were isolated and then fragmented. The resulting product ions were fragments of the 373 m/z precursor ion and created the GC/MS-MS product ion spectrum shown in Figure 4. The use of MS/MS in this case improved selectivity and enabled reliable confirmation of the presence of chlordane in the sample.

In another application, an ion trap GC/MS-MS system was used to analyse Bis(2-ethylhexyl)phthalate (DEHP) in serum. Recently, there have been many cases of DEHP being found in drinks, the consumption of which results in this harmful compound entering the human body. EI GC-MS analysis of phthalates typically generates a non-specific full scan spectrum with a prominent m/z ratio of 149. Using positive chemical ionisation (PCI) with ammonia generates a prominent [M⁺¹] at 391 m/z. This method involves the ion being isolated and fragmented in the ion trap GC/MS-MS instrument, delivering improved specificity and confident identification and quantitation of phthalates. Figure 5 shows the calibration curve for DEHP in serum.

[caption id="attachment_27070" align="alignleft" width="300" caption="Figure 5: DDT analysis in contaminated surface water"][/caption]

Triple quadrupole GC-MS technology allows scientists to further increase analytical specificity and achieve highly precise targeted screening and quantitation of low level analytes in very complex matrices. Sample matrix effects are dramatically reduced, making peaks easy to integrate. The technology can be used in selected reaction monitoring (SRM), multiple reaction monitoring (MRM), highly selective reaction monitoring (H-SRM) and ultra-selective reaction monitoring (U-SRM) modes, depending on the particular application needs. Hundreds of compounds can be analysed in a single injection by operating the system in the timed-SRM mode. This function automatically adjusts timing on the SRMs to provide consistent points across the peak even with partial overlap of these segments.

The plot in Figure 6 shows a full scan chromatogram of dichlorodiphenyltrichloroethane (DDT) in contaminated surface water, generated using a triple quadrupole GC-MS instrument. Due to matrix interferences, quantitation of DDT using this method is not possible. The chromatogram shown in the middle is from the same sample analysed using SIM. Matrix interferences have been reduced, but not enough to reliably quantitate the DDT. The bottom chromatogram is free of matrix interferences. This has been achieved by using SRM techniques. By monitoring the transition from the DDT precursor ion at 234.94 m/z to the product ion at 164.96 m/z, all matrix interferences have been eliminated from the data file. The only practical limit to the number of compounds is how many you can realistically separate on a column.

[caption id="attachment_27071" align="alignright" width="300" caption="Figure 6: The complete data file of coeluting pesticide analysis"][/caption]

Coeluting pesticides were analysed using timed-SRM on a triple quadrupole GC-MS system. The bottom chromatogram in Figure 7 shows the complete data file. By zooming into one region of less than one minute, it becomes evident that there are a lot of compounds eluting simultaneously. Timed-SRM allowed the instrument to rapidly optimise itself for each compound in order to handle the heavy workload in a timely fashion. This enabled researchers to focus on other important activities in the laboratory without worrying about the outcome of this analysis. In addition, excellent reproducibility and precision were achieved.

Timed-SRM allowed the instrument to analyse many compounds in the same injection and achieve excellent precision. Table 2 shows a subset of the compounds in the method. These compounds were analysed at 10pg on column with 10 replicate injections.

There are a variety of GC-MS technologies available and each one is suited to different environmental analysis laboratories depending on the complexity of the samples to be analysed. Single quadrupole GC-MS and ion trap GC-MS can both perform precise and reproducible full scan analyses, whereas MS/MS analyses are enabled by both ion trap GC-MS and triple quadrupole GC-MS. GC-MS is just one step in the path of reporting data and it is important to pay attention to other important parameters, including sample preparation. Autosamplers can significantly improve sample preparation efficiency and eliminate errors associated with manual techniques.

The author: Eric Phillips, Method and Application Development Manager, Thermo Fisher Scientific, Austin, Texas