Wavelength Tech Forum: MS


Joining us for this discussion are Greg Wells, Varian; Scott Kuzdzal, Shimadzu Scientific Instruments; and O. David Sparkman,  Pacific Mass Spectrometry Facility, University of the Pacific.

With the premier event in mass spectrometry taking place later this month, it is only fitting that this month’s technical forum is dedicated to MS. With hyphenated techniques such as ICP-MS and IM-MS continuing to make advances and find more widespread use, this workhorse technique is leading the way in modern-day materials analysis.

Joining us for this discussion are Greg Wells, Varian; Scott Kuzdzal,Shimadzu Scientific Instruments; and O. David Sparkman, Pacific Mass Spectrometry Facility, University of the Pacific.

What improvements have been made to mass spectrometers in the last few years to make them more efficient?

Wells: Most improvements have been in the area of LC–MS and have been confined to the ionization and atmospheric pressure ionization interface. Thermally assisted electro-spray, larger sampling orifices accompanied by larger vacuum pumps, and rf means of ion capture (ion funnels and ion guiding structures in the API) all contribute to producing more ions from the liquid and transporting them more efficiently into the vacuum chamber and mass analyzer. The most significant advance in GC–MS has been the development of supersonic molecular beam sample inlet coupled with electron ionization that produces enhanced molecular ion of compounds. The latter is the effect of super cooling the vibrational modes of the molecule to prevent excessive fragmentation that often prevents the detection of molecular ions in a spectrum.

Kuzdzal: Mass spectrometers have become more sensitive and faster in response to faster HPLC analysis requirements. Today’s small, cost-effective mass spectrometers are extremely reliable and provide extensive information for chromatographic peaks as narrow as 100 milliseconds. In addition to their enhanced performance characteristics (greater sensitivity, accuracy, and resolution), they are extremely easy to operate and integrate well with most analytical workflows. These rugged instruments are capable of multiple ionization and dissociation techniques, expanding their utility in the most demanding of applications.

Sparkman: Two areas that have significantly advanced the field of mass spectrometry are vacuum systems and detector technology involving electron multipliers. Vacuum technology has advanced in both the fore pump capability and in the turbo pump performance. The adding of roots and scroll pumps as potential replacements for the oil-based rotary vane pump have greatly reduced mass spectrometer maintenance and downtime. The rotary-vane fore pump (or mechanical pump) has been an Achilles tendon of the mass spectrometer. These pumps are usually placed out of site because of their noise and the fact that they generate heat. As a consequence, the oil is often not changed on a regular basis, may evaporate, or leak so that the pump will run dry and fail. Of course, it always fails at the most inappropriate time. The oil in the rotary vane pump is the place where un-ionized analyte accumulates and, when it is remembered to change the oil, presents a disposal issue. The scroll and roots are dry pumps and go a long way to solve these problems. These two pumps also have some disadvantages such as initial and maintenance cost but, on the whole, are probably better than the rotary vane pump.

Other than the advances in the area of split-flow technology, which is now beginning to dominate the needs for differential pumping, it is hard to be specific about turbo pump improvements. There are a lot fewer postings on the mass spectrometry forums about turbo problems, indicating longer life and better performance. In my own lab, we just don’t have turbo problems, and pumps are easily lasting more than five years. I often say that it is not possible to say whether the needs of mass spectrometry have driven the improvements in turbo pumps or the improvements in turbo pumps have led to better mass spectrometers; however, the end result has been improvements in the area of reduced instrument maintenance and more operation time.

Probably due to the number of manufacturers of electron multipliers (EM), new products are introduced every year that are longer lasting and more stable. The manufacture of EMs is still to some extent an art, but the understanding of the technology has really advanced these devices, especially over the last five to ten years.

What is the role of MS in the ever-growing biofuels industry?

Wells: MS allows the identification and profiling of components of biofuels such as triglycerides and fatty acid methyl esters. Fingerprints of biofuels are important for production quality control, control of undesired components that cause engine fouling, blends from multiple sources, and production yields. A new ASTM method now exists for measuring bio-fuel contamination of jet fuel by GC–MS by measuring the fatty acid methylesters (FAME) components.

Kuzdzal: Applications of MS in biodiesel are showing value in determining the levels of mono-, di-, and triglycerides in the fuels. This is important in determining the quality of the fuel. For example, LC–MS can provide fast screening without needing saponification or derivitization of the sample so it can be used for fast screening and monitoring the manufacturing process in biodiesel plants. Higher quality fuels will have lower ratios of triglycerides. Traditionally, this was a difficult application because of the sample preparation required. Now, LC–MS can provide direct analysis of these samples with little more than sample filtration.

It has been said that sample preparation is the most critical and most challenging task in MS analysis. How have manufacturers addressed that issue recently?

Wells: New generations of sample preparation products and separation media that separate the matrix from the components of interest and are packaged in form factors for automated use, have simplified and helped to mistake-proof sample preparation. These have been tailored to compound classes or classes of matrix (i.e., selective phospholipid removal for bioassays have helped to reduce ionization suppression effects often accompanying electrospray ionization of these types of samples).

Kuzdzal: There are a number of different aspects to this question. In the case of HPLC and LC–MS, instruments have been designed to be easy to maintain and clean. In addition, over a dozen techniques have been developed for the analysis of solid samples. While there is no technique that works for all types of materials and mass ranges, some of the recent developments allow convenient analysis of many types of materials with no sample prep required.

For analysis of liquid-type biological samples, a number of techniques have become available for removal of salts and other specific interferences. HPLC columns are available for fast removal of proteins from plasma samples to eliminate the possibility of sample loss or contamination from the sample-handling process. In other areas, comprehensive multidimensional HPLC has dramatically increased the number of peaks per sample that can be analyzed in a single run. This approach has been automated through innovative software to allow even the most complex samples to be analyzed reliably and quickly now.

In the area of MALDI-MS, today’s mass spectrometers accept industry standard microtiter plate formats (96, 384, and 1536 samples), making automated processing of samples much easier, and allowing for higher throughput. Integration with automated sample-spotting devices has made interrogation of even the most challenging samples routine. For example, a chemical inkjet printer (CHIP-1000) can be used to precisely and accurately spot MALDI matrix directly on tissue sections for analysis of intact biological samples.

Sparkman:More multiple-tasking autosampler/sample introduction systems have found their way into system integrating. These systems have mainly been produced by CTC, their OEMs, and Tekmar. The instrument companies are embracing these third-party products better than they previously did; therefore, their interface is more seamless.

How is MS being used to detect contaminants in food products? Is this an application in which you see potential for growth? Why?

Wells:The area of food safety continues to be one of the strongest growth markets – everyone eats, everyone is concerned about acute and chronic exposure to residues in food, adulterants, and naturally occurring toxins. Contamination can occur from long-term exposure of animal and aquatic life-forms in the food chain that can accumulate contaminants. These concerns have spawned the need and the will to bear the cost of food safety testing to control the quality of the food supply worldwide.

Kuzdzal:The high sensitivity and specificity of mass spectrometers used with HPLC provide excellent low-level detection of contaminants in food products, especially in situations where multiple components coelute in HPLC separations. MS also allows researchers to rapidly detect specific contaminants and also to perform structural elucidation of unknown contaminants. MS will continue to play a larger role in food safety assessment, from the detection and identification of food contaminants, ranging from synthetic organic contaminants such as pesticides and naturally occurring toxins, to identification of complex microorganisms including bacteria and fungi. New software is starting to show up to allow for rapid microorganism identification and “fingerprinting.”

Sparkman:One of the main food contaminants is due to agricultural chemicals used in getting better crop yields. GC–MS-MS selected reaction monitoring (SRM) has brought back an interest in MS-MS for gas chromatography due to its high selectivity that is resulting in low detection limits for potentially harmful substances.

Now that potential problems have been brought to the attention of the public (worldwide), more food testing is going to be carried out by both government agencies and the industry itself. GC–MS-MS will see a growth for food testing.

What impact has increased speed and sensitivity had on software and data interpretation? Have the instruments become too fast and sensitive for most software systems?

Wells:The desire for faster analysis has pushed the need for both hardware and software improvements. More samples per unit time require more data to be processed and interpreted per unit time. Increased sensitivity has driven the need for increased selectivity to better discriminate between the compounds of interest and the background matrix.

Kuzdzal:As MS instruments have become faster, the impact of large amounts of data has grown significantly. However, many MS instruments have real-time computers embedded inside the instrument to allow for some signal processing on the fly. This has made it possible for most PCs to keep up with the large amounts of data without overwhelming the limitations of communication speeds. Today’s software is also much more integrated with the analysis workflow.

Sparkman:Data are being produced faster, but the time required for a person to evaluate the data is not changing. A person can read a chromatogram produced in 50 seconds no faster than they can read one produced in 25 minutes. An instrument’s ability to produce higher signals from less sample have been complicated by the fact that instruments can now produce more signal from components that are not of interest; the matrix, the background. The decrease in detection limits has led to trying to balance between what is detectable and what is harmful. In many cases, the evaluation of true health risks has not caught up to the decreased detection limit.

It is true that software systems have to be improved to handle nosier data, which often results from lower detection limits and faster acquisition; but the instrument manufacturers are aware of this and are doing a fairly good job of maintaining a good balance for faster acquisition with lower detection limits and the ability of their software to process these data.

How has the implementation of computer-controlled automation in MS expanded the use of the technology?

Wells:Computer automation has allowed the routine operation of complex instruments and methods by less skilled operators. It also has allowed the linking of more complex sample-handling and preparation methods to the sample analysis in a seamless manner.

Kuzdzal:Automation with MS instruments is opening up a number of new applications. Software allows for faster method development including optimization of parameters for sensitivity, quantitation, and calibration. In addition, the software is more closely coupled between the MS detector and the rest of the components (usually HPLC) to insure that all components are ready and able to function at maximum efficiency. Essentially, most of the recent software makes it harder to make a mistake or leave out some important step. Software for walk-up/walk-away use allows for “wake-up” functions, can run samples based on priority requirements, and can even call you on a cell phone or email you when it is done or needs maintenance. Automation software is making it easier for everyone to get the most out of their instruments.

Sparkman:Computer-controlled automation in mass spectrometry has lessened the need for highly skilled operators. The good news is that this has greatly increased the pool from which to draw operators. The bad news is that many of these new operators don’t know what to question when something changes in the data. An example is a recent experience I had with someone reporting the crash of a program that had always worked with samples they were analyzing. After some searching, it was discovered that a threshold value of 100 had always been employed during the data acquisition. Somehow, the threshold had been changed to 0. The operator knew to inject the sample using a specific method. It never occurred to the operator to check to see if there had been any changes in the method. This has now led to a new requirement for the development of the next revision of the software.

Because of the high degree of computer-controlled automation, the operator is less motivated to understand the method, which can lead to errors if for some unknown reason the method is altered.

What do you think?

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