Sample preparation reduces sample complexity and renders samples into a format amenable to downstream analysis. Sample prep is most necessary for complex, multicomponent samples containing substances that interfere either with the MS (e.g., through ion suppression) or, in GC/LC-MS, the chromatography. Interferences are common in biological samples due to the large molecular diversity and wide concentration dynamic range. Prep is also required for foods (due to their complexity) and environmental samples (low concentration target analytes).
We often hear of chromatography being the “prep” step for LC/GCMS, but that is only partly true. As Vivek Joshi, Ph.D., senior research scientist at EMD Millipore (Danvers, MA) notes, prep is often required even with chromatography as the first step. “In fact, really complex samples might require multiple sample preparation techniques for enriching particular components. On the other hand, with simple particle- free solutions, ‘dilute and shoot’ methods might work as well.”
Some MS techniques require less rigorous sample prep than others. Atmospheric-pressure chemical ionization (APCI) has been known since the early 1980s but is emerging as a popular approach to MS. In APCI, samples are ionized directly from HPLC effluent, without splitting off a large fraction to waste. In this technique, the mobile phase is heated above 400º C, and swept by a nitrogen flow into an ionizing corona discharge at atmospheric pressure vs. the high vacuum required by other ionization methods. Unlike more common electrospray, ionization occurs in the gas phase. APCI allows the use of nonpolar HPLC solvents. On the downside, this technique involves hard ionization, resulting in more fragment ions.
According to PerkinElmer’s Andrew Tyler, modified APCI-like techniques also allow the direct analysis of organic molecules from dry or solid samples. “You can basically wave a sample on a dipstick in front of the machine and generate data from it. APCI is great for seeing if your compound is there or not, without running chromatography.”
Numerous papers have been published on APCI for direct MS measurement of a wide range of analytes with little or no sample preparation. Quantifying drugs of abuse on currency, cholesterol variants from dry blood spots, plasticizers in toys, plasma drug levels, and chemical warfare agents are just a few of the possibilities. Matthias Jecklin at Switzerland’s ETH Zurich wrote in Rapid Communications in Mass Spectrometry that a variant of APCI was “a powerful high throughput tool for the investigation of very low amounts of pesticides in fruit juices and on fruit peel/vegetable skin.”
Because it is a rapid technique, APCI is an excellent screen for the presence or absence of target analytes. Samples that come back positive could then be subjected to corroborative GC-MS or HPLC-MS afterward.
In MS or GC/LC-MS, the sample preparation method must match the workflow as well as the analysis objectives. For example, prep can easily become a bottleneck when conducting rapid MS methods. “If your sample prep isn’t rapid and convenient, you lose the benefit of rapid analysis,” observes Robert “Chip” Cody, Ph.D., Mass Spectrometry product manager for JEOL (Peabody, MA). Laboratory automation (dispensing and labware handling) relieves this bottleneck and provides consistency as well.
In contrast to ambient ionization methods, techniques such as electrospray and MALDI (matrix-assisted laser desorption/ionization) require sample prep to remove interferences from salts. Many kits and consumables on the market have been designed for specific matrices such as pond water or biological fluids. Analysts employ solid phase extraction, microextraction fibers, and related products consisting of pipette tips and chromatography media. Even with DARTTM (direct analysis real time), it often makes sense to remove the matrix before introducing the sample.
Because of its gentle ionization, MALDI is widely used in biomedical research. For example, scientists at University Hospital (Essen, Germany) use MALDITOF to identify bacteria in urine in 30 minutes. MALDI’s kid-glove treatment preserves the bacterium’s ribosomal proteins, plus lipids and polysaccharides.
“MALDI-TOF MS results are independent of assumptions about the putative genus of your isolate,” lead researcher Dr. Frank Mosel explains. “You can use the very same protocol for any sample, which is not true for standard methods. And the MS method is very fast and inexpensive.”
Sample prep for MS is a deep subject since so many permutations of samples, matrices, ionization techniques, and MS detector combinations are possible. Generally, sample prep needs to serve overall workflow objectives, which encompasses basic objectives in MS: to provide a sample that is appropriate for the analysis.
When JEOL introduced DART in 2005, the company touted the technique as one that required little or no sample preparation. “We found many interesting applications for DART,” comments Robert Cody. “But after several years of trying to run everything that way, we modified our position.” JEOL currently holds that the right sample preparation method for the analysis objectives, even with DART, provides analysts with the best “mileage” from MS.
DART is a plasma ion source that strips surfaces of analyte ions and sweeps them into the spectrometer. Analysts place raw samples into the ionization chamber where analytes are protonated. Anything qualifies as a sample: a shred of plastic, a fork, a swab from a fruit or piece of meat, a pharmaceutical tablet—as long as it fits within the gap between the source and the MS inlet. An interesting demonstration of DART ionization can be found at http://bit.ly/YkVMhP.
Two other ambient ionization techniques have been commercialized. DESI (desorption electrospray ionization), which resembles conventional electrospray ionization, also works with nonconventional substrates and requires minimal sample prep. LAESI (laser ablation electrospray ionization), developed at George Washington University and commercialized by Protea Bio (Morgantown, WV), generates 2-D maps of surfaces, including biological samples.
Many MS methods can proceed without sample prep with DART “ambient ionization” ion sources. “Nevertheless, sample prep can greatly expand the applications of the source and the benefit of rapid analysis with DART is not lost if the sample prep is rapid, inexpensive, and convenient,” according to Robert Cody. “We have found benefits in combining techniques such as solid-phase microextraction and in-situ derivatization with DART.”