primer is then extended by a number of bases that are characteristic of the particular mutation. The mass value of the predesigned products is far more analytically definitive than signals from fluorescence-based assays, which treat the hybridization characteristics as the "be all and end all" of detection. Also, because a particular mutation yields a known mass, problems with the reaction could easily be detected when an unexpected mass was encountered. Little says that they have not needed to use this particular advantage of MS because of the "high fidelity" of the polymerase enzymes used. In the experiments that Little described, a MALDI matrix was predispensed on the chip and nanoliter quantities of diagnostic material were placed on top using a piezoelectric pipettor (a tool that resembles a single element of an inkjet printer) or by a simple pin transfer. He says that the matrix material can also be mixed with the analyte solution or placed on the chip after the DNA is dispensed. Little presented data showing the reproducibility of the technique and its use in population studies. The same analyte solution was transferred with die piezoelectric pipettor into chip wells and analyzed reproducibly 100 times. When the samples do not necessitate washing the pipettor between transfers, the total time is only a few minutes. For parallel analysis of different samples, they used a 16-pin tool that was dipped into the samples and transferred to the chip. He showed PROBE reaction products for a 15-person population study using this method. Little described MALDI on a chip as an "enabling technology" for gene-hunting and pharmaceutical companies. Currently, the most powerful use of MALDI on a chip is in typing applications (which Little says will be particularly important for large-scale clinical studies) and microsatellite (sizing) analysis. "Diagnostic" or "signature" sequencing on a chip on a large scale is still further down the road.
The incredible shrinking mass spectrometers With field-portable instruments, the smaller the better, and portable mass spectrometers are no exception. Maria C. Prieto of Lawrence Livermore National Laboratory presented results on an ion cyclotron resonance mass spec-
trometer that fits on one side of an 18 x 12 x 3.5-in. briefcase. The instrument, which was developed by Dan Dietrich and Bob Keville, consists of an open-endcap cylindrical Penning trap, a piezoelectrically actuated inlet valve, and an ion-pump vacuum system, all of which are miniaturized. Power is provided by four 12-V laptop computer batteries. The analyzer cell is located in a 0.44-T permanent magnet. Prieto and co-workers characterized the performance of the analyzer cell by studying the variation of the ion cloud radius with applied rf voltage (needed for ion excitation). The ionization time needed to produce the same number of ions was documented for different trapping and compensation electrode voltages. The system currently achieves mass resolution of 500-100 at mid 10-7 torr pressures with a magnetic field homogeneity of ~ 1000 ppm. Another miniaturized instrument presented by Scott Ecelberger of The Johns Hopkins University Applied Physics Laboratory has been dubbed 'Tiny TOF". The instrument is designed for chemical and biological warfare agent detection, using biomarkers developed at the University of Maryland-Baltimore County and an analyzer developed at The Johns Hopkins University School of Medicine. Tiny TOF consists of a 9-cm coaxial TOF analyzer and a shoebox-sized vacuum chamber. A 600-ps pulsed N2 2aser is ssed for the UV MALDI experiments. The mass spectrometer, sensitive to the subpicomole level, has a mass range > 11,000 m/z znd resolution of 300-1000. Also seen at ASMS was the quadrupole array mass spectrometer developed by the Jet Propulsion Laboratory. (Anall Chem. 1997,69,335 A-36 A)
Using buffers with MS The conventional wisdom says that the buffered mobile phases used in HPLC are incompatible with MS analysis because they contaminate the ion source, create chemical noise, and reduce the analyte signal. Duncan Bryant and Sean Burnside of SmithKline Beecham (U.K.) demonstrated that the conventional wisdom might not always be right. They tested various nonvolatile and low-volatility buffers with the pharmaceutical compound eprosartan, including sodium dihydrogen phosphate, ammonium
dihydrogen phosphate, sodium acetate, and orthophosphoric acid. In each case, a 1- to 50-mM buffer gradient containing 1 ppm eprosartan entered the mass spectrometer at 100 uL/min. Sodium dihydrogen phosphate was usable up to 20 mM. At higher concentrations, the background chemical noise increased greatly, and the eprosartan signal decreased. The sodium acetate was completely unusable, and suppression occurred at concentrations as low as 5 mM. The orthophosphoric acid had the widest range, usable up to almost 50 mM. They also tested several flow rates, using sodium dihydrogen phosphate as the test buffer. They determined that the system is optimized in the range of 220 uL/min. At flow rates > 25 uL/min, they observed analyte signal suppression and a high level of chemical noise.
Low-pressure electrospray? Electrospray is considered an atmospheric pressure ionization technique, but does it have to be? According to results presented by Edward Sheehan and Ross Willoughby of Chem-Space Associates (Pittsburgh, PA), under the proper conditions electrospray ionization can be performed at pressures as low as 10"4 torr. They divided a plot of discharge onset voltage versus pressure into three regions. At intermediate pressures, the voltage was not high enough to overcome the threshold for cone-jet formation. However, at very low and atmospheric pressures, the discharge onset voltage was above the required minimum. At atmospheric pressure, there is enough heat to prevent the electrospray cone-jet from freezing. However, at reduced pressures, the entrance aperture needed to be heated to prevent freezing. They demonstrated low-pressure ESI with the protein myoglobin and caffeine. The ESI spectrum of myoglobin contained the characteristic multiply charged envelope, which was centered around the +11 ion. The spectrum of caffeine showed the predominant protonated molecular ion at m/z 195. By changing the voltage difference between the desolvation region and the exit aperture, they were able to fragment the m/z 195 ion. They said that several questions remain, including the optimum conditions, the ion-sampling efficiency, and the analytical use of the method.
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