MSACL 2016 US Abstract

Combining Microfluidic Separations with High Pressure Mass Spectrometry for Clinical Diagnostic Applications

J. Scott Mellors (Presenter)
908 Devices Inc. and University of North Carolina

Authorship: J. Scott Mellors1, Mike Goodwin1, Erin Redman2, Mac Gilliland2, Christopher D. Brown1, J. Michael Ramsey2
(1) 908 Devices Inc., Boston, MA (2) University of North Carolina, Chapel Hill, NC

Short Abstract

We are attempting to move LC-MS analyses out of the core lab by combining microfluidic capillary electrophoresis with high pressure mass spectrometry. Combining these two miniaturized technologies yields a system that can fit on the benchtop of nearly any lab and can be operated by a non-expert. The integration of sample injection, separation and electrospray ionization in a single glass microchip enables extremely fast and efficient separations; while operation of miniaturized ion traps at high pressure (~1 Torr) removes the need for large and expensive pumping systems. We are developing fully automated assays on this platform for a range of applications. Of particular relevance are newborn screening for inborn errors of metabolism; and monitoring pain management. Recent results will be presented for both of these applications.

Long Abstract

A fantastic range of analytical challenges can be met with current mass spectrometry instrumentation; but the current paradigm of large, powerful instruments necessarily limits their deployment to a relatively small number of core labs and well equipped specialty labs. The addition of a conventional liquid chromatography (LC) system only adds to the size and complexity of the system. We are developing a simpler alternative to typical LC-MS instrumentation by combining microfluidic capillary electrophoresis with high pressure mass spectrometry (HPMS). HPMS uses miniaturized cylindrical ion traps and high frequency RF to enable MS to be performed at pressures (~1 Torr) that are orders of magnitude greater than conventional MS. High pressure operation eliminates the need for turbo pumps or differential pressure chambers within the instrument. The whole instrument, including a small rough pump, can be miniaturized to handheld size. Microfluidic capillary electrophoresis can be paired with HPMS to provide the separation power of LC with much simpler instrumentation, and the combined system can be packaged into a small benchtop instrument.

Microfluidic CE-MS has been under development in the Ramsey lab at UNC for many years. The integration of sample injection, separation, and electrospray ionization (ESI) in a single glass microchip yields extremely fast and efficient separations. For the work described here, the surfaces of the microfluidic channels are coated using a 2-step process (chemical vapor deposition of aminopropylsilane and covalent attachment of polyethylene glycol) to prevent analyte-surface interactions and suppress electroosmotic flow (EOF). With no EOF, charged anlaytes migrate electrophoretically down the separation channel before being driven out of the corner of the microchip via the integrated ESI emitter. In the current work, neutral and negatively charged components of the sample do not migrate toward the detector. This specificity for positively charged analytes allows samples to be analyzed from complex matrices with very minimal sample prep. Typically, samples are simply diluted to an appropriate concentration and filtered to remove particulates. The separation background electrolyte (BGE) is typically 50% methanol, acidified with formic acid to a pH of 2.2. This low pH BGE is ideal for sensitive and stable ESI. We have recently developed a novel sample injection method that eliminates injection bias and enables inline sample focusing via transient isotachophoresis (tITP). Samples that contain a relatively high concentration of leading electrolyte (e.g. >50 mM ammonium acetate) will undergo tITP when voltage is applied. We can achieve a concentration factor of greater than 10x using integrated tITP.

The utility of this method for newborn screening for errors of metabolism was demonstrated by simulating a test for phenylketonuria (PKU). Blood from a healthy individual was spiked with phenylalanine to concentrations of 0, 100, 300, 500, and 1000 µM. 100 µL aliquots of the blood were then spotted onto filter paper and allowed to dry. 5 mM circles of the dried blood spots were then removed with a biopsy punch and placed in the top reservoir of a centrifugal filter (0.45 µm nylon). 200 µL of BGE containing 100 mM ammonium acetate was added to extract polar metabolites from each dried blood spot. After 10 minutes at room temperature the extract was collected by spinning on a centrifuge for 60 seconds. These samples were then analyzed by pipetting directly into the sample well of a microfluidic CE-MS device. The complete separation, including a 30 second sample loading step, was completed in less than 2.5 minutes. The average CE peak width was approximately 1 second, yielding a peak capacity of approximately 60 in the 1-minute period between the elution of the first and last analyte peak. While the current version of the HPMS instrument does not yield high resolution mass spectra (~5 Da FWHM), the combined separating power of the CE and HPMS fully resolves all of the amino acids and enables easy identification of the phenylalanine peak.

A similar experiment was performed to simulate the application of this technology to monitoring pain management drugs. For this experiment, morphine was spiked into urine at a range of concentrations. The microchip CE-HPMS method was virtually identical to the method used for the PKU simulation. For this experiment the sample prep consisted simply of diluting the urine 10x with BGE and filtering through a centrifugal filter. The detailed results of both of these experiments will be presented.

References & Acknowledgements:

Financial Disclosure

Salaryyes908 Devices
Board Memberno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above: