Two High Speed Separation Alternatives to HPLC for Ultra High Throughput Mass Spectrometry

Scenarios where millions of samples need to be rapidly analyzed by mass spectrometry are prevalent in drug discovery. High throughput screening (HTS) of large libraries and the maintenance of the quality of these libraries are examples where such throughputs are justifiable. The implementation of such throughputs has been largely achieved by using a form of flow injection analysis where nL volume samples are dispensed from microtiter well plates using sound waves to acoustically eject for transport into a mass spectrometer using an Open Port Interface (OPI) referred to as acoustic ejection mass spectrometry (AEMS). One sample per second is routinely being used but speeds as high as 6 per second have been demonstrated and 20 per second shown to be feasible, approximately matching the time it takes to blink an eye. One limitation, compared to LC/MS, is the lack separations to ameliorate ionization suppression as well as to separate structural isomers that cannot be distinguished by MS or MS/MS. To match the duty cycle of AEMS millisecond separations are required and samples must be pre-purified in high throughput batch mode to eliminate matrix ionization suppression effects.
Described here are separation technologies compatible with the AEMS duty cycle. Differential ion mobility using clustering agents is used to separate isomers from the metabolome based on the differences in their gas phase ion molecule chemistries in 10 msec. No other form of ion mobility has been successful separating these classes of compounds typified by the citrate – isocitrate isomeric pair. Additionally, a novel magnetohydrodynamic means for rapid batch-wise sample preparation to ameliorate ionization suppression has been developed. Novel ferrimagnetic magnetic beads are spun at high frequency using rotating magnetic fields. The turbulent fluid flow conditions created increase extraction and reaction rates by 10-fold over other forms of mixing used to reduce diffusion times.