No standards involved, but are all problems solved?

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  • Published: Aug 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Laboratory Informatics / Chemometrics & Informatics
thumbnail image: No standards involved, but are all problems solved?

Availability of standards can be an issue for LC-MS

LC-HRMS enables the detection of hundreds of compounds in a single run, but generally relies on reference standards of these compounds being run under the same conditions. However, there are many cases, such as analysing drug metabolites, novel mixtures of natural products or new designer drugs, where standards may not be available, and their synthesis can be a time-consuming and expensive undertaking. Libraries of MS spectra exist, but fragmentation patterns can vary depending on the instrument and conditions, particularly with the ESI (electrospray ionisation) usually used with LC-MS.

Data-independent acquisition (DIA) is a mass spectrometry technique in which all ions in a given mass range are fragmented and analysed. The Zurich analysts used a variety of DIA that employed UHPLC (ultra-high performance liquid chromatography) in combination with an IMS/TOF (i.e. ion mobility spectrometry/time of flight) mass spectrometer. A technique known as MSE was employed, in which alternating high and low fragmentation energy scans are acquired. The high energy scans detect fragment ions, while those of low energy detect precursor ions. The product and precursor ions can be linked by having the same ion mobility drift times. Chromatographic peak deconvolution techniques were also used to link precursor and product ions.

UHPLC-IMS-TOF used to detect multiple veterinary compounds

Extracts from cattle liver and tilapia fish were spiked with 98 veterinary compounds. The UHPLC employed a Water Acquity UPLC system, fitted with a Phenomenex Kinetex C18 column eluted with varying amounts of acetonitrile and water (plus formic acid). Mass spectrometry was carried out with a Vion IMS QTof instrument with ESI.

UNIFI software (version 1.7) was used for data processing. Up to a million ‘chromatographic features’ can be extracted by the software; these may be matrix compounds or ‘compounds of interest’ (in this case veterinary compounds). The software selected those signals where high and low energy peaks were associated with the same retention time and peak shape, while also having a plausible loss of mass going from precursor to product ions. Around 100,000 ‘candidate compounds’ were produced in this manner.

Further signals were eliminated by only taking through precursor and product ions with identical drift times in the IMS portion of the mass spectrometer, since such times are known to be identical when a product ion arises from a particular precursor. A combination of peak deconvolution and drift time filtering gave much cleaner spectra. This form of DIA gave comparable results to DIA using another instrument, an Orbitrap mass spectrometer.

MassFragment software was used to estimate the likely fragmentation of a range of veterinary compounds. Comparison to the ‘cleaned’ experimental data gave a large number of ‘false positives’ for high m/z compounds. Further refinement was possible by comparing the m/z values and CCS (collision cross section) values of potential compounds. The veterinary compounds were found on or near a straight line going across the graph of CCS against m/z (i.e. there was a linear positive correlation between the two variables). Potential compounds away from the correlation line could be discarded. Following this final criterion, the number of ‘false positives’ was reduced to only six. 94 out of 98 compounds were detected in blank solvent, while 94 compounds were identified in the fish extract and 90 in the liver extract, all at 200 μg/L, somewhat fewer were detected at lower concentrations.

DIA using IMS detects many compounds without using standards

The paper has shown that a combination of data ‘filtration’ techniques permits a large number of compounds to be correctly identified using IMS and DIA. This technique and other similar DIA methods still require some further improvement before standards can be reliably dispensed with, although this point may not be far off.

Related Links

Rapid Communications in Mass Spectrometry, 2017, 31, 1147-1157. Kaufmann et al. Practical application of in silico fragmentation based residue screening with ion mobility high-resolution mass spectrometry.

Wikipedia, Data-independent acquisition

Wikipedia, Ion-mobility spectrometry - mass spectrometry

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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