10 February / March 2021
analysis). Incremental advances in mass spectrometry technology and informatics have unleashed the potential of high- specificity, data independent non-targeted screening Three-dimensional resolution comprised of m/z, chromatographic and ion mobility separation is illustrated for Passiflora edulis in Figure 3. UPLC-IM- MS provides an alternate strategy to the 2-D separation techniques discussed by Cao et al and Zhu et al [28,29]. Retention time and drift time (DT) aligned spectra provide specificity akin to that of directed MSMS, however the necessity to perform preselection of target analyte mass using the quadrupole is negated. A CCS value is also generated for all IM resolved precursor analytes, which can enable isomeric species to be distinguished. Observed and expected CCS (Å2
Figure 4. Chromatographic resolution and cycle time comparison A) HPLC and B) UPLC. Inset expanded HPLC/UPLC peak II. C) Mass resolution comparison for quadrupole and oa-TOF-MS leucine enkephalin [M+H]+
m/z 556.2766.
challenge of sample complexity has been overcome, using the LC-MS technology advances, in particular, the coupling of ultra-performance liquid chromatography with ion mobility mass spectrometry (UPLC- IM-MS), which provides three dimensions of separation; m/z, chromatographic and ion mobility resolution. The commercialisation of IM instrumentation (Waters Corporation, Milford, MA, USA - SYNAPT (2006)) [9,10] has led to a continuous increase in publications citing the use of IM [11,12]. IM is a property of gas phase ions relating to their velocity in a buffer gas relative to an applied electric field, where the IM separation depends on an ion’s mass, charge and shape [13]. Dwivedi et al illustrated a factor of 2 to 10 increase in peak capacity using LC, MS, and IM, depending on the MS and IM resolution [14,15]. An increase in analytical strategies which use collision cross section (CCS) values as an additional descriptor to aid identification have taken place, in combination with MS measurement and tr
, they provide a complementary metric [16-18]. Development in chromatographic and mass resolution separation strategy have been employed, to reach an end point where using ion mobility, speciation profiling of Passiflora is extended beyond reliance upon only two known critical isomer pairs (see Figure 2). A ‘known-unknown’ compound is defined as an analyte or feature that is consistently detected and characterised by multi-metric analytical measurements in and across multiple samples [19]. Incorporation of ‘knowns’,
‘known-unknowns’ and identification of ‘known-unknowns’, makes it possible to obtain a comprehensive component marker fingerprint to control the quality of phytomedicines and profile the phytochemical makeup of medicinal plants [20-23].
Experimental. Sample information:
Preparation of the voucher specimens of all plant materials employed in this study, P. incarnata, P. edulis, P. caerulea and P. alata has previously been described [8]. Extraction was with ethanol/water (2:1 v/v), 1 g plant/10 mL solvent, according to the Brazilian Pharmacopoeia procedure [24].
Chromatographic and Mass Spectrometry Conditions.
High performance liquid chromatography (HPLC)-oa-TOF-MS, UPLC-IM-MS experimental and processing parameters have been described previously elsewhere [8,25-27].
Results and Discussion
Profiling of Passiflora variants highlights the challenges of complex sample analysis, which are common to numerous areas of research including metabolomics, metabolite identification and food safety (veterinary drug residues, pesticide residues
alongside tr (ppm).
) values are routinely reported (min) and m/z accurate mass
The ability to achieve higher mass resolutions is a major factor in overcoming the challenges of complex sample analysis, since it facilitates the separation of isobaric species which improves m/z specificity. Increases in oa-TOF-MS mass resolution from > 5000 FWHM to > 50000 FWHM are illustrated at m/z 556 [M+H]+ for the TOF-MS system lock mass leucine enkephalin in Figure 4 and compared to unit resolution of a quadrupole MS instrument. The introduction of UPLC facilitated a 66% reduction in total analysis cycle time to 17 mins (50 min using HPLC), for the analysis of Passiflora species (see Figure 4) and chromatographic base peak widths were sharpened from ~ 1min to ~ 0.1 min (for the 17 min UPLC gradient used). The enhanced peak capacity [5] provides further component separation and correspondingly cleaner retention time aligned product ion spectra in MSE
experiments. UPLC
improves S/N due to the reduction in band broadening, and thus an increase in sensitivity. Extracts analysed using UPLC- IM-MS were diluted 40:1 compared to those used for HPLC-oa-TOF-MS.
Distinctive fragment ion distributions were identified during our initial HPLC-MS investigations using positive mode ESI [8]. At uncharacteristically high collision induced dissociation (CID) collision energies, the isomer pairs formed differentiating isomeric fragment ions. Accurate mass measurement confirmed the elemental measurement and reaffirmed the discovery of characteristic information that could be used to differentiate 6-C and 8-C glycoside isomers, as illustrated in Figure 5 for isoorientin and
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