Jack Cunniff, Philip Tiller, Michael Harvey,
and Adrian Land,
Natural Product Analysis
The data presented here can be acquired using the Thermo Finnigan LCQ
Series of ion trap mass spectrometers.
For more than five thousand years tea has been used in China as an herbal
remedy. In recent years pharmacological studies have supported some of these
health claims. Flavonoids, a class of polyphenolic compounds, found in tea,
plants and many fruits have shown some promising results related to anti-cancer
as well as antiinflammatory and anti-allergy properties. For these reasons,
there has been renewed interest in both detecting and characterizing these
In many instances, flavonoids exist as their glycosylated conjugate. In
a typical MS/MS experiment, glycosylated moieties may be cleaved from the
molecular precursor with a resulting MS/MS spectrum which is difficult to
relate back to a hypothetical structure. The primary reason for the difficulty
is the fact that first generation fragment ions may undergo further fragmentation,
which can result in a very complex spectrum. Third and fourth generation
product ions can not readily be distinguished from second generation product
The LCQ Series of mass spectrometers can perform MSn, so precursor ions
are isolated before a subsequent MS/MS experiment is performed. Due to this
isolation step, fragmentation spectra are generally less complex and ambiguous
than traditional MS/MS spectra. Through successive MS/MS steps, product
origins can be assigned. As the following experiments will show, the LCQ
may remove glycosylated side chains in a controlled, step-by-step manner
using the power of MSn. Eventually, a core structure is exposed. A final
fragmentation spectrum of the core structure yields a classic fingerprint
which may then be referenced against a library MS/MS spectrum of the base
component. In this way, a compound may be unequivocally classified.
This experiment demonstrates:
The utility of MSn
for structural determination and unambiguous classification
of a suspected flavonoid compound.
The utility of MSn
for the unambiguous determination of product origin.
Thermo Finnigan LCQ fitted with ESI probe
Infusion: 3 L/min using integrated syringe pump
Capillary temp: 150 C
Needle voltage: +4.2 kV
Sheath gas: 45 units
Auxiliary gas flow: 10 units
Apiginin (Figure 1) (mw 270.2) is a base component of a class of flavonoids.
The MS/MS spectrum of apiginin is rich in detail (Figure 2). Because the
LCQ imparts energy solely onto the precursor molecule, variations in collision
energy generally have very little impact on the relative ratios of the product
ions. The MS/MS spectrum of apiginin will be used as our library
spectrum or fingerprint for the classification of Compound X
whose full-scan mass spectrum is shown in Figure 3.
Figure 1. Structure and full-scan mass spectrum of apiginin.
Figure 2. Full-scan MS/MS (m/z 271) spectrum of apiginin.
Figure 3. Full-scan mass spectrum of Compound X.
From the spectrum in Figure 3, it is clear that compound X has an [M+H]+
ion at 579 and an [M+Na]+ ion at 601. The MS/MS spectrum of the [M+H]+ ion
of Compound X (Figure 4) indicates a loss of 146 and also exhibits an
ion of m/z 271the m/z of the [M+H]+ ion of apiginin. The loss of 146 is
often indicative of the loss of a deoxy-hexose sugar and the m/z 271.1 ion
is indicative of the base component: apiginin.
At this point it is possible to do MS3
on the 271.1 ion and compare the
spectrum with that of the MS/MS spectrum of apiginin. Before we do this,
however, we shall first endeavor to determine whether the m/z 271 ion is
related to the m/z 433 ion. To accomplish this, an MS3
experiment was performed
on the m/z 433 ion and the spectrum is illustrated in Figure 5. The hypothesis
that the m/z 271 originated from the m/z 433 ion has been confirmed. The
difference in the two masses (162 amu) is also indicative of the loss of
a hexose sugar.
Figure 4. Full-scan MS/MS (m/z 579) mass spectrum of Compound X.
Figure 5. Full-scan MS3 (579>433>) mass spectrum of Compound X.
Figure 6. Full-scan MS4 (579>433>271>) mass spectrum of Compound
Up to now, the only evidence that Compound X belongs to the flavonoid
class is the presence of the m/z 271 ion. This is far from compelling evidence.
Compelling evidence would be achieved if the MS/MS of this m/z 271 ion yields
a spectrum which is similar to the MS/MS spectrum of apiginin. This hypothesis
is tested via the MS4
experiment of the m/z 271 ion. The spectrum
(Figure 6) is an exact match of the spectrum generated by the MS/MS experiment
of apiginin (Figure 7). Compound X is a flavonoid! In fact, it is the
flavonoid rhoifolin (apiginin 7-0-neohesperidoside).
Figure 7. Comparison of full-scan MS/MS (271) spectrum of apiginin with
the full-scan MS4 (579>433>271>) mass spectrum of Compound X.
Electrospray ionization of an unknown compound provides a soft ionization
technique allowing the generation of an intact pseudomolecular ion at m/z
579. The MS2
experiment yielded two major ions which were indicative
of the loss of a deoxyhexose sugar (m/z 433) and a product mass which was
the same as a base component of flavonoids (m/z 271). MS3
of the m/z 433
ion proved that the m/z 271 ion was a product of the m/z 433 ion and also
indicated the loss of a hexose sugar. The MS4
experiment on the m/z 271
ion resulted in a fragmentation spectrum which was a direct match with the
fragment spectrum of apiginin. The direct match indicates that the unknown
compound belongs to the flavonoid class.
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