6. Examine the consistency of the isotopic pattern between product ions and molecular ion, taking advantage of different software tools. For example, starting from a tandem MS
experiment and selecting a precursor ion, the SmartFormula3D™ yields a list of product ions with their elemental composition and exact mass, the fragment loss, mass error and
isotopic pattern matching. Only those fragment ions with the
same isotopic pattern as the precursor ion should be selected as
true fragment ions derived from the molecular ion.
7. Compare the in silico and experimental data of the tentatively
identified carotenoids with experimental data of authentic standards (if available) to increase the accuracy of the identification (see Note 11).
4 Notes
1. Purity of the solvent is a critical issue to get the appropriate
signal-to-noise ratio and avoid the presence of interferences in
the MS analysis. Our experience has shown that even those
LC-MS-grade solvents need to be checked in the applied experimental conditions. If none of them yields the required standard, then the use of pesticide residue analysis (PRA)-grade
solvents is advised.
2. Avoid the extensive use of plastic lab supplies as contaminants
dissolve with the extract and appear as artifacts in the MS
analysis.
3. The extracting solvent and the ratio of tissue and solvent
depend on the pigment polarity range and concentration in
the tissue, as well as on the kind of matrix for extraction. It is
convenient to perform some preliminary tests to establish
amount of solvent and number of extractions. Methanol and
acetone are frequently applied in this step, although solvent
mixtures with water are also used. In any case, the user should
consider safety and ease of use in the solvent selection.
4. If the sample lacks esterified carotenoids, chemical hydrolysis
step could be applied at this point to remove interference with
chlorophylls and triacyl glycerides [5].
5. The filtering rules are independent and complementary physicochemical properties available at the experimental and theoretical level, including chromatographic behavior, UV-visible
spectrum, m/z values (for both [M+H]
+ and the product ions)
and isotopic patterns (for both [M+H]
+ and the product ions)
that
once
organized
and
compared
(experimental vs. theoretical values) allow the characterization
within a database of targeted compounds.
142
Antonio Pe ´ rez-Ga ´ lvez et al.
experiment and selecting a precursor ion, the SmartFormula3D™ yields a list of product ions with their elemental composition and exact mass, the fragment loss, mass error and
isotopic pattern matching. Only those fragment ions with the
same isotopic pattern as the precursor ion should be selected as
true fragment ions derived from the molecular ion.
7. Compare the in silico and experimental data of the tentatively
identified carotenoids with experimental data of authentic standards (if available) to increase the accuracy of the identification (see Note 11).
4 Notes
1. Purity of the solvent is a critical issue to get the appropriate
signal-to-noise ratio and avoid the presence of interferences in
the MS analysis. Our experience has shown that even those
LC-MS-grade solvents need to be checked in the applied experimental conditions. If none of them yields the required standard, then the use of pesticide residue analysis (PRA)-grade
solvents is advised.
2. Avoid the extensive use of plastic lab supplies as contaminants
dissolve with the extract and appear as artifacts in the MS
analysis.
3. The extracting solvent and the ratio of tissue and solvent
depend on the pigment polarity range and concentration in
the tissue, as well as on the kind of matrix for extraction. It is
convenient to perform some preliminary tests to establish
amount of solvent and number of extractions. Methanol and
acetone are frequently applied in this step, although solvent
mixtures with water are also used. In any case, the user should
consider safety and ease of use in the solvent selection.
4. If the sample lacks esterified carotenoids, chemical hydrolysis
step could be applied at this point to remove interference with
chlorophylls and triacyl glycerides [5].
5. The filtering rules are independent and complementary physicochemical properties available at the experimental and theoretical level, including chromatographic behavior, UV-visible
spectrum, m/z values (for both [M+H]
+ and the product ions)
and isotopic patterns (for both [M+H]
+ and the product ions)
that
once
organized
and
compared
(experimental vs. theoretical values) allow the characterization
within a database of targeted compounds.
142
Antonio Pe ´ rez-Ga ´ lvez et al.