SLs quantification [12]. Nowadays, the improvement of the
LC-MS/MS techniques have made them the fastest and most
sensitive method to identify and quantify SLs.
Nevertheless, almost all published methods do not take into
account the matrix effect, which should be corrected by the use of
an internal standard (IS) [1, 13–16]. For instance, Jamil and coworkers quantified orobanchol and 2
0 -epi-5-deoxystrigol using an IS
[17]. Also, the use of deuterium labeled SLs have been proposed as
an alternative, however, it is limited because of the limited availability of deuterium labeled SLs [18]. Recently, our research group
have published the development, optimization and validation of a
simple, fast, and sensitive analytical method for the simultaneous
quantification of seven natural SLs (7-oxoorobanchyl acetate [19],
solanacol [20], orobanchol [21], strigol [22, 23], fabacyl acetate
[24], orobanchyl acetate [25], and 5-deoxystrigol [7]) by UHPLCMS/MS using (Æ)-GR24 as single IS [26] directly in root exudates
and extracts, without additional sample preparation [27]. This
method provides a useful tool for research in all the fields related
to SLs, both for studies related to their function as hormones, and
signaling molecules in the rhizosphere. However, due to the low
availability of SLs standards just few research groups in the world
have developed validated analytical methods for the SLs
quantification.
Herein, it is described a protocol for the development of
LC-MS/MS methods for the quantification of SLs, using GR24
as IS, in roots exudates and extracts. Validation of the quantitative
analytical method was carried out following the ICH
recommendations [28].
2 Materials
1. Distilled and ultrapure water.
2. Acetone (HPLC grade).
3. Filter paper.
4. 200 mg C18 solid phase extraction (SPE) cartridges.
5. Ethyl acetate (HPLC grade).
6. PTFE syringe filter (0.22 μm).
7. MeOH (LC-MS grade or HPLC grade filtered by a 0.22 μm
membrane filter).
8. Solvent A: water, 0.1% formic acid (see Note 1).
9. Solvent B: MeOH, 0.1% formic acid.
202
Carlos Rial et al.
LC-MS/MS techniques have made them the fastest and most
sensitive method to identify and quantify SLs.
Nevertheless, almost all published methods do not take into
account the matrix effect, which should be corrected by the use of
an internal standard (IS) [1, 13–16]. For instance, Jamil and coworkers quantified orobanchol and 2
0 -epi-5-deoxystrigol using an IS
[17]. Also, the use of deuterium labeled SLs have been proposed as
an alternative, however, it is limited because of the limited availability of deuterium labeled SLs [18]. Recently, our research group
have published the development, optimization and validation of a
simple, fast, and sensitive analytical method for the simultaneous
quantification of seven natural SLs (7-oxoorobanchyl acetate [19],
solanacol [20], orobanchol [21], strigol [22, 23], fabacyl acetate
[24], orobanchyl acetate [25], and 5-deoxystrigol [7]) by UHPLCMS/MS using (Æ)-GR24 as single IS [26] directly in root exudates
and extracts, without additional sample preparation [27]. This
method provides a useful tool for research in all the fields related
to SLs, both for studies related to their function as hormones, and
signaling molecules in the rhizosphere. However, due to the low
availability of SLs standards just few research groups in the world
have developed validated analytical methods for the SLs
quantification.
Herein, it is described a protocol for the development of
LC-MS/MS methods for the quantification of SLs, using GR24
as IS, in roots exudates and extracts. Validation of the quantitative
analytical method was carried out following the ICH
recommendations [28].
2 Materials
1. Distilled and ultrapure water.
2. Acetone (HPLC grade).
3. Filter paper.
4. 200 mg C18 solid phase extraction (SPE) cartridges.
5. Ethyl acetate (HPLC grade).
6. PTFE syringe filter (0.22 μm).
7. MeOH (LC-MS grade or HPLC grade filtered by a 0.22 μm
membrane filter).
8. Solvent A: water, 0.1% formic acid (see Note 1).
9. Solvent B: MeOH, 0.1% formic acid.
202
Carlos Rial et al.