with a weight of 1/nx (R
2
> 0.99), being “n” the calibration
level.
8. Calculate the quantification (LOQ) and detection (LOD) limits for each SL, analyzing nine replicates and considering ten
and three times the standard deviation of baseline noise
respectively.
9. Study the precision of the method by analyze a sample with all
the SLs in an intra- and interday assay (n ¼ 9). Repeatability
analyzing nine replicates of the same sample in the same day
and the intermediate precisions injecting the same sample on
three different days. They are expressed using the relative standard deviation percentage (RSD %) of the area and the retention times and they are calculated as (relative standard
deviation/mean) Â 100.
3.2.2 Analysis
of Samples
1. Dissolve the exudates or the roots extracts obtained above with
MeOH to achieve a ratio of 1/1 g/L.
2. Filter all the samples through a PTFE syringe filter (0.22 μm).
3. Transfer 1 ml to a 1.5 ml glass vial for LC-MS.
4. Add the IS GR24 to each sample to give a final concentration of
10 μg/L.
5. Inject 5 μL of each sample onto the UHPLC-MS/MS three
times using the same parameters described above.
6. Calculate the concentration of the SLs present in each sample
introducing the ratio of the area of the SL/area of IS in the
calibration curves.
7. Calculate the RSD % of the concentration for each SL in each
sample.
4 Notes
1. MeOH is used for the analysis because it helps to the proton
adduct formation. However, SLs are more stable on acetonitrile (ACN). For this reason, it is recommended prepared a
stock solution of SLs at 1000 or 100 mg/L in ACN.
2. Hydroponic or semihydroponic culture is recommended to
reduce the interferences produced by the soil, for instance,
use as support a mixture of vermiculite, sand, and clay (1:1:1)
and half-strength Hoaglands as nutrient solution.
3. In the cases where SLs amounts are too small for the direct
infusion in the mass spectrometer, the optimization of the mass
transitions could be done by using the product scan mode in
the mass spectrometer by injection of 5 μL aliquot in the
LC-MS/MS. In this mode the mass spectrometer filters the
Quantification of Strigolactones
205
2
> 0.99), being “n” the calibration
level.
8. Calculate the quantification (LOQ) and detection (LOD) limits for each SL, analyzing nine replicates and considering ten
and three times the standard deviation of baseline noise
respectively.
9. Study the precision of the method by analyze a sample with all
the SLs in an intra- and interday assay (n ¼ 9). Repeatability
analyzing nine replicates of the same sample in the same day
and the intermediate precisions injecting the same sample on
three different days. They are expressed using the relative standard deviation percentage (RSD %) of the area and the retention times and they are calculated as (relative standard
deviation/mean) Â 100.
3.2.2 Analysis
of Samples
1. Dissolve the exudates or the roots extracts obtained above with
MeOH to achieve a ratio of 1/1 g/L.
2. Filter all the samples through a PTFE syringe filter (0.22 μm).
3. Transfer 1 ml to a 1.5 ml glass vial for LC-MS.
4. Add the IS GR24 to each sample to give a final concentration of
10 μg/L.
5. Inject 5 μL of each sample onto the UHPLC-MS/MS three
times using the same parameters described above.
6. Calculate the concentration of the SLs present in each sample
introducing the ratio of the area of the SL/area of IS in the
calibration curves.
7. Calculate the RSD % of the concentration for each SL in each
sample.
4 Notes
1. MeOH is used for the analysis because it helps to the proton
adduct formation. However, SLs are more stable on acetonitrile (ACN). For this reason, it is recommended prepared a
stock solution of SLs at 1000 or 100 mg/L in ACN.
2. Hydroponic or semihydroponic culture is recommended to
reduce the interferences produced by the soil, for instance,
use as support a mixture of vermiculite, sand, and clay (1:1:1)
and half-strength Hoaglands as nutrient solution.
3. In the cases where SLs amounts are too small for the direct
infusion in the mass spectrometer, the optimization of the mass
transitions could be done by using the product scan mode in
the mass spectrometer by injection of 5 μL aliquot in the
LC-MS/MS. In this mode the mass spectrometer filters the
Quantification of Strigolactones
205