5. Transfer organic phase to a glass vial.
6. Repeat steps 3–5 with the same roots three times and join the
organic phases.
7. Evaporate the organic solvent in a rotary evaporator and store
the samples at À80
C until their analysis.
3.2 LC-MS/MS
Analysis
This method was optimized using a Bruker EVOQ Triple Quadrupole Mass Spectrometer, an ACE Excel 1.7 column (Advanced
Chromatography Technologies Ltd., Aberdeen, Scotland) and following the ICH recommendations [28]. The parameters described
herein should be optimized for others mass spectrometers and
columns.
3.2.1 LC-MS/MS Method
Calibration
and Optimization
1. Dissolve the SL with MeOH to achieve a concentration of
10 mg/L.
2. By direct infusion on the mass spectrometer in positive mode,
determine the most intense SL adduct ([M + H]
+ or
[M + Na]
+ ), their most stable fragments and the collision
energy to achieve the maximum signal for them (see Note 3).
When more than one fragments are obtained, the most intense
is used as quantifier ion and the second as qualifier ion to
improve the identification of the SLs (see Notes 4 and 5).
3. Prepare a diluted stock solution of 100 μg/L for each SL with
10 μg/L of GR24 in all of them (it is used as internal standard
(IS)).
4. The calibration curve of each SL is prepared by serial dilution of
the 10 mg/L stock solution prepared above with a solution of
10 μg/L of GR24 in MeOH.
5. Filter all the samples through a PTFE syringe filter (0.22 μm)
(see Note 6).
6. Inject 5 μL of each level of the calibration curve onto the
UHPLC-MS/MS three times with an electrospray ionization
source (ESI) in positive mode and a C18 (100 Â 2.1 mm,
1.7 μm particle size) UHPLC column maintained at 40
C.
Use as mobile phase solvent A and B with the flow rate set to
0.3 mL/min, with linear gradient as follows: 0–0.5 min, 50%
B; 0.5–5 min, to 100% B; 5–7 min, 100% B; 7–7.5 min, to 50%
B; 7.5–10.5 min, 50% B. Set the instrument parameters as
follows: spray voltage +4500 V, cone temperature 300
C,
cone gas flow 15 psi, heated probe temperature 400
C, heated
probe gas flow 15 psi, nebulizer gas flow 55 psi, and collision
pressure 2.0 mTorr (see Note 7).
7. Construct each calibration curve by plotting the peak area ratio
(y ¼ area of the standard/area of the IS) versus the ratio of their
concentrations (x ¼ concentration of the calibration curve
level/10 μg/L) and fitting the curves to a linear function
204
Carlos Rial et al.
6. Repeat steps 3–5 with the same roots three times and join the
organic phases.
7. Evaporate the organic solvent in a rotary evaporator and store
the samples at À80
C until their analysis.
3.2 LC-MS/MS
Analysis
This method was optimized using a Bruker EVOQ Triple Quadrupole Mass Spectrometer, an ACE Excel 1.7 column (Advanced
Chromatography Technologies Ltd., Aberdeen, Scotland) and following the ICH recommendations [28]. The parameters described
herein should be optimized for others mass spectrometers and
columns.
3.2.1 LC-MS/MS Method
Calibration
and Optimization
1. Dissolve the SL with MeOH to achieve a concentration of
10 mg/L.
2. By direct infusion on the mass spectrometer in positive mode,
determine the most intense SL adduct ([M + H]
+ or
[M + Na]
+ ), their most stable fragments and the collision
energy to achieve the maximum signal for them (see Note 3).
When more than one fragments are obtained, the most intense
is used as quantifier ion and the second as qualifier ion to
improve the identification of the SLs (see Notes 4 and 5).
3. Prepare a diluted stock solution of 100 μg/L for each SL with
10 μg/L of GR24 in all of them (it is used as internal standard
(IS)).
4. The calibration curve of each SL is prepared by serial dilution of
the 10 mg/L stock solution prepared above with a solution of
10 μg/L of GR24 in MeOH.
5. Filter all the samples through a PTFE syringe filter (0.22 μm)
(see Note 6).
6. Inject 5 μL of each level of the calibration curve onto the
UHPLC-MS/MS three times with an electrospray ionization
source (ESI) in positive mode and a C18 (100 Â 2.1 mm,
1.7 μm particle size) UHPLC column maintained at 40
C.
Use as mobile phase solvent A and B with the flow rate set to
0.3 mL/min, with linear gradient as follows: 0–0.5 min, 50%
B; 0.5–5 min, to 100% B; 5–7 min, 100% B; 7–7.5 min, to 50%
B; 7.5–10.5 min, 50% B. Set the instrument parameters as
follows: spray voltage +4500 V, cone temperature 300
C,
cone gas flow 15 psi, heated probe temperature 400
C, heated
probe gas flow 15 psi, nebulizer gas flow 55 psi, and collision
pressure 2.0 mTorr (see Note 7).
7. Construct each calibration curve by plotting the peak area ratio
(y ¼ area of the standard/area of the IS) versus the ratio of their
concentrations (x ¼ concentration of the calibration curve
level/10 μg/L) and fitting the curves to a linear function
204
Carlos Rial et al.