3. Add 1.1 g of solid CaCl 2
. 2H 2 O and 500 μL of a 100 mM
EDTA solution (pH 7.5), close the vial and mix gently.
4. Ultrasonicate for 5 min.
5. Transfer 1 mL of the homogenized mixture to the bottom of a
10 mL screw-cap headspace vial and close hermetically (see
Notes 18 and 19).
3.2.3 HS-SPME GC-MS
Analysis
1. Preincubate de vials at 80
C for 10 min with 500 rpm agitation
by means of the CombiPAL autosampler (see Note 20).
2. Insert the SPME holder in the vial, and expose the 65 μm
DVB/PDMS fiber to the headspace for volatile extraction at
80
C for 20 min with 500 rpm agitation.
3. Introduce the fiber for 1 min in the gas chromatograph injection port at 250
C for desorption of volatile compounds.
Sample desorption is performed in splitless mode.
4. Clean the fiber in the Fiber Conditioning Module with Helium
flow at 250
C for 5 min to prevent cross-contamination.
5. Gas chromatography conditions:
– Constant Helium flow of 1.3 mL/min.
– Oven temperature program: 120
C for 1 min, 7
C/min
ramp until 250
C, then held at 250
C for 5 min.
Chromatographic time is 25.5 min. Total run time,
including oven cooling, is around 30 min.
6. Mass spectrometry conditions:
– Transfer line 260
C, ionization source 230
C, quadrupole
150
C.
– Electron impact ionization (EI), electron energy 70 eV.
– Scan mode acquisition in the m/z range 35–250 (6.2 scans
per second) (see Note 11).
3.2.4 Compound
Identification
and Quantitation
Do exactly as indicated in Subheading 3.1.4, except that retention
time and m/z for integration of compounds are those in Table 2.
4 Notes
1. A variety of fiber coatings exists, and the most adequate should
be selected depending on the chemical nature of the most
important target compounds in the experiment. In the case of
volatile apocarotenoids, a PDMS coating would also be a good
option due to the chemical nature of these compounds.
2. Solid CaCl 2
. 2H 2 O is available in different presentations. It
must be as a fine powder so that can be rapidly dissolved.
172
Jose ´ L. Rambla and Antonio Granell
. 2H 2 O and 500 μL of a 100 mM
EDTA solution (pH 7.5), close the vial and mix gently.
4. Ultrasonicate for 5 min.
5. Transfer 1 mL of the homogenized mixture to the bottom of a
10 mL screw-cap headspace vial and close hermetically (see
Notes 18 and 19).
3.2.3 HS-SPME GC-MS
Analysis
1. Preincubate de vials at 80
C for 10 min with 500 rpm agitation
by means of the CombiPAL autosampler (see Note 20).
2. Insert the SPME holder in the vial, and expose the 65 μm
DVB/PDMS fiber to the headspace for volatile extraction at
80
C for 20 min with 500 rpm agitation.
3. Introduce the fiber for 1 min in the gas chromatograph injection port at 250
C for desorption of volatile compounds.
Sample desorption is performed in splitless mode.
4. Clean the fiber in the Fiber Conditioning Module with Helium
flow at 250
C for 5 min to prevent cross-contamination.
5. Gas chromatography conditions:
– Constant Helium flow of 1.3 mL/min.
– Oven temperature program: 120
C for 1 min, 7
C/min
ramp until 250
C, then held at 250
C for 5 min.
Chromatographic time is 25.5 min. Total run time,
including oven cooling, is around 30 min.
6. Mass spectrometry conditions:
– Transfer line 260
C, ionization source 230
C, quadrupole
150
C.
– Electron impact ionization (EI), electron energy 70 eV.
– Scan mode acquisition in the m/z range 35–250 (6.2 scans
per second) (see Note 11).
3.2.4 Compound
Identification
and Quantitation
Do exactly as indicated in Subheading 3.1.4, except that retention
time and m/z for integration of compounds are those in Table 2.
4 Notes
1. A variety of fiber coatings exists, and the most adequate should
be selected depending on the chemical nature of the most
important target compounds in the experiment. In the case of
volatile apocarotenoids, a PDMS coating would also be a good
option due to the chemical nature of these compounds.
2. Solid CaCl 2
. 2H 2 O is available in different presentations. It
must be as a fine powder so that can be rapidly dissolved.
172
Jose ´ L. Rambla and Antonio Granell