3.5 Characterization
of FAME (Up to C 30 )
and Pyrolysis Products
from MAME (C 22 –C 26 )
by GC-MS (Gas
Chromatography
Coupled to Mass
Spectrometry)
1. Dissolve total FAME and MAME (from Subheading 3.3, step
10) in petroleum ether at an approximate concentration of
10 mg/mL.
2. Inject 1 μL in the GC apparatus and run (100–290
C, 10
C/
min) to observe the GC profile of FAME and alcohols (see
Fig. 2).
3. Perform the identification by electron impact fragmentation.
Lipids are characterized by their molecular weight and their
fragmentation patterns (see Fig. 2).
3.6 Characterization
and Quantification of
MAME by HPTLC (HighPerformance TLC)
1. Dissolve total FAME and MAME (Subheading 3.3, step 10) in
CHCl 3 at 0.1–1 mg/mL.
2. Spot 10 μL/spot (1–10 μg) on the TLC plate in the form of
5 mm bands using a CAMAG ATS4 Automatic TLC sampler
4 equipped with nitrogen.
3. Program the CAMAG ADC2 Automatic Developing Chamber
as follows:
l
Tank saturation with saturation sheet: 10 min with 20 mL
diethyl ether (10%) in petroleum ether (v/v).
l
Development: Plate preconditioning 5 min with 20 mL
diethyl ether (10%) in petroleum ether (v/v).
l
Drying time: 5 min.
l
Five successive runs are required for the separation of α-,
methoxy-, and keto-MAME (see Note 11).
4. Stain the HPTLC plate by two successive immersions in the
primulin solution, for 3 s at speed 2 with a CAMAG Chromatogram Immersion Device III.
5. Dry under a hood and observe at 365 nm.
6. Analyze the TLC by CAMAG TLC Scanner. The chromatogram of each column track is extracted, and peaks are
integrated by winCATs software for quantitative analyses.
Best quantifications are done when the calibration points are
done on the same plate.
3.7 Characterization
and Quantification of
Free Mycolic Acids by
UPLC-MS (UltraPerformance Liquid
Chromatography
Coupled to Mass
Spectrometry)
A UPLC-MS-ESI-QTOF apparatus equipped with a 2.1 inner
diameter (ID) Â 150 m, 3.5 μm XBridge C18 column (Waters)
heated to 45
C was used with a binary or ternary solvent system
and a flow rate of 320 μL/min (see Note 12).
1. Dissolve synthetic C 32 corynomycolic acid (internal standard)
in the base mix (see Subheading 2.7, item 8) in order to obtain
a solution at 0.01 μg/μL.
2. Dissolve non esterified mycolic acids resulting from saponification of whole cells (Subheading 3.3, step 8a) in base mix
(Subheading 2.7, item 8) in a 1.5 mL vial containing cyclohexane to obtain 0.5 μg/μL of mycolic acids (see Note 13).
Lipid and Lipoarabinomannan
127
of FAME (Up to C 30 )
and Pyrolysis Products
from MAME (C 22 –C 26 )
by GC-MS (Gas
Chromatography
Coupled to Mass
Spectrometry)
1. Dissolve total FAME and MAME (from Subheading 3.3, step
10) in petroleum ether at an approximate concentration of
10 mg/mL.
2. Inject 1 μL in the GC apparatus and run (100–290
C, 10
C/
min) to observe the GC profile of FAME and alcohols (see
Fig. 2).
3. Perform the identification by electron impact fragmentation.
Lipids are characterized by their molecular weight and their
fragmentation patterns (see Fig. 2).
3.6 Characterization
and Quantification of
MAME by HPTLC (HighPerformance TLC)
1. Dissolve total FAME and MAME (Subheading 3.3, step 10) in
CHCl 3 at 0.1–1 mg/mL.
2. Spot 10 μL/spot (1–10 μg) on the TLC plate in the form of
5 mm bands using a CAMAG ATS4 Automatic TLC sampler
4 equipped with nitrogen.
3. Program the CAMAG ADC2 Automatic Developing Chamber
as follows:
l
Tank saturation with saturation sheet: 10 min with 20 mL
diethyl ether (10%) in petroleum ether (v/v).
l
Development: Plate preconditioning 5 min with 20 mL
diethyl ether (10%) in petroleum ether (v/v).
l
Drying time: 5 min.
l
Five successive runs are required for the separation of α-,
methoxy-, and keto-MAME (see Note 11).
4. Stain the HPTLC plate by two successive immersions in the
primulin solution, for 3 s at speed 2 with a CAMAG Chromatogram Immersion Device III.
5. Dry under a hood and observe at 365 nm.
6. Analyze the TLC by CAMAG TLC Scanner. The chromatogram of each column track is extracted, and peaks are
integrated by winCATs software for quantitative analyses.
Best quantifications are done when the calibration points are
done on the same plate.
3.7 Characterization
and Quantification of
Free Mycolic Acids by
UPLC-MS (UltraPerformance Liquid
Chromatography
Coupled to Mass
Spectrometry)
A UPLC-MS-ESI-QTOF apparatus equipped with a 2.1 inner
diameter (ID) Â 150 m, 3.5 μm XBridge C18 column (Waters)
heated to 45
C was used with a binary or ternary solvent system
and a flow rate of 320 μL/min (see Note 12).
1. Dissolve synthetic C 32 corynomycolic acid (internal standard)
in the base mix (see Subheading 2.7, item 8) in order to obtain
a solution at 0.01 μg/μL.
2. Dissolve non esterified mycolic acids resulting from saponification of whole cells (Subheading 3.3, step 8a) in base mix
(Subheading 2.7, item 8) in a 1.5 mL vial containing cyclohexane to obtain 0.5 μg/μL of mycolic acids (see Note 13).
Lipid and Lipoarabinomannan
127