tetrabutylammonium hydroxide (TBH) at 70
C, concluded
that this was not sufficient to cleave the wax-ester-MA, TBH
giving a mixture of keto and wax inseparable on TLC [56].
(b) Methylation.
Methylation of fatty acids by diazomethane is an efficient
method to convert free acids obtained by saponification into
methyl esters; however, diazomethane is a toxic gas and has
been recently forbidden. Alternative to diazomethane, to still
use with caution, exists:
l
Treatment with TMS diazomethane (Aldrich) 2 M solution
in hexane. Methanol is required in the solvent to suppress
the production of acyl silane artifacts.
l
Treatment with Iodomethane.
l
Conditions should be carefully tested with standards to
avoid methoxylation of some chemical functions.
11. For 1-D TLC, two plates using different solvent systems are
necessary: (1) dichloromethane (one run) and (2) petroleum
ether/diethyl ether 9:1 (v/v) four subsequent runs. These are
complementary and systematically used for the definition of
the MAME composition of a given strain. Some of the MAME
are not separated in dichloromethane, e.g., methoxy- and ketoMA display the same migration but are well separated in petroleum ether/diethyl ether 9:1 (v/v) with four or five runs
(Fig. 1a, b). When several runs are needed, stop the migration
always at the same level and let the plate dry 5 min at air
between each run [52, 55].
For 2-D TLC (which is also informative in the case of
complex patterns of species of mycobacteria [67] or of strains
of BCG from different origins [68]: first direction petroleum
ether/acetone (95:5, v/v, three runs) and second direction
toluene/acetone (97:3, v/v). Between each run, the plate is
let to dry 5 min under the hood.
12. ESI-QTOF-MS method: the cone voltages have to be optimized at 40 V for corynomycolic acid (0–6 min, range m/z
200–400) and at 90 V for all the mycolic acids (6–45 min,
range m/z 1000–2000), independently of their length and
type. ESI-QTOF-MS-MS method: the cone voltage has to be
optimized at 40 V and the collision energy at 25 eV. The
quantification of corynomycolic acid is based on the m/z
255 fragment corresponding to the C16 acid released from
fragmentation of molecular ion 495 of corynomycolic acid
(internal standard) whereas a cone voltage of 90 V and a
collision energy of 55 eV has to be used for the other types of
mycolic acids, whose quantification has to be based on the m/z
367 and 395 fragments corresponding to the C 24 and C 26 acids
fragmentation, respectively), giving a time range adjusted to
the retention time of each one.
Lipid and Lipoarabinomannan
145
C, concluded
that this was not sufficient to cleave the wax-ester-MA, TBH
giving a mixture of keto and wax inseparable on TLC [56].
(b) Methylation.
Methylation of fatty acids by diazomethane is an efficient
method to convert free acids obtained by saponification into
methyl esters; however, diazomethane is a toxic gas and has
been recently forbidden. Alternative to diazomethane, to still
use with caution, exists:
l
Treatment with TMS diazomethane (Aldrich) 2 M solution
in hexane. Methanol is required in the solvent to suppress
the production of acyl silane artifacts.
l
Treatment with Iodomethane.
l
Conditions should be carefully tested with standards to
avoid methoxylation of some chemical functions.
11. For 1-D TLC, two plates using different solvent systems are
necessary: (1) dichloromethane (one run) and (2) petroleum
ether/diethyl ether 9:1 (v/v) four subsequent runs. These are
complementary and systematically used for the definition of
the MAME composition of a given strain. Some of the MAME
are not separated in dichloromethane, e.g., methoxy- and ketoMA display the same migration but are well separated in petroleum ether/diethyl ether 9:1 (v/v) with four or five runs
(Fig. 1a, b). When several runs are needed, stop the migration
always at the same level and let the plate dry 5 min at air
between each run [52, 55].
For 2-D TLC (which is also informative in the case of
complex patterns of species of mycobacteria [67] or of strains
of BCG from different origins [68]: first direction petroleum
ether/acetone (95:5, v/v, three runs) and second direction
toluene/acetone (97:3, v/v). Between each run, the plate is
let to dry 5 min under the hood.
12. ESI-QTOF-MS method: the cone voltages have to be optimized at 40 V for corynomycolic acid (0–6 min, range m/z
200–400) and at 90 V for all the mycolic acids (6–45 min,
range m/z 1000–2000), independently of their length and
type. ESI-QTOF-MS-MS method: the cone voltage has to be
optimized at 40 V and the collision energy at 25 eV. The
quantification of corynomycolic acid is based on the m/z
255 fragment corresponding to the C16 acid released from
fragmentation of molecular ion 495 of corynomycolic acid
(internal standard) whereas a cone voltage of 90 V and a
collision energy of 55 eV has to be used for the other types of
mycolic acids, whose quantification has to be based on the m/z
367 and 395 fragments corresponding to the C 24 and C 26 acids
fragmentation, respectively), giving a time range adjusted to
the retention time of each one.
Lipid and Lipoarabinomannan
145