Improvement of the Sensitivity for the Determination of
Anilines by LC-MS/MS
Nadin Epperlein1, Madeleine Adolf3, Melinda Bittner1, Berthold Kettner1, Alfredo Montes-Nino2, Karl Speer3
1 ILAU GmbH, Unterwöhrn 15, D-83543 Rott am Inn, GERMANY, E-mail: berthold.kettner@gmx.de, nadin.epperlein@ilau.de
2 Laboratorios Microbioticos s/c/ Ltda Residue Laboratory, Av. Santa Isabel 2116, Caixa Postal 6175, Campinas, Estado de Sao Paulo, CEP 13083-970, BRAZIL
3 TU Dresden, Institut für Lebensmittelchemie, Bergstraße 66, D-01062 Dresden, GERMANY, E-mail: karl.speer@chemie.tu-dresden.de
Introduction
We recently developed a method for the separation of 17 anilines with LC-MS/MS [1]. Unfortunately, the sensitivity of the LS-MS/MS API 3200
(Applied Biosystems) is not suitable for the determination of all the anilines in a matrix at lower ranges. Only 3-chloro-4-methoxyaniline,
3-chloro-4-methylaniline, 4-chloro-2-methylaniline and 4-(4-chlorophenoxy)-aniline can be determined at the level of 0.01 mg/kg.
By means of a derivatization we want to improve the necessary sensitivity for the determination of the anilines even at the range of 0.01 mg/kg.
After the derivatization there should be another fragmentation characteristic in the MS/MS, which is much more sensitive than the MRM-transition
of the underivatized aniline.
Derivatization of the Anilines
We chose the derivatization with acetanhydride for an acetylation of the amino
group of the anilines (figure 1). As shown in table 1, the sensitivity of the single
anilines increases about the factor 1 000 and even more after the derivatization.
aniline (e.g. 3-chloroaniline)
O
+ acetanhydride
+ sodium acetate
+ chloroform
reflux for 1 h
C
NH2
HN
O
CH3
O
O
+
+
H3C
O
CH3
H3C
Cl
OH
Cl
Figure 1: Derivatization with acetanhydride using the example of 3-chloroaniline
+ iced water
stirring for 1 h
Table 1: Increase of the sensitivity after derivatization with acetanhydride
Name of the aniline/
Name of the acetanilide after
derivatization
4-chloroaniline/
4-chloroacetanilide
3-chloroaniline/
3-chloroacetanilide
4-chloro-2-methylaniline/
4-chloro-2-methylacetanilide
3-trifluoro-methylaniline/
3-trifluoro-methylacetanilide
4-trifluoro-methoxyaniline/
4-trifluoro-methoxyacetanilide
3,4-dichloroaniline/
3,4-dichloroacetanilide
3,5-dichloroaniline/
3,5-dichloroacetanilide
4-bromo-3-chloroaniline/
4-bromo-3-chloroacetanilide
Sensitivity of the
aniline
Sensitivity of the
Factor of the increase
generated acetanilide
of the sensitivity
extraction with 3 x 10 ml methylene chloride
2.96·104
3.29·107
1 110
2.80·104
3.17·107
1 130
1.10·105
6.70·107
610
9.00·103
1.53·107
1 700
2.60·104
3.71·107
1 430
8.19·103
1.13·107
1 380
4.06·103
7.17·106
1 770
1.01·104
1.27·107
1 260
1
remove methylene chloride
complete the residue to 10 ml with acetanhydride
solution of the derivatized aniline
(e.g. 3-chloroacetanilide)
Scheme of the derivatization with acetanhydride
Results
5
The derivatization of the anilines with acetanhydride leads
to an increase of the sensitivity of more than the factor
1 000 during the determination with LC-MS/MS (API 3200,
Applied Biosystems). The exception is 4-chloro-2methylacetanilide with an increase factor of 610.
2 3
4
6
7
8
Figure 2: MRM-chromatogram 1) 4-chloro-2-methylacetanilide, 2) 4-chloroacetanilide, 3) 3-chloroacetanilide, 4) 3-trifluoromethyl-acetanilide, 5) 4-trifluoromethoxyacetanilide, 6) 3,4-dichloroacetanilide,
7) 4-bromoacetanilide, 8) 3,5-dichloroacetanilide
Acknowledgement
We would like to thank Mr. Ulrich Sigmund1 for his excellent technical assistance
and his untiring encouragement during the development of this method.
The separation of the generated acetanilides can be carried
out only with an already developed LC-MS/MS method [1]
for the separation of the 17 anilines. This method uses a
Luna PFP (2) column (Phenomenex®) and an optimized
gradient of the solvents introduced by Alder [2]. With these
conditions, the separation of the analytes (including four
isomer pairs) is completed within 45 minutes (figure 2).
With these results – the HPLC separation and the here
presented derivatization – it is now possible to determine
the 17 anilines with LC-MS/MS at a range of less than
0.01 mg/kg. The determination of the anilines with
LC-MS/MS by means of a derivatization is much more
comfortable than the determination of the anilines by
GC-MS, due to the robustness and the longevity of the used
HPLC column and the sensitivity and selectivity of the
used LC-MS/MS (much lower LOD and LOQ are achievable!).
References
[1]
Adolf, M., Untersuchungen zur Bestimmung von Anilinen mit LC/MS/MS-Kopplung beispielhaft an Karotten, Wissenschaftliche Abschlussarbeit, TU Dresden, Institut für Lebensmittelchemie, AK Prof. Karl Speer, 30.01.2009,
paper in progress
[2]
Klein, J., Alder, L., Applicability of Gradient Liquid Chromatography with Tandem Mass Spectrometry to the Simultaneous Screening for about 100 Pesticides in Crops, Journal of AOAC International 86 (5), 1015-1037 (2003)
2nd LAPRW, Santa Fe, Argentina, June 8-11, 2009