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DOI 10.1007/s00216-003-2377-0
O R I G I N A L PA P E R
route for AZ-A under different conditions of pH and temperature. The data show that at room temperature and pH
between 4 and 5 the product degrades into two preferential forms that are hydrolyzed to a single product over time
and as a function of pH change.
Keywords Neem Triterpenoids Azadirachtin-A
Degradation Kinetics HPLCMSMS
Introduction
The oil extracted from seeds of neem (Azadirachtin indica
A juss) has been the subject of considerable research. After isolation of this oil in seminal work by Morgan and
Butterworth in 1968 [1], research on its properties has
consistently shown the efficacy of azadirachtin as an insecticide [2, 3, 4, 5]. When applied to larvae it causes
mortality at different stages of their development and malformations (in particular reduced longevity and fertility of
adults). The 50% lethal dose (LD50) varies with species
from 1 to 4 g azadirachtin per gram of insect weight [2].
The industrial use of neem extracts has now become a reality as a result of these ecotoxicological characteristics,
with the appearance on the market of a variety of commercial formulations for varied applications: protection of
plants and stock [6]. Most products are in the form of emulsifiable solutions, among which are Neem-Azal F, NeemAzal T, and Neem-Azal T/S. All formulations on the market indicate the quantity of the active ingredient only, azadirachtin-A, which is generally found in company of eight
other compounds (Fig. 1) in varying proportions.
Several publications have dealt with different techniques
for the separation and identification of products contained
in neem extracts [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16].
Separation in these studies involved reversed-phase liquid
chromatography (RP-HPLC) [6, 8, 9, 11, 12, 13, 14, 15,
16], using octadecylsilane (C18) columns and detection by
ultraviolet absorption. These coupled methods are effective for separation and detection of compounds present in
the oil extracted from neem but are insufficient to identify
754
Fig. 1 Chemical structures of
the compounds present in neem
extracts
755
Preparation by semi-preparative chromatography. The commercial product was purified by semi-preparative chromatography using an HP 1050 instrument (HewlettPackard) equipped with a
quaternary pumping system and manual Rheodyne 7525 injection
valve with 100-L loop and coupled to a Kratos Spectroflow 757
diode-array UV detector and an HP 3396A integrator-recorder.
Separation was achieved on a 250 mm10 mm Interchrom Uptisphere ODSB column (Interchim). The mobile phase was water
and acetonitrile with an elution gradient of 16 to 100% acetonitrile
in 40 min at a flow-rate of 4.4 mL min1. Detection was at the
wavelength =215 nm. After several repeated injections of the
same fraction, azadirachtin-A was recovered and evaporated under
a stream of nitrogen. To eliminate solvent traces for further NMR
analysis azadirachtin-A was then treated with a vacuum pump (at
15 mm Hg) for several hours.
Characterization of chemical species isolated
Liquid chromatography (LCUVMS)
Analyses were carried out with an HP1100-MSD system. The
chromatograph was an HP 1100 equipped with a 250 mm3 mm
Interchim Uptisphere ODSB column maintained at 40 C and fitted
with a 10 mm3 mm pre-column. UV detection was performed at
=215 nm. The mobile phase was water and acetonitrile with an elution gradient of 16% to 100% acetonitrile in 40 min at a flow-rate
of 0.4 mL min1. Samples were diluted in acetonitrile and filtered
through a 0.45 m pore diameter Teflon membrane before analysis.
Mass detection involved two ionization modes at available atmospheric pressure APCI and ESI using two polarities (positive or
negative). The acquisition conditions for mass spectra were thoroughly optimized. The flushing gas was ultrapure nitrogen at a pressure of 55 psig, a temperature of 350 C and a flow-rate of 12 L min1.
The mass-range detected was between 200 and 800 m/z, with fragmentor voltages of +80 V and +100 V for ESI and APCI, respectively, depending on the polarity of the ions detected. In APCI
mode vaporizer temperature was 480 C and the discharge current
at the corona needle was set at 35 A under negative APCI conditions and at 8 A in positive mode. Ion-extraction voltages applied
to the transfer capillary in the two ionization sources with negative
and positive polarity were 2500 V and 3500 V.
To enhance ionization a T-connector was placed between UV
detector output and the MSD source input. When using negative
polarity a mixture of water, ammonium formate, and aqueous ammonia solution at pH 10.5 was added before the sources (APCI and
ESI) of the mass spectrometer at flow-rates of 0.6 and 0.2 mL min1,
respectively. In positive polarity, a mixture of water and formic
acid, pH 3.7, was added under the same conditions as above.
Nuclear magnetic resonance (NMR)
Liquid state 13C NMR spectra (50 MHz) were recorded with a Bruker
Avance 200 spectrometer equipped with a 5-mm 1H/13C probe.
Samples were dissolved in deuterated chloroform [13C(CDCl3)=
77 ppm] and analyses were conducted at room temperature. Acquisition conditions were: pulse angle 30, spectral width 12,000 Hz,
acquisition time 1.41 s, and repetition interval 2 s. The number of
accumulations was set at 16,000 to obtain a good signal/noise ratio.
Experimental design and monitoring of degradation kinetics
The degradation of azadirachtin-A was studied under different
conditions of pH, light, and temperature.
Preparation of solutions
Several solutions of azadirachtin-A were prepared at different pH.
Depending on the target pH they were buffered and adjusted to the
final value with phosphoric acid (pH 20.1), acetic acid (pH 40.1
or 60.1), or sodium carbonate (pH 80.1)
756
Kinetic changes during temperature variation
This study was performed in a dark room to limit photodegradation. Degradation of azadirachtin-A was followed at 40, 50, and
70 C.
The disappearance of azadirachtin-A was followed by means of
liquid chromatography with determination of the concentration of
residual active ingredient using the external standard method.
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Table 1 Retention times, peaks in ESI and APCI mass spectra, and attributions for Neem compounds
Compounds
Azadirachtin I
Retention
time
(min)
ESI mode
APCI mode
m/z(ESI) Attribution
17.1
641a
[M+Na]+
617a
[MH]
ndb
617a
501
[M+H(H2O+tigOH)]+
599
557
517
[M(H+H2O)]
[M(H+AcOH)]
[M(H+tigOH)]
Azadirachtin H
17.9
685a
645
627
545
[M+Na]+
[M+HH2O]+
[M+H2H2O]+
[M+H(H2O+tigOH)]+
661a
643
601
561
[MH]
[M(H+H2O)]
[M(H+AcOH)]
[M(H+tigOH)]
nd
661a
Azadirachtin D
20.3
699a
677
541
[M+Na]+
[M+H]+
[M+H(2H2O+tigOH)]+
675a
643
615
[MH]
[M(H+MeOH)]
[M(H+AcOH)]
nd
675a
Azadirachtin A
21.3
743a
721
685
585
[M+Na]+
[M+H]+
[M+H2H2O]+
[M+H(2H2O+tigOH)]+
719a
687
659
[MH]
[M(H+MeOH)]
[M(H+AcOH)]
703a
719a
Azadirachtin B
21.9
685a
645
627
545
[M+Na]+
[M+HH2O]+
[M+H2H2O]+
[M+H(H2O+tigOH)]+
661a
643
599
[MH]
[M(H+H2O)]
[M(H+H2O+CO2)]
nd
nd
Deacetylnimbin
28.8
521a
467
449
[M+Na]+
[M+HMeOH]+
[M+H(MeOH+H2O)]+
nd
499a
nd
Deacetylslannin
29.3
577a
555
523
[M+Na]+
[M+H]+
[M+HMeOH]+
nd
555a
nd
Nimbin
31.0
563a
509
481
[M+Na]+
[M+HMeOH]+
[M+HAcOH]+
nd
541a
nd
Slannin
32.1
619a
597
459
[M+Na]+
nd
[M+H]+
[M+Na(AcOH+tigOH)]+
597a
nd
aBase
bNot
peak
detected
758
Table 2 13C NMR chemical shifts (, ppm) of the three compounds isolated (solvent CDCl3, reference TMS)
Number of
carbon atoma
2
3
4
5
6
8
9
10
11
12
13
14
15
17
19
20
22
23
24
25
27
28
29
30
32
34
36
38
41
44
47
48
49
50
51
104.2
44.9
45.6
74.5
73.9
73.0
53.1
37.1
50.4
69.1
67.1
29.8
70.7
173.3
52.7
70.1
68.6
48.7
83.6
108.8
76.5
25.1
107.5
147.0
21.4
171.8
52.6
169.5
166.2
18.4
20.8
128.8
137.5
14.2
11.9
aSee
79.5
43.9
44.1
73.6
74.3
73.3
53.4
35.2
51.3
69.5
67.7
32.1
71.4
174.1
52.7
69.4
66.6
48.9
83.6
109.1
76.1
25.1
107.5
146.7
21.2
173.4
52.4
b
166.9
18.1
b
128.6
138.5
14.2
11.9
101.0
48.5
43.3
76.2
74.0
70.0
52.3
37.1
47.7
73.0
67.1
30.8
72.2
b
b
73.0
69.8
48.2
83.5
108.6
77.2
25.3
107.5
147.2
20.6
173.5
52.7
169.8
166.7
18.6
20.6
128.7
138.2
14.4
11.9
bCarbon
t1/2(min)
2
4
6
8
Darkness
Sunlight
UV
14032
141133
188278
75357
14097
142413
63360
62053
340
1274
1410
1050
Ea
(kJ mol1)
1160
385
116
1.00105
3.00105
1.00104
66.9
40
50
70
23104
11552
1650
5.00107
1.00106
7.00106
79.9
40
50
70
23104
2888
1155
5.00107
4.00106
1.00105
83.4
40
50
70
38508
11552
1444
3.00107
1.00106
8.00106
77.7
pH
Temperature (C)
40
50
70
t1/2(min)
Effect of temperature
The effect of temperature on degradation kinetics was
studied at the same pH. We noticed that the rate of disappearance of azadirachtin-A increased with temperature in
dependently of pH. For a given temperature half-life values
tended to increase as pH increased, except for the combination (50 C, pH 6) (Table 4). These results show that the
disappearance kinetics of azadirachtin-A depend on media pH and on temperature.
Activation energy calculations
The curves of the disappearance of azadirachtin-A vs.
time in all the experiments were exponential. Based on
Influence of light
Three experiments were carried out to determine the influence of light on the degradation kinetics of azadirachtin-A. The first was at room temperature and in daylight,
the second with UV irradiation, and the third in darkness.
The influence of these conditions was also examined at
pH 2, 4, 6, and 8. The half-lives measured (Table 3) show
that kinetics of disappearance are higher under UV illumination than in daylight or darkness at the four pH values
used. When the influence of daylight and darkness was
examined, no notable variations were observed at pH 2, 4
and 8; at pH 6, however, disappearance in daylight was
three times faster than in the dark.
759
these experimental data, it can be deduced that the disappearance of azadirachtin-A is first order (apparent
order).
The experimental data enable the apparent rate constant (k1) to be determined graphically by plotting the
variation of ln (Cr) vs. time. By applying Eq. (1) to the experimental data, we could determine the resulting rate
constants and activation energies (Table 4):
(1)
. = $H (D 57
where Ea is the activation energy (kJ), R the ideal gas constant (J), and T the temperature (K).
Fig. 4 Structures of azadirachtin-A and its degradation products
760
Fig. 5 Chromatograms obtained from degradation products: (A) pH 2, (B) pH 4 and 6,
(C) pH 8
761
762
Conclusions
An HPLC method has been developed for separation of the
major products of neem. Use of this method with preparative or the equivalent semi-preparative columns enabled
the pure products to be isolated. These were identified by
use HPLCMS in conjunction with two modes of ionization, ESI and APCI. As shown in this work, the best technique for characterizing all the compounds in the neem
extract is positive ESI. This ionization method renders all
molecules detectable and identifiable. It is also the most
sensitive in terms of the total ion-current formed. The results obtained with positive APCI are comparable with
those published by Schaaf et al. [21] and show the fundamental importance of the choice of the ionization source
and polarity for identification and analysis of neem products. Some of the major compounds in extracts could be
analyzed by liquid state 13C NMR. The data obtained agreed
with results obtained by HPLCMS and confirm that neem
extracts contain several compounds whose activities can be
amplified to different extents in complex mixtures. It was
concluded from kinetic studies carried out in this work
that the process of degradation of azadirachtin-A is accelerated by ultraviolet radiation, irrespective of the pH of
the medium (Table 3). This process is more rapid in basic
medium (pH 8) and highly acidic medium (pH 2), for which
the lowest half-reaction times were recorded. In addition,
the effect of daylight is low in comparison with darkness,
except at pH 6. The study of pH variations showed that
the kinetics of disappearance of azadirachtin-A are slowest at pH 6. This was confirmed by the activation energies
calculated as a function of pH (Table 4).
The identification of breakdown products by HPLCMS
and HPLCMSMS in different controlled media showed
that the disappearance of the starting product azadirachtin-A varies with pH. At pH 4, 6, and 8, its degradation is
initiated by hydrolysis of ester functions and the formation of products P6 and P7, confirming the hypothesis put
forward by Sundaram [11]. This hydrolysis is followed by
dehydration to yield product P8. At pH 2, we identified
five breakdown products, four of which are two-by-two
positional isomers ((P1 and P3), (P2 and P4)). These results
enable us to propose different degradation reactions for decomposition of this product in controlled environments.
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