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Determination of azadirachtin and fatty acid

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Article in Analytical and Bioanalytical Chemistry January 2003

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Anal Bioanal Chem (2002) 374 : 11991204
DOI 10.1007/s00216-002-1638-7
O R I G I N A L PA P E R
Nutan Kaushik
Determination of azadirachtin and fatty acid methyl esters
of Azadirachta indica seeds by HPLC and GLC
Received: 19 March 2002 / Revised: 7 October 2002 / Accepted: 11 October 2002 / Published online: 12 November 2002
Springer-Verlag 2002
Abstract A simple and economical method has been de-
veloped to estimate the azadirachtin content and fatty acid
composition of neem kernels. Neem kernels are crushed
and soaked overnight in ethanol. The extract obtained is
analysed by HPLC after filtering through a 0.22 m mem-
brane. The peaks are separated using acetonitrilewater
(40:60) 1 mL min1 as the mobile phase on an RP-18 col-
umn and monitored at 214 nm. For the determination of
fatty acid composition, the fatty acids are directly transmeth-
ylated in the kernel powder by heating with methanol
acetyl chloridebenzene (20:1:4, v/v) for 1 h in a water
bath. The fatty acid methyl esters (FAMEs) obtained are
extracted in hexane and analysed using GLC. The separa-
tion of the FAMEs is achieved using an RH-Wax column
using temperature programming, 170200 C at 2 min1.
The peaks are detected using an FID. Both the methods do
not require any clean up or defatting of seeds. This results
in faster, easier and more economical sample preparation.
Keywords Neem Azadirachta indica Azadirachtin
Fatty acids HPLC GLC
Introduction
The neem (Azadirachta indica) tree yields a wide range of
triterpenoids such as azadirachtin, nimbin, salanin, azadirach-
tol, etc., and 3050% oil. These triterpenoids are com-
monly known as limonoids. Azadirachtin is the most im-
portant limonoid present in the neem seed and is reported
to have antifeedant, growth disrupting, and ovicidal activ-
ity against a variety of insect pests [1, 2, 3, 4, 5, 6, 7, 8, 9,
10]. Although many isomers of azadirachtin, Aza-A to
Aza-K [11, 12, 13, 14], have been reported in the litera-
ture, azadirachtin-A is the most important and is used as a
N. Kaushik ()
Bioresources and Biotechnology Division, TERI,
Habitat Place, Lodhi Road, New Delhi 110 003, India
e-mail: kaushikn@teri.res.in
standard to express the activity of neem seed, extract and
its formulations.
While azadirachtin content determines the value of the
seeds, the fatty acid composition determines the quality of
the oil. Neem oil comprises four important fatty acids of
which two, palmitic and stearic, are saturated. Oleic acid
is a mono-unsaturated acid and linoleic acid is a poly-un-
saturated fatty acid. The proportion of each fatty acid in
the oil establishes its fatty acid composition.
The neem tree occurs throughout India. According to
an estimate, there are about 20 million trees in the country
[15]. However, individual neem trees may vary in chemi-
cal make-up because of genetic and environmental fac-
tors. The studies carried out by Ermel et al. [16], Ren-
gasamy et al. [17], Kumar et al. [18] and Kaushik and
Singh [19] confirm the natural variability of azadirachtin
content in seeds obtained from individual trees. There-
fore, it is important to test the azadirachtin content of
the neem seed not only to monitor quality, but also to
select high azadirachtin yielding trees. The fatty acid
composition is an important indicator of the quality of the
oil.
Several chromatographic and non-chromatographic meth-
ods have been reported for the estimation of azadirachtin
content in neem seed kernel, oil and its formulations.
Among the non-chromatographic methods, the colorimet-
ric method developed by Dai et al. [20] can be utilized to
determine the total azadirachtin-related limonoids in
neem seed kernel extracts but the azadirachtin content
cannot be separately determined. Shutz et al. [21] devel-
oped an Enzyme Linked Immuno Sorbent Assay (ELISA)
for determination of azadirachtin.
Ermel et al. [22] employed Thin Layer Chromatogra-
phy (TLC)fluorescence to determine the azadirachtin
content in the neem seed kernel. Johnson and Morgan
[23], Huang and Morgan [24] and Ambrosino et al. [25]
reported Supercritical Fluid Chromatography (SFC) for
the qualitative and quantitative determination of azadi-
rachtin in crude neem seed extracts. Although, this tech-
nique requires no clean-up of the samples, it cannot be
employed for the routine determination of azadirachtin
1200

content. It is better suited to the large-scale extraction of
azadirachtin from neem seeds.
High Performance Liquid Chromatography (HPLC)-
based methods reported for estimation of azadirachtin in
neem kernel, oil and formulations differ either in extrac-
tion methodology or clean-up procedure. Hull et al. [26]
described the use of HPLC for quantification of azadi-
rachtin in new formulations using C18 solid-phase extrac-
tion (SPE) cartridges for clean-up of the sample. How-
ever, Azam et al. [27] employed column chromatography
for the clean-up of samples of emulsifiable and solution
concentrate of neem.
Kumar and Parmar [28] analysed azadirachtin in neem
oil by partitioning it in 45% aqueous methanol and hexane.
The aqueous methanol was used to determine the azadi-
rachtin content by HPLC. Ramesh and Balasubramanian
[29] reported a method for the extraction of azadirachtin,
nimbin and salanin from neem oil using a graphitized car-
bon black column instead of liquidliquid partitioning fol-
lowed by column clean-up.
A method reported by Tejavathi et al. [30] for the esti-
mation of azadirachtin content in neem kernel involves
extractions with a sonicator and clean-up with a C-18 Sep-
Pak cartridge. A multi-step sample preparation method fol-
lowing the methodology of Schroder and Nakanishi [31]
has been reported by Deota et al. [32]. The method re-
ported by Schaaf et al. [33] based on HPLC- Mass Spec-
trophotometry (MS) is sensitive enough to detect the
azadirachtin in ng mL1, but is not suitable for routine
analysis since HPLCMS is not available in all laborato-
ries.
Various combinations of HPLC conditions have been
reported to achieve the separation of neem limonoids on
reversed-phase columns. A summary is presented in Table 1.
For the evaluation of the fatty acid composition of neem
oil, Kumar and Parmar [28] reported a complex method
for preparing methyl esters involving saponification, hy-
drolysis and partitioning followed by diazomethane ester-
ification. The process is too cumbersome for routine use.
The methods reported so far involve multistep sample
preparation and are difficult for routine monitoring of the
azadirachtin content and fatty acid composition in the
neem seed kernel. The present paper describes a faster
and more economical method involving single-step sam-
ple preparation for the determination of azadirachtin con-
tent and fatty acid composition in the neem seed kernel
using HPLC and Gas Liquid Chromatography (GLC), re-
spectively.
Experimental
Apparatus
Azadirachtin estimation was performed using a Waters LC Module
I quaternary automated liquid chromatograph equipped with au-
toinjector, high-sensitivity tunable UV and photodiode array de-
tectors and Novapak RP-18 column (3.9 mm150 mm). The chro-
matograms and data were acquired and processed with the Waters
Millennium 2010 Chromatography Manager version 2.1 software.
The photodiode array spectrum was recorded on a Waters 996
Photodiode Array Detector.
Fatty acid methyl ester separation was achieved on a Hewlett
Packard gas chromatograph (HP6890) equipped with an FID and
RH-wax column (30 m0.53 mm).
Chemicals and materials
HPLC grade acetonitrile was procured from Merck (India). Ethanol
was obtained by distilling spirit. Azadirachtin standard (96%) was
procured from Trifolio-M (Germany). Standards of fatty acid
methyl esters were procured from Sigma (USA). Acetyl chloride
and dichloromethane were from Merck (India); benzene, hexane,
acetone, ethyl acetate and acetonitrile from Qualigens (India); and
dehydrated methanol was from Bengal Chemicals (India). Water
was from a Milli-Q water purification system (Millipore, USA).
Neem seeds were collected from Gual Pahari, Haryana, India.
The samples were prepared in Borosil screw-capped centrifuge
tubes (15 mL). A thermostatic serological water bath was used for
heating the samples. A REMI Revolutionary Research Centrifuge
(Model R-23) which could accommodate 36 tubes was used for
centrifugation of the samples. Samples were filtered through Swin-
nex polypropylene 25-mm filter holders (Millipore, USA) contain-

Table 1 Summary of HPLC

conditions reported for separa- Mobile phase Flow rate Wavelength Reference
tion of azadirachtin and other monitored (nm)
neem limonoids
1 Acetonitrile-water (30:70) 1 mL min1 254 [34]
2 Methanol-water (60:40) a 215 [35]
3 Methanol-water (65:35) 1 mL min1 214 and 250 [17]
4 Acetonitrile-water (28:22) 1 mL min1 215 [26]
5 Acetonitrile-water (gradient) initially 1 mL min1 210 [36]
20% acetonitrile increased linearly
to 35% acetonitrile for 22.5 min.
6 Acetonitrile-water (40:60) for 5 min. a 217 [30]
followed by linear gradient to 100%
acetonitrile in 3 min.
7 Methanol-water (70:30) 1 mL min1 214 and 250 [28]
8 0 to 15 min, 32% (acetonitrile -water); 1 mL min1 217 [23]
15 to 20 min, 32 to 55% (acetonitrile: water);
20 to 50 min (55% acetonitrile)
anot reported 9 Methanol Acetonitrile-water (35+15+50) 1 mL min1 a [29]

ing Durapore 0.22 m filters. Azadirachtin samples were filtered
into Waters autosampler vials (4 mL). The mobile phase for HPLC
was filtered through a Millipore sample clarification kit fitted with
Durapore 0.45 m, 47 mm filters (Millipore, USA). A Hewlett
Packard 1 L syringe was used to inject the Fatty Acid Methyl Es-
ters (FAMEs) into the chromatograph.
Determination of azadirachtin content
Optimization of extraction procedure
Effect of temperature. To determine the effect of temperature, if
any, and also to determine the extraction efficiency of cold extrac-
tion, 1 g seed powder from five individual trees was taken in three
replicates in 15 mL centrifuge tubes for each treatment. Distilled
ethanol (6 mL) was added to each tube. The tubes were screw-
capped and kept in two sets. The first set of samples was kept in a
water bath at 50 C for 2 h and the second set left overnight in the
solvent.
The tubes of the first set were taken out from the water bath af-
ter 2 h and allowed to cool to room temperature. The tubes were
then centrifuged at 5000 rpm for 10 min. The supernatant was
transferred into a new tube and the residue re-extracted twice with
ethanol (26 mL) and the upper layers were combined. The pooled
extracts were combined and the final volume was made up to
25 mL in a volumetric flask. A part of this sample (4 mL) was fil-
tered into an autosampler vial through a 0.22 m membrane in a
Swinnex filter holder. The vials were then tightly capped and
stored in a refrigerator until analysis.
The contents of the second set, kept overnight in the solvent,
were centrifuged at 5000 rpm the next morning and the same pro-
cedures followed as described above for the first set.
Optimization of sample size. To see the effect of a sample size on
accuracy of estimation of azadirachtin content of the seeds col-
lected from individual trees, samples were prepared in triplicate in
two sets. In the first set 1 g kernel was taken while in the second
set 1 g kernel was crushed to powder and from this powder 40 mg
of the sample was taken. The seeds were crushed to a fine powder
and then processed to estimate the azadirachtin content.
Extraction efficiency of the solvent. Samples were prepared in
ethanol, HPLC grade acetonitrile, cold water and hot water to de-
termine the effect of solvent on extraction efficiency.
Fig. 1 Chromatogram of
azadirachtin in standard (A)
and neem seeds samples (B)
obtained using HPLC
1201
Defatted vs. non-defatted kernel powder To estimate the loss of
azadirachtin during defatting, the azadirachtin content was deter-
mined in the seed kernel powder without defatting and also after
defatting. For this, samples were prepared in two sets. In the first
set, samples were prepared by soaking the seed kernel powder in
ethanol. In the second set, the seed kernel powder was first ex-
tracted with hexane for removal of oil and then the residual pow-
der was extracted with ethanol for estimation of azadirachtin.
Preparation of standard solution
A standard solution of azadirachtin (1000 g mL1) was prepared by
dissolving 10 mg of the compound in 10 mL of HPLC grade aceto-
nitrile. Serial dilutions were made in the range of 10010 g mL1
to plot the calibration curve. The standard solutions were stored at
20 C.
High performance liquid chromatography
The sample (10 L) was injected into the HPLC using an autoin-
jector. The separation of azadirachtin was achieved using acetoni-
trilewater (40:60) 1 mL min1 and the peaks monitored at 214 nm.
Online degassing was done with helium by using an online de-
gasser.
Determination of fatty acid composition
Fatty acid methyl esters (FAMEs) preparation
The dried seeds (200 mg) were methylated as per the method of
Kaushik and Agnihotri [37] using methanolacetyl chlorideben-
zene (20:1:4, v/v) as methylating reagent. Neem seeds (200 mg)
were crushed and transferred into the centrifuge tube. The methyl-
ating agent (1 mL) was added and the tubes were heated in a water
bath for 1 h. The fatty acid methyl esters thus prepared were sepa-
rated out in n-hexane.
Gas liquid chromatography
Temperature programming was carried out to achieve the separa-
tion of stearic and oleic acids. Operation conditions used were:
1202

Fig. 2 Photodiode array spec-

trum of azaditachtin: (A) map;
(B) 3D view

oven temperature, 170200 C at 2 min1; injector temperature, Table 2 Effect of temperature on the azadirachtin content of
240 C and detector temperature, 250 C. The carrier gas flow rate neem kernel
was 5 mL min1 and make up flow rate was 25 mL min1. The sam-
ple (0.1 L) was injected into the GC. The results were processed Azadirachtin content (g g1 of the kernel)a
using HP Chem station software.
Cold extraction Water bath (50 C)
4212 4028
Results and discussion 3090 2970
7276 7040
High performance liquid chromatography
3360 3540
The HPLC chromatogram of azadirachtin-A standard and 3870 3835
of neem seed sample is given in Fig. 1. The azadirachtin aValues are the mean of three replicates determined in five samples
eluted at a retention time of 2.756 min and elution time
was affected by the proportion of acetonitrile present in Table 3 Effect of sample size on azadirachtin estimation
the mobile phase. With a 95:5 wateracetonitrile system
azadirachtin could not be eluted from the RP-18 column. Azadirachtin content (g g1 of neem kernel)a
However, when the proportion of acetonitrile was varied 1g 40 mg
from 5 to 30%, the peak of azadirachtin could be detected.
The Photo Diode Array (PDA) spectrum of azadi- 1650 1540
rachtin indicated maximum absorbance between 200 and 1380 1040
214 nm (Fig. 2) and beyond this no absorbance was 1310 1420
recorded. Thus, selection of wavelength between this 1170 980
range will give the best estimation and wavelengths like 930 1120
254 nm or 250 nm as used by Yamasaki et al. and Re- aValues are the mean of three replicates determined in five samples
gasamy et al. should not be used for azadirachtin determi-
nation.
The acetonitrilewater (40:60) solvent system is strong time required for analysis and also loss of solvents. How-
enough to elute azadirachtin from the RP-18 column. ever, it was observed that one washing for 10 min with
However, other limonoids could not be eluted. To avoid 100% acetonitrile at the end of the day was sufficient to
clogging, column washing is required. Hull et al. [26] clean the column.
suggested column cleaning using tetrahydrofuran (THF)
for 5 min at the end of each run. The column needs to be Sample preparation
re-equilibrated with the mobile phase. Tejavathi et al. [30]
also recommend washing the column with 100% acetoni- To evaluate the effect of temperature and also to make
trile at the end of each run. This means an increase in the sample preparation simple, the extraction efficiency of the
 1203

Table 4 Extraction efficiency

of different solvents Solvent Azadirachtin content (g in 2 mL)

1st Extraction 2nd Extraction 3rd Extraction Total

(g in 6 mL)
Ethanol 39.82 10.95 6.34 57.11
(69%)a (19%) (11%)
Acetonitrile HPLC grade 44.05 11.26 5.9 61.21
(71%) (18%) (9%)
Cold water 22.33 8.87 7.11 38.31
(58%) (23%) (18%)
aFigures in parentheses repre-
Hot water 20.85 8.91 9.84 39.6
sent percent of the total
azadirachtin extracted (52%) (22%) (24%)

Table 5 Azadirachtin content in seed and meal samples ter (Table 4). Since HPLC grade acetonitrile is very ex-
pensive, ethanol is the best choice for azadirachtin extrac-
Azadirachtin (g g1 of the kernel)a
tion. About 6971% azadirachtin is extracted in the first
Seed Meal extraction, about 1819% in the second and 911% in the
third extraction. Three extractions are sufficient to com-
5595 5099
pletely remove azadirachtin from the neem seed.
5167 3792
5972 3794
The literature suggests that azadirachtin is partially
6621 5307
soluble in neem oil [28]. To evaluate the loss of azadi-
8372 5572 rachtin during the de-oiling of samples prior to its estima-
tion, azadirachtin content was determined in whole seeds
ameans of three replicates determined in five samples as well as de-oiled neem seeds of the same samples. It is
evident from Table 5 that some loss (833%) of azadi-
rachtin occurs during de-oiling. In order to estimate the
method involving extraction of azadirachtin using a water total azadirachtin content of the seed, more accurately, it
bath maintained at 50 C for 2 h was compared with the is therefore preferable to use the whole seed.
extraction efficiency of the method where crushed seeds
soaked in the solvent were left overnight for extraction.
The results are presented in Table 2. It is evident that there Determination of fatty acid composition
is no significant difference in the extraction efficiency of
the two methods. No loss of azadirachtin was observed by The separation of fatty acid methyl esters obtained with
heating seeds at 50 C for 2 h and complete extraction of the RH-Wax column is presented in Fig. 3. Good separa-
the azadirachtin can be achieved by leaving the crushed tion of all the four major fatty acids could be achieved
kernels in the solvent overnight. Dai et al. [20] also reported
complete extraction of azadirachtin by cold extraction.
Large sample sizes require more solvent, more space
and result in problems with routine analysis. The effect of
sample size on accuracy of azadirachtin estimation was
studied to determine whether smaller samples could be
used. Sample size did not appear to have a significant ef-
fect on the accuracy of azadirachtin estimations (Table 3).
Thus a 40-mg sample of crushed seeds can be used for
sample preparation from 1 g seed powder.
Initially various solvents were run through the HPLC
to determine whether they caused interfering peaks. The
HPLC analysis of these solvents shows that SQ grade ace-
tonitrile, dichloromethane (methylene chloride), ethyl ac-
etate and acetone cannot be utilized for azadirachtin ex-
traction because they also give peaks at the same retention
time as azadirachtin. Only ethanol, methanol, HPLC
grade acetonitrile and water were found suitable for
azadirachtin analysis by HPLC. Therefore, the compara-
tive extraction efficiency of ethanol, HPLC grade acetoni-
trile and water were tested.
The extraction efficiency of these solvents followed Fig. 3 Separation of fatty acid methyl esters of neem oil obtained
the order HPLC acetonitrileethanol>hot watercold wa- with an RH-Wax column
1204
Table 6 Fatty acid composition of neem seeds
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