Documente Academic
Documente Profesional
Documente Cultură
Journal of Environmental
Science and Health,
Part B: Pesticides, Food
Contaminants, and
Agricultural Wastes
Publication details, including instructions
for authors and subscription information:
http://www.tandfonline.com/loi/lesb20
High performance
liquid chromatographic
method for the analysis
of azadirachtin in two
commercial formulations
and neem oil
a a
K.M.S. Sundaram & J. Curry
a
Forest Pest Management Institute ,
Forestry Canada , P.O. Box 490, 1219 Queen
Street East, Sault Ste. Marie, Ontario, P6A
5M7, Canada
Published online: 21 Nov 2008.
To cite this article: K.M.S. Sundaram & J. Curry (1993) High performance
liquid chromatographic method for the analysis of azadirachtin in two
commercial formulations and neem oil, Journal of Environmental Science and
Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 28:2,
221-241, DOI: 10.1080/03601239309372824
This article may be used for research, teaching, and private study
purposes. Any substantial or systematic reproduction, redistribution,
reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access
and use can be found at http://www.tandfonline.com/page/terms-
and-conditions
J. ENVIRON. SCI. HEALTH, B28(2), 221-241 (1993)
Forestry Canada
Forest Pest Management Institute
P.O. Box 490, 1219 Queen Street East
Sault Ste. Marie, Ontario, Canada
P6A 5M7
ABSTRACT
was purified by Florisil® column cleanup, eluted with ethyl acetate and
221
detection at 210 nm. The method was found to be sufficiently rapid and
3 µg/g.
INTRODUCTION
placed on the use of selected natural products derived from plants and on
environment.
Among the botanical insecticides, extracts from the seeds of the neem
activity on a wide variety of insect pests, and have been intensively studied
1989; Schmutterer, 1990). Recent studies (Thomas et al, 1992) have shown
that neem extract is also active against the larvae of spruce budworm,
The active principles in the neem seed extract that have been isolated
(W.R. Grace and Co., Conn., Cambridge, MA) and Azatin® EC (AgriDyne
Tech. Inc., Salt Lake City, Utah). If neem formulations are going to be
required to control pests and to determine the stability and shelf-life of the
formulations.
phase in gas Chromatographie columns, the preferred method for the analysis
224
"„
\
/
o
i—i
2
4
o
00
Ë
m
vn
u
2
E
•S
<
o*
op
sí
SUNDARAM AND CURRY
LIQUID CHROMATOGRAPHIC METHOD 225
al, 1979; Schneider and Ermel, 1986; Yamasaki et al, 1986) for the
separation and isolation of AZ from neem seed kernels. Stokes and Redfern
(1982) and Barnby et al. (1989) used HPLC techniques to screen the
Downloaded by [University of Toronto Libraries] at 08:05 19 November 2014
and in neem oil respectively. However, these methods were not sensitive
and the limit of quantification reported for AZ was around 50 pg/g (Isman
et al, 1990).
quantification capability unless large sample sizes are used, which in turn
transparent mobile phase, while ensuring at the same time that there is no
formulations listed above and in a fresh neem oil sample received from
226 SUNDARAM AND CURRY
extraction and cleanup procedures and the HPLC instrumental aspects used
Downloaded by [University of Toronto Libraries] at 08:05 19 November 2014
gave better sensitivity (lower detection limits) and precision for the
APPARATUS
system and dual pumpheads with common drive, which gave stable
The four columns used were: Regis Spherisorb Hi-Chrom Rev. ODS2
10 urn, 200 x 4.6 mm i.d.; and HP LiChrosorb RP-18, 10 um, 200 x 4.6
L4V 1M8).
Downloaded by [University of Toronto Libraries] at 08:05 19 November 2014
(2) Water:Methanol, using flow rates of 1.0 mL/minute and 1.5 mL/minute.
The oven temperature was varied from 30°C to 80°C and the injector
volumes used were 20, 50 and 100 uL. Quantification was done with a
210, 215 and 220 nm with a detector setting in the range of 1.6 x 10*3 to
2.56 x 10*2 AU/cm. The slope sensitivity used during the study was 0.20
and the average baseline noise and baseline drift were found to be 1.6 x 10"4
Electric Co., Blue Island, IL U.S.A.); Buchler rotary evaporator Model No.
PTFE-1GN (Buchler Instruments, Inc., Fort Lee, NJ, 07025 U.S.A.); and
Associates Inc., South Berlin, MA, 01549 U.S.A.). The filters used were
Acrodisc® LC13 PVDF 0.2 um and Nylaflo® Nylon Membrane Filter 0.2
um (Gelman Sciences Inc., Rexdale, Ont. M9W 5X6). Distilled water used
228 SUNDARAM AND CURRY
REAGENTS
XL35126) were gifts from W.R. Grace and Co., Conn., 62 Whitmore
3.0 % of AZ respectively.
Neem Oil: A fresh sample of oil obtained by crushing neem seeds collected
in use in folk medicine since ancient times) was used in this study.
Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178. A stock
25°C. Aliquots of this stock solution were diluted with the mobile
in the range 1.0 - 25 ug/mL. The diluted stock solution (9.5 ug/mL),
(nv t_oi x)
uiu 013 eousqiosqv
LIQUID CHROMATOGRAPHIC METHOD
229
230 SUNDARAM AND CURRY
purified (Sundaram, 1990) and used. Silica gel (70-230 mesh ASTM,
prior to use.
Ontario), filtered through a Nylaflo filter, 0.20 ¿im pore size. Water
above.
EXTRACTION OF AZADIRACHTIN
chloride solution were added to each separatory funnel, shaken well for
ten minutes and the phases were allowed to separate. The aqueous
extraction with the solvent did not increase the AZ content. The
Neem oil: The extraction of AZ from the neem oil was very similar to the
Downloaded by [University of Toronto Libraries] at 08:05 19 November 2014
Use of different ratios of methanol and water instead of the 1:1 ratio
COLUMN CLEANUP
The crude extracts of the two formulations and neem oil required
(15 cm x 0.8 cm i.d.) plugged with a small wad of glass wool at the bottom
adsorbents, viz., Florisil, silica gel, aluminum oxide (neutral and acidic) and
of column with 10 mL of ethyl acetate and loading the column with 1.0 mL
of the crude extract. Florisil and silica gel columns retained most of the
the other two adsorbents gave either inconsistent recoveries and/or poor
cleanup. Elution with different solvents and solvent mixtures did not
improve the situation. Between Florisil and silica gel, the former gave
(Meyer N-Evap) and the residue was taken in acetonitrile for HPLC
Prior to the use of Florisil and silica gel columns for cleanup of crude
spiking the preconditioned columns with AZ standard and eluting them with
ethyl acetate.
234 SUNDARAM AND CURRY
HPLC ANALYSIS
1. Column selection:
formulations and neem oil were optimized by trial and error. Standard
into each of the four reverse phase columns for a fixed set of
was poor. Similarly the HP LiChrosorb RP-8,10 um, 200 x 4.6 mm i.d.
was not effective unless a double column assembly was used for better
AZ was adequate, but the run time was higher (retention time for AZ of
column gave broad peaks for the AZ in the two formulations and gave
Í
poor resolution for the analyte in neem oil, even after coupling two
5 ion, 15 cm x 4.6 mm i.d. was the best among the four columns
investigated and selected as the most suitable column for this study. The
present in the matrices. Deviation in R.T.s for each injection was less
than 1% but noticeable variations (about 5%) did occur from day to day.
late eluters.
2. Solvent system:
reequilibration was necessary after each run. For the run time of 35 min,
3. UV absorption by AZ:
X ^ for the analyte is 208 nm. Earlier workers (Uebel et al, 1979;
baseline noise. Our findings showed that wavelengths higher than 210
nm gave lower detection limits and the increase in baseline noise at 210
4. Oven temperature:
drift in baseline and gave artificially high concentration levels for the
LIQUID CHROMATOGRAPHIC METHOD 237
coextractive peak gradually separated from the AZ peak, whose R.T. was
similar to that for the standard, and at 80cC the resolution of the two
5. Flow rate:
Two flow rates, 1.0 mL/min and 1.5 mL/min, were investigated.
As expected, the higher flow rate lowered the R.T. of AZ. This flow
rate was acceptable for the LiChrosorb RP-8 column, since it was 10 urn
this study and the higher flow rate increased column pressure, giving
problems. Therefore a flow rate of 1.0 mL/min was used in this study
resolution.
6. Injection volume:
in the neem oil was low, while at 100 uL, peak broadening occurred
injection volume was decreased until good resolution was obtained. This
7. Quantification ofAZ:
Hg/mL (0.05 to 1.25 ng). A very good linear relationship (R2 = 99.9 %)
was observed in the range tested with a y-intercept near zero. Fifty uL
fixed as 3 ¡ig/g (3 ppm) for the formulations and neem oil. Similarly the
Margosan-0 and Azatin were respectively 7% and 4% and for the neem
Margosan-0 (Fig. 2B), Azatin (Fig. 2C) and neem oil (Fig. 2D). The
Downloaded by [University of Toronto Libraries] at 08:05 19 November 2014
computed from the calibration curve are respectively 0.31 ± 0.02, 3.24
+ 0.12 and 0.13 + 0.02 for Margosan-O, Azatin and neem oil (n for the
such as humidity, climate, sunlight, storage time, seed drying, soil pH,
etc. (Uebel et al., 1979; Bambarkar, 1990) and varied from below 50
Hg/g to 4026 ng/g (Isman et al, 1990). Our value of 1300 pg/g (0.13%)
CONCLUSIONS
formulations and neem oil after repeated solvent extraction, partition and
the literature (50 ug/g) (Isman et al., 1990). The analytical scheme on
materials.
possible by using the sensitive diode array detector currently available for
ACKNOWLEDGEMENTS
The authors are grateful to W.R. Grace and Co., Conn., 62 Whitmore
Ave., Cambridge, MA 02140 and AgriDyne Inc., 417 Wakara Way, Salt
Lake City, Utah 84108 for supplying respectively Margosan-O® and Azatin®
REFERENCES
Bambarkar, S., Pesticides, 24(1). 36-40 (1990).
Barnby, M.A., Yamasaki, R.B. and Blocke, J.A., J. Econ. Entomol., 82(1),
58-63 (1989).
Isman, M.B., Koul, O., Luczynski, A. and Kaminski, J., J. Agric. Food
Chem., 38. 1406-1411 (1990).
Schneider, B-H and Ermel, K. Proc. 3 r d Int. Neem Conf., Nairobi (1986),
pp 161-170.
Stokes, J.B. and Redfern, R.E., J. Environ. Sci. Health, A17(1), 57-65
(1982).
Thomas, A.W., Strunz, G.M., Chaisson, M. and Chan, T.N., Etomol. Exp.
Appl., 62, 37-46 (1992).
Uebel, E.C., Warthen, Jr., J.D. and Jacobson, M., Jour. Liquid Chromatogr.,
2(6). 875-882 (1979).
Warthen, Jr., J.D., Stokes, J.B., Jacobson, M. and Kozempel, M.F., Jour.
Liquid Chromatogr., 7(3), 591-598 (1984).
Yamasaki, R.B., Klocke, J.A., Lee, S.M., Stone, G.A. and Darlington, M.V.,
J. Chromatogr., 356, 220-226 (1986).