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Journal of Environmental
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Contaminants, and
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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

To link to this article: http://dx.doi.org/10.1080/03601239309372824

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J. ENVIRON. SCI. HEALTH, B28(2), 221-241 (1993)

HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC


METHOD FOR THE ANALYSIS OF AZADIRACHTIN IN TWO
COMMERCIAL FORMULATIONS AND NEEM OIL
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Key Words: Natural Products, Azadirachtin, HPLC analysis, neem


formulations, neem oil

K.M.S. Sundaram and J. Curry

Forestry Canada
Forest Pest Management Institute
P.O. Box 490, 1219 Queen Street East
Sault Ste. Marie, Ontario, Canada
P6A 5M7

ABSTRACT

An improved high performance liquid Chromatographic method is

reported to determine azadirachtin (AZ) content in neem formulations and

neem oil. Aqueous methanol solutions of samples were extracted

sequentially with hexane and dichloromethane. The dichloromethane layer,

containing AZ, was taken to dryness and reconstituted in ethyl acetate. It

was purified by Florisil® column cleanup, eluted with ethyl acetate and

injected into the liquid Chromatograph. Separation was performed with a

reversed-phase Spherisorb C-18 ODS 5 µm column with an

221

Copyright © 1993 by Marcel Dekker, Inc.


222 SUNDARAM AND CURRY

acetonitrile/water gradient system. The eluent was monitored with UV

detection at 210 nm. The method was found to be sufficiently rapid and

reproducible for use in the analysis of AZ in neem formulations and neem

oil. Limits of quantification and detection were respectively 6 µg/g and


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3 µg/g.

INTRODUCTION

There is continuing demand for the development of environmentally

acceptable alternatives to more toxic products for the control of forest

insects and other pests. As alternatives, considerable emphasis is being

placed on the use of selected natural products derived from plants and on

various groups of insect pathogens that would be less harmful to the

environment.

Among the botanical insecticides, extracts from the seeds of the neem

tree, Azadirachta indica A. Juss (Meliaceae) show considerable insecticidal

activity on a wide variety of insect pests, and have been intensively studied

as an alternative to toxic chemical insecticides (Saxena, 1989; Jacobson,

1989; Schmutterer, 1990). Recent studies (Thomas et al, 1992) have shown

that neem extract is also active against the larvae of spruce budworm,

Choristoneura fumiferana (Clem.), a destructive defoliator of spruce-fir

forests in eastern Canada. Helson (1992) also suggested that neem-based

insecticides, such as Margosan-O®, might have potential for forestry use.


LIQUID CHROMATOGRAPHIC METHOD 223

The active principles in the neem seed extract that have been isolated

so far are a mixture of seven structurally-related tetranortriterpenoids, known

as azadirachtins A to G (Rembold, 1989). Among them, the predominant

compound (about 85%) with clearly demonstrated insecticidal activity is


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azadirachtin-A (AZ-A) ( C ^ H ^ O ^ ) (Fig. 1) (Rembold, 1989; Schmutterer,

1990). Azadirachtins are reported to be unstable when exposed to air, heat,

moisture and sunlight (Larson, 1989).

Two neem formulations containing azadirachtin are commercially

available for horticulture use in North America, including Margosan-0

(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

used in large-scale insect control programs, suitable analytical methods are

necessary to quantify the AZ content in them (especially in view of its

reported instability), to standardize potency, to establish dosage levels

required to control pests and to determine the stability and shelf-life of the

formulations.

Analytical methodology for the quantization of AZ is still in the

developmental stage relative to established synthetic pesticides. Due to its

thermal instability (Larson, 1989), and possible adsorption to the stationary

phase in gas Chromatographie columns, the preferred method for the analysis

of AZ is by high performance liquid chromatography (HPLC) using UV


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224

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SUNDARAM AND CURRY
LIQUID CHROMATOGRAPHIC METHOD 225

detection. HPLC methods have been described in the literature (Uebel et

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
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residual amounts of AZ after its exposure to sunlight and UV radiations

respectively. Warthen et al (1984) and Isman et al (1990) developed

HPLC methods for the estimation of AZ in neem extracts and formulations

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).

The analysis of AZ by HPLC using UV detection is somewhat

problematic because the molecule has no strong UV absorbing

chromophores to obtain adequate specificity, detection sensitivity and

quantification capability unless large sample sizes are used, which in turn

cause peak broadening. To maximize sensitivity, detection of AZ has to be

done at a low wavelengths (around 210 ran) using an appropriate UV-

transparent mobile phase, while ensuring at the same time that there is no

concomitant increase in baseline drift. Gas Chromatographie methods are

unsuitable because of the thermal instability of AZ. In this paper we

describe an HPLC method for the assay of AZ in the two commercial

formulations listed above and in a fresh neem oil sample received from
226 SUNDARAM AND CURRY

India after solvent extraction of AZ from the sample matrix, followed by

solvent partitions, Florisil® minicolumn cleanup of the impure AZ and

eventual analysis of the insecticide using UV detection at 210 ran. The

extraction and cleanup procedures and the HPLC instrumental aspects used
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gave better sensitivity (lower detection limits) and precision for the

quantification of AZ in formulations and neem oil than the methods reported

earlier in literature. The methodology may also serve as a useful analytical

tool for future users of this insecticide.

MATERIALS AND METHODS

APPARATUS

(a) HPLC instrumentation: Hewlett-Packard (HP) 1084B HPLC equipped

with a variable wavelength detector (190 to 600 run, model 79875A),

variable volume injector and automatic sampler (model 79842),

microprocessor and electronic integrator (model 79859B). The

instrument employed an automatic degassing system, dual solvent

system and dual pumpheads with common drive, which gave stable

and reproducible flows. A Hewlett-Packard LC terminal (model

79850B) provided the chromatogram, area, area %, retention time, etc.,

for each peak.

The four columns used were: Regis Spherisorb Hi-Chrom Rev. ODS2

Octadecyl II, 5 pm, 15 cm x 4.6 mm i.d. (Regis, Morton Grove, IL 60053


LIQUID CHROMATOGRAPHIC METHOD 227

U.S.A.); HP Hypersil ODS, 5 um, 100 x 2.1 mm i.d.; HP LiChrosorb RP-8,

10 urn, 200 x 4.6 mm i.d.; and HP LiChrosorb RP-18, 10 um, 200 x 4.6

mm i.d. (all three columns from Hewlett Packard, Mississauga, Ontario

L4V 1M8).
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Two mobile phase systems were examined: (1) WatenAcetonitrile and

(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

constant reference wavelength of 430 nm and three sample wavelengths of

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

AU and 4.8 x 10"4 AU respectively.

The shaker, rotary evaporator and nitrogen evaporator used in this

study were respectively, Magni Whirl® Constant temperature bath (Blue M

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

Meyer N-EVAP® Analytical Evaporator, Model No. I l l (Organomation

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

in the study was purified further by using a Milli-Q® Water System

(Millipore Canada Ltd., Mississauga, Ont. L4V 1M5).

REAGENTS

Formulations: Margosan-O® and Azatin® EC (prototype formulation No.


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XL35126) were gifts from W.R. Grace and Co., Conn., 62 Whitmore

Ave., Cambridge, MA 02140 and AgriDyne Inc., 417 Wakara Way,

Salt Lake City, Utah 84108, respectively. According to the label

information, the two formulations are reported to contain 0.3 and

3.0 % of AZ respectively.

Neem Oil: A fresh sample of oil obtained by crushing neem seeds collected

around Kanthayapalayam in S. India (where neem products have been

in use in folk medicine since ancient times) was used in this study.

Azadirachtin: Analytical standard (>95% purity), was purchased from

Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178. A stock

solution of AZ was prepared in methanol (0.5 mg/mL) and stored at -

25°C. Aliquots of this stock solution were diluted with the mobile

phase immediately before use, to give working solutions containing AZ

in the range 1.0 - 25 ug/mL. The diluted stock solution (9.5 ug/mL),

on injection (0.475 ug in 50 uL), gave a distinct single peak (Fig. 2A)

with retention time 20.6 min, which is assumed to be due to the

presence of AZ-A only.


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LIQUID CHROMATOGRAPHIC METHOD
229
230 SUNDARAM AND CURRY

Column Packings: Florisil® (Roridin Co., Pittsburgh, PA, calcined at

650°C, PR grade, 60 - 100 mesh), carbon powder (acid washed, 60-

mesh, J.T. Baker Inc., Phillipsburgh, NJ 08865), Whatman CF-11®

cellulose powder (Whatman Ltd., Maidstone Kent, England) were


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purified (Sundaram, 1990) and used. Silica gel (70-230 mesh ASTM,

Merck Reagents, Darmstadt, Germany) and aluminum oxide (90-mesh

ASTM, 70-230 mesh, neutral and acidic, Merck Reagents, Darmstadt,

Germany) were heated to 120°C overnight and stored in a desiccator

prior to use.

Chemicals: Sodium sulphate anhydrous granular, ACS grade (BDH Ltd.)

was purified (Sundaram, 1990), dried for 8 h at 300°C and stored in

a desiccator prior to use. Sodium chloride was ACS grade (Fisher

Scientific) and used as received.

Solvents: Acetonitrile, dichloromethane, ethyl acetate, hexane and methanol

were HPLC (Omnisolv®) grade obtained from BDH Inc. (Toronto,

Ontario), filtered through a Nylaflo filter, 0.20 ¿im pore size. Water

was deionized, purified by a Milli-Q Water System and filtered as

above.

EXTRACTION OF AZADIRACHTIN

Formulations: In addition to AZ, both formulations contain various

additives to increase their field performance. Each formulation was


LIQUID CHROMATOGRAPHIC METHOD 231

thoroughly shaken in a mechanical shaker (Magni Whirl) and a sample

size equivalent to 1.0 g of the formulation was transferred to a 100 mL

capped centrifuge tube. After adding 50 mL methanol, the tube was

shaken for 20 min and centrifuged at 1000 g for 10 min. Aliquots of


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samples (3.0 mL containing 0.06 g of Azatin and 30 mL containing

0.60 g of Margosan, giving both 1.8 mg AZ) were transferred to two

250 mL separatory funnels. To the separatory funnel containing

Azatin, 27 mL methanol was added to give a volume of 30 mL. Equal

volumes of deionized water and hexane and 5.0 mL of 5% sodium

chloride solution were added to each separatory funnel, shaken well for

ten minutes and the phases were allowed to separate. The aqueous

methanolic phase containing AZ was separated and partitioned twice

more with hexane to remove nonpolar and lipoid impurities. Most of

the methanol in the aqueous phase was removed in vacuo at 30°C

using a rotary evaporator (Buchler) and the aqueous layer was

extracted thrice with dichloromethane, each time using 60 mL. Further

extraction with the solvent did not increase the AZ content. The

pooled organic layer, containing the AZ was dried by passing it

through a column (3 cm diam. x 3 cm length) of anhydrous sodium

sulphate. The column was rinsed twice with 10.0 mL of

dichloromethane and these rinses were added to the extract. It was


232 SUNDARAM AND CURRY

evaporated to dryness at 30°C (Buchler) and the residue was dissolved

in exactly 6.0 mL of ethyl acetate for column cleanup, so that 1.0 mL

corresponded to either 0.1 g of Margosan or 0.01 g of Azatin.

Neem oil: The extraction of AZ from the neem oil was very similar to the
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formulations except the following changes. Step (c) below is optional

and is required only to improve the quality of HPLC analysis.

(a) 2.0 g of oil was extracted using 50 mL of methanol;

(b) 30.0 mL of methanolic solution was used in the

water/hexane partition and

(c) the residues of AZ from dichloromethane evaporation

were partitioned once with hexane/acetonitrile (10/30

mL)(to remove artifactual bands and to improve HPLC

separation) and the AZ residues in the polar layer were

dissolved, after removing the acetonitrile, in 6.0 mL of

ethyl acetate for column cleanup.

Use of different ratios of methanol and water instead of the 1:1 ratio

in the extraction procedure did not result in increased recoveries of AZ from

the matrices. Substituting acetonitrile for the alcohol enhanced the

extraction of coextractive impurities causing diffículties in column cleanup.

COLUMN CLEANUP

The crude extracts of the two formulations and neem oil required

minicolumn cleanup to remove the bulk of the coextractives present in them


LIQUID CHROMATOGRAPHIC METHOD 233

prior to HPLC analysis. The column consisted of a disposable pasteur pipet

(15 cm x 0.8 cm i.d.) plugged with a small wad of glass wool at the bottom

and 5 cm of adsorbent sandwiched and setded uniformly between the glass

wool and 1 cm of anhydrous sodium sulphate (Sundaram, 1990). Different


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adsorbents, viz., Florisil, silica gel, aluminum oxide (neutral and acidic) and

carbon powder-cellulose mixture were tried after preconditioning each type

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

coextractive impurities when eluted with 15 mL of ethyl acetate, whereas

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

better recovery and consistently cleaner eluates than the latter.

Consequently, Florisil column cleanup was preferred over silica gel in

subsequent analysis. The eluate containing AZ was evaporated to dryness

(Meyer N-Evap) and the residue was taken in acetonitrile for HPLC

analysis. The concentration of each sample was adjusted to the

predetermined linear range of the UV detector.

Prior to the use of Florisil and silica gel columns for cleanup of crude

extracts, the elution patterns of AZ in these columns were established by

spiking the preconditioned columns with AZ standard and eluting them with

ethyl acetate.
234 SUNDARAM AND CURRY

RESULTS AND DISCUSSION

HPLC ANALYSIS

1. Column selection:

The final operating parameters to analyze the AZ content in the


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formulations and neem oil were optimized by trial and error. Standard

solutions with increasing concentrations of AZ were injected separately

into each of the four reverse phase columns for a fixed set of

instrumental parameters and HPLC response was examined. The

parameters were altered in sequence and according to the exploratory

runs to obtain the best HPLC response.

The HP Hypersil RP-ODS, 5 um, 100 x 2.1 mm i.d. column,

because of its dimensions developed appreciable back pressure and

column plugging after few injections, consequently the separation of AZ

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

resolution, which required an increased flow rate (1.5 mL/min). Under

such conditions the column assembly performed well and separation of

AZ was adequate, but the run time was higher (retention time for AZ of

23.2 min). The HP LiChrosorb RP-18, 10 um, 200 x 4.6 mm i.d.

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

similar columns. The Regis Spherisorb Hi-Chrom ODS 2 Octadecyl II,


LIQUID CHROMATOGRAPHIC METHOD 235

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

resolution of AZ in the three matrices was good with an average

retention time (R.T.) of 20.6 min. The chromatograms (Fig. 2) were


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sharp, narrow and symmetrical with maximum separation efficiencies

from solvent front and interfering peaks due to endogenous materials

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.

Frequent flushing of the column with mobile phase was necessary to

maintain its performance, to minimize band tailing and to remove the

late eluters.

2. Solvent system:

Isocratic and gradient elution procedures using two binary solvent

mobile phases (methanol/water and acetonitrile/water) were investigated

to optimize the analyte separation. Isocratic elution with either phase

was found to be unsuitable because of poor resolution of the analyte

although the baseline drift was minimal.

The methanol/water gradient system gave poor and

nonreproducible separation of the analyte from all three matrices, with

considerable baseline drift. The acetonitrile/water gradient system was

found to be suitable for the separation of AZ giving sharp peaks and

good resolution without much baseline drift except that column


236 SUNDARAM AND CURRY

reequilibration was necessary after each run. For the run time of 35 min,

the gradient program began with 20 % acetonitrile in water increasing

continuously to 35 % acetonitrile at 22.5 min. The column was flushed

by increasing the acetonitrile concentration to 60 % over 2.5 min and


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maintaining this for 10 min.

3. UV absorption by AZ:

A UV scan of the standard in acetonitrile (Shimadzu UV-Visible

Recording Spectrophotometer model UV160U, Shimadzu Sei. Instr. Inc.,

7102 Riverwood Drive, Columbia, Maryland 21046) showed that the

X ^ for the analyte is 208 nm. Earlier workers (Uebel et al, 1979;

Warthen et al, 1984; Yamasaki et al, 1986) usually used higher

wavelengths (214 to 254 nm) for sample analysis to avoid enhanced

baseline noise. Our findings showed that wavelengths higher than 210

nm gave lower detection limits and the increase in baseline noise at 210

nm was not significant. So 210 nm was chosen as the most suitable

wavelength for the analysis of AZ in this study.

4. Oven temperature:

The oven temperature was found to have considerable influence

on the separation of AZ in the formulations. At ambient temperature, a

coextractive peak eluting with the same R.T. as AZ caused increased

drift in baseline and gave artificially high concentration levels for the
LIQUID CHROMATOGRAPHIC METHOD 237

analyte due to band overlap. With an increase in oven temperature, the

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

bands was complete with appreciable band spacing. The optimum


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column temperature was therefore fixed at 80°C.

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

pore size. The Regis Spherisorb ODS 2, 5 um column was selected in

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

and provided optimum band spacing during analysis with good

resolution.

6. Injection volume:

Injection volumes ranging from 20 uL to 100 jiL were examined.

Usually at 20 uL, the response of the analyte in the matrices, especially

in the neem oil was low, while at 100 uL, peak broadening occurred

with increased detector noise resulting in poor peak resolution. The

injection volume was decreased until good resolution was obtained. This

occurred with a 50 uL injection volume (analyte mass ranged from 0.05


238 SUNDARAM AND CURRY

to 1.25 ng in 50 pL), which gave better detection limits and a good

separation of the analyte.

7. Quantification ofAZ:

The concentration of AZ in the three matrices was determined


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from the calibration curve obtained by plotting peak area of

chromatograms versus AZ concentrations, by injecting 50 uL of AZ

standard solutions in triplicate, ranging in concentrations from 1.0 to 25

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

injections of 1.0 jig/mL (0.05 ug) standard had a peak height of 9 mm

(0.00144 AU) with a baseline noise level of 3 mm (0.00048 AU). The

standards below this level are not detectable because of appreciable

baseline noise. This phenomenon became significant especially when the

sample extracts were injected. Therefore the minimum limit of detection

(MLD) of AZ in the samples is defined in this study as the concentration

that gives three times peak-to-peak baseline noise and is conservatively

fixed as 3 ¡ig/g (3 ppm) for the formulations and neem oil. Similarly the

limit of quantification (LOQ) of AZ was estimated arbitrarily as twice

the MLD or 6 ng/g (6 ppm). The intra-assay coefficient of variation for

Margosan-0 and Azatin were respectively 7% and 4% and for the neem

oil it was 13% (n=9).


LIQUID CHROMATOGRAPHIC METHOD 239

8. Concentrations of AZ in the formulations and neem oil:

Figure 2 illustrates the time course of AZ in the standard (50 fiL

injection containing 5.0 ng/mL)(Fig. 2A) and the purified extracts of

Margosan-0 (Fig. 2B), Azatin (Fig. 2C) and neem oil (Fig. 2D). The
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mass percent concentrations (g/100g) of AZ in the three matrices

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

three matrices is 5). The concentrations of AZ found in the two

formulations agree with the values reported on the label.

The AZ content of neem oil is influenced by a number of factors

such as humidity, climate, sunlight, storage time, seed drying, soil pH,

extraction method from kernels, geographical location, time of harvest,

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%)

is in agreement with some values reported by Isman et al. (1990).

CONCLUSIONS

This paper describes an improved reverse phase HPLC method using

a UV detector at 210 nm for the quantification of AZ in commercial

formulations and neem oil after repeated solvent extraction, partition and

column cleanup. The method adequately eliminates the coextractive

impurities and is an accurate, efficient and reliable means to quantify AZ in


240 SUNDARAM AND CURRY

neem products at a higher sensitivity (6 ng/g) than the methods reported in

the literature (50 ug/g) (Isman et al., 1990). The analytical scheme on

which this method is based could be applicable, with minor modifications,

to the assay of AZ in various forestry and agricultural matrices, and to


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examine the persistence and environmental fate of this chemical in these

materials.

Further refinement to enhance the sensitivity of AZ quantification is

possible by using the sensitive diode array detector currently available for

HPLC, combining HPLC with immunoassay technique and by fluorescence

labelling of AZ with suitable compounds. A resurgence of interest will

emerge if AZ becomes a successful candidate material for forest insect

control programs in Canada.

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®

EC formulations used in this study. The excellent technical assistance

provided by Linda Sloane is acknowledged with thanks.

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).

Helson, B., For. Chron., 68(3). 349-354 (1992).


LIQUID CHROMATOGRAPHIC METHOD 241

Isman, M.B., Koul, O., Luczynski, A. and Kaminski, J., J. Agric. Food
Chem., 38. 1406-1411 (1990).

Jacobson, M. (ed.) Focus on Phytochemical Pesticides - Vol. 1: The Neem


Tree CRC Press, Inc., Boca Raton, Florida, U.S.A. (1989), pp.178.

Larson, R.O., The Commercialization of Neem. In Focus on Phytochemical


Pesticides - Vol. 1: The Neem Tree Jacobson, M. (ed.), CRC Press,
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Inc., Boca Raton, Florida, U.S.A. (1989), pp 155-168.

Rembold, H. Azadirachtins, their Structure and Mode of Action. In


Insecticides of Plant Origin Arnason, J.T., Philogene, B. and Morand,
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Received: October 20, 1992