81

3.1 Determination of Phenylurea Herbicides by SPE-HPLC-UV

and its application to soft drink and tap water samples

3.1.1 Introduction

The phenylurea herbicides are widely used in a broad range of herbicide formulations and

also for non-agricultural use; consequently their residues are frequently detected as major

water contaminants in the areas where these are used extensively [1]. Diuron and Linuron

are both substituted urea compounds which are soluble in water and can migrate in soil

and enter the food chain [2]. These herbicides are of significant toxicological risk to

humans and wild life. Diuron is used in cotton growing areas, fruit crops, etc. and is rated

as the 3rd

most hazardous pesticide for ground water resources. These herbicides are also

applied on railway tracks to maintain quality and provide a safer working environment

[3] but this may lead to groundwater contamination as their leaching potential is

significant. Phenylureas enter the environment through different pathways like spray

drift, runoff from treated fields and leaching into groundwater. Most of the excess

material penetrates into the soil, where it is subjected to the action of microorganisms [4]

and degradation as studied by Canonica et al [5]. Phenylureas are photo chemically

unstable as discussed by Khodja et al [6], but these can persist in water for several days

or weeks depending on the temperature and pH. Cases of incidental pesticide pollution of

water reservoirs [2-4 7-13] have become more numerous in recent years. Phenyl urea

residues can be found in water sources, processed products and on the crops where these

are applied. In India, most of the soft drink bottling plants use surface water i.e. from

canals and rivers which have high chances of pesticides contamination. The water

treatment measures used are insufficient for complete removal of these pesticide residues

82

which have been found to be above permissible limits. The evidence for the above stated

facts was provided in Centre for Science and Environment (CSE), New Delhi report of

2003 that found several pesticide residues in many soft drink samples of leading

international brands procured from all over India. The CSE, New Delhi, findings were

further affirmed by a Joint Parliamentary Committee (JPC) setup to verify the facts. In

2006 again CSE conducted another round of tests and yet again found pesticides in soft

drink samples. Keeping this in mind, the present work has a lot of importance as it

involves the determination of phenyl urea herbicides in soft drink samples and tap water.

Therefore, it is imperative that sensitive, selective and efficient methods for herbicide

analysis should be designed. The common analytical methods used are HPLC-UV [2 – 4,

7-9], SPME-HPLC [10], diode array [11], immunosorbent trace enrichment and HPLC

[12, 14], LC-MS [15, 16], GC-MS [13], capillary electrophoresis [17, 18, 19], photo

chemically induced fluorescence [20, 21] and derivative spectrophotometry [22]. A

useful review is presented by Sherma [23] on the use of TLC and its modified versions

for the analysis of these herbicides. SPE of phenylurea herbicides has been reported in

literature by several workers [24-29]. The SPE of soft drinks has been extensively

reported [30-36]. As the use of polar and degradable pesticides is becoming rampant, it is

an urgent requirement that more sensitive analytical methods must be developed for their

residual analysis in various matrices. HPLC technique has several advantages over GC

technique as it can be used for simultaneous analysis of thermally unstable, non-volatile,

polar and neutral species without a derivative step. Due to thermally unstable nature of

phenyl urea herbicides the direct application of GC to these compounds is not possible

and derivatization prior to the detection is needed. For this reason, high performance

liquid chromatography (HPLC) with UV absorption or fluorescence detection [7-10] is

83

preferred over GC. Hence HPLC is gaining high popularity and preference as pesticide

analyzing technique.

The present work gives a simple and sensitive HPLC-UV method for the analysis of

phenyl urea herbicides namely monuron, diuron, linuron, metazachlor and metoxuron and

it involves a single step pre-concentration by Solid Phase Extraction (SPE).

Diuron

Linuron

Metazachlor

Monuron

Metoxuron

Figure 3.1.1 Molecular structure of Phenylurea herbicides

3.1.2 Experimental

3.1.2.1 Equipment and Reagents

The HPLC system used included a Dionex P680 HPLC pump, a Dionex 4.6 X 250 mm

C18 5µm RP analytical column (Acclaim) and a Dionex UVD 170U detector operated at a

84

wavelength of 210 nm coupled to a Chromeleon computer program for the acquisition of

data. Monuron, diuron, linuron, metoxuron and metazachlor (figures 1-5) pesticide

standards were obtained from Reidel-de-Haen (Germany). HPLC grade acetonitrile and

methanol were obtained from J.T. Baker (USA). All the solvents were filtered through

Nylon 6.6 membrane filters (Rankem) using filtration assembly (Perfit, India) and

sonicated before use. Triply distilled water was used for all purposes.

3.1.2.2 Standard preparation

Stock solutions were prepared in a mixture of 50:50 MeOH: water. All the solutions

were stored below 4 oC stored under refrigeration.

3.1.2.3 Sample preparation

The solid phase extraction of the tap water and soft drink samples were performed using

SPE system VISIPREPTM

SPE Vacuum Manifolds, (Supelco, Bellefonte, PA, USA) and

C18 cartridges from J. T. Baker were used. The SPE cartridges were attached to the

solvent recovery assembly and connected to a vacuum pump. The conditioning was done

by using 1ml each of Acetonitrile, Methanol and triply distilled water.

3.1.2.3.1 Cold drink samples

The presence of phenylurea herbicides was studied in three different types of locally

purchased soft drinks (viz. coke, mirinda and limca). These were filtered with nylon 6.6

membrane filters, degassed by sonicating for 30 minutes. The samples were then spiked

with the metoxuron, monuron, diuron, metazachlor and linuron at a concentration of 5 ng

mL-1

. 20 mL of these samples were passed through the C18 SPE cartridges under vacuum

85

and eluted with 1.5 mL of acetonitrile. The eluants were further used for the HPLC-UV

analysis. The sample blanks were also prepared similarly.

3.1.2.3.2 Tap water sample

The tap water sample was taken from lab. It was filtered and then degassed with an

ultrasonic bath. The sample was spiked with metoxuron, monuron, diuron, metazachlor

and linuron at a concentration of 5 ng ml-1

each. 50 mL sample of the tap water

containing mixture of herbicides was pre-concentrated using C18 SPE cartridges. 1.5 mL

acetonitrile was used for the elution and the eluant was subjected to HPLC-UV analysis.

The sample blanks were prepared by the same method.

3.1.3 HPLC determination of phenylurea herbicides

3.1.3.1 Procedure

Aliquots of mixture of five herbicides were taken having concentrations of 5 ppb to 500

ppb. These mixtures were analysed at an optimum wavelength of 210 nm. Mobile phase

is an important factor in HPLC analysis as it interacts with solute species of the sample.

Hence, the composition of the mobile phase was carefully selected as ACN : Water in the

ratio 60:40 and the flow rate was set at 1ml/min. All measurements were carried at

ambient temperature. The calibration curves for all five herbicides were prepared and the

curves were linear in the range studied.

3.1.4 Results and discussion

3.1.4.1 HPLC-UV studies

The separation of these herbicides was studied using direct injection of samples and

86

various parameters like effect of flow rate, selection of suitable wavelength and

composition of mobile phase were optimized. The composition of mobile phase was

ACN: Water (60:40). At higher flow rates than 1.0 mL/min, the separations were not up

to the baseline and with lower flow rates peak tailing was observed, so the flow rate was

optimized to 1.0 mL/min. The wavelength for detection was selected from the UV

absorption spectra of the five herbicides and it was selected as 210 nm.

3.1.4.2 Preparation of calibration curve

The calibration curves were constructed for the detection of monuron, linuron, diuron,

metoxuron and metazachlor in the range of 5–500 ppb under the optimized conditions

using the HPLC with UV detection. The calibration curves were linear over this range.

Various characteristics of HPLC-UV like: regression equation, working range, RSD, etc.

are summarized in Table 1.The LODs of the phenylurea herbicides were calculated using

3.3 X S/m (S = Standard Deviation, m = slope of calibration curve), which are in the

range 0.82 to1.29 ng/mL. Characteristic chromatogram with HPLC-UV detection at 210

nm are shown in figures 3.1.2 for the separation of these herbicides.

3.1.4.3 Recoveries, repeatability and LODs

The method Detection Limits were calculated for these herbicides as per the ICH

Harmonised Tripartite Guidelines (www.ich.org/LOB/media/MEDIA417.pdf). The

method LOQs can be calculated by using 10 x S/m. The accuracy (% recovery) and

precision (% RSD) of HPLC-UV method was evaluated for each analyte by analyzing a

standard of known concentration (5ng/ml) five times and quantifying it using the

calibration curves. Method optimization and validation parameters are presented in tables

87

3.1.1 and 3.1.2. Good linearity and repeatability were observed for all the compounds

studied (with correlation coefficient > 0.99). The method gives satisfactory results when

used to quantify these herbicides in soft drink samples and tap water sample (Table 3.1.2)

with %age recoveries varying in the range 75 % to 90.1 %.

3.1.4.4 Applications

The phenylurea herbicides were studied in various soft drink and tap water samples and

no interfering peaks appeared at the retention times of these herbicides in the spiked

samples. The tap water, Coke, Mirinda and Limca (figures 3.1.3, 3.1.4) samples were

spiked with metoxuron, monuron, diuron, metazachlor and linuron at a concentration of 5

ng mL-1

. The analytical validation for the simultaneous quantification of metoxuron,

monuron, diuron, metazachlor and linuron has been performed with good recovery. The

recoveries obtained are very good in all the cases. Hence, this method can be used to

detect the presence of these harmful herbicides in the soft drinks and water samples.

3.1.5 Conclusion

The objective of the current study is to develop a simple, isocratic, reproducible, specific

and highly sensitive method for quantitative and qualitative determination of phenylurea

herbicides. The present method is rapid as compared to several reported methods [37, 38,

39]. The proposed method can determine phenylurea herbicides up to very low

concentrations. The present paper describes the application of HPLC to the separation

and quantitative determination of five phenylurea herbicides and feasibility of the method

developed was tested by simultaneous determination of these herbicides in different

brands of soft drinks and tap water samples. Good linearity and repeatability were

88

observed for all the compounds studied (with correlation coefficient > 0.99). It is hoped

that the results of the present study contribute to the increase of scientific knowledge in

the field of pesticide residue analysis in various food and environmental samples.

89

Figure 3.1.2: HPLC-UV chromatogram of mixture containing 5 ppb each of the phenyl urea

herbicides a: metoxuron, b: monuron, c: diuron, d: metazachlor, e: linuron

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12 14

Peak H

eig

ht

(mA

U)

Retention Time (min)

A B

C D

E

90

Figure 3.1.3: HPLC-UV chromatograms of Coke spiked with a mixture of phenylurea

herbicides containing 5 ppb of each obtained after preconcentration by SPE

Figure 3.1.4: HPLC-UV chromatograms of Limca spiked with a mixture of phenylurea

herbicides containing 5 ppb of each obtained after preconcentration by SPE

0

1

2

3

4

0 5 10 15

Ab

so

rba

nc

e (

mA

U)

Retention Time (min)

B C E A D

0

1

2

3

4

0 5 10 15

Ab

so

rban

ce (

mA

U)

Retention time (min)

B C E A D

91

Figure 3.1.5: HPLC-UV chromatograms of Tap water spiked with a mixture of phenylurea

herbicides containing 5 ppb of each obtained after preconcentration by SPE.

Figure 3.1.6: HPLC-UV chromatograms of Mirinda spiked with a mixture of phenylurea

herbicides containing 5 ppb of each obtained after preconcentration by SPE.

0

1

2

3

4

0 5 10 15

Ab

so

rban

ce (

mA

U)

Retention Time (min)

`

A

B E C D

0

1

2

3

4

0 5 10 15

Ab

so

rban

ce (

mA

U)

Retention time (min)

B C E A D

92

Table 3.1.1: Analytical figures of merit obtained under optimum conditions

Characteristic Metoxuron Monuron Diuron Metazachlor Linuron

Regression

Equation 0.0016x+0.0712 0.0014x+0.0308 0.0035x+0.128 0.0017x+0.083 0.002x+0.1664

R2 0.992 0.994 0.992 0.992 0.993

Retention Time

(min) 4.3 4.9 7.25 8.68 12.4

Linear Range

(ng/mL) 5-500 5-500 5-500 5-500 5-500

LOD=3.3 x S/m

(ng/mL) 0.92 0.82 0.93 1.28 1.29

LOQ=10 x S/m

(ng/mL) 2.76 2.46 2.79 3.84 3.87

Recovery%*

(R.S.D.) 81.0 (2.4) 85.4 (3.0) 91.1 (3.0) 88.2 (3.2) 92.3 (5.0)

* Amount of phenylurea herbicides taken 5 ng/mL each (n=5)

93

Table 3.1.2: Analytical figures of merit obtained using various samples

Phenylurea

herbicides Metoxuron Monuron Diuron Metazachlor Linuron

Samples tested

Tap Water

Linear Range

(ng/mL) 5-500 5-500 5-500 5-500 5-500

LOD (ng/mL)

0.92 0.84 0.91 1.30 1.35

LOQ (ng/mL)

2.76 2.52 2.73 3.90 4.05

Recovery* %

(R.S.D.) 80.6 (4) 84.2 (3.1) 90.1 (4) 87.1 (4) 76.3 (5)

Limca

Linear Range

(ng/mL) 5-500 5-500 5-500 5-500 5-500

LOD (ng/mL)

0.95 0.89 0.99 1.39 1.41

LOQ (ng/mL) 2.85 2.67 2.97 4.17 4.23

Recovery*%

(R.S.D.) 79.4 (4) 83.1 (4) 87.8 (4.2) 87.8 (4.5) 75.4 (5.1)

Coke

Linear Range

(ng/mL) 5-500 5-500 5-500 5-500 5-500

LOD (ng/mL) 0.95 0.90 1.0 1.37 1.40

LOQ (ng/mL) 2.85 2.70 3.0 4.11 4.20

94

Recovery*%

(R.S.D.) 77.5 (4.6) 80.2 (4.7) 88.6 (5) 85.3 (5) 77.3 (4.8)

Mirinda

Linear Range

(ng/mL) 5-500 5-500 5-500 5-500 5-500

LOD (ng/mL) 0.96 0.89 0.99 1.37 1.42

LOQ (ng/mL) 2.88 2.67 2.97 4.11 4.26

Recovery*%

(R.S.D.) 81.1 (3.4) 83.4 (3.2) 87.6 (4) 85.1 (4.3) 77.3 (5.3)

* Samples spiked at 5 ng / mL, n=5

95

3.2 Extraction of a Natural Pesticide Azadirachtin from Neem

tree parts and its determination in fruit, vegetable and tea

samples using SPE-HPLC

3.2.1. Introduction

Synthetic chemical insecticides are being used extensively as they offer ready solutions to

manage food production and human health but they also pose serious health hazard.

Thus, the need of the hour is to develop such feasible alternatives which offer effective

pest control but with fewer health risks [40]. One such option is azadirachtin, a natural

pesticide derived from the Neem tree (azadirachta indica). The Neem tree which is

widely distributed across India has been observed to contain variable amounts of Az due

to variation in genetic and environmental factors [41-45]. Venkateswarlu et al [46]

observed that mycorrhizal inoculation in Neem enhances the Az content in Neem seed

kernels. Az is credited with low toxicity towards the non-target and beneficial organisms,

low damage to environment and with short lifetime in water and soil, thus, it is a suitable

case for inclusion in integrated control programmes. Azadirachtin is present in not only

the seeds of oval ripe fruits, but also in the twigs, leaves and barks of Neem tree [47-50].

Sujanya et al [51] have studied the in vitro production of Az from cell suspension

cultures. Az production from hairy root cultures was studied by Satdive et al [52]. Neem

tree is the rich source of not only the Az but over 300 other compounds can also be

isolated from it, one-third of which are tetranorterpenoids [48] including Az, nimbin,

salanin, azadirachtor etc. and 30-50 % oil [53]. The most important and potent of these,

Az is reported to have antifeedant, insect repellent and ovicidal properties [45,53].

96

Martinez has given a useful account of the pests that can be controlled by Neem

Figure 3.2.1 Molecular Structure of Azadirachtin

formulations [54]. The potency of Az against several pathogens has been studied [55, 56,

57, 58] by several researchers. Jenkins et al [59] analysed the effect of storage conditions

on the efficacy of Az. azadirachtin has been found to mimic the insect hormones and

block the larval development [45] of insects thus inhibiting their growth. Vervek and

Wright [60] observed that many active components that are present along with Az deliver

a synergistic effect on the biological activity of Az as compared to when the equivalent

amount of pure Az is used. HPLC technique is very commonly applied to determine the

amount of Az in Neem kernels whereby. Ruch [61] has given a preview of the HPLC

method for Az determination. Menezes et al [62] determined Az in fish and pond water

samples. Kaushik employed HPLC and gas-liquid chromatography (GLC) [53]

techniques for determination of azadirachtin and fatty acid methyl esters in Neem seeds.

Barrek et al [63] analysed Neem oils by LC-MS and studied degradation kinetics of Az

A. Schaaf et al [64] developed a new method for Az by HPLC and atmospheric pressure

chemical ionization mass spectrometry. Thoeming et al [65] have studied the persistence

97

of Az and related compounds in soil and bean plants. Dai et al [66] optimized a

microwave assisted process (MAP) for Az extraction which could greatly reduce the time

required to extract Az and other limonoids from Neem. Caboni et al determined the Az

residues on strawberries and olives [67,68]. The stability of Az in aqueous and organic

solvents has been studied by Jarvis et al [69]. Sarais et al [70] developed a simple and

selective method for Az content in fruits and vegetables using LC- Electrospray

ionization tandem mass spectrometry. Sannino [71] has described an HPLC/tandem mass

spectrometric method for Az determination in fruits and vegetables. HPLC-MS/MS

detection (Selected Reaction Monitoring; SRM) [72] was also applied to determine Az

content. Dai et al used spectromphotometry [73] and multivariate calibration [74] to

determine total azadirachtin related limonoids. Ambosino et al [47] used a new

supercritical extraction methodology to extract Az A from kernels, followed by

evaluation using HPLC and LC/MS. Thompson et al employed HPLC/DAD technique

for analyzing Az in water sediments [75]. Agrawal et al [76] have presented an HPTLC

and packed column supercritical fluid Chromatographic separation of azadirachtin

components from Neem seed extracts. Sanguanpong has developed a method for the

Neem based extract processing [77]. Govindachari et al [78] were successful in isolating

new azadirachtin components; H & I by HPLC.

Ramesh et al [79] have mentioned the use of graphitized carbon black SPE methods for

concentration of active Neem components like azadirachtin A and B. Jarvis and Morgan

[80] display the use of SPE technique for the purification of triterpenoids and rapid

extraction of azadirachtin from tissue culture extracts. Ruch [81] has recommended the

use of SPE for determination of azadirachtin in water samples, soil and plant materials.

SPE was performed on Neem seed kernel, bark and leaf extracts and the amount of

98

azadirachtin was determined using HPLC-UV. In a similar manner the azadirachtin

content in tomato, carrot, brinjal, grapes and tea samples is described. The analytical

procedure involves extraction with methanol followed by the SPE. In the present work

C18 cartridges have been used for performing SPE (solid phase extraction) of Neem and

food samples. The extracts were analysed by HPLC-UV on a C18 column using

ACN/water as mobile phase in an isocratic flow. Studies were carried out at fortification

levels of 0.05-1 mg/kg and the mean recoveries were obtained in the range 70-102%. The

method was also used to determine the azadirachtin content from the Neem seed kernel,

leaves and bark extracts.

3.2.2 Experimental

3.2.2.1 Instruments

Solid phase extraction system VISIPREPTM

SPE Vacuum Manifolds,

(Supelco,

Bellefonte, PA, USA) with C18 cartridges (J.T. Bakers, U.S.A) was used for the

extraction of the analytes. HPLC system consisted of Dionex P680 HPLC pump, a

Dionex (Acclaim 120) C18 reversed-phase analytical column (4.6 x 250 mm, 5 μm) and a

Dionex UVD170U detector operated at a wavelength of 217 nm. Chromatographic data

was collected and recorded by Chromeleon software.

3.2.2.2 Materials and Reagents

Pesticide standard azadirachtin was obtained from Supelco (Bullefonte, PA, USA),

gradient grade ACN and Methanol were obtained from J.T. Baker Chemicals, USA.

Triply distilled water was used for all purposes. All the solvents were filtered through

99

Nylon 6.6 membrane filters (Rankem) using filtration assembly (Perfit, India) and were

sonicated before use.

3.2.2.3 Standard solutions

Stock solutions were prepared by dissolving the 1 mg of the standard azadirachtin in10

mL of methanol and the solution was diluted as per requirement with methanol. All the

solutions were stored in a refrigerator.

3.2.3 Procedure

3.2.3.1 Extraction of azadirachtin from Neem Tree Parts

3.2.3.1.1 Extraction of azadirachtin from neem seed kernels

The Neem seeds were deshelled and kernels were used for the extraction of azadirachtin.

5 g of powdered Neem seed kernels were stirred with 60 ml hexane to remove the oil.

The defatted residue was extracted with 3 x 10 ml portions of methanol by stirring. The

extract was filtered and the filtrate was collected. Methanol was evaporated from this

extract by rotary evaporator and the residue containing azadirachtin was re-dissolved in

water to make the volume 10 ml.

3.2.3.1.2. Extraction of azadirachtin from neem leaves and barks

The Neem leaves and bark were extracted using the same procedure as for neem seed

kernels without the defatting step with hexane.

3.2.3.2 Solid Phase Extraction of azadirachtin

The solid phase extraction of the neem tree part extracts was performed using the SPE

100

system VISIPREPTM

SPE Vacuum Manifolds, (Supelco, Bellefonte, PA, USA) and C18

cartridges from J. T. Baker were used. The C18 cartridges were attached to the solvent

recovery assembly and connected to a vacuum pump. The conditioning was done with

1mL each of Acetonitrile, Methanol and triply distilled water. The aqueous extracts from

Neem tree parts were pre-concentrated using Solid Phase Extraction (SPE). The analytes

were eluted with 3 mL of methanol.

3.2.3.3 Preparation of food samples

3.2.3.3.1. Preparation of tomato, grapes, brinjal and carrot samples

The azadirachtin content was determined in tomatoes, grapes, brinjal and carrot samples.

Tomatoes, grapes, brinjal and carrot were homogenized with a food blender. The Az

standard solutions were added to 20 g of tomatoes, grapes, brinjal and carrot samples.

These samples were mixed and allowed to stand for 15 minutes, 5 g of NaCl was added

to 20 g of homogenate and stirred at a high speed. The samples were extracted with 15 ml

of methanol and extracts were collected in beaker. The extracts were concentrated by

room temperature evaporation of methanol to 10ml. The extracts were pre-concentrated

using the SPE and the residue was finally eluted with Methanol. This sample was injected

into the HPLC system for analysis. Triple replicates at each fortification level were

prepared. Limit of quantification (LOQ) was defined as amount equal to ten times the

method noise, which included the instrument noise and the background signal contributed

by the matrix blank. Calibration curves were used to quantify the amount obtained in

these samples.

101

3.2.3.3.2 Preparation of Tea samples

Tea samples were prepared by mixing standard Az solution with 5 g of tea leaves and

stirring it with 15 ml Methanol. The process was repeated with 2 x 15 ml portions of

methanol and the extracts were combined. The rest of the procedure followed was same

as that followed for the other food samples.

3.2.3.4. Procedure for solid phase extraction

The solid phase extraction was optimized by passing 10 ml of the spiked food extracts

through the C18 cartridges. The SPE cartridges (octadecyl) were first conditioned with 1

mL each of ACN, Methanol and then with triply distilled water. The water extracts of the

food samples were passed through the cartridges under pressure. The analytes were then

eluted with 3 mL of methanol.

3.2.4 Results and Discussion

3.2.4.1 Optimization of SPE

SPE columns were used for the pre-concentration of neem tree extracts as well as for

food sample extracts. The elution took place efficiently after the conditioning of the

cartridges with 1 mL each of acetonitrile, methanol and triply distilled water in this order.

The aqueous extracts from various samples were then passed through the cartridges under

pressure and the elution was done with 3 mL of methanol.

3.2.4.2 HPLC determination of azadirachtin A and B

The ACN/Water, isocratic elution allowed the chromatographic separation of

azadirachtin components. The run time was 15 minutes and the retention times of Az A

102

and Az B were 10.6 and 11.2 minutes respectively. It was observed that after the clean up

with SPE, no interfering peaks were observed near the retention times of the Az A and

Az B. Characteristic chromatogram showing separation of Az A and Az B is given in

figure 3.2.2.

3.2.4.3 Linearity

Standard calibration curves of azadirachtin were found to be linear in the range 0.05 to 5

mg/kg with a correlation coefficient of 0.999. Characteristics of the calibration curve are

summarized in Table 3.2.1.

3.2.4.4 Method Validation

3.2.4.4.1 Extraction of Az from Neem seeds, leaves and bark

The Neem seed kernels, leaves and bark were stirred with methanol and SPE was

performed to obtain the extracts. These extracts were analysed through HPLC to

determine the amount of Az A and Az B present in the Neem tree parts. The Neem tree

parts had an average amount of Az A and Az B as: seed kernels (Figure 3.2.3) 4.81

mg/kg and 2.25 mg/kg, leaves (Figure 3.2.4) had 1.11 mg/kg and 0.71 mg/kg respectively

and the bark (Figure 3.2.5) contained an average amount of 0.540 mg/kg of Az A (Table

3.2.2).

3.2.4.5. Application of method to azadirachtin determination in food samples

The HPLC method was successfully used to determine the amount of azadirachtin A and

B in the spiked samples of brinjal (Figure 3.2.6 a and 3.2.6 b), tomatoes (Figure 3.2.7 a

and 3.2.7 b), tea leaves (Figure 3.2.8 a and 3.2.8 b), grapes (Figure 3.2.9 a and 3.2.9 b)

103

and carrot (Figure 3.2.10 a and 3.2.10 b) after performing their solid phase extraction.

3.2.4.6. Recoveries

The recovery of Az from the spiked samples shows good recoveries (Table 3.2.3) in case

of fruits and vegetable samples (>80 %) and more than 70 % for tea samples and the R.

S. D. values ranging between 5 and 10 %. The LOD was determined as the sample

concentration which produces that produces a peak with height three times the level of

baseline noise.

3.2.5 Conclusions

In the present work SPE technique has been used to extract azadirachtin A and B from

Neem tree parts like seed kernels, leaves and barks followed by their Liquid

Chromatographic determination. Further the method has been successfully validated by

the determination of azadirachtin A and B from tomato, grapes, brinjal, carrot and tea.

The LOQs for azadirachtin A and B are 29.7 µg / kg.

104

Figure 3.2.2: HPLC chromatogram of AZ standard at 217 nm, with mobile phase

ACN/Water (40/60) at a concentration of 50 µg / kg.

0

0.5

1

1.5

2

2.5

3

3.5

4

0 2 4 6 8 10 12 14

Peak H

eig

ht

(mA

U)

Retention Time (min)

Az A Az B

105

Figure 3.2.3: HPLC chromatogram of neem seed kernel extract. Rest of the

conditions same as in figure 3.2.1

Figure 3.2.4: HPLC chromatogram of neem leaves extract. Rest of the conditions

same as in figure 3.2.1

0

50

100

150

200

0 5 10 15

Pe

ak H

eig

ht

(mA

U)

Retention Time (min)

Az B

Az A

0

10

20

30

40

50

0 2 4 6 8 10 12 14 16

Retention Time (min)

Peak H

eig

ht

(mA

U)

AZ AAZ B

106

Figure 3.2.5: HPLC chromatogram of neem bark extract. Rest of the conditions

same as in figure 3.2.1

0

10

20

30

40

50

0 2 4 6 8 10 12 14

Peak H

eig

ht

(mA

U)

Retention Time (min)

Aza A

107

Figure 3.2.6 (a): HPLC chromatogram

of blank brinjal sample. Rest of the

conditions same as in figure 3.2.1

Figure 3.2.6 (b): HPLC chromatogram of

brinjal spiked at 50 µg / kg. Rest of the

conditions same as in figure 3.2.1

Figure 3.2.7 (a): HPLC chromatogram

of blank tomato sample. Rest of the

conditions same as in figure 3.2.1

Figure 3.2.7 (b): HPLC chromatogram of

tomato sample spiked at 50 µg / kg. Rest

of the conditions same as in fig 3.2.1

0

1

2

3

4

5

0 2 4 6 8 10 12 14

Retention Time (min)

Peak H

eig

ht

(mA

U)

0

1

2

3

4

5

0 2 4 6 8 10 12 14

Retention time (min.)

Peak H

eig

ht

(mA

U)

Az A Az B

0

1

2

3

4

5

0 2 4 6 8 10 12 14

Retention Time (min.)

Peak H

eig

ht

(mA

U)

0

1

2

3

4

5

0 2 4 6 8 10 12 14

Retention Time (min.)

Peak H

eig

ht

(mA

U)

Az AAz B

108

Figure 3.2.8 (a): HPLC chromatogram

of blank tea sample. Rest of the

conditions same as in figure 3.2.1

Figure 3.2.8 (b): HPLC chromatogram of

tea sample spiked at 50 µg / kg. Rest of

the conditions same as in figure 3.2.1

Figure 3.2.9 (a): HPLC chromatogram

of blank grapes sample. Rest of the

conditions same as in figure 3.2.1

Figure 3.2.9 (b): HPLC chromatogram of

grapes spiked at 50 µg / kg. Rest of the

conditions same as in figure 3.2.1

-1

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14

Retention Time (min)

Peak H

eig

ht (m

AU

)

0

1

2

3

4

5

0 2 4 6 8 10 12 14

Retention Time (min.)

Peak H

eig

ht

(mA

U) Az A Az B

0

1

2

3

4

5

0 2 4 6 8 10 12 14

Retention Time (min.)

Peak H

eig

ht

(mA

U)

0

1

2

3

4

5

0 2 4 6 8 10 12 14

Retention Time (min.)

Peak H

eig

ht

(mA

U)

Az A Az B

109

Figure 3.2.10 (a): HPLC chromatogram of blank carrot sample. Rest of the

conditions same as in figure 3.2.1

Figure 3.2.10 (b): HPLC chromatogram of carrot sample spiked at 50 µg / kg. Rest

of the conditions same as in figure 3.2.1

0

1

2

3

4

5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Retention Time (min)

Peak h

eig

ht

(mA

U)

0

1

2

3

4

5

0 2 4 6 8 10 12 14

Retention time (min.)

Peak H

eig

ht

(mA

U)

Az AAz B

110

Table 3.2.1 HPLC characteristics of Azadirachtin A and B on C18 column

Characteristic Az A Az B

R2 0.9993 0.9991

Regression Equation 0.2154 x + 0.0027 0.2127 x + 0.0197

Slope 0. 2154 0.2127

Working Range (µg / kg) 50 – 500 50 – 500

Retention Time (min.) 10.7 11.3

Selectivity 0.9081

Retention factor 4.684 5.158

LOD (µg/kg, S/N=3) 9 9

LOQ (µg/kg, 3.3 x S/N) 29.7 29.7

Table 3.2.2 Extraction of Azadirachtin from Neem seeds, leaves and bark

Amount of Azadirachtin extracted from Neem Parts (in mg/kg)

Neem Seed

Kernel Neem Leaves Neem Bark

Az A

4.72

4.89

Az A

1.14

1.08

Az A

0.55

0.53

Az B

2.16

2.29

Az B

0.67

0.75

Az B Not found

111

Table 3.2.3 Comparison for the determination of Azadirachtin in tomato, brinjal,

grapes, carrot and tea leaves

Sample Azadirachtin

Added* (µg / kg) Recovery % RSD (%)

Tomato

100

200

90

85

8.2

6.7

Brinjal

100

200

85

80

8.4

6.2

Carrot

100

200

102

92

10.5

8.5

Grapes

100

200

91

87

8.2

6.1

Tea Leaves

500

700

75

70

11.2

10.4

*Each measurement is a mean of three values

112

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