HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE

DETERMINATION OF ALUMINIUM PHOSPHIDE (ALP) PESTICIDE IN STORED

MAIZE GRAIN

Osho, Kemi, *Ayejuyo, O.Olusegun, Kazeem Basheeru and Akinrinade E. Olumide

Department of Chemistry, University of Lagos, Akoka, Nigeria.

*Correspondence Author: oayejuyo@unilag.edu.ng

ABSTRACT

Maize grains are important source of food for human and animal population. A common method

for the protection of grains from pest infection is the use of aluminum phosphide (Alp) pesticide

sold under the brand JustoxinR in Nigeria. A simple and suitable HPLC technique for the

determination of Alp pesticide applied on maize was developed and validated. The HPLC system

achieved a good separation on a Zorbax Eclipsed XDB C18 reversed-phase Column (5µm, 150

mm x 4.6 mm), maintained at room temperature (25ºC) using isocratic mobile phase consisting of

methanol: milliQ water (50:50 v/v) at a flow rate of 1.0 mL/ min. 20 mL of injection volume was

used at a run time of 15 min and a detection wavelength of 195 nm. The chromatographic

conditions indicated a precision, RSD of 0.911-0.932%. The method showed a good linear

relationship at the working standard range of 5 – 50 µg/mL. The coefficient of determination was

0.995. The LOQ and LOD were determined as 7.47 µg/mL and 2.46 µg/mL respectively. The

recoveries after fumigation were between 77.4% and 120.1%. The study showed increase in the

amount of Alp pesticide residue applied on maize grain stored for 7, 10 and 14 days respectively.

Key words: Aluminium phosphide (Alp), pesticide residues, maize grain, high performance liquid
chromatography
Introduction
Food crops are essential to keep the stability of population in country by providing able work

force, feedstock for animal feeds and foreign exchange. The human populations are therefore

threatened by pest and rodents who continuously feed on food crops to the verge of total

destruction if not properly controlled. Pesticide is often applied in order to prevent the destructive

activities of pests. Maize, a commonly grown and one of the most staple food crops in Nigeria, is

usually protected from pests by the application of Aluminium phosphide (Alp) pesticide normally

considered to be cheap and effective in the control of pests (Bond, 1989; Musshoff et al., 2008;

USEPA, 2001). Alp is a synthetic compound which produces a toxic gas, phosphine, when in

contact with the moisture in the atmosphere (WHO, 2007). Alp pesticide contains Alp and suitable

additives to control the evolution of hydrogen phosphide (phosphine gas) and to prevent self-

ignition (APVMA, 2004). The use of Alp involves direct application of the solid in the form of

tablets, pellets or paper bag in an air tight container (Bond, 1989; Moghhadamnia, 2012).

Application by hand is allowed and the effective dosage for bulk fumigation in upright storage is

1-2 g/m3 for minimum exposure of 5 days at 21-25 oC or 4 days at 26oC above (Bond, 1989). The

most important factors are the aeration and fumigation time (Ali et al., 2010).

Alp is highly poisonous; though, self poisoning is the mostly reported case but accidental

poisoning is uncommon. Farmers are exposed occupationally during fumigation but poisoning is

rare (Anger et al., 2000; Mehrpour et al., 2012; Musshoff et al., 2008; WHO, 2007). There is an

increasing concern on the environmental risk of Alp pesticide in food. Food is wholly implicated

as a source of Alp since it is insoluble in water. USEPA (2001) has rated it as one of the highest

risk posing chemicals and recommended a limit of 0.0113 mg/kg/day for a chronic reference dose

and 0.018 mg/kg/day for acute reference dose.

 According to AVPMA (2004) the active constituent in Alp pesticide can be determined by

spectroscopic tests (IR spectrum, NMR, mass spectra), Chromatography (HPLC or GC retention

time with reference compound) or any other suitable test method. Determination of Alp in wheat

kernels by spectrometric method has been reported. The phosphine gas was generated to the

headspace and determined spectrophotometrically by reaction with silver nitrate (Longobardi et al,

2008). The phosphine gas was also determined with micro-gas chromatography (Longobardi et al,

2008). Zheng et al. (2014) determined phosphine gas from Alp applied on agaric by gas

chromatograph equipped with nitrogen-phosphorous detector. In another reported work involving

rice, Zhang et al. (2011) dissolved the phosphine gas in toluene and then analysed Alp using flame

photometric detector. Headspace Gas Chromatography was also employed by Graver et al. (2001)

and Misumi et al. (2008). Carlson and Thompson (1998) converted phosphine gas into

orthophosphate and then isolated the phosphate by ion chromatography with eluent conductivity

detection. Reports on HPLC methods are rare and most of these methods are technical and also

require set of equipment which are not commonly found. This study introduces a simple technique

for the determination of Alp in fumigated maize grain. The storage capacity of Alp pesticide in

relation with harmful residue was investigated in air tight containers.

Material and Methods

Sample location

The samples were collected from a local farmer at Funbi-Fagun Crescent along Ondo-West

Local Government Secretariat, Ondo State, Nigeria (Fig. 1). Ondo State has a vast farmland

and well known for agricultural production. The location was carefully selected based on the fact

that there was no previous use of commercial fertilizer or pesticide applications.

Fig 1: Ondo State Governmenet, Nigeria

Source: Oyedotun and Obatoyinbo (2012)

Chemical and reagents

Aluminium phosphide pesticide (Alp) under the brand JustoxinR was purchased from a retailed

shop in Lagos, Nigeria.

Aluminium phosphide standard, HPLC grade methanol and milliQ water were supplied by a

standard laboratory, Swiss Pharma Nig. Ltd in Lagos, Nigeria.

Methodology
The study was structured in three stages. The first stage was aimed at evaluating the presence of

Alp pesticide residue in a maize grown in a natural farmland devoid of pesticides or any related

chemicals. In the second stage, sub samples of the maize were stored in an air-tight container (Fig.

2) for fourteen days in a way to mimic the common storage pattern by the farmers. The contents

were then treated with the Alp (Justoxin R). Alp pesticide residue was determined at predetermined

days. In the third stage, rather than whole storage in a container, the maize was treated separately

and stored in three different containers. Alp pesticide residue was determined in each of the

containers at fixed days.

Sample collection

Dried yellow maize (Zea mays) ears were purchased immediately after harvesting from local

farmers. Some monitoring and consistent inspections were done to ascertain the integrity of no

pesticide usage on the maize plants. The kernels were carefully removed from their cobs; the

cleaned ones were completely picked out from the damaged grains, silk and the other associated

foreign materials.

Simulated Storage Design

About 8 g of the maize kernel was further dried and crushed to a fine powder with pre-cleaned

pestle and mortar. The moisture content was then determined by weighing 2 g of milled grain and

dried in an oven at 105ºC for 2 hours. The grain moisture content was recorded as 11 %, this

indicated a suitable condition for storage.

Five (5) air-tight containers with 700 g maize capacity were each filled with 500 g maize grains

and covered. Then, 0.005 g Alp pesticide, properly wrapped in an envelope, was placed in the
centre of each of four containers while the secluded container was used for method blank. Each

container was properly labeled. The first container was kept and opened on 7 th, 10th and 14th day

for sub-sampling. The second, third and fourth containers were kept and opened individually on

7th, 10th and 14th days respectively for sub-sampling.

Preparation of standard solution

1mg/mL (1000 ppm) of the stock solution of Alp pesticide was prepared by dissolving 20 mg of

the commercial pesticide in 20 ml of methanol. The solution obtained was then sonicated for 10

mins and later centrifuged to give the clear portion. Aliquot working standards of 5, 10, 20, 40 and

50 mg/mL were prepared from the solution obtained after sonication.

Sample preparation

Maize kernel was picked randomly from the stored samples. Alp pesticide residues were extracted

with 10 mL methanol in approximately 1.0 g of the maize kernel by mechanical agitation for about

20 mins. The extract was filtered and kept for HPLC analysis. The procedure was repeated for all

the treated samples.

Method Blank determination

1g of untreated stored maize was also weighed. The maize kernel was then sonicated with 10 mL

of methanol for 20 mins to extract Alp pesticide residue in a similar procedure as described for the

standard and the treated samples. The extract was filtered and analysis was performed with HPLC

system using the same chromatographic conditions.

Chromatographic conditions

The HPLC system involves water Alliance 2695 of SM4 4267A auto sampler and UV 2489

detector. The chromatographic elution was performed on a zorbax eclipse XDB C 18 reversed phase

column (150 mm x 4.6 mm, 5 µm). Different ratios of solvents were prepared and used as mobile

phase at different flow rates with the aim of getting a better peak resolution with an optimum flow

rate. All solvents used were filtered through 0.45µm membrane and degassed prior to use. The

best isocratic condition was obtained at the injection volume 20 µl, solvent mixture of methanol:

milliQ water (50:50 v/v), a constant flow rate of 1.0 ml/min and run time of approximately 15

mins. The wavelength of the system after adjustment was monitored at 195 nm. The column was

maintained at room temperature (25oC). The resolution obtained is shown in Fig 3.

Method Validation

The procedure was validated as outlined in International Conference on Harmonisation, (ICH,

2005)

Accuracy

After storage for 7 days, approximately 1 g of the maize kernel was weighed and dozed with 15

mg/L of the standard solution. The recovery of the standard used was then determined after the

extraction employing the same chromatographic conditions earlier established.

Precision

Three replicate analyses were done on 1g of the treated samples after 7 days. The procedure was

repeated for samples left for 10 and 14 days. The intra-day precision was determined as RSD%
Three triplicate determinations of another 1 g of the 7-day treated sample was determined at 2 days

interval to establish the inter-day precision. The results were calculated in RSD%.

Linearity Range

The area response of the working standard was plotted against the concentration using Microsoft

office excel 2007 software to generate a calibration curve. The linearity is shown in Fig.4.

Limit of Detection and Limit of Quantification

The limit of detection (LOD) and the limit of quantification (LOQ) of Alp pesticide residue were

determined as 3.3 σ / S and 10 σ / S respectively, where σ is the standard deviation of the response

and S is the slope of the calibration .

Quality Control/Assurance

Reagents used were of analytical and HPLC grades. A solvent blank was run at the end of

chromatographic determination and a needle wash was done to ensure that there was no carry over

from the previous run.

Results and Discussion

Alp pesticide was not detected in the method blank indicating that there was minimal level of

contamination in the procedure used and insignificant level of pesticide in the maize kernel (Table

1). The accuracy as indicated by the recovery studies was between 77.4 and 120.1 % (Table 2).

This was close to the 83.4%-112.6% value in agaric (Zheng et al., 2014), 76.8 to 106.5% values in

rice (Zhang et al., 2011) and 85.0 – 100.0% in wheat (Rangaswamy, 1984). The method indicated a
good linear relationship of the area response to the working standard within a linear range of 5 to

50 µg/mL. This was better in range than the 0.012–1.2 mg/L value reported by Zhang et al. (2011).

Similar correlation coefficient, 0.995, was obtained (Fig. 4) compare to the 0.999 in Zhang et al.

(2011), and 0.995 reported in (Zheng et al., 2014) at a working range of 0.05-50 mg. The LOQ and

LOD determined were 7.47 µg/mL and 2.46 µg/mL respectively. The method showed good intra-

day and inter-day precisions, the RSD% determined in both cases were less than 1. The proposed

method is simple, accurate and suitable for the determination of Alp pesticide on maize grain.

The treated maize kernels contain Alp pesticide residue level which generally increased

over the days of storage (Table 1). This is expected as the pesticide is released into the grain

gradually to prevent infection of pest and hence preserving the integrity of maize grain. Bond

(1989) reported that the increase was supposed to have reduced after about 5 days. The continuous

increase with the number of days as observed could pose serious health challenges in the

environment. The amount of residue determined when the kernels were opened intermittently,

were lower than when they were stored separately and opened at once at the specific days (Table

1) as well shown in ( Fig. 5 and Fig. 6). This was probably due to the disturbance of phosphine-air

layer and escape of pesticide into the atmosphere when the containers were opened. There could

be a significant level of exposure risk to workers who are in charge of grain storage especially

when there is constant opening of the storage bags.

Phosphine gas is poisonous, but Alp pesticide is still preferred because of the short

life span of the gas in air and the residue left is not toxic (USEP, 2001). This study revealed that

there could be a substantial amount of the Alp pesticide residue on maize crops capable of posing

serious health risks to human and animal population who feed on them. Local farmers, who

indulge in the fumigation of maize grain, could also be exposed to the pesticide and the exposure
is further compounded by the fact that most of the local farmers show low level of education and

incompetency in the application of pesticides as they lack the basic protective equipment (Dahiru

et al., 2014; Ugwu et al., 2015). There is need for regulation and close monitoring of pesticide

usage in local and modern agricultural practices. Farmers should be educated and trained on the

proper usage and the effect of wrong pesticide practices to themselves and the general community.

The study further buttressed the recommendation of USEPA (2001) that the application of Alp

pesticide must be done with nose mask and other necessary gadgets. Consumers need to avoid

eating raw and treated agricultural commodity before washing or cooking. There exists some data

gaps on the current trend or levels of alp pesticide in agriculture produce in our markets. There is

therefore a need for investigation of alp pesticide residue on the produce sold in market and the

toxicity implications of Alp pesticide usage on crops and in cooked grains.

Conclusion

The proposed HPLC method is simple and suitable for a routine determination of Aluminium

phosphide (Alp) pesticide applied on maize grain. Farmers and other handlers of pesticides should

be trained and educated on the toxicity implications of Alp pesticide on crops. There is need for

consistent and adequate monitoring of pesticide producers claim on labels and the usage on food

crops to prevent health risks of the pesticides on human and animal populations. There is also need

for further investigation of Alp pesticide application on raw and cooked food in the market.
Fig 2: Treated maize grains

Fig. 3: Chromatogram of extracted Alp residue

 100000

90000
f(x) = 1710.93x + 2755.67
80000 R² = 1

70000

60000
Peak Area

50000

40000 Linear ()

30000

20000

10000

0
0 10 20 30 40 50 60
Conc. (ppm)

Fig 4: Linearity curve of the working standard

Table 1: Level of Aluminium phosphide pesticide residues

Storage condition Days of storage Concentration

(ppm)
Interval opening 7 8+0.19
( same container) 10 15+0.21
14 28+0.16
Open only after 7 12+0.23
(different 10 18+0.17
containers) 14 30+0.29
Method blank 7 N.D
10 N.D
14 N.D

N.D. – Not detected

Table 2: Recovery of spiked fumigated samples

Days of storage Amount of standard spiked Recovery (%)

(ppm)
7 15 77.4
7 15 120.1
7 15 89.7

Table 3a: Intra-day precision of the method adopted

Days of storage RSD (%)

7 0.911
10 0.964
14 0.981

Table 3b: Inter-day precision of the method adopted at 2 days interval

Days of storage Days of interval RSD (%)

7 0 0.911
10 2 0.945
14 4 0.932

30
f(x) = 2.39 exp( 0.18 x )
25 R² = 0.99

20
Conc. (ppm)

15

10

0
6 7 8 9 10 11 12 13 14 15
Days of exposure

Fig 5: Alp pesticide residue of treated samples open in the same container
 35
30
f(x) = 4.83 exp( 0.13 x )
Concentraton (ppm)

25 R² = 1
20
15
10
5
0
6 7 8 9 10 11 12 13 14 15
Days of storage

Fig 6: Alp pesticide residue of treated sample kept in different containers

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