American Journal of Anal yt ical Chemistry, 2011, 2, 32-36
doi:10.4236/ajac.2011.228121 Published Online December 2011 (http://www.SciRP.org/journal/ajac)
Analysis of Pesticide Raid® in Feed of Wistar Rat by
High-Pressure Liquid Chromatography (HPLC)
Albert C. Achudume
Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife, Nigeria
Received November 21, 2011; revised December 24, 2011; accepted December 31, 2011
Abstract
The distribution of pesticide by-product in tissues of wistar rats were analyzed using high pressure liquid
chromatography. The limit of detection of the HPLC was 0.1 µg. Results show bioaccumulation factor of
pesticide “Raid®” in lipid, up to three times that of the feed at the first concentration and gradually de-
creased as the concentration increased in the muscle > (0.7), brain > (0.5) and liver > (0.3) as indicated in the
text. At higher concentration of 961 µg/g, bioaccumulation factor decreased in the lipid to 1.2 and 0.6 in the
muscle, 0.03 in the brain and 0.08 in the liver respectively. High Pressure Liquid Chromatography (HPLC)
analysis of raid extract suggests the presence of micprothrin and palethrin. The implications are numerous,
but simply put that accidental ingestion of chlorinated hydrocarbon as in “Raid®” may involve convulsions,
collapse and coma after only brief excitation and ataxia at the onset.
Keywords: High Pressure Liquid Chromatography, Pesticide, Raid®, Chlorinated Hydrocarbon,
Bioaccumulation
1. Introduction
Pesticides have been used to boost food production to a
considerable extent and to control vectors of disease [1].
However, these advantages of great economic benefits
sometimes come with disadvantages when subjected to
critical environmental and human health considerations.
The chronic effects of pesticides from food intake on
human health are not well defined, but there is increasing
evidence of carcinogenetic [2], teratogenic [3] and ge-
netotoxic [4], as well as disruption of hormonal functions
[5]. Analysis of these pesticides and their residues h ad in
the past aided objective re-evaluation and reassessment
of these substances on a benefit-risk analysis basis and
their subsequent withdrawal from use when found to be
hazardous to human health and the environment.
Residues of pesticides and heavy metals enter the food
system. This occurs more often than one might think.
Additionally, residues of pesticides found in the air in-
side the home and on floors and other interior surfaces
contribute to non-occupational pesticide exposure [6].
For example, any potentially harmful substances that
have no set tolerance thresh may not necessarily be
harmful. This means that if there is any toxin in food
supply or water, such food cannot be ban unless it exists
in quantities over th e tolerance threshold no matter what.
There are few studies of compliance to pesticide use
[7,8].
The quality and sophiscation of analysis have grown
and very minute quantities of these pesticides and their
residues can be analyzed with high degree of specify,
precision and accuracy. Therefore, it is important and
utmost necessary to develop methods for analysis of pes-
ticides residues in environmental samples. In the light of
the above, and in view of pesticide related adverse reac-
tions, the study, presents chromatographic and absorp-
tion spectra from a common pesticide Raid® in tissu e of
rat as it affects basal metabolism using high pressure
liquid chromatograph.
2. Materials and Methodology
2.1. Test Facility
Tests with Wistar rats were conducted in a room tem-
perature exposure facility (23˚C). Thirty-five animals
weighing between 180 - 210 were caged in perforated
aluminum chambers (38 × 55 × 35 cm). Five animals
were placed in each of the chambers containing sawdust
shaves, and a light: dark cycle of approximately 12 h.

A. C. ACHUDUME33
Water was given ad libitum, temperature was maintained
at 24˚C ± 1˚C. Air temperature was ±2˚C of the water
temperature. Protocols describing the use of animals in
accordance with National Research Council (NRC) [8]
and World Medical Association (WMA) [9] guide on the
care and use of laboratory animals.
2.2. Test procedures
Wistar rats were obtained from the animal breeding fa-
cility of College of Health Sciences, Obafemi Awolowo
University, Ile-Ife, Nigeria. Technical grade “RAID”
(Johnson Wax, Nigeria), was dissolved in corn oil and
with (in a rotary mixer) 2:98 (w/w) the co mmercial feed,
Purina 5000, for 10 min: the control diet contained corn
oil in the same proportion. Animals were fed raid-spiked
commercial feed at measured concentrations of 25.0 ±
2.4, 54.0 ± 9.2, 108.0 ± 12.5, 216.0 ± 14.6, and 430.0 ±
20.2 and 961.0 ± 80.6 µg/g “Raid” for 10 days, increas-
ing 5 - 10 g/day and ending with 96 g/rat. Two rats from
different chambers receiving the same concentration of
raid in the food were selected and tissue samples were
dissected out (lipid, muscle, brain and liver) at test ter-
mination. The symptom-li mited sensation tests were per-
formed after an overnight fast [10]. Mean values were
calculated from the two rats sampled from each concen-
tration of insecticide-raid.
2.3. Test Extraction
In the toxicity persistence test, “Raid” was extracted with
1:1 diethyl ether-petroleum ether [11]. This compound
was identified and quantified by high-pressure liquid
chromatography (HPLC), using UV1-202nm@01, Base
(Amersham Pharmacia Biotech), serial number 90042,
mobile phase was Methanol: H2O (1:1). The limit of de-
tection of the HPLC was 0.1 µg .The dimension of the
column C18, 4.6 × 250 mm and particle size 5 micron.
Flow rate was 1 ml/min, the injection volume 100 µl and
a wavelength of 202 nm was used for detection. The
values were quantified by standard calibration curve.
2.4. Analytical Procedures
The brain, muscle and liver homogenate fractions were
prepared by the method described by [12] and resus-
pended in 0.15 M KCl to an appropriate final concentra-
tion of 20 mg of protein per millimeter (approximately)
0.5 g of tissue. Liver tissue lipid s were extracted by ch lo-
roform/methanol ratio (2:1) following the method of
Folch [13]. Fractions of each tissue homogenate were
used for the determination of Glucose-6-phosphatase
(G-6-Pase) and Lactate dehydrogenase respectively, to
test for inhibition of metabolic enzymes required to aid
digestion. Glucose-6-phospatase was determined using
aliquot of each homogenate (20%), incubated with 1 ml
of P-nitrophenyl phosphate (NPP) for 15 min and the
reaction was stopped by addition of 2 ml of 0.2 N NaOH;
absorbance was measured at 450 nm as described by [14].
Lactate dehydrogenase was determined by the method of
[15]. Protein was determined by [16] method. Experi-
ments were conducted in a completely randomized man-
ner with five replicates. These were repeated and mean
data of two experiments is presented. Data were sub-
jected to one-way analysis of variance and significance
of treatments from control was tested at 5% level of sig-
nificance followed by Dunnet’s test. Further, data were
also subjected to determination of correlation coefficient
between raid concentration and the observed response.
The statistical analysis was performed using SPSS soft-
ware version 10.0.
3. Results and Discussion
Bioaccumulation factor of pesticide “Raid®” was ob-
served in lipid, up to three times that of the feed at the
first concentration and gradually decreased as the con-
centration increased (Table 1) while accumulation factor
in the muscle (0.7), brain (0.5) and liver (0.3) was about
the indicated number times that of the feed. At higher
concentration of 961 µg/g, bioaccumulation factor de-
creased in the lipid to 1.2 and 0.6 in the muscle, 0.03 in
the brain and 0.08 in the liver. Using the mean of insecti-
cide in feed, the tissues accumulate the insecticide in
ascending order: brain < liver < muscle < lipid. The ef-
fects increased with increasing insecticide concentration
in the feed an d showed a significant deference compared
to control (P < 0.05) at a concentration of 430 µg/g.
Table 2 indicates the estimated detectable levels of
toxicity in rat tissues exposed to the insecticide “Raid®”.
Tissue insecticide concentration increased in lipid, mus-
cle, brain and liver, respectively. The insecticide accu-
mulation in these organs may therefore indicate that the
active ingredients are also absorbed resulting in the de-
creased enzymes formation, which should accelerate
immediate metabolic processes, but instead, remained
complexed in the tissue organs. Additionally, the reten-
tion time (within 10 min.) may be increased because of it
easy solubility and lower excretion rate leading to bio-
accumulation. The brain shows insignificant decreases in
the enzymes Glucose-6-phosphatase and Lactic acid de-
hydrogenase, while significant decreases were noticeable
in the muscle and liver. The tissues concentrations were
the sums of the two components.
High Pressure Liquid Chromatography (HPLC) analy-
sis of raid extract suggests the presence of micprothrin

A. C. ACHUDUME 34
Table 1. Tissue total raid concentrations and bioaccumulation factors (BAF) in Wister Rats exposed to insecticide “Raid”.
Mean ± SD insecticide “Raid” in feed (µg/g) Raid concentration in Wister rats (µg/g) a and bioaccumulation factor (BAF)b
Lipid Muscle Brain Liver
0.00 - - - -
25.0 ± 2.4 72.5 17.5 12.5 7.5
(2.9) (0.7) (0.5) (0.3)
54.0 ± 9.2 86.4 21.7 16.4 9.4
(1.6) (0.4) (0.3) (0.2)
108.2 ± 12.5 172.8 30.4 19.5 10.8
(1.6) (0.3) (0.2) (0.10)
216.2 ± 14.6 280.8 45.8 22.9 19.8
(1.3) (0.2) (0.1) (0.09)
430.0 ± 20.6 324.0 86.4 25.8 37.3
(0.8) (0.2) (0.06) (0.09)
961.2 ± 70.5 1153.2 576.6 28.8 76.9
(1.2) (0.6) (0.03) (0.08)
aMean raid concentration at test termination (~steady state value); bBAF = bioaccumulation factor ‘Raid’ in tissue (µg/g)/feed raid concentration (µg/g).
Table 2. Index of toxicity in rat tissues exposed to insecticide “Raid”.
Indicators Brain IU/L Muscle IU/L Liver IU/L
Control 0.28 ± 0.02 1.15 ± 0.20 2.54 ± 0.1
Glucose-6-phospatase 0.25 ± 0.05 0.35 ± 0.09* 0.15 ± 0.03*
Control 0.22 ± 0.04 0.10 ± 0.01 1.94 ± 0.02
Lactate dehydrogenase 0.19 ± 0.08 0.15 ± 0.11* 0.07 ± 0.08*
*Significantly different P < 0.05 from control.
and palethrin. Figure 1(a) shows the chromatographic
profile of the raid insecticides. Figure 1(b) shows the
chromatographic profile of standards (0.025%). The high-
est peak obtained from “Raid®” extract is most likely
micprothrin. This peak possessed a retention time (9.25)
min. For the identification of raid, co-chromatography
with authentic standards in different chromatographic
conditions was used. Two different peaks were eluted
within 10 min and were well resolved (Figure 1(a)), but
unidentified peak was eluted within a retention time of
early eluting of 2 min. The HPLC analysis failed to de-
tect the unidentified component in “Raid®”. The reason
is not clear, but raid may have undergone a structural
change because of combination, hence different absorb-
ance property [17]. The fine tuning of the mobile phase
composition depended on the choice of the internal stan-
dard. Most internal standard compounds were rejected
because of interfering components. Only micprothrin and
palethrin had acceptable chromatographic characteristics.
The implications are numerous, but simply put that ac-
cidental ingestion of chlorinated hydrocarbon as in
“Raid®” may involve convulsions, collapse and coma
after only brief excitation and ataxia at the onset.
Other workers have contrasted neuritic outgrowth of
chlorpyrifos, diazinon and organophosphate pesticides
including parathion and concluded that all have adverse
effects on brain development and systemic toxicity [18].
In recent study, Walz, [19], shows that glyphosate, the
active ingredient in Roundup, causes birth defects in
frogs and chicken embryos at far lower levels than used
in agricultural and garden applications. The malforma-
tions primarily affected the skull, face, midline and spi-
nal cord. Other independent scientific research has also
found that glyphosate causes endocrine disruption, de-
velopmental and reproductive toxicity, DNA damage,
neurotoxicity and cancer [3,18]. Many of these effects

A. C. ACHUDUME35
(a)
(b)
Figure 1. Chromatograms and absorption spectra from analysis of “Raid” extract in tissue of rat. Analysis of micprothin and
palethrin (a) and standard peak (b).
were apparent at much lower doses than the typical lev-
els of pesticide residues found in food [20,21]. Yet de-
spite the evidence [which strongly support the National
Campaign for Sustainable Agriculture, (a diverse part-
nership of individuals and organizations), cultivating grass
roots efforts to engage in policy development processes
that may result in food, agricultural systems and rural
communities that are healthy, environmentally sound, pro-
fitable, humane and just [22], pesticides continue to be
found in agr icultural produce and lurking in tap water.

A. C. ACHUDUME 36
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