PAPER www.rsc.org/methods | Analytical Methods

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A simple assay for analyzing residues of carbaryl insecticide in buffalo meatby liquid chromatography–photodiode array detection

Ashim K. Biswas,†*a Napa Kondaiah,a Anne Seeta Ram Anjaneyulu,a Gadam Setty Raob

and Ram Prakash Singhc

Received 15th December 2009, Accepted 18th January 2010

First published as an Advance Article on the web 3rd February 2010

DOI: 10.1039/b9ay00301k

A simple and sensitive liquid chromatographic (LC) method was developed for determination of

carbaryl residue in buffalo meat samples. This method is based on a solid-phase extraction technique

followed by high-performance liquid chromatography (HPLC)–photo-diode-array (PDA) detection.

Meat samples (0.5 g) were deproteinized by adding acetonitrile followed by centrifugation and

filtration. The analyte was separated on a reverse-phase (RP-C18) column using isocratic elution.

Acetonitrile along with water appears to be an excellent extractant as recovery of the analyte in spiked

sample at maximum residue level (MRL) was 98.5%, with coefficient of variation (CV) of 4.97%. The

limit of detection (LOD) and limit of quantification (LOQ) of the method was 0.015 and 0.03 mg g�1,

respectively. The linearity of the carbaryl was 0.9992. Excellent method repeatability and

reproducibility were also observed by intra- and inter-day assay precision. For robustness, the method

was employed to analyze 122 buffalo meat samples, and intensities for the insecticide were found to be

unaffected by the sample matrices interference.

Introduction

Carbaryl insecticide is one of the most extensively used chemicals

in agriculture for crop protection, as an ectoparasiticide, and for

regular household practices.1 The reason for this is that it proves

to have high and broad insecticidal efficacy but low toxicity

towards a variety of warm-blooded animals. In addition, this

compound is less persistent than organochlorine pesticides and

produces fewer or no toxicological products.2 However, the

presence of residues of carbaryl in meat is of toxicological and

regulatory concern as it could be an acetyl cholinesterase inhib-

itor and cause allergic hypersensitivity reactions in human

beings. Therefore, in recent years, both legislators and consumers

have shown increased interest in the safety of food products.

Events such as the appearance of pesticide residues in food of

animal origin have impelled governments in the United States,

the European Union, Japan, India, and many other developed

and developing countries in the world.3 The Codex Alimenta-

rious Commission (CAC) and United States Department of

Agriculture (USDA) set the maximum residue limits (MRLs) of

0.1 mg g�1 carbaryl in cattle meat. As the established tolerance for

carbaryl insecticide in meat is low and metabolism in animals is

high,4 the analytical method for monitoring carbaryl residues in

meat is required to be simple, precise, inexpensive, and capable of

detecting residues below the MRL.

aDivision of Livestock Products Technology, Indian Veterinary ResearchInstitute, Izatnagar, Bareily, 243122, UP, India. E-mail: biswaslpt@gmail.com; Fax: +91-161-2400822; Tel: +91-161-2414025bDivision of Pharmacology and Toxicology, Indian Veterinary ResearchInstitute, Izatnagar, Bareily, 243122, UP, IndiacDivision of Livestock Products Technology, Division of Post HarvestTechnology, CARI, Izatnagar, Bareilly, 241 122, UP, India

† Present Address: Department of Livestock Products Technology,COVS, GADVASU, PAU Campus, Ludhiana-141 004 (Punjab), India

This journal is ª The Royal Society of Chemistry 2010

Methods for analysis of carbaryl insecticide by gas chroma-

tography (GC) have proved to be problematic because of their

polarity and heat labile characteristics. Liquid chromatography

(LC) with flurogenic labeling technique was also mentioned.5 LC

method involved a reverse-phase separation followed by a post

column based hydrolysis that liberated methylamine, which

further reacted with o-phthalaldehyde (OPA)-mercaptoethanol

to form a highly fluorescent isoindole.6,7 Post column derivati-

zation with fluorescence detection in LC and liquid chromatog-

raphy-mass spectrometry (LC-MS) has high sensitivity and

selectivity, but the required instrumentation is complicated and

expensive. Further, all of the above methods were developed

using high amounts of toxic solvents, require exhaustive cleanup

techniques, and their applications are relied on only by crop

residue analysis.8,9

The method described here in this study requires only a little

toxic solvent and involves simple sample extraction and cleanup

steps. The sample was deproteinized with acetonitrile, cleanup on

aminopropyl-bonded silica cartridge and determined by photo-

diode-array (PDA) detector. The method was partially validated,

and this validated method was used for the determination of

carbaryl residue in buffalo meat samples.

Materials and methods

Chemicals and reagents

Pure standard of carbaryl (1-napthyl methyl carbamate; assay

99.7%) was obtained from Sigma-Aldrich (USA). Aminopropyl

bonded silica cartridges (3 cm3, 500 mg) and twelve-port vacuum

manifold were procured from Supelco Co., USA. LC grade

acetonitrile, methanol and water were obtained from E. Merck

and Rankem (India). Deionized water was also obtained by

a Milli-Q water purification system (Millipore, France) and was

Anal. Methods, 2010, 2, 393–396 | 393

Fig. 1 Calibration curve for carbaryl in buffalo meat.

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filtered using 0.45 mm cellulose filter prior to use. All other

reagents were used of high analytical quality grade.

Standard preparation

The standard stock solution at 1 mg mL�1 free base concentra-

tion of carbaryl standard prepared by dissolving pure standard in

HPLC grade water and solution was maintained at 4 �C in the

dark. Working standard solutions of 320, 160, 80, 40, 20, 10, 5,

2.5, 1.25, 0.62 and 0.3 mg mL�1 carbaryl were prepared daily in

mobile phase [a mixture of acetonitrile–water (50 : 50, v/v)] and

stored at 4 �C.

Sample collection

A total of 122 buffalo meat samples composed of 92 Longissimus

dorsi (LD) and 30 silver sides (SS) were collected from four

different export meatpacking plants located across the country.

Samples were collected over a 12 month period. The samples

were collected from the deboning table where the chilled

carcasses were cut, deboned, trimmed and packed. About 200 g

of buffalo meat was cut aseptically from LD or SS randomly at

different periods of deboning operations and transferred to self-

sealing colorless low density polyethylene (LDPE) bags. The

bags were labeled and blast frozen (�40 �C) and brought to the

laboratory under frozen conditions in a foam box containing

chiller packs. Both types of samples were stored at�20 �C before

analysis, separately.

Sample preparation, extraction and cleanup

Frozen meat samples were thawed overnight in a refrigerator

(4 � 1 �C). The muscle samples were made into small cubes with

scissors after external fat and fascia were trimmed off. The finely

cut samples were blended in a high speed (15 000 rpm) tissue

blender (York Scientific Industries Pvt. Ltd., New Delhi,

S.No.293) for 2 min. Ten grams of blended sample was taken into

a 100 mL polypropylene centrifuge tube, and 10 mL of Milli-Q

water was added; the mixture was homogenized for 1.5 min using

an Ultra-Turrex T25 tissue homogenizer (Janke and Kenkel,

IKA, Labor Technik, USA).

For extraction, 0.5 g of meat homogenate was spiked with

50 mL of the working standard solution in a glass test tube. Then

1.5 mL of acetonitrile was added to it and the tube was held for

15 min at room temperature (27 � 1 �C), and vortexed for

1.5 min. The mixture was kept for 5 min undisturbed, 1 mL of

water was added and again vortexed at high speed for 1 min and

finally centrifuged at 3000 rpm for 15 min in a refrigerated

centrifuge (Biofuge, Heraeus, USA). Supernatant was collected

into a separate test tube and cleanup of this sample extract was

performed on aminopropyl-bonded silica cartridge precondi-

tioned with 3 mL of methanol and 2 mL of water. The sample

extract was passed through the cartridge under low vacuum at

a flow rate of 3 mL min�1. The cartridge was then washed with

methanol, sorbent bed was dried and finally the analyte was

eluted with 2.5 mL of dichloromethane in a graduated tube. To

dry up this eluted fraction, it was evaporated under a gentle

stream of nitrogen at 40 �C. That is to accelerate the evaporation

step a heating module was used to heat the aluminium block

containing graduated tubes. The residue dissolved in mobile

394 | Anal. Methods, 2010, 2, 393–396

phase to a final volume of 0.5 mL. The aliquot was filtered using

0.22 mm nylon filter and directly injected into the LC system.

HPLC-PDA conditions

For the analysis of carbaryl, a high-performance liquid chro-

matograph (Shimadzu Corp., Kyoto, Japan) composed of an

LC-10 AT quaternary gradient pump, a Rheodyne manual loop

injector with a 20 mL loop, a column oven CTO-10AS vp, and

a PDA detector was employed. Separation of carbaryl was

achieved using a reverse phase octyldecylsilane C18 (RP-C18)

stainless steel column; 250 � 4.6 mm i.d., 5 mm particle size,

100 A� pore size, (Phenomenex, Torrence, CA) with matching

guard column as stationary phase and a mixture of acetonitrile–

water (50 : 50; v/v) as mobile phase. The eluent was monitored at

a wavelength of 220 nm with a flow rate of 0.9 mL min�1 at

a column oven temperature of 35 �C. The data collected were

analyzed with class-vp 6.12 version software, taking into account

the peak heights of analyte.

Fortification of blanks and preparation of calibration curve

Blank homogenates of buffalo meat were prepared as described

above. A working standard containing 320 mg mL�1 of carbaryl

was prepared from the 1 mg mL�1 stock solutions kept at 4 �C.

From this working standard different dilutions were made to

spike the homogenates. Blank homogenates of 0.5 g were spiked

with working standards to obtain final concentrations 2.0, 1.0,

0.5, 0.25, 0.125, 0.062, 0.031 and 0.015 mg g�1 of carbaryl and

extracted as described previously and injected into the HPLC

system. Calibration curves were plotted by taking peak height to

the respective concentrations. This curve was used to quantify

the residues of carbaryl in the buffalo meat samples analyzed.

Analytical recovery and precision

Analytical recovery was determined by spiking carbaryl to blank

meat homogenates to yield concentrations of 0.02, 0.1, and

0.50 mg g�1of carbaryl and then analyzed. The amount of pesti-

cide found by the assay method for each concentration was

estimated using a linear regression equation after calibration of

standard curve (y ¼ 0.0962x + 0.0029, r2 ¼ 0.9992; Where, y ¼peak height, x ¼ carbaryl concentration, and r2 ¼ correlation

coefficient) considering peak heights (Fig. 1). Five determinants

were made for each concentration, and the percent recovery was

calculated. Both intra- and interday assay precisions were also

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determined by analyzing three spiked concentrations of 0.02, 0.1,

and 0.5 mg g�1, five sets each with blank. However, intraday assay

precision was determined at three occasions at least 6 h apart,

whereas interday precision was determined at least 24 h apart for

three successive days. The lowest and highest concentrations of

standard routinely used were 0.02 and 0.50 mg g�1, respectively.

The limit of detection (LOD) and limit of quantification (LOQ)

for carbaryl was 0.015 and 0.031 mg g�1.

Fig. 2 Liquid chromatogram of carbaryl spiked (A and B) and blank (C)

buffalo meat homogenates. Samples spiked at 1.0 mg g�1 (A) and 0.031 mg

g�1 (B) concentration.

Limits of detection (LOD) and limits of quantification (LOQ)

The limit of detection (LOD) and limit of quantification (LOQ)

of the method was done as per proposed guidelines of CAC with

slight modification. The limit of detection (LOD) was determined

from injection of working standards and was defined as the

amount corresponding to mean value plus three times the stan-

dard deviation for the blank sample. So, consideration was given

only when the first condition was satisfied. The detection limit

was computed with a signal to noise-ration of 3 (S/N-3). The

limit of quantification (LOQ) measured on the fortified tissue

sample from where standard calibration curve was satisfied. For

measurement, the peak height to average background noise was

determined. The background noise estimates were based on the

peak-to-peak baseline near the analyte peak. LOQ was then

calculated on the basis of minimal accepted value of the signal to

noise ration of 6 (S/N-6).

Fig. 3 Effects of different solvents on extraction efficiency of carbaryl.

Results and discussion

Optimization of HPLC conditions

Chromatographic separation of carbaryl was carried out with

isocratic elution of acetonitrile–water (50 : 50 v/v) at the flow rate

of 0.9 mL min�1 and at column oven temperature of 35 �C. At the

initial stage of separation it has been observed that a slight

change in the ratio of acetonitrile and water changed the polarity

of the mobile phase reasonably and thereby peak resolution and

retention time. The retention time of carbaryl was approximated

to be 8.0 min. Many analysts reported various mobile phase

profiles during separation of carbaryl compound in foods.2,5,8,9

Mobile phase containing acetonitrile : water, 40 : 60 v/v showed

satisfactory result on isocratic elution.2 A photo-diode-array

(PDA) detector set at a wavelength of 220 nm was used for the

detection of carbaryl compound. The majority of literature

focused on LC with fluorogenic detection techniques after post-

column derivatization.5,8,10 Post-column derivatization with

fluorescence detection in LC had higher sensitivity and selec-

tivity, but the required processing step and instrumentation are

complicated and expensive. Moreover, all the methods were

employed in crop residue analysis such as fruits, vegetables and

juices, which contain high amounts of chromophoric compo-

nents that could interfere with carbamate residues analysis.8,9 In

contrast to crop residues analysis, PDA detector response at

220 nm for meat was more than sufficient as only little interfering

substances were observed in the chromatogram (Fig. 2). It has

been observed that UV-spectrum below 210 nm wavelength

matrices interference is higher, with increased peak resolution

and sharpness. Sharpness of peaks above 230 nm was unac-

ceptable. So, PDA detector was set at 220 nm wavelength.

This journal is ª The Royal Society of Chemistry 2010

Sample extraction and cleanup

Acetonitrile along with water was considered to be an effective

solvent for extraction of carbaryl in buffalo meat samples,

leaving over 99% of fat and fiber behind (Fig. 3). Pre-extraction

permits larger sample sizes to be taken and thus assured more

representative sampling while removing a substantial amount of

fat and other interfering substances present in meat. Use of water

in extraction cell acts as an efficient medium for extraction of

carbaryl,9 even a very small amount of amino acids may be

present.2 But, these overwhelming interfering substances can be

considerably reduced by the use of guard column, efficient clean-

up and subsequent filtration. Therefore, sufficient reduction in

the background signal was observed in the chromatogram. It has

also been observed that cleanup of the sample extract with

aminopropyl-bonded silica cartridge adds extra specificity to the

reversed-phase mode of LC separation due to their strong feature

in the normal-phase mode of solid phase extraction (SPE).

Various workers reported extraction of carbaryl with acetone,

acetonitrile, methanol and methylene chloride in their multi-

residue analysis.5,8,9,11 The use of methanol as an extraction

solution has been shown to have 15% more carbamate residues in

the sample extract.11 Other analysts reported use of methanol–

water or acetonitrile–water as an extraction solution in

Anal. Methods, 2010, 2, 393–396 | 395

Table 1 Distributions of carbaryl residues in export buffalo meat sam-plesa

Sample type Mean/mg g�1 Median/mg g�1

Sample showeddetectable residues (%)

LD 0.045 0.022–0.069 3.1SS 0.028 0.027–0.029 6.89Overall 0.036 0.022–0.069 4.1

a LD ¼ Longissimus dorsi; SS ¼ Silver side.

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combination with SPE cleanup or supercritical fluid extraction

has better acceptability for the recovery of moderately polar

carbaryl component from sample matrix.12,13

Validation of analytical methodology

The analytical method was validated by evaluating % recovery,

precision, linear dynamic range, sensitivity, limit of detection

(LOD), and limit of quantification (LOQ) of the analytes.

Results showed (Fig. 4) that the recovery range of the insecticide

at different concentrations is good. The excellent recovery mainly

occurred due to complete extraction of moderately polar insec-

ticide.14 As the cleanup was conducted with SPE columns,

unwanted interfering substances due to this might be avoided

with an increased mean recovery value. Higher recovery value

might also be due to repeated extractions.12 The coefficients of

variation (CVs) were excellent for all of the three spiked

concentrations (Fig. 4). In fact, the average recovery and CVs is

more than sufficient than mentioned in the Codex guidelines.

Results of precision study indicated that intra- and inter-day

precisions were adequate with the coefficients of variation ranged

from 2.9 to 19.6 and 3.8 to 9.4%, respectively. Method stan-

dardized showed the linear dynamic range (0.03 to 1.0 mg g�1) of

the detector response for the pesticide with the average correla-

tion coefficient of 0.9992 (Fig. 1). The limit of detection (LOD)

and limit of quantification (LOQ) of the method was 0.015 and

0.03 mg g�1, respectively.

Application in real samples

Results for residue data of pesticides in meat are shown in

Table 1. The residual concentration of carbaryl pesticide in LD

muscle ranged from 0.022 to 0.069 mg g�1 with the statistical

mean of 0.045 mg g�1. However, no sample showed residues

above the MRL of CAC, although 3 samples (3.1%) were posi-

tive for carbaryl. In contrast to LD muscle, mean residual

concentration in SS muscle was 0.028 mg g�1 but only 2 samples

(6.89%) out of 30 were positive for carbaryl residues. These

results indicate that only very few samples contain carbaryl

pesticide in buffalo meat, though market reports indicate use of

this component in regular house hold practices, crop protection

or even in animal husbandry practices as ectoparasiticide

Fig. 4 Recovery and precision data of carbaryl spiked into buffalo meat

samples.

396 | Anal. Methods, 2010, 2, 393–396

measures. Low incidence of this chemical component might be

due to sufficient clearance time pre-slaughter or may be degraded

very quickly in the environment.

Conclusion

HPLC coupled with photo-diode-array detector and acetoni-

trile–water as the extraction medium was successfully employed

to simple and sensitive determination of carbaryl residues in

meat. In comparison to the pretreatment methods mentioned

previously, the proposed HPLC method is environmentally

friendly and inexpensive and easily determined. In addition,

analysis was accomplished with high sensitivity and specificity.

Therefore the proposed method will be useful and practical in

future residue monitoring of carbaryl in meat.

Acknowledgements

Authors of this manuscript are thankful to In-charge National

Referral Lab (Residue monitoring) for providing sufficient

facilities for sample analysis. We are also equally thankful to

Director of Indian Veterinary Research Institute, and Agricul-

tural and Processed Food Export Development Authority, Govt.

of India for their financial support.

References

1 EMEA, European Medicines Agency, 2004. Available at: http://www.emea.in.int/.

2 R. J. Argauer, K. I. Eller, M. A. Ibrahim and R. J. Brown, J. Agric.Food Chem, 1995, 43, 2774–2778.

3 M. Takino, K. Yamaguchi and T. Nakahara, J. Agric. Food Chem,2004, 52, 727–735.

4 JECFA, Fifty-eight report of the Joint FAO/WHO Expert Committeeon Food Additives, 2002, 907.

5 M. J. Page and M. French, J. AOAC-Int, 1992, 75, 1073–1083.6 R. J. Bushway, J. Chromatogr, 1988, 457, 437–444.7 G. S. Nunes, M. P. Marco, M. L. Riberio and D. Barcelo,

J. Chromatogr. - A, 1998, 823, 109–120.8 G. Ozhan, S. Topur and B. Alpertunga, J. Food Prot, 2003, 66, 1510–

1513.9 S. Bogialli, R. Curini, A. D. Carcia, M. Nazzari and D. Tamburro,

J. Agric. Food Chem, 2004, 52, 665–671.10 B. D. McGarvey, J. Chromatogr, 1989, 481, 445–451.11 R. T. Krause, J. Assoc. Off. Anal. Chem, 1985, 68, 726–733.12 A. Kok and M. Hiemstra, J. AOAC-Int, 1992, 75, 1063–1072.13 J. W. Wong, M. G. Webster, C. A. Halverson, M. J. Hengel,

K. K. Ngim and S. E. Ebeler, J. Agric. Food Chem, 2003, 51, 1148–1161.

14 D. M. Holstege, D. L. Scharberg, E. R. Tor, L. C. Hart andF. D. Galey, J. Assoc. Off. Anal. Chem, 1994, 77, 1263–1274.

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