A New Validated HPLC method for determination of Cyantraniliprole and its metabolite residues in tomato fruit

 

Botsa. Parvatamma,¹*  Tentu. Nageswara Rao²

¹Department of Organic Chemistry, Gayathri PG College, Vizianagaram, Andhra Pradesh, India.

²Department of Chemistry, Krishna University, Machilipatnam, Andhra Pradesh, India.

*Corresponding Author E-mail: tentu6581@rediffmail.com

A simple and inexpensive method was developed using solid-phase extraction, together with high performance liquid chromatographic method with UV detection for determination of Cyantraniliprole and its metabolite (J9Z38) residues.  The evaluated parameters include the extracts by silica gel packed column using acetonitrile and water solvents. The method was validated using fruit samples spiked with Cyantraniliprole and its metabolite (J9Z38) at different fortification levels (0.01 and 0.1 µg/g). Average recoveries (using each concentration six replicates) ranged 83-94%, with relative standard deviations less than 2%, calibration solutions concentration in the range 0.01-10.0 µg/mL and limit of detection (LOD) and limit of quantification (LOQ) were 0.003µg/g and 0.01µg/g respectively. Finally the fruit residue samples were analyzed by HPLC.

 

 

INTRODUCTION:

Cyantraniliprole is the second xylem-systemic active ingreadient in the new anthranilic diamide class Field studies were conducted to determine the efficacy of Cyantraniliprole for managing Bemisia tabaci biotype B and in interfering with transmission of tomato yellow leaf curl virus by this whitefly^1-3. Cyantraniliprole Applied As Soil treatments (200 SC) provided excellent adult whitefly control, TYLCV suppression and reduced oviposition and nymph survival, comparable to current standards.  Cyantraniliprole to be used in a whitefly management program ⁴.

 

Field evaluations of soil drench treatments conformed the suppression TYLCV transmission demonstrated in the greenhouse studies. Cyantraniliprole is diamide insecticide. Damides affect ryanodine receptors in insect muscle, causing paralysis. Cyantraniliprole formulated products applied as a seed treatment, as a foliar spray or as a soil derench, it controls, suppresses or reduces damage caused by insect pests of field, tree fruit, tree nut and bush berry crops and greenhouse and outdoor ornamentals. Cyantraniliprole is used to control insect pests in fruit crops, tree nuts, oil seed crops, cotton, grapes, rice, vegetables, ornamentals and turf around the world⁵.

 

This study has been undertaken to develop an improved method for analysis of Cyantraniliprole and its metabolite J9Z38 to determine residue retention in tomato fruit.

 

EXPERIMENTAL:

Standards, Reagents and samples

The analytical standards of Cyantraniliprole (99.5%) and J9Z38 (96.6%) were obtained from Sigma Aldrich. Acetonitrile and HPLC water were purchased from rankem, New Delhi and silica gel sorbet was supplied from Merck Limited and tomato fruits were purchased from local market.

Standard stock solutions

The cyantraniliprole and metabolite- J9Z38 standard stock solutions were individually prepared in acetonitrile at a concentration level 1000 µg/mL and stored in a freezer at -18°C. The stock standard solutions were used for up to 3 months. Suitable concentrations of working standards were prepared from the stock solutions by dilution using acetonitrile, immediately prior to sample preparation.

 

Sample preparation

Representative 20.0 g portions of tomato fruit fortified with 0.1 mL of working standard solutions. The sample was allowed to stand at room temperature for one hour, before it was kept at refrigerator condition, until analysis.

 

Extraction procedure

Weighed 20g of fruit sample in to a 500 ml Stoppard conical flask and extracted with 100 mL of acetonitrile + water (90:10 v/v) using an end-over-end mechanical shaker for about 30 minutes and filtered.  Extraction was repeated twice with 50 ml of acetonitrile + water (90:10 v/v).  Combined filtrate was passed through anhydrous sodium sulfate and concentrated near to dryness at 50°C using rotary vacuum evaporator and the contents were re-dissolved in 20mL of same extracted solvent.

 

Clean-up procedure

A chromatographic column was packed with 5 g of silica gel (column chromatography grade) using acetonitrile.  The above residual extract were poured over the column and allowed to percolate for 5 min⁶. Then eluted the residues with 100 ml of acetonitrile + water (80:20 v/v) mixture.  Concentrated the eluate to near dryness using rotary vacuum evaporator then re-dissolved in 20 mL of acetonitrile. The sample was filtered through 0.45 µm filter and analyzed by HPLC.

 

Chromatographic separation parameters

The HPLC-UV system used, consisted shimadzu high performance liquid chromatography with LC- 20AT pump and SPD-20A interfaced with LC solution software, equipped with a reversed   phase C18 analytical column of 75 mm x 4.6 mm and particle size 3.5 µm (Zorbax  SB -C18)  Column temperature was maintained at 30°C. The injected sample volume was 20µL. Mobile Phases A and B was 1 L of Milli Q water containing 0.1 ml of  1 M formic acid + 0.1 ml of 0.1 M ammonium formate and 1 L of Methanol (HPLC grade) containing 0.1 ml of 1 M formic acid + 0.1 ml of 0.1 M ammonium formate (50:50 (v/v)) with program. The flow- rate used was kept at 0.7 mL/min. A detector wavelength was 254 nm. The calibration curve method was used for determination of Cyantraniliprole and its metabolite (J9Z38) residues in fruit.

Mobile phase composition program:

Time (min)	A Conc (%)	B Conc-(%)
0	50	50
7	10	90
13	10	90
15.0	50	50

 

Method validation

Method validation ensures analysis credibility. In this study, the parameters accuracy, precision, linearity and limits of detection (LOD) and quantification (LOQ) were considered. The accuracy of the method was determined by recovery tests, using samples spiked at concentration levels of 0.01 and 0.1 mg/kg. Linearity was determined by different known concentrations (0.01, 0.1, 0.5, 1.0, 5.0 and 10.0 µg/mL) were prepared by diluting the stock solution. The limit of detection (LOD, µg/mL) was determined as the lowest concentration giving a response of  3 times the baseline noise defined from the analysis of control (untreated) sample. The limit of quantification (LOQ, µg/mL) was determined as the lowest concentration of a given  a response of 10 times the baseline noise^7,8.

 

RESULTS AND DISCUSSION:

Specificity

Aliquots of cyantraniliprole and metabolite- J9Z38 standard solutions, spiking sample solution, fruit control, extracted solvents and mobile phase solvents were assayed to check the specificity. There were no matrix peaks in the chromatograms to interfere with the analysis of residues shown in (Figure 1 and 2). Furthermore, the retention times of Cyantraniliprole and metabolite of Cyantraniliprole (J9Z38) were constant at 4.3 ± 0.2 and 5.5 ± 0.2, minutes.

 

Fig.1. Representative Chromatogram of tomato fruit control

 

Fig.2. Representative Chromatogram at fortification level of 0.01 µg/g

 

Linearity

Preparation of Cyantraniliprole standard stock solution

 Accurately weighed 10.05 mg of reference standard of tembotrione (Purity 99.5%) in 10 mL volumetric flask and dissolved in acetonitrile, sonicated and made upto the mark with the same solvent. The concentration of the stock solution was 1000 µg/mL.    

 

Preparation of metabolite of Cyantraniliprole (J9Z38)              

Accurately weighed 10.35 mg of reference standard of metabolite of Cyantraniliprole (J9Z38) (Purity 96.6%) in 10 mL volumetric flask and dissolved in acetonitrile, sonicated and made upto the mark with the same solvent. The concentration of the stock solution was 1000 µg/mL.                           

Preparation of Calibration solutions

Different known concentrations of standard solutions (0.01, 0.1, 0.5, 1.0, 5.0 and 10.0 µg/mL) were prepared in acetonitrile by diluting the above stock solutions. The serial dilution details were presented in Table 1. These standard solutions were directly injected into a HPLC. A calibration curve has been plotted of concentration of the standards injected versus area observed and the linearity of method was evaluated by analyzing six solutions ⁹. The peak areas obtained from different concentrations of standards were used to calculate linear regression equations. These were Y=6648.62X + 3.41 and Y=4849.46X + 42.06 with correlation coefficients of 0.9999 and 1.0000 for Cyantraniliprole and metabolite of Cyantraniliprole (J9Z38) respectively. A calibration curve showed in (Figure 3). 

 

Table 1. Serial dilutions of linearity standard solutions

Stock solution concentration (µg/mL)	Volume taken from stock solution (mL)	Final make up volume (mL)	Obtained concentration (µg/mL)
1000	1.000	10	100
100	1.000	10	10
100	0.500	10	5
100	0.100	10	1
10	0.5	10	0.5
10	0.1	10	0.1
1	0.1	10	0.01

 

Fig.3. Representative Calibration curve of Cyantraniliprole and metabolite (J9Z38)

 

Accuracy and Precision

Recovery studies were carried out at 0.01 and 0.1 µg/mL fortification levels for cyantraniliprole and metabolite- J9Z38 in fruit. The recovery data and relative standard deviation values obtained by this method are summarized in Table 2.

 

These numbers were calculated from four (6) replicate analyses of given sample (tembotrione and tembotrione–dihydroxy) made by a single analyst on one day. The repeatability of method satisfactory (RSDs<2 %).

 

 

Table 2. Recoveries of the Cyantraniliprole and metabolite (J9Z38) from fortified

Tomato fruit sample (n=6)

 

 

Detection and Quantification Limits

The limit of quantification was determined to be 0.01 µg/mL. The quantitation limit was defined as the lowest fortification level evaluated at which acceptable average recoveries (83-94%, RSD<2%) were achieved. This quantitation limit also reflects the fortification level at which an analyte peak is consistently generated at approximately 10 times the baseline noise in the chromatogram. The limit of detection was determined to be 0.01 µg/mL at a level of approximately three times the back ground of control injection around the retention time of the peak of interest.

 

Storage Stability

A storage stability study was conducted at refrigerator condition ( 5 ± 3°C ) and Ambient temperature (25 ± 5°C) of  0.1 µg/g level fortified fruit samples were stored for a period of 30 days. Analysed for the contents of Cyantraniliprole and metabolite J9Z38 before storing and at the end of storage period ¹⁰. The percentage dissipation observed for the above storage period was only less than 3% for Cyantraniliprole and metabolite J9Z38 showing no significant loss of residues on storage. The results are presented in Table 3 and 4.

 

 

 

Table3. Storage stability Details at refrigerator condition (5 ± 3°C)

 

             

Table 4. Storage stability Details at ambient Temperature (25 ± 2°C)

 

 

CALCULATIONS

The concentration of acetaminophen in the samples analyzed by HPLC was determined directly from the standard curve.

Y = mx + c

Where,

Y = peak area of standard (mAU*sec)

m = the slope of the line from the calibration curve

x = concentration of injected sample (mg/L)

c = ‘y’ intercept of the calibration curve

 

The recovered concentration or Dose concentration was calculated by using the formula:

Recovered concentration or Dose concentration	=	(x-c) X D X 100
		m X P

Where,

m = the slope of the line from the calibration curve

x = sample area of injected sample (mAU*sec)

c = ‘y’ intercept of the calibration curve

D = Dilution Factor

P = Purity of Test item

 

Recovery	=	Recovered Concentration	×	100
		Fortified Concentration

 

 

CONCLUSIONS:

This paper describes a fast, simple sensitive analytical method based on SPE-HPLC-UV simultaneous determination of cyantraniliprole and metabolite of cyantraniliprole (J9Z38)  residues in tomato fruit The SPE extraction procedure is very simple and inexpensive method for simultaneous determination of Cyantraniliprole and metabolite J9Z38 residues in tomato fruit. The mobile phase composition was showed good separation and resolution and the analysis time required for the chromatographic determination of the Cyantraniliprole and metabolite J9Z38 were very short (around 15 min for a chromatographic run). Satisfactory validation parameters such as linearity, recovery, precision and very low limits were obtained and according to the SANCO guidelines ¹⁴. Therefore, the proposed analytical procedure could satisfactorily be useful for regular monitoring of Cyantraniliprole and metabolite J9Z38 residues on a large number of leaf, seed, oil, fruit, water and soil samples ^11-13.

 

ACKNOWLEDGEMENT:

The authors are thankful to the Dr. Gowtham Prasad, S.V.V University, Hyderabad for his keen interest and help.

 

REFERENCES:

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8.        Tentu. Nageswara Rao, T. Srinivasa Rao and G. Silpa. Determination of lymecycline and tetracycline residues in bovine milk followed by matrix solid-phase dispersion coupled to  highperformance liquid chromatography with ultraviolet detection. World Journal of Pharmaceutical Research. 1(5); 2012: 1281-1290. 

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11.     Muccio AD, et al. Application of solid-phase extraction and liquid chromatography–mass spectrometry to the       determination of neonicotinoid pesticide residues in fruit and    vegetables.  Journal of Chromatography A. 1108; 2006: 1-6.

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Received on 06.06.2015         Modified on 17.06.2015

Accepted on 20.06.2015         © AJRC All right reserved