Core Programme Clusters

Chemical Safety

Monitoring of Pesticide Residues in Sugar Samples

Foreword

Acknowledgement

Activity Undertaken

Summary Report

1.      Background

A report on analysis of pesticide residues in soft drinks was conducted by Centre for Science and Environment (CSE), an NGO based in Delhi which stated presence of pesticides in the samples of 12 soft drinks brands procured by it from open market in Delhi. A serious concern was raised over the finding of pesticide residues in soft drinks. A Committee of 15 members of Parliament was constituted as Joint Committee on Pesticide Residues in and safety standards for soft drinks, fruit juices and other beverages. The major ingredient of soft drinks is water which account for 86%-92% of the total soft drink composition. Besides water, soft drinks contain sugar varying from 5 to 10%, carbon dioxide, acid like citric acid, phosphoric acid and malic acid which are added in the concentrate. It has been stated that there is possibility of pesticides entering into the beverages through sugar. A need was felt to fix the MRL’s of pesticides used on sugar. Generally, MRL’s are fixed based on the residue data generated under supervised trials generated under different agro climatic conditions. MRL’s are also fixed based on the monitoring data.

2.      Objectives

Fixation of Maximum Residue Limit of different pesticides in sugar based on monitoring data/ with Limits of quantitation(LOQ).

Pesticides Monitored in Market Sugar

The following pesticides are considered for monitoring study as listed below

3.      Standardization of different analytical grade pesticides in Gas-Chromatograph equipped with Electron Capture Detector and Nitrogen Phosphorus Detector

The analytical grade pesticides under monitoring study were purchased from USA made of Accu Standard along with the purity certificate and the purity percentage is listed below:

Stock standard solution: 10 mg of each individual pesticide standard was taken in a 100 ml volumetric flask and dissolve in hexane, acetone or methanol depending upon each individual compounds solubility. Thus, 100 PPM stock solution of each individual pesticide standard was prepared.

Working standard of individual pesticide: 1-10 PPM working standard solution was prepared using dilution technique. Individual pesticides (lindane, fipronil, cypermethrin) were injected into gas chromatograph equipped with Electron Capture Detector (ECD), dichlorovos, monocrotofos, phorate, atrazine, metribuzin, chlorpyrifos and quinalfos were injected into gas chromatograph equipped with Nitrogen Phosphorus Detector (NPD), Carbofuran and Carbaryl in Gas Chromatograph – Mass Spectrometer (GC-MS) and Imidacloprid in High Performance Liquid Chromatography (HPLC) and their retention time was recorded maintaining the same instrument condition for each individual compound.

Mixed standard solution: Depending upon character, retention time and sensitivity of each compound of instrument, mixed standard solutions was prepared at different concentration. Thus,   two mixtures were prepared containing organo-phosphorus, and triazine herbicides    ( 0.01- 5.0 ppm) for analysis in GC-NPD, organocarbamates (0.1 – 1.0 ppm) in GC-MS and another mixture was prepared containing lindane, fipronil, cypermethrin ( 1ppb- 1ppm) for analysis in GC-ECD. Only Imidacloprid (0.1ppm - 5.0ppm) was prepared for analysis in HPLC. However, all these pesticides were also standardized in Gas-Chromatography- Mass Spectrometer.

4.      Standardization of mixed standard solutions using Multi-residue method in Gas Chromatograph equipped with Electron Capture Detector and Nitrogen Phosphorus Detector and Gas Chromatograph – Mass Spectrometer

The mixed standard solutions were injected into gas chromatograph and retention times of all the pesticides were verified from the records of individual pesticide retention time.

5.      Gas Chromatographic/HPLC/GC-MS parameters for determining pesticides using Multi-residue method

Table-01: Gas Chromatographic Parameters For Multiresidues Of  Organochlorine (Lindane, Fifronil) And Synthetic Pyrethroid (Cypermethrin) Pesticides

Table-02: Gas Chromatographic Parameters For Multiresidues Of Organophosphorus (dichlorovos, monocrotofos, phorate, chloropyrifos and quinalfos) insecticides and two triazine (atrazine and metribuzin) herbicides

Table-03: HPLC Parameters For Imidacloprid

Table-04: GC- MS parameters for carbofuran and carbaryl

6.      Limit of detection

The mixed pesticide standard solutions of different concentrations using dilution technique (10 PPM, 5 PPM, 1PPM, 0.5 PPM, 0.25 PPM, 0.1 PPM, 0.05 PPM, 0.01 PPM, 0.005 PPM and 0.001 PPM) were prepared for determining of LIMIT OF DETECTION.

Table-05:  The Limit Of Detection Of Different Pesticides In Gc-Ecd

Table-06: The Limit Of Detection Of Different Pesticides In Gc-Npd

Graph-01

Graph-02

Table-07: The Limit Of Detection Of Different Pesticides In Gc-Ms

Table-08: The Limit Of Detection Of Different Pesticide In HPLC

7.      Recovery experiment

A recovery experiment was conducted in order to standardize the efficiency of the proposed methodology. After determination of Limit of detection (LOD) of individual pesticides, the sugar samples (20g, 50g and 100g) was fortified with mixture of pesticides in such a way so that double the concentration of LOD is maintained  irrespective of sugar weight  and kept for an hour. The sample was dissolved with 50 ml HPLC grade water using magnetic stirrer for about 10 minutes. Then it was transferred to 1 lt. separatory funnel and partitioned with 50 ml HPLC grade hexane: dichloromethane (1:1) for two times followed by HPLC ethyl acetate (50 ml x 2). The organic layer was passed over anhydrous sodium sulfate (activated) and concentrated using rotary vacuum evaporator (~ 40°C). The final volume was made up with HPLC hexane (1 ml) for estimation of organophosphorus, carbamate and herbicides. For organochlorine and synthetic pyrethroid the final volume was 10 ml. Sugar sample without fortification was also tested as control sample. All the solvents used were also analysed in GC-ECD, NPD, GC-MS and in HPLC. The analysis was carried out in three replicates.

8.      Sampling

Sugar samples were divided mainly into two categories viz. Factory sugar outlet samples and market sugar. Factory sugar samples were of mainly Small (S), Medium (M) and Large (L) size. A representative and valid sugar samples   (2 kg) were collected randomly from different sugar factories of the country. For market sugar, representative and valid (1kg) sugar samples were collected randomly from five to six vendors of each market of different geographic locations. Collection site of different sugar samples are listed in Table-09 and Table-10.

9.     Storage condition of samples before analysis

Sugar samples were extracted immediately after collection as far as practicable. Extracts were cleaned up immediately after extraction as far as possible but in some cases the extracts were also stored in the refrigerator to a minimum period of time before final analysis.

10.  Method of analysis

Principle

Sugar samples were dissolved in water and partitioned with Hexane : Dichloromethane (1:1) followed by  further partition with ethyl - acetate and concentrated in rotary vacuum evaporator for final analysis in HPLC and GLC equipped with ECD and NPD and GC-MS.

Reagents and Apparatus

i.        Analytical grade pesticides

ii.      HPLC Hexane LiChrosolv (EM)

iii.      HPLC Acetone LiChrosolv (EM)

iv.     HPLC Dichloromethane LiChrosolv (EM)

v.      HPLC Ethyl acetate LiChrosolv (EM)

vi.     HPLC Acetonitrile LiChrosolv (EM)

vii.    HPLC Methanol, SRL

viii.  Rotary vacuum evaporator

ix.     Magnetic stirrer

x.      Separatory funnel

xi.     Anhydrous sodium sulfate

xii.    Non-absorbent cotton

xiii.  Funnel

xiv.  Volumetric flask, Borosil, Certified

xv.   Graduated centrifuge tube (25 ml)

xvi.  µL – Pipette, ISO 9001-14001 Certified

Working procedure

Extraction and Clean up of Sugar Samples

Individual sugar samples was mixed well and representative 20g sugar was taken in three replicates by quartering technique and was weighed accurate to 0.01g into a 250 ml beaker.  30 ml HPLC grade water was added and placed on a magnetic stirrer for about 10 minutes, ensuring the sugar samples have completely dissolved in water. 10 ml of hexane : dichloromethane (1:1) was added to the beaker and was transferred to 1 lt. separatory funnel. The beaker was rinsed with 40 ml hexane : dichloromethane (1:1) for several times and this rinse was transferred to the separatory funnel for partitioning. Partition was carried out using ten strokes of the separatory funnel. The bottom layer was passed through a funnel containing anhydrous sodium sulfate (activated) into a 250 ml pear shaped flask. The step was repeated once more. The partition was further done in the similar way using HPLC ethyl acetate (50 ml x 2). All the organic layer was passed over anhydrous sodium sulfate (activated) and concentrated using rotary vacuum evaporator (~40⁰C) to about 1 ml for estimation of organophosphorus, carbamate and herbicides. For organochlorine and synthetic pyrethroid the final volume was 10 ml. The method adopted was the partial modification of “Multiresidue Method for the Gas Chromatographic Determination of Pesticides in Honey after Solid Phase Extraction Cleanup” by Christer Jansson, Journal of AOAC International, Vol. 83 (3), page 714-719, Year: 2000.

Final analysis and calculation

A stock solution containing analytical grade pesticides mixture of lindane, fipronil, cypermethrin injected into GC- ECD,  dichlorovos, monocrotofos, phorate, atrazine, metribuzin, chlorpyrifos and quinalfos injected into GC- NPD, carbofuran and carbaryl injected into GC-MS  and imidacloprid injected into HPLC, as an external standard, for which the retention time was recorded. Then 1-2 µl of each cleaned up test samples was injected into the GC- ECD, GC-NPD, GC-MS and HPLC for the respective mixture of pesticides.  Theresidue was identified by comparing the retention time of sample peaks with that of mixture of standard.

The amount of residues in ppm (µg/g) was calculated as follows:

Where,

A₁= Area of sample in the Chromatogram

A₂= Area of standard in the Chromatogram

V₁= Total volume of sample in ml

V₂ = Injected volume of the sample in µl

C =Concentration of analytical standard in ng

W = Weight of the sample in gm

Rf = % mean recovery factor

11.  Results of recovery experiments

Recovery studies were carried out in order to establish the reliability of the analytical methods and to know the efficiency of extraction and cleanup steps for the present study by fortifying double the concentration of LOD of mixture of analytical standards in such a way so that double the concentration of LOD is maintained irrespective of sugar weights. The results of the recovery study revealed that the recovery percentage varied from 71.2-147% in 20g sugar, 61-120% in 50g sugar and 45.0 – 90.0% in 100g sugar with low Relative Standard Deviations indicating the suitability of the method. Based on the recovery percentage, the method proposed to carry out the analysis of sugar samples with 20g for best results as well as for easy handling. The Results of the recovery experiments are shown in Table 11-14.

Table-11:  Results Of Recovery Of Pesticides From Sugar (20g) Analyzed By The Proposed Method

Table-12: Results Of Recovery Of Pesticides From Sugar (50g) Analyzed By The Proposed Method

Table-13: Results Of Recovery Of Pesticides From Sugar (100g) Analyzed By The Proposed Method

Table-14: Summary Of The Recovery Results

Analysis Graph

12.  Limit of quantitation

The limit of quantitation of different pesticides based on recovery experiment are as follows:

13.  Monitoring results of pesticide residues in different sugar samples

Table–15: Results Of Monitoring Of Pesticide Residues (Lindane, Fipronil, Cypermethrin, Dichlorovos, Monocrotofos, Phorate, Atrazine, Metribuzin, Chlorpyrifos, Quinalfos, Carbofuran, Carbaryl and Imidacloprid) in market sugar samples.

Table–16: Results Of Monitoring Of Pesticide Residues (Lindane, Fipronil, Cypermethrin, Dichlorovos, Monocrotofos, Phorate, Atrazine, Metribuzin, Chlorpyrifos, Quinalfos, Carbofuran, Carbaryl and Imidacloprid) in factory sugar samples.

14.  Conclusion

Multiresidue method for the determination of pesticides in sugar involves dissolution of sugar in water followed by liquid-liquid partitioning and gas chromatographic determination. Thirteen pesticides (lindane, fipronil, cypermethrin, dichlorovos, monocrotofos, phorate, atrazine, metribuzin, chlorpyrifos, quinalfos, carbofuran, carbaryl and imidacloprid) were determined by the method. Recovery from sugar (20g), spiked at 0.01-10 µg/g, ranged from 71.2 to 147% with a relative standard deviation (RSD) of 1.06 – 3.25%. It is a quick and less time consuming method that gives good recoveries of all the thirteen pesticides tested. All the 200 sugar samples (factory outlet and market) were analyzed by the proposed methodology and residues were Below Detectable Limit (BDL).