Title of Invention

A METHOD FOR THE ESTIMATION OF MULTI RESIDUE IN MILK & MILK PRODUCTS

Abstract A method for the estimation of multi residue in milk and milk products comprising the step of adding enhydrous sodium salt, activated silica gel, Florisil to the milk sample, subjecting the said mixture to the step of mixing to form a homogenized flowable materials; packing the said homogenized material in a glass column; eluted the residues with solvent mixture of acetone and dichloromethane in 1:1 ratio, concentrating the eluant and is divided into two equal parts; concentrating both fractions to complete dryness in gas manifold evaporator, adding n-hexane to one fraction for recovering organochlorines and synthetic pyrethroids and adding ethylacetate to the other fraction for recovering organo phosphate and carbamates from milk.
Full Text Still further object of this invention is to propose method of extraction and clean-up in a single step and the residue can be estimated simultaneously and efficiently. STATEMENT OF THE INVENTION
This invention relate to a method for the estimation of multi residue in milk and milk products comprising the step of adding enhydrous sodium salt, activated silica gel, Florisil to the milk sample, subjecting the said mixture to the step of mixing to form a homogenized flowable materials;
packing the said homogenized material in a glass column;
eluted the residues with solvent mixture of acetone and dichloromethane in 1:1 ratio,
concentrating the eluant and is divided into two equal parts;
concentrating both fractions to complete dryness in gas manifold evaporator,
adding n-hexane to one fraction for recovering organochlorines and synthetic pyrethroids and adding ethylacetate to the other fraction for recovering organo phosphate and carbamates from milk.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS;
Fig.l: The technology flow chart of protocol from milk and Butter/Ghee.
Fig. 2: Standard chromatogram of organochlorine and Synthetic pyrethroid
insecticides employing GC-ECD system with capillary column.
Fig.3: Standard chromatogram of organophosphates insecticides employing
GC-NPD system with capillary column.
Fig.4: Standard chromatogram of organophosphates insecticides employing
GC-NPD system with capillary column.
Fig 4: Standard chromatogram of organophosphates insecticides employing GC-NPD system with capillary column.
Rg.5: Chromatogram showing mik recovery for OC and SP.
Rg.6: Chromatogram showing milk recovery for OP end carbamate compound!.
Rg 7 Chromatogram showing butter recovary for OC and SP.
Rg.6: Chromatogram showing mik racovary for OP and carbamate compounds.
Rg.9i Chromatogram showing mik racovary for OP and carbamate compounds.
DETAILED DESCRIPTION OF THE INVENTION
Repid analyse proceures using capillery gas chromagrapn with election capture and nitrogen phoaphorous detections for simultaneously determinetlon of residues of organochlorine, synthetic pyrethroid, orgenophosphete and carbamate group of pasticides in mik and mik products ska butter and ghee (clarified butter fat) ware developed. Fortified max samples Q 0.001-1.0 µgg'1 subjected to extration-cum-clean-up using column chromatography gave satisfactory recoveries (>60%) for different groups of pesticides. Butter/ghee samples fortified at the levels of 0.001 -10 µgg'1 were extracted by liquid-liquid Derationing and column chromatography. Recoveries ranged between 75-125% for el group of pesticides as shown in figure 7 and Tables. With respect to existing procedures, the methods showed acceptable smite of detection (0.002 to 0.20µgg'1) dapanding on the pesticides and matrices while minimizing environmental concerns, time and labor. The methods are easy to standerize and is suitable for automation.
These methlos reduce/curteil the earier tedious and time consuming steps for extraction and clean-up. Less amount of solvents are used. In a single run on
gas Iiquid chramatograph (GLC) many numbers of pesticide's residues can be identified quafltativefy and quantitatively both. For clean-up concentrated sulphuric acid is not used. Because on adopting the proposed methods, extraction and clean-up can be performed in a single step and the residues of orgenochlorine and synthetic pyrethroids on GC-ECD and of orgenphosphate and carbamates on GC-NPD can be estimated simultaneously and efficiently as shown in figures 2, 3 & 4 respectively.
A stepwise methodology along with recoveries has been provided in detail and also shown in figure 1.
a) Solvents I lexane, n-hexane, acetone, dichtoromothane, acetonitrile, and ethyl acetate (Analytical Grade from Merck).
b) Adsorbents and other chemicals-Sillca gel (60-120 mesh), Florist (90-100 mesh), activated charcoal, anhydrous sodkim sulphate, sodium chloride.
c) Reference standards: HCH and its isomers, DOT and its metaboites, endoeurfan (A, B and sulphate), heptachlor, aldrin, chiordane, cypermethrin, fenvaierate, deltamethrin, chtorpyriphos, matethion, monocfotophos, dimethoats, methyi-perathion and quinalphos of Analytical Grade from Environmental Protection Agency (EPA), North Carolna, USA.
AM the solvente to be used were doubly glees distilled before use. Pre washed the adsorbent sflca gel (60-120 mesh) first wtth acetone fotowed by olchloromethene and activated at 120*C for 1.5 h.
MILK
Extraction CUM Clean Up Using Column Chromatography
• Took 10ml representative sample of milk from homogenized 1L bulk sample.
• To the mlk added 10g anhydrous sodkim sulphate, 15g pre washed and activated silica gel (60-120mesh), 5g Florisil (90-100mesh) and mixed them thoroµghly to make it flow able.
• Packed this homogenized material compactly in a glass column (60cmx 22mm) fitted with PTFE stop cock in between two layers of anhydrous sodium sulphate. Eluted the pesticide residues with 150 ml solvent mixture of acetone and dichloromethane (1:1 v/v) at flow rate of 4mVmin.
• Concentrated the ekient to 10ml on Rotary Vacuum Rash Evaporator and divided In to two equal parts. One part for crganochtorines and synthetic pyrethroids whereas second part for organopnosphates and carbamates.
• Concentrated the both fractions to complete dryness using gas manifold evaporator.
• Meticulous care has been taken to remove traces of Achtoromethane from the extract used for estimation of OC/SP insecticides.
• Made the final volume to 2ml in n-hexane for OC/SP inaecickies and in ethyl acetate for OP and carbamates for injecting Into gas chromatograph.
BUTTER Extraction
• Took 5g representative sample of butter/ghee from 500g bulk sample.
• Dissolved the representative sample in 40 ml hexane and partitioned with equal volume of 1% aqueous acetonitrile.
• Diluted the ecetonitrile phase 4-5 times with 10% NaC1 solution and partitioned thrice (50, 40, 40 ml) with hexane followed by Iiquid-liquid partitioning thrice (50,40,40 mf) with dichloromethene.
• Combined the organic phases, passed throµgh a bed of anhydrous sodium sulphate and concentrated to 10 ml on rotary flash vacuum evaporator.
• Concentrated the both fractions to complete dryness using gas manifold evaporator.
• Meticulous cmn be taken to remove traces of olchloromethane from the concentrated extract used for estimation of OC and SP insecticides.
• Made the final volume to 2ml in n-hexane for OC and SP insecticides and in ethyl acetate for OP and carbamates for injecting into gas chromatograph.
NOTE: No clean up was required In butter/ghee as no interfering peaks
were observed during analysis.
RECOVERY EXPERIMENT
For authenticity of methods, recovery experiments were conducted at the fortification levels of 0.001 to 1.0 µgm1-1 /µg g-1 according to the sensitivity of each pesticide of the different chemical groups in each commodity i.e. milk and butter/ghee in tiplicate. Known concentration of each pesticide under study was added to max and butter samples separately and kept the samples overnight and processed the fortified samples as per methods given above. Control samples were processed along with spiked ones. Control samples were also processed along with spiked ones.
The recoveries of OC, SP, OP and carbamates in milk ranged from 77-112, 74-106 and 89-95%, where as in butter ranged from 75-105, 79-98, 117-125 and 76%, respectively Limite of detection (LOD) were 0.001 µgg"1 for OC, 0 005µgg1 for SP, for OP 0.01 µgg"1 and 0.05µgg1 for carbamate compounds as shown in figures 5, 6, 8 & 9. Recoveries were considered satisfactory for aft the insecticides in both the cornmodities with the proposed methods. Retention times and peak areas of the studied pesticides were comparable with the relative standerds as snown in tables.
Oas liquid chromatograph (OLC) equipped with capiliary column using electron capture (ECO) and nitrogen phosphorous (NPD) detectors were used for analysis. Various parameters are given below:
GLC Parameters for Analysis of OC and SP pesticides as shown in figure 5
GLC Mode* : HP5890A Detector : ECD NI*3
Column : Capllary column, HP-5, 30m X 0.32mm id. x 0.25 µm film
thickness of 5% diphenyl and 95% dimethyl polysaiaxane Temperature (°C) Programming:
(Equation Removed)
Injection port temperature : 280*C
Detector temperature : 300°C
Gas flow rate : Total flow 60ml/mJn wMh spit ratio
1 10 (Through column 2ml)
GLC Parameters for the Extimation of OF and Carbermate Pesticldes as shown in figure C.
GLC model : HP509OA
Detector : NPD
Column : Capilary column, HP-1,10m x 0.53mm Id. x 2.65 µm film
Thickness of 100% dimethyl pofrtieoxane
Temperature (c) Programming.
(Equation Removed)
\Inpction port temperature : 250°C
Detector temperature : 275*C
Gas flow rate : H2 :1 8ml/mim
O? : 130mVmin N2 1.8ml/min
TABLE 1: Retention timet observed for different OC end SP insecticides (ECD-63NI-GLC system) (Table Removed)
TABLE 2: Retention times observed for OP and carbamates insecticides (NPD-GLC system)
(Table Removed)
TABLE 3: Percent recoveries of different groups of pesticides in milk
(Table Removed)
•Mean of three replicates in each approach
*Spiking levele for OC: 0 001-0 1 µgml1; SP: 0.1-0.2 µg ml-1; OP: 0 1-0.5 µgml1;
Carbamates : 0.5-1.0 µgmf1.
TABLE 4: Percent recoverises of different groups of pesticides in Butter
(Table Removed)
*M*an of three replicates in each approach
•Spiking levels for OC: 0.001-0.1 µgmf1; OP: 0.1-0.5 µgmr1; Carbamates : 0.5-
1.0µgmr1



WE CLAIM
1. A method for the estimation of multi residue in milk and milk
products comprising the step of adding enhydrous sodium salt,
activated silica gel, Florisil to the milk sample, subjecting the said
mixture to the step of mixing to form a homogenized flowable
materials;
packing the said homogenized material in a glass column;
eluted the residues with solvent mixture of acetone and dichloromethane in 1:1 ratio,
concentrating the eluant and is divided into two equal parts;
concentrating both fractions to complete dryness in gas manifold evaporator,
adding n-hexane to one fraction for recovering organochlorines and synthetic pyrethroids and adding ethylacetate to the other fraction for recovering organo phosphate and carbamates from milk.
2. A method as claimed in claim 1, wherein the sodium salt is sodium sulphate.

Documents:

480-DEL-2006-Abstract-(12-07-2012).pdf

480-del-2006-Abstract-(19-09-2013).pdf

480-del-2006-abstract.pdf

480-DEL-2006-Claims-(12-07-2012).pdf

480-del-2006-Claims-(19-09-2013).pdf

480-del-2006-claims.pdf

480-DEL-2006-Correspondence Others-(12-07-2012).pdf

480-del-2006-Correspondence Others-(19-09-2013).pdf

480-del-2006-Correspondence-Others-(06-11-2013).pdf

480-del-2006-Correspondence-Others-(07-02-2013).pdf

480-del-2006-Correspondence-Others-(15-04-2013).pdf

480-del-2006-correspondence-others.pdf

480-del-2006-correspondence-po.pdf

480-DEL-2006-Description (Complete)-(12-07-2012).pdf

480-del-2006-description (complete).pdf

480-del-2006-drawings.pdf

480-del-2006-form-1.pdf

480-del-2006-form-18.pdf

480-del-2006-form-2.pdf

480-del-2006-form-3.pdf

480-del-2006-form-5.pdf

480-DEL-2006-GPA-(12-07-2012).pdf

480-del-2006-gpa.pdf

480-del-2006-ur-Abstract-(19-09-2013).pdf

480-del-2006-ur-Claims-(19-09-2013).pdf


Patent Number 258152
Indian Patent Application Number 480/DEL/2006
PG Journal Number 50/2013
Publication Date 13-Dec-2013
Grant Date 10-Dec-2013
Date of Filing 22-Feb-2006
Name of Patentee CCS HARYANA AGRICULTURAL UNIVERSITY
Applicant Address HISAR-125004
Inventors:
# Inventor's Name Inventor's Address
1 SHASHI SINGH C/O.CCS HARYANA AGRICULTURAL UNIVERSITY HISAR-125004.
2 T S KATHPAL C/O.CCS HARYANA AGRICULTURAL UNIVERSITY HISAR-125004.
3 BEENA KUMARI C/O.CCS HARYANA AGRICULTURAL UNIVERSITY HISAR-125004.
4 JAGDEEP SINGH C/O.CCS HARYANA AGRICULTURAL UNIVERSITY HISAR-125004.
PCT International Classification Number A23C
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA