PYRIPROXYFEN TECHNICAL
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1 WHO/IS/TC/715/2001 TECHNICAL TECHNICAL 1. Specification 1.1 Description Interim specification WHO/IS/TC/715/2001 The material shall consist of pyriproxyfen together with related manufacturing impurities. It shall be in the form of a white to light yellow solid or colorless to yellow clear liquid at ambient temperature, free from visible extraneous matter or added modifying agents. 1.2 Chemical and physical requirements The material, sampled from any part of the consignment (see method WHO/M/1.R1), shall comply with the requirements of section 1.1 and with the following requirements Identity test Where the identity of the active ingredient is in doubt, then it shall comply with at least one additional test Pyriproxyfen content (g/kg basis) The pyriproxyfen content shall be declared (not less than 950 g/kg) and, when determined by the method described in section 2.1, the mean measured content obtained shall not be lower than the declared content ph of aqueous dispersion The ph value of aqueous dispersion, determined by the CIPAC method MT 75 (CIPAC Handbook F, p.205) shall lie in the range 5.5 to 6.5 at 30 ± 2 C Material, insoluble in acetone The material insoluble in acetone, determined by the CIPAC method MT 27 (CIPAC Handbook F, p.27), shall not be higher than 1 g/kg Acidity The acidity of the material, determined by the CIPAC method MT31 (CIPAC Handbook F, p.96), shall not be higher than 1 g/kg calculated as H 2 SO 4. Page 1 of 9
2 WHO/IS/TC/715/2001 TECHNICAL Water content The water content, determined by the method WHO/M/7.R1 (equivalent to the CIPAC method MT 30.5 CIPAC Handbook J, p.120), shall not be higher than 1 g/kg. 1.2 Packing and marking of packages The pyriproxyfen technical shall be packed in suitable clean containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following: Manufacturer s name Pyriproxyfen technical Batch or reference number, and date of test Net weight of contents Date of manufacture and the following minimum cautionary notice. Pyriproxyfen is an insect growth regulator. Avoid getting on mouth, skin, in eyes or clothing and wear protective clothing. If eyes are splashed, immediately flush eyes and continue for 15 minutes with large amounts of water. Get medical attention. If skin is contacted, remove all contaminated clothing at once. Thoroughly wash skin with soap and water. Get medical attention. Container should be stored in a cool, dry, well ventilated area and away from heat and flame. Keep containers closed when not in use. Keep containers out of reach of children and well away from foodstuffs and animal feeds and their containers. 2. Methods of determining chemical and physical properties 2.1 Pyriproxyfen content Outline of method The sample is dissolved in methanol containing di-isopropyl phtalate as an internal standard. Separation is carried out by high-performance liquid chromatography, using a silica gel column and a mixture of acetonitrile and water as the mobile phase Apparatus 1. Liquid chromatograph. The instrument should be one that is designed for use with stainless steel columns and that is equipped with a pumping system able to generate more than 17 Mpa pressure and a UV spectrophotometric detector able to measure UV absorbance at 254 nm. 2. Liquid chromatographic column. The column should be a stainless steel tube 25 cm long and 4 mm in internal diameter, packed with 5 µm octadecylsilanized silica gel (LiChrosorb RP-18) or equivalent. Page 2 of 9
3 WHO/IS/TC/715/2001 TECHNICAL Reagents Pyriproxyfen standard. Analytical standard of known purity Internal standard. Analytical reagent grade of di-isopropyl phtalate Methanol. Analytical reagent grade Acetonitrile. Analytical reagent grade Mobile phase. A mixture of acetonitrile and water (65:35) Preparation of calibration solutions Internal standard solution. Weigh about g of di-isopropyl phtalate into a 250 ml volumetric flask, dissolve and dilute to volume with methanol. Store under refrigeration, remove from refrigerator and allow to reach room temperature naturally before using. Pyriproxyfen calibration solutions. Weigh ( to the nearest 0.1 mg) approximately 30 and 50 mg quantities of pyriproxyfen standard into separate vials. Add by pipette 10 ml of the internal standard solution. Label these solutions as A and B. The solution B is the working calibration solution for liquid chromatography. The solutions A is used to guard against weighing error in the preparation of the calibration solution. The supply of the solution B can be replenished from time to time without preparing new supplies of the solution A Equilibration of the system Pump sufficient mobile phase through the column to equilibrate the system. When the column has come to equilibrium at the conditions described below, inject 5 µl aliquots of the calibration solution B until a constant response is obtained. To meet this requirement, the response ratios (area of pyriproxyfen peak/area of the internal standard peak) of three consecutive injections must agree to within 2% Operating conditions for high-performance liquid chromatography The conditions given below are typical values and may have to be adjusted to obtain optimum results from a given apparatus. Column temperature Flow rate Wavelength Retention times Pyriproxyfen Internal standard Ambient 1.0 ml/min 254 nm 18 minutes 7.4 minutes Linearity check Inject triplicate aliquots of appropriate volume of the calibration solutions A and B into the liquid chromatograph, determine the response ratio for each injection, and average the resulting ratios for each solution. Divide the average response ratio for each solution by the corresponding pyriproxyfen content (in mg) and compare the resulting response factors. These factors should agree to within 2%. Failure to meet this requirement indicates either a weighing error in the preparation of one of the calibration solutions or instrument difficulties, which must be corrected before proceeding with the analysis of samples. Page 3 of 9
4 WHO/IS/TC/715/2001 TECHNICAL Sample preparation Weigh (to the nearest 0.1 mg) in duplicate a quantity of the sample containing about 50 mg of pyriproxyfen directly into vials. Add by pipette 10 ml of the internal standard solution. The sample should be warmed at 40 ± 2 C and thoroughly mixed before weighing Analysis of sample Inject duplicate aliquots of appropriate volume of the calibration solution B. Calculate the response ratios by dividing the area (or height) of the pyriproxyfen peak by that of the internal standard peak. Response ratios should agree to within 2%. Average the duplicate response ratios obtained with the calibration solution B. Inject duplicate aliquots (same volume as that used in the preceding step) of each of the sample solutions. The precision considerations discussed in the preceding step apply here also. Average the duplicate response ratios for each sample solution. After duplicate injections of two sample solutions, inject duplicate aliquots of the calibration solution B. Average the response ratios of the calibration solution injections immediately before and after the sample solutions. Use the average to calculate the pyriproxyfen content of the two sample solutions. Each determination of response ratio should give a yield equal to within 2% of the previously determined ratio Calculation For each injection the response ratio r is given by the equation: r = Area of pyriproxyfen peak Area of internal standard peak Pyriproxyfen content(g/kg)= Where: r 2 x m 1 x P r 1 x m 2 r 1 = average response ratio for the calibration solution B r 2 =average response ratio for the sample solution m 1 =mass(mg) of pyriproxyfen standard in the calibration solution B m 2 =mass(mg) of sample taken P=purity of pyriproxyfen standard(g/kg) Page 4 of 9
5 Interim specification WHO/IS/GR/715/ Specification 1.1 Description The material shall consist of granules containing technical pyriproxyfen complying with the requirements of WHO specification WHO/IS/TC/715/2001 together with clay and any necessary formulants. It shall be on the form of dry, free flowing, essentially non dusty granules, free from visible extraneous matter and hard lumps. 1.2 Chemical and physical requirements The material, sampled from any part of the consignment (see method WHO/M/1.R1), shall comply with the requirements of section 1.1 and with the following requirements Pyriproxyfen content (g/kg basis) The pyriproxyfen content shall be declared and, when determined by the method described in section 2.1, shall not differ than the declared content by more than the following amounts: Declared content Up to 25 g/kg Permitted tolerance ± 15% of the declared content The mean measured content obtained shall not be lower than the declared content Apparent density The apparent density shall be not less than 0.90 g/ml and not more than 1.0 g/ml when tested by the method described in the CIPAC method MT58.3 (CIPAC Handbook F, p.175) Sieving Not less than 95% of the granules shall pass through a 1.0 mm sieve and not more than 5% shall pass through a 300 µm sieve when tested by the method described in section Dustiness Dustiness, determined by the CIPAC method MT 171 (CIPAC Handbook F, p.425), shall not exceed 0.5 mg Attrition resistance Attrition resistance, determined by the CIPAC method MT 178 (CIPAC Handbook H, p.302), shall be more than 98%. Page 5 of 9
6 Storage stability The content of pyriproxyfen, when determined by the method described in the section 2.1, shall be lower than 4.7 kg/kg after storage at 54 C ± 2 C for 2 weeks according to the CIPAC method MT 46 (CIPAC Handbook F, p.148). The determined average active ingredient content shall not be lower than 95% relative to the determined average content found before storage and physical properties must comply with the requirements of sections 1.2.3, and Packing and marking of packages The pyriproxyfen granules shall be packed in suitable, clean, airtight containers, as specified in the order. All packages shall bear, durably and legibly marked on the container, the following. Manufacturer s name Pyriproxyfen granules Batch or reference number, and date of test Net weight of contents Date of formulation Instruction for use and the following minimum cautionary notice. Pyriproxyfen is an insect growth regulator. If swallowed, keep patient prone and quiet, call a physician. If eyes are splashed, immediately flush eyes and continue for 15 minutes with large amounts of water. Get medical attention. If skin is contacted, remove all contaminated clothing at once. Thoroughly wash skin with soap and water. Get medical attention. Keep containers out of children and well away from foodstuffs and animal feeds and their containers. 2. Methods of determining chemical and physical properties 2.1 Pyriproxyfen content Outline of method Pyriproxyfen is extracted from the sample with methanol and di-isopropyl phtalate is added as an internal standard. Separation is carried out by high-performance liquid chromatography, using silica gel column and a mixture of acetonitrile and water as the mobile phase Apparatus 1. Liquid chromatograph. The instrument should be one that is designed for use with stainless steel column and that is equipped with a pumping system able to generate more than 17 Mpa pressure and a UV spectrophotometric detector able to measure UV absorbance at 254 nm. 2. Liquid chromatographic column. The column should be a stainless steel tube 25 cm long and 4 mm in internal diameter, packed with 5 um octadecylsilanized silica gel (LiChrosorb RP-18) or equivalent. Page 6 of 9
7 2.1.3 Reagents Pyriproxyfen standard. Analytical standard of known purity Internal standard. Analytical reagent grade of diisopropyl phtalate Methanol. Analytical reagent grade Acetonitrile. Analytical reagent grade Mobile phase. A mixture of acetonitrile and water (65:35) Preparation of calibration solutions Internal standard solution. Weigh g of diisopropyl phtalate into 250 ml volumetric flask, dissolve and dilute to volume with methanol. Store under refrigeration, remove from refrigerator and allow to reach room temperature naturally before using. Pyriproxyfen calibration solutions. Weigh(to the nearest 0.1 mg) approximately 50 mg of pyriproxyfen standard into 50 ml volumetric flask, dissolve and dilute to volume with methanol. Pipette exactly 3 and 5 ml of this solution into separate conical flasks, add exactly a 10 ml aliquot of the internal standard solution and mix well to make calibration solutions. Label these solutions as A and B. The solution B is the working calibration solution for liquid chromatography. The solutions A is used to guard against weighing error in the preparation of the calibration solution. The supply of the solution B can be replenished from time to time without preparing new supply of the solution A Equilibration of the system Pump sufficient mobile phase through the column to equilibrate the system. When the column has come to equilibrium at the conditions described below, inject 5 µl aliquots of the calibration solution B until a constant response is obtained. To meet this requirement, the response ratios(area of pyriproxyfen peak/area of the internal standard peak) of three consecutive injections must agree to within 2% Operating conditions for gas-liquid chromatography The conditions given below are typical values and may have to be adjusted to obtain optimum results from a given apparatus. Column temperature Flow rate Wavelength Retention times Pyriproxyfen Internal standard Ambient 1.0 ml/min 254 nm 18.0 minutes 7.4 minutes Linearity check Inject triplicate aliquots of appropriate volume of the calibration solution A and B into the liquid chromatograph, determine the response ratio for each injection, and average the resulting ratios for each solution. Divide the average response ratio for each solution by the corresponding pyriproxyfen content ( in mg ) and compare the resulting response factors. These factors should agree to within 2%. Failure to meet this requirement indicates either a weighing error in the preparation of one of the calibration solutions or Page 7 of 9
8 instrument difficulties, which must be corrected before proceeding with the analysis of samples Sample preparation Sampling. Homogenize the bulk material before taking at least 100 g as a sub-sample for analysis. Preparation of the sample solutions. Homogenize the material by the method given here above for sampling. Weigh ( to the nearest 1 mg ) 2 g quantity of sample into 50 ml screw vial, add 20 ml of the internal standard solution and 10 ml of methanol into the vial. Extract pyriproxyfen from the sample with ultrasound for 10 minutes. Filter the extract through membrane filter to prepare sample solution Analysis of sample Inject duplicate aliquots of appropriate volume ( as determined under section ) of the calibration solution B. Calculate the response ratios by dividing the area ( or height ) of the pyriproxyfen peak by that of the internal standard peak. Response ratios should agree to within 2%. Average the duplicate response ratios obtained with the calibration solution B. Inject duplicate aliquots ( same volume as that used in the preceding step ) of each of the sample solutions. The precision considerations discussed in the preceding step apply here also. Average the duplicate response ratios for each sample solution. After duplicate injections of two sample solutions, inject duplicate aliquots of the calibration solution B. Average the response ratios of the calibration solution injections immediately before and after the sample solutions. Use the average to calculate the pyriproxyfen content of the two samples. Each determination of response ratio should give a yield equal to within 2% of the previously determined ratio Calculation For each injection the response ratio r is given by the equation: r = Area of pyriproxyfen peak Area of internal standard peak Pyriproxyfen content (g/kg) = r 2 x m 1 x P r 1 x m 2 Where: r1 = average response ratio for the calibration solution B r 2 =average response ratio for the sample solution m 1 = mass (mg) of pyriproxyfen standard in the calibration solution B m 2 = mass (mg) of sample taken P = purity of pyriproxyfen standard (g/kg) Page 8 of 9
9 2.2 Sieving test Place 100 g of the sample (weighed to the nearest 0.1 g) on a clean, dry, 1.0 mm sieve, nested over a 300 µm sieve and a receiver. Shake the sieves by hands in a rotary horizontal direction and vertically by tapping on a hardwood surface for three minutes. Disconnect the sieves and brush away any material on the underside of each sieve on to the sieve or receiver below it. Weigh the material on the receiver, 300 µm sieve(w) and 1.0 mm sieve. Calculate % of material passing through the 1.0 mm sieve and remaining on the 300 µm sieve by the following equation. % = W x Page 9 of 9
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