GLENMARK GENERICS LIMITED, GOA QUALITY CONTROL DEPARTMENT STANDARD TEST PROCEDURE METHODOLOGY FOR DETERMINATION OF EXTRAN RESIDUES IN SWAB SAMPLES
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1 GRADE : IN - HOUSE PAGE No. : 1 of 5 Instruments Required: (a) Analytical Balance (b) Spectrophotometer, Pharo 100 Spectroquant [EQC 372] Materials Required (a) Purified water (b) Phosphate test kit from Merck, Germany -Catalogue.No.14848, consisting of following components Component_1: Reagent Po4-1 [Liquid] Component_2: Reagent Po4-2 [Solid] (c) Extran MA02 Neutral from Merck specialities (I) Ltd, Catalogue.No (d) Swab stick from Tex wipe Instrument settings Measurement mode : Concentration Method id : Selection Cuvette Wavelength Ordinate mode : From method list : 20mm : 715nm : Absorbance Allow 15 minutes to stabilize the instrument, after the instrument start up
2 GRADE : IN - HOUSE PAGE No. : 2 of 5 Blank preparation Take 1 swab [intact/pure swab stick available] in a test tube add 5ml of water and sonicate for 5minutes. Test preparation Take 1 swab [given from manufacturing for analysis] in a test tube add 5ml of water and sonicate for 5minutes. [.1] Standard stock preparation for Batch suitability Weigh accurately 2.2g of Extran MA 02 Neutral, into a 1000ml cleaned & dried volumetric flask. Dilute up to the volume with purified water and mix. Extran Solution [110µg of Extran w.r.t swab] Pippete 10.0ml of the above Extran stock solution to 1000ml with purified water and mix. Standard preparation Pippete 5.0ml of the above Extran solution in a test tube. [*] Procedure To each of test tubes [Blank, Standard and Test preparation] add the following components in sequence. 1 Reagent Po4-1 5 drops 2 Reagent Po4-2 1 level blue µ spoon [in the cap of Po4-2 bottle].1-batch suitability need to performed, only when the Lot of Extran is changed in manufacturing area. Obtain a representative sample [about 10ml] of Extran MA02 Neutral, from the Production / manufacturing area. If lot of Extran remains the same, Skip the "Standard stock preparation for Batch suitability" proceed directly to the Procedure[*] avoiding standard preparation
3 GRADE : IN - HOUSE PAGE No. : 3 of 5 Add and shake gently until the reagent is completely dissolved. Leave it to stand for 5min (Reaction time). Filter the solutions [Blank, Test & Standard] using 0.45µ Nylon membrane filter. Perform Blank value correction. Determine the concentration of Extran in Test preparation and Standard preparation (if applicable) Acceptance criteria for Standard preparation In cases, If batch suitability is performed, the instrument readout for Extran concentration, should be in the range of NMT 110 ± 5.00µg of Extran/swab. If Batch suitability, is above the specified tolerance levels then recalibrate the instrument with the available Extran lot as per procedure mentioned in.2 Limit of Detection Limit of Quantitation 9.61µg of Extran/swab 33.14µg of Extran/swab Range of Analytical method 33.14µg to µg of Extran/swab. Limit NMT 110µg of Extran/swab..2-Recalibration need to performed, only when the Batch suitability is not met the acceptance criteria [.2] Standard stock preparation for Recalibration Weigh accurately 2.2g of Extran MA 02 Neutral, into a 1000ml cleaned & dried volumetric flask. Dilute up to the volume with purified water and mix. Blank preparation Pippete 5.0ml of Purified water to a test tube
4 GRADE : IN - HOUSE PAGE No. : 4 of 5 Prepare calibration solutions as per the below table. Standard Volume [in ml] of Extran concentration levels Extran Stock solution [ in µg w.r.t swab] to be taken Calibration solutions Pippete 5.0ml of each above standard levels into individual test tube. level '0' of standard. Procedure to be followed Pipette as per mentioned volume of Extran stock solution into another cleaned & dried 1000ml volumetric flask, containing 20ml of Water & make up to the volume with Water & mix. [Standard level 5 Limit level Procedure To each of test tubes [Blank, and Standard] add the following components in sequence. 1 Reagent Po4-1 5 drops 2 Reagent Po4-2 1 level blue µ spoon [in the cap of Po4-2 bottle] Add and shake gently until the reagent is completely dissolved. Leave it to stand for 5min (Reaction time). Perform Blank correction measurement & enter the obtained blank value at the
5 GRADE : IN - HOUSE PAGE No. : 5 of 5 Perform duplicate measurement of each standard levels & carry out Regression analysis for the obtained data values. X-variable : Extran concentration in µg/swab :Y-variable : Measured Absorbance Acceptance criteria for recalibration Following parameters should be satisfied from regression analysis. Correlation coefficient [R 2 ] NLT Slope [m] 0.03 ± 1 % b intercept [%b] NMT ± 2.0% Standard error [S r ] NMT 0.25µg of Extran /Swab Coefficient of variation of method [CV m ] NMT 2.0% If all the above regression parameter are within the acceptance criteria, proceed to determine the amount of Extran in Test preparation as mentioned in procedure [*] Notes on photometric measurement (i) Hold the bottle vertically [90 position] while adding reagent Po4-1 reagent (ii).colour of measurement solution, remains stable for at least 60min after the end of reaction time Precaution[s] (i).use only phosphate free detergent to rinse the glassware (Volumetric flasks & pipettes).other wise fill the glass ware with Hydrochloric acid (approx 10%) and leave it to stand for at least 8 hours before use. (ii).all glassware's [Cuvette & test tubes] coming into contact with the coloured solution must be cleansed with Sodium hydroxide solution (approx 0.4%) and leave to stand for minimum 1hour
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