新西兰双氰胺检测方法.doc

AsureQuality Limited Wellington LaboratoryWC 180Version 1 Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by Liquid Chromatography Tandem Mass Spectrometry LC MS MS Page 1 of 16Jan 2013 QA Controlled Document Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by High Performance Liquid Chromatography Tandem Mass Spectrometry LC MS MS 1 CONFIDENTIALITY STATEMENT This method is the property of AsureQuality Ltd and cannot be copied used or distributed without the written permission of the Wellington Laboratory Business Manager 2 SAFETY AND HEALTH MSD sheets of all chemicals solvents and standards must be understood by the analyst before commencing the method Avoid inhaling vapors or chemicals Use the assigned protective clothing and equipment at all times Ensure that you are familiar with the location of fire extinguishers first aid kit and any safety equipment that may be required Refer to the Laboratory s Hazard Register for Significant Hazards identified and the control methods specified to minimise these hazards 2 1 SPECIFIC HAZARDS AND CONTROL METHODS Not Applicable 3 DEFINITIONS 3 1 ABBREVIATIONS DCDDicyandiamide HPLC High Performance Liquid Chromatography IR Ion Ratio IS Internal Standard KTP Key Technical Personnel LC MS MS Liquid Chromatography Tandem Mass Spectrometry LIMSLaboratory Information Management System LOD Limit of Detection LOQ Limit or Level of Quantification LORLimit of Reporting M Molecular Mass MRM Multiple Reaction Monitoring MSD sheets Material Safety Data sheets PVDFPolyvinylidene Fluoride Q1 Quantification ion AsureQuality Limited Wellington LaboratoryWC 180Version 1 Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by Liquid Chromatography Tandem Mass Spectrometry LC MS MS Page 2 of 16Jan 2013 QA Controlled Document Q2 Secondary quantification check ion QC Quality Control RR Relative Response RS Recovery Standard RT Retention Time SOP Standard Operating Procedure U Uncertainty of Measurement with 95 confidence interval WS Working Standard 3 2 DEFINITION OF TERMS Analyte This is the component of a sample that has to be detected identified and quantified The term analyte includes where appropriate derivatives formed from the analyte during analysis Analytical samples Samples required to be analysed quantitatively for the presence of target analytes External Standard A solution in the HPLC solvent which has been fortified with known amounts of analytes and internal standard Internal Standard IS Compound added at the beginning of the analysis which is similar to the target analytes in chemical composition and behaviour Limit of Detection LOD That concentration of analyte which gives an instrument signal significantly different from the blank Commonly defined as the mean blank level plus 3SD of blank measurements Limit or Level of Quantification LOQ Regarded as the lower limit for precise quantitative measurements Commonly defined as either the mean blank level plus 10SD of blank measurements or 3LOD Commonly is represented by the lowest concentration calibration standard so that samples cannot be quantified below this standard by definition Limit of Reporting LOR The practical limit of residue quantification at or above the LOQ The LOR must be no lower than the lowest calibration standard a limit specified by a regulatory body or client Matrix Blank Recovery Blank A blank prepared from the same type as the matrix of interest Matrix Recovery Sample Recovery Sample A sample prepared from the matrix blank to which is added known amounts of analytes and internal standard in order to establish the efficacy of the method In this method they are also used as calibration samples to quantify analytes in analytical samples Reagent blank determination This is the complete analytical procedure applied with omission of the test portion or using an equivalent amount of suitable solvent in place of the test portion Relative Response RR Ratio of the response of an analyte to its internal standard Recovery Standard RS Compound added at the end of the analysis AsureQuality Limited Wellington LaboratoryWC 180Version 1 Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by Liquid Chromatography Tandem Mass Spectrometry LC MS MS Page 3 of 16Jan 2013 QA Controlled Document 4 INTRODUCTION AND OVERVIEW 4 1 INTRODUCTION Food contaminant analysis in agricultural products is of vital importance for the safety of our food and to safeguard our export markets 4 2 SCOPE The method has been validated in milk and milk powders It can be used for other similar matrices provided that it is demonstrated that the method performance values are met Additional validation and records that may be required will be according to the relevant validation procedure of the laboratory NOTE Refer to the relevant validation procedure for validation actions required if additional matrices analytes to those already validated are required to be used by the method 4 3 METHOD SUMMARY Samples of milk or reconstituted dairy powder are diluted with acetonitrile sodium chloride is added and after centrifugation filtration and addition of a recovery standard the resulting acetonitrile solution is analysed by LC MS MS 4 4 ANALYTES Analytes and Internal Standards are summarised in Table 1 TABLE 1 Test Articles Analytes and Internal Standards Test ArticlesCAS Number Analytes Dicyandiamide 2 cyanoguanidine 461 58 5 M 84 Cyanuric Acid108 80 5 M 129 Melamine108 78 1 M 126 Internal Standards 13C3 15N3 Melamine Recovery Standard M 132 13C3 Melamine1173022 88 2 M 129 13C3 Cyanuric Acid201996 37 4 M 132 M molecular mass 4 5 STRUCTURES Not Applicable AsureQuality Limited Wellington LaboratoryWC 180Version 1 Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by Liquid Chromatography Tandem Mass Spectrometry LC MS MS Page 4 of 16Jan 2013 QA Controlled Document 5 REFERENCED DOCUMENTS AM 006 Analytical Methods Unidentified Analytical Responses Investigation and Record Keeping AM 013 Chemicals Reagents and Solutions AsureQuality Limited Validation Report VR 290 Determination of Cyanuric Acid and Melamine in Milk and Powdered Milk products by High Performance Liquid Chromatography Tandem Mass Spectrometry LC MS MS October 2008 AsureQuality Limited Validation Report VR 441 Determination of Dicyandiamide Cyanuric Acid and Melamine in Milk and Powdered Milk products by High Performance Liquid Chromatography Tandem Mass Spectrometry LC MS MS May 2010 AsureQuality Limited Validation Report VR 499 Determination of Cyanuric Acid and Melamine in Milk and Powdered Milk products by High Performance Liquid Chromatography Tandem Mass Spectrometry LC MS MS Supplementary validation to VR 290 January 2011 EQ 004 Working Standards Preparation Checking and Records EQ 043 Equipment Procedure for LC MS MS EQ 058 Primary Standards Ordering Storage and Records QC 025 Uncertainty of Measurement in Analytical Results QC 026 Control Charts Procedure QC 028 Quality Control Blind Spike Intra Laboratory Check Sample Scheme QC 029 Quality Control Procedures for Analytical Results SR 012 Sample Preparation 6 REAGENTS Refer to AM 013 DO NOT USE EXPIRED CHEMICALS REAGENTS OR SOLUTIONS All reagents and chemicals must be of such a grade that they do not interfere with the analytical process Use the stated grade or equivalent 6 1 CHEMICALS Acetic acid analytical grade Acetonitrile pesticide grade Ammonium acetate analytical grade Deionised water Dichloromethane analytical grade Sodium chloride analytical grade 6 2 SOLUTIONS HPLC Solvent A Into a 1000 mL Schott bottle add 0 77 g of ammonium acetate and 1000 mL of deionised water Stir until the solid is dissolved AsureQuality Limited Wellington LaboratoryWC 180Version 1 Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by Liquid Chromatography Tandem Mass Spectrometry LC MS MS Page 5 of 16Jan 2013 QA Controlled Document Store at room temperature Stable for 2 weeks HPLC Solvent B Into a 2000 mL Schott bottle add 1 54 g of ammonium acetate and 60 mL of deionised water Stir until the solid is dissolved Using a 2000 mL measuring cylinder add 1940 mL of acetonitrile Warm the bottle to approximately 50oC water bath to aid complete dissolution of ammonium acetate Store at room temperature Stable for 2 weeks 7 STANDARDS Refer to SOPs EQ 004 and EQ 058 Upon receipt an orange Waiting Approval label is placed on the standard Once the standard has been found to be acceptable as determined in EQ 058 the orange label is overlaid by a green QC Released label dated and initialled NOTE Do not use expired primary standards or standard solutions Do not use standards which have not been released approved for use by a Senior Scientist Scientist approved by the Technical Manager or the Technical Manager 7 1 PRIMARY STANDARDS Melamine is available from Acros Organics 99 Catalogue number 22048 Cyanuric acid is available from Aldrich 98 Catalogue number 185809 25G Dicyandiamide is available from Aldrich 99 Catalogue number 76609 13C315N3 Melamine Recovery Standard is available from Cambridge Isotope Laboratories 100 g mL in water Catalogue number CNLM 8150 1 2 13C3 Melamine Internal Standard VETRANAL 10 mg is available from Sigma Aldrich Catalogue number 32666 10MG 13C3 Cyanuric Acid Internal Standard VETRANAL 10 mg is available from Sigma Aldrich Catalogue number 32679 10MG 7 2 SECONDARY STANDARDS 7 2 1 Unlabelled Melamine and Cyanuric Acid Standards 100 g mL Weigh accurately 10 0 mg of standard into a 125 mL polypropylene bottle and add 100 g of deionised water by weight to give a 100 g mL solution Cap and shake thoroughly to dissolve the compound Store at room temperature Solutions are stable for two years 7 2 2 Dicyandiamide Standard 1 mg mL Weigh accurately 10 0 mg of standard into a 10 mL calibrated volumetric flask Add deionised water shake to dissolve and make up to volume Transfer to a 20 mL screw cap vial and cap tightly Store at room temperature in the dark Solution is stable for six months 7 2 3 Recovery Standard 1 g mL Remove 1 2 mL of the primary standard solution of 13C315N3 melamine with a calibrated Hamilton syringe and dilute to 120 mL in a 125 mL polypropylene pottle with water to give a 1 g mL solution AsureQuality Limited Wellington LaboratoryWC 180Version 1 Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by Liquid Chromatography Tandem Mass Spectrometry LC MS MS Page 6 of 16Jan 2013 QA Controlled Document Secondary standards have an expiry date of two years from the date of preparation of solution or the expiry date of the primary standard whichever is earlier These standards are to be stored at room temperature 7 2 4 Internal Standards 100 g mL Weigh accurately 10 0 mg of standard into a 125 mL polypropylene bottle and add 100 g of deionised water by weight to give a 100 g mL solution Cap and shake thoroughly to dissolve the compound Store at room temperature Solutions are stable for two years 7 3 WORKING SOLUTIONS Working standard solutions are prepared as required and have a general expiry date as that of the secondary standard solution it is prepared from Working standards are stored at room temperature 7 3 1 Analyte Working Standard Solutions 10 g mL Individual working standard solutions for melamine and cyanuric acid are prepared from each secondary standard solution by adding 10 mL of the secondary standard solution to a 100 mL calibrated volumetric flask and make up to volume with deionised water For dicyandiamide the working standard solution is prepared by adding 250 L of the secondary standard solution to a 25 mL calibrated volumetric flask and making up to volume with deionised water NOTE Working standard solutions are added individually to the recovery samples Refer to section 10 1 1 1 7 3 2 External Standard Solution 0 05 g mL An external standard solution 0 05 g mL is prepared by adding 5 L each of solutions 7 3 1 and 7 3 3 and 50 L of 7 2 3 to a 2 mL HPLC vial and adding 925 L acetonitrile capping the vial and shake to mix the solution 7 3 3 Internal Standard Working Solutions 10 g mL Individual internal standard working solutions are prepared from each secondary standard solution by adding 10 mL of the secondary standard solution to a 100 mL calibrated volumetric flask and make up to volume with deionised water 8 APPARATUS NOTE Where specific equipment is listed other brands or models may be used provided that they have equivalent performance 8 1 LABORATORY EQUIPMENT Balance Electronic analytical Balance pan balance for weighing samples to 2 decimal places Centrifuge capable of centrifuging the 50 mL pp tubes at 3500 rpm Dispenser with adaptor for 4 L bottles of solvent Filters 17 mm 0 45 m PVDF and 2 mL plastic syringes Pasteur pipettes Polypropylene pottle 125 mL AsureQuality Limited Wellington LaboratoryWC 180Version 1 Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by Liquid Chromatography Tandem Mass Spectrometry LC MS MS Page 7 of 16Jan 2013 QA Controlled Document Positive displacement pipettes Repeat dispenser Schott bottles 1000 mL and 2000 mL Shaker Horizontal reciprocal Shaker Orbital Tubes Polypropylene 50 mL conical Ultrasonic bath Vials 2 mL and capped with teflon lined caps for HPLC autosampler Vortex mixer Water bath capable of operating at 70 C 8 2 ANALYTICAL INSTRUMENTATION HPLC column TOSOH Bioscience LLC TSKgel Amide 80 150 mm x 2 1 mm column with 3 m particle size Instrument Agilent Model 1200 HPLC interfaced with an Applied Biosystems Model 4000 QTrap Mass Spectrometer controlled by computer with Analyst Software or equivalent 9 SAMPLING AND SAMPLE PREPARATION 9 1 GENERAL Batches on the LC MS MS will be given an unique batch number This number will be entered into the appropriate instrument logs and sample worksheets A batch using this method will be named FMC 06 xxx where xxx is a unique number from 001 to 999 Refer to Attachment WC 180 1 9 2 PREPARATION OF A TEST SAMPLE Samples of dairy products are analysed as received and a test portion is weighed directly from the sample container Samples of unhomogenised milk are agitated gently to evenly distribute the fat and a test portion is immediately weighed into a 50 mL tube with the aid of a positive displacement pipette Homogenised milk is weighed directly from the sample container Refer to SR 012 9 3 PREPARATION OF A TEST PORTION Weigh test portions into 50 mL polypropylene conical tubes For milk powders whole milk powder skim milk powder and buttermilk powder liquid milk butter and anhydrous milk fat AMF a test portion of 5 0 0 1 g is to be weighed For all other dairy products such as caseinate casein cheese colostrum lactoferrin milk protein concentrate nutritional products whey protein concentrate and whey protein isolate a test portion of 2 0 0 05 g is to be weighed 10 PROCEDURE AsureQuality Limited Wellington LaboratoryWC 180Version 1 Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by Liquid Chromatography Tandem Mass Spectrometry LC MS MS Page 8 of 16Jan 2013 QA Controlled Document NOTE 1 Unless values for weight and volume have either a sign or the word exactly then they are to be taken as approximate values NOTE 2 All samples are tested singly as either 5 g or 2 g test portions of the supplied sample Deviations from this may be where on repeat testing there was insufficient sample or a confirmation that require less sample to minimize background interferences 10 1DETERMINATION 10 1 1Procedure 10 1 1 1Samples and blanks are fortified according to the schedules in Table 2 and Table 3 using the working standard solutions prepared in section 7 TABLE 2 Fortification of Blanks Recoveries and Samples 5 g test portions SampleVolume of Analyte WS L Volume of IS WS L Concentration Analyte mg kg Concentration IS mg kg Matrix Rec Blank 010000 2 Rec 1251000 050 2 Rec 2501000 10 2 Rec 31001000 20 2 Rec 42501000 50 2 QC Sample501000 10 2 Samples010000 2 Reagent Blank010000 2 NOTE Wait for 10 minutes before proceeding with extraction after fortification TABLE 3 Fortification of Blanks Recoveries and Samples 2 g test portions SampleVolume of Analyte WS L Volume of IS WS L Concentration Analyte mg kg Concentration IS mg kg Matrix Rec Blank 010000 5 Rec 1101000 050 5 Rec 2201000 10 5 Rec 3401000 20 5 Rec 41001000 50 5 QC Sample201000 10 5 Samples010000 5 Reagent Blank010000 5 NOTE Wait for 10 minutes before proceeding with extraction after fortification AsureQuality Limited Wellington LaboratoryWC 180Version 1 Determination of Dicyandiamide Cyanuric Acid and Melamine in Dairy Products by Liquid Chromatography Tandem Mass Spectrometry LC MS MS Page 9 of 16Jan 2013 QA Controlled Document 10 1 2Procedure Butter and AMF samples 10 1 2 1Add 10 mL of water and 20 mL of dichloromethane shake vigorously by hand for 20 seconds to mix 10 1 2 2Place the tubes in an ultrasonic bath for 15 minutes shaking by hand every 5 minutes 10 1 2 3Centrifuge the tubes after shaking at 3500 rpm for 10 minutes 10 1 2 4Transfer 5 mL of the top water layer into a 50 mL polypropylene tube containing 5 mL of acetonitrile 10 1 2 5Add one level 1 25 mL spoonful of sodium chloride to the tubes shake vigorously by hand then on the horizontal shaker for 10 minutes 10 1 2 6Centrifuge the tubes after shaking at 3500 rpm for 10 minutes 10 1 2 7Transfer 1 0 mL of the supernatant solution to a 2 mL HPLC vial containing 50 L of the recovery secondary standard solution 10 1 2 8Mix the vial contents with the tip of a clean Pasteur pipette and filter the solution through a PVDF filter into a clean HPLC vial and cap 10 1 2 9Analyse the samples by LC MS MS 10 1 3Procedure Liquid Milk Samples 10 1 3 1Place the tubes in a 70 C water bath for 30 minutes 10 1 3 2Remove the tubes from the water bath and immediately add 5 0 mL of acetonitrile 10 1 3 3Place on the horizontal shaker and shake for 10 minutes 10 1 3 4Add one level 1 25 mL sp