Agilent Rapid Screening of Melamine Cyanuric Acid in Milk Products Using Agilent J W HP-5ms GC Column Agilent 7890A/5975C GC/MSD with Column Backflushing Manual
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1. was obtained for the two compounds with in the range of 10 to 200 ug g with a correlation coefficient higher than 0 9996 Abundance 3e 08 2 8e 08 2 6e 08 2 4e 08 2 2e 08 2e 08 1 8e 08 1 6e 08 1 4e 08 1 2e 08 1e 08 8e 07 6e 07 4e 07 2e 07 Scan mode Cyanuric acid tri TMS To check the applicability of the method a powdered infant formula blank matrix spiked with 40 ug g of targeted ana lytes was analyzed Excellent recoveries were obtained with 96 1 for cyanuric acid and 95 6 for melamine see Table 3 Melamine tri TMS T 4 50 5 00 5 50 6 00 6 50 7 00 Time Abundance 1 1e 07 1e 07 9000000 8000000 SIM mode 7000000 6000000 5000000 4000000 3000000 2000000 1000000 7 50 8 00 8 50 9 00 T a oa er re a 4 50 5 00 5 50 6 00 6 50 7 00 Time Figure 4 TICs of powdered infant formula sample 7 50 8 00 8 50 9 00 3500000 3000000 2500000 v 2 2000000 o E 2 1500000 1000000 500000 i 282 9 315 0 402 9 439 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m z 4500000 4000000 3500000 3000000 o 2 2500000 Z 2000000 lt 1500000 1000000 500000 0 362 0 393 1 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m z Figure 5 Mass spectra of cyanuric acid tri TMS derivative top and melamine tri TMS derivative bottom Table 3 Recovery of Spiked Sample Concl2. 99 cyanuric acid 345 330 188 MS temperature Scan mode SIM mode Backflush Conditions Device 3 way splitter P N G3183B Restrictor 0 706 m x 180 pm id Outlet PCM P N G1530 63309 2 psi 60 psi for post run 2 psi for post run Outlet pressure Inlet pressure Quantitative ion Sample Preparation Extraction 0 5 g of sample powder or liquid was weighed into a 20 mL polypropylene centrifuge tube 5 mL of methanol was added The sample was capped vortex mixed and then sonicated for 10 minutes After the sample was centrifuged at 4 000 rpm for 6 minutes the supernatant fluid was filtered through a 0 45 um PTFE filter into a glass GC vial Derivatization 40 uL of the above extract was transferred into a glass GC vial The extract was evaporated to dryness under a stream of nitrogen at approximately 70 C 50 uL of pyridine and 50 pL of BSTFA were added The sample was vortex mixed and incu bated at 70 C for 30 minutes Results and Discussion 7890A 5975C GC MSD with a Backflush System for Milk Extracts Milk extract usually contains many low volatile or nonvolatile constituents These compounds may stay near the front of the column until the oven temperature is high enough to move them through the column In this application a three way splitter with makeup gas was employed to perform the backflush The device has makeup gas supply tubing and four connectors one connector for the analytical column an
3. Authors Min Cai Chongtian Yu Yun Zou and Wei Luan Agilent Technologies Shanghai Co Ltd 412 Ying Lun Road Waigaoqiao Free Trade Zone Shanghai 200131 China Rapid Screening of Melamine and Cyanuric Acid in Milk Products Using Agilent J amp W HP 5ms GC Column and Agilent 7890A 5975C GC MSD with Column Backflushing Application Note Food Safety Abstract A rapid screening method for the determination of melamine and cyanuric acid in milk products is developed using Agilent 7890A 5975C GC MSD along with Agilent J amp W HP 5ms GC columns With the backflushing by capillary flow technologies this method eliminates the time consumption for column bakeout after elution of target compounds so as to significantly shorten the GC run time from more than 70 minutes to 14 5 minutes Good linearity was obtained within the range of 10 to 200 ug g with correlation coefficients greater than 0 9996 The recoveries of both target compounds are greater than 95 RE Agilent Technologies Introduction The contamination of food with melamine and cyanuric acid has attracted much attention all over the world For the analy sis of milk products one of the major problems is the pres ence of less volatile and nonvolatile matrix components such as protein and fat These components might contaminate the analytical system if the sample is introduced without selec tive sample preparation The usual way to remove the matrix is to bake the co
4. d three connectors for the restrictor tube connecting to three available detectors Since only an MSD is used as a detector in this application the first two connectors were capped in the direction of makeup flow the third connector was for the column in and the last connector for the restrictor out to the MSD This flow configuratin was used to avoid peak broaden ing due to improper flow sweeping The length and internal diameter of the restrictor tubing is 0 706 m and 0 18 mm 7683B 0 706 m x 180 um restrictor Auto sampler ae ett 5975C MSD ayuya Figure 1 30 m x 250 um x 0 25 pm Agilent HP 5ms respectively The schematics of the GC MS system configura tion and tubing connection for the three way splitter are shown in Figure 1 First a milk extract was analyzed in a typical mode without backflush by programming the oven to 300 C to ensure that late eluters were eluted It took more than 70 minutes to elute all the constituents in the extract Figure 2A Then the extract was analyzed with a 5 minute backflush Figure 2B The backflush was accomplished by increasing the pressure in the outlet and decreasing the inlet pressure The column flow was reversed to push the heavy constituents through the column inlet and out of the split vent Figure 2C shows a 70 minute blank solvent run after the backflush The blank run shows that the column was clean after backflushing except for some peaks coming
5. from the vial septa Instead of baking the column at 300 C for 55 minutes the heavy matrix compo nents were effectively removed from the column through backflushing This reduced the run time from 70 minutes to 14 5 minutes or a 4 8 fold increase in speed For a complex matrix even baking for a long time cannot thor oughly remove the high boiler which may result in peak retention time shift in the following injection In Figure 3 two consecutive runs of the extract with 5 minute backflushing are shown Excellent retention time and peak area repeatabili ty were obtained with no evidence of carryover no emerging ghost peaks and no increasing baseline This demonstrated 3 way splitter with make gas 2 ports capped Column in Capped Capped Aux EPC in Schematics of GC MS system configuration and tubing connection for three way splitter le 08 g 8e 07 6e 07 A Analysis without backflush and baking the column for more than 50 min at 300 C 60 00 65 00 55 00 45 00 50 00 45 00 50 00 55 00 60 00 65 00 45 00 55 00 60 00 65 00 50 00 S 4e 07 2 2e 07 0 5 00 10 00 15 00 20 00 25 00 30 00 35 00 40 00 f Time le 08 5min BOA g 8e 07 backflush B Analysis with backflush 6e 07 S 4e 07 PINS E 0 5 00 10 00 15 00 20 00 25 00 30 00 35 00 40 00 Time le 08 g 8e 07 geen C Blank run after backflush S 4e 07 7 2 2e 07 0 5 00 10 00 15 00 20 00 25 00 30 00 35 00 40 00 Ti
6. lumn at high temperature which often takes much longer than the sample run time for the analysis of interest For example the application of a milk extract of FDA method 1 usually takes about 70 minutes Selective extrac tion or selective sample introduction is not easy since the target compounds cover a broad volatility and polarity range Moreover for a routine QC analysis laboratories want to reduce the typical cycle time by 25 to 60 minutes to improve productivity The Agilent 7890A 5975C GC MSD system meets this demand by using backflushing and faster cooling In this application note a modified U S FDA method is devel oped using a standard split splitless inlet and Agilent Capillary Flow Technology A three way splitter device with a makeup gas is placed at the end of the column and connected to MSD with a restrictor thereby allowing column outlet pres sure to be controlled with auxiliary electronic pneumatic con trol EPC By decreasing the column inlet pressure and increasing the column outlet pressure after the last peak of interest eluted from the column the column flow is reversed and the matrix interference especially high boiler can be removed out of the inlet end of the column 2 Experimental Standards and Reagents The standards and reagents used in the experiment are listed in Table 1 Stock solutions of melamine and cyanuric acid Table 1 Standards and Reagents Standard Melamine Sigma Aldrich gt 99 p
7. me Figure 2 Time savings using backflush that backflushing is a perfect solution to avoid both high tem perature baking and retention time shift from run to run Meanwhile the faster oven cooling down capability of the Agilent 7890A allows for shorter cycle time Additional time savings can be realized by using the three way splitter used in this application so that the liner and column can be changed without venting the MSD 3e 08 2 8e 08 2 6e 08 2 4e 08 2 2e 08 2e 08 1 8e 08 1 6e 08 1 4e 08 1 2e 08 1e 08 8e 07 6e 07 4e 07 2e 07 Abundance had l 6 50 Time _ J 6 00 7 00 Figure 3 Figure 4 shows the total ion chromatograms TIC of a spiked milk powder sample Synchronous SIM scan was used to monitor ions of interest with high sensitivity SIM mode and to simultaneously acquire library searchable scan data in one run This helped simplify the process of confirming positive or negative results Figure 5 shows the mass spectra of cyanuric acid tri TMS derivative 6 335 minutes and melamine tri TMS derivative 7 341 minutes respectively Wlah a 7 50 8 00 Overlay of total ion chromatogram spectra of two consecutive runs of a powdered infant formula extract Linearity and Recovery A matrix blank milk sample is employed for a linearity experi ment 0 5 g of milk samples were spiked with four levels of cyanuric acid and melamine 10 20 80 and 200 ug g Excellent linearity
8. n 5989 1716EN Oct 13 2004 For More Information For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2008 Published in the USA October 8 2008 5989 9866EN 7 Agilent Technologies
9. urity Cyanuric acid Sigma Aldrich gt 99 purity Solvent Methanol Fisher Scientific HPLC grade Pyridine Fisher Scientific Certified A C S Reagent Silylating BSTFA with Supelco reagent 1 TMCS BSTFA bis trimethylsilyl trifluoroacetamide TMCS Trimethylchlorosilane each at a concentration of 1 000 ug mL were separately pre pared in methanol Stock solutions of standards are stored in the refrigerator Instrument The experiment was performed on an Agilent 7890A gas chro matograph equipped with a split splitless capillary inlet an Agilent 5975C GC MSD with Triple Axis Detector and an Agilent 7683B automatic liquid sampler ALS The split split less inlet is fitted with a long lifetime septum P N 5183 4761 and split injection liner P N 5188 4647 Injections are made using a 10 uL syringe P N 9301 0714 The instrument conditions are listed in Table 2 Table 2 Gas Chromatograph and Mass Spectrometer Conditions GC Conditions Column HP 5ms 30 m x 0 25 mm x 0 25 um P N 19091S 433 EPC split splitless 250 C 1 pL split ratio at 3 1 Inlet temperature Injection volume Carrier gas Helium constant flow mode 1 3 mL min Oven program 75 C 1 min 30 C min to 300 C 1 min Post run 280 C hold for 5 min backflushing duration Transfer line 280 C MS Conditions MS El SIM scan Solvent delay 4 2 min 230 C source 150 C quad Mass range 40 to 450 amu lon melamine 342 327 171
10. usions RI Spiked level Measured Recovery The work described here is a rapid screening and quantitation Compound min pg g level pg g eae DE ae RN method for the analysis of melamine and cyanuric acid in milk Cyanuric acid 6 319 40 38 42 96 1 g i g tri TMS products that provides excellent linearity and recovery Using Melamine 7 333 40 38 23 95 6 Agilent 7890A 5975C GC MSD combined with backflushing tri TMS the analysis time was cut down to five times shorter than conventional method This method is fast and suitable for quality control of milk products for the determination of Real Sample Analysis melamine and cyanuric acid A brand of liquid milk was analyzed with backflush using the previously described method The two targeted compounds were identified in less than 10 minutes with cyanuric acid at 34 90 ug g and melamine at 3 72 ug g see Figure 6 9 5e 07 9e 07 8 5e 07 8e 07 7 5e 07 7e 07 6 5e 07 6e 07 5 5e 07 5e 07 4 5e 07 4e 07 3 5e 07 3e 07 2 5e 07 2e 07 1 5e 07 1e 07 5000000 Melamine tri TMS Abundance Cyanuric acid tri TMS Figure 6 Total ion chromatogram of a contaminated brand of liquid milk References 1 U S Food and Drug Administration GC MS Method for Screening and Confirmation of Melamine and Related Analogs Version 2 May 7 2007 2 Mike Szelewski New Tools for Rapid Pesticide Analysis in High Matrix Samples Agilent Technologies publica tio
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