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Agilent Rapid Screening Confirmation of Melamine its Analogs in Baby Formula Soy Products Using Triple Quadrupole GC/MS Backflushing Manual

1. Standard Melamine Sigma Aldrich gt 99 purity Cyanuric acid TCl America gt 98 0 Ammelide TCl America gt 98 0 Ammeline TCl America gt 95 0 Internal Sigma Aldrich 98 standard Solvent Diethylamine Sigma Aldrich SigmaUltra DEA grade Pyridine Fisher Certified A C S Scientific reagent Acetonitrile Fisher HPLC grade Silylating reagent BSTFA with Sigma Aldrich Derivatization 1 TMCS grade SYLON BFT t DACP 2 6 Diamino 4 chloropyrimidine BSTFA bis trimethylsilyl trifluoroacetamide TMCS Trimethylchlorosilane Instruments The experiment was performed on an Agilent 890A gas chro matograph equipped with a split splitless capillary inlet an Agilent 7000A Series Triple Quadrupole GC MS with Triple Axis Detector and an Agilent 683B automatic liquid sampler ALS The split splitless inlet was fitted with a long lifetime septum p n 5183 4761 and a deactivated splitless single taper injection liner p n 5181 3316 Injections were made using a 10 pL syringe p n 9301 0714 The instrument conditions are listed in Table 2 Table 2 GC Run Conditions Gas Chromatograph and Mass Spectrometer Conditions Column Two 15 m x 0 25 mm x 0 25 pm HP 5ms columns p n 19091S 431 Inlet temperature 280 C Inlet pressure 12 9 psi Helium constant flow mode 1 2 mL min 25 psi at 0 5 min 100 C 1 min hold 10 C min to 210 C Carrier gas Pulsed splitless Oven program Column velocity 41 cm s Inj
2. Author Stephan Baumann Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA 95051 USA Rapid Screening and Confirmation of Melamine and its Analogs in Baby Formula and Soy Products Using Triple Quadrupole GC MS and Backflushing Application Note Food Abstract A rapid method for the screening and confirmation of melamine ammelide ammeline and cyanuric acid in baby formula and soy meal was developed using the Agilent 7890A 7000A Series Triple Quadrupole GC MS and backflushing with a Purged Ultimate Union The extraction and derivatization procedures are the same as those used in the FDA GC MS method Excellent linearity R2 gt 0 99 was obtained in the range of 0 16 to 2 5 ppm with run times less than 15 minutes E Agilent Technologies Introduction The adulteration of food with melamine has quickly become an international problem as it has been detected in baby for mula produced in the US chocolates distributed in Canada biscuits sold in the Netherlands condensed milk in Thailand and eggs in Hong Kong In response many countries have established allowable limits for melamine with the FDA maxi mum residue limit MRL as 1 part per million ppm for infant baby formula and 2 5 ppm for other products The FDA GC MS screening method 1 is capable of detecting melamine and its analogs ammeline ammelide and cyanuric acid at 2 5 ppm However the FDA import alert of February 2009 requires that a test
3. Injecting Analysis Parameters The parameters used in the analysis of melamine and its analogs as well as the internal standard are shown in Table 3 Table 3 Analysis Parameters Triple Quadrupole GC MS Dwell time Collision energy Compound RT SRM ms EV Melamine 12 467 327 171 20 17 342 285 150 20 342 213 150 22 Ammelide 10 801 344 171 50 22 344 214 50 15 329 171 50 20 Ammeline 11 748 328 171 50 25 343 214 50 20 343 171 50 30 Cyanuric acid 9 613 345 215 50 8 345 188 50 12 330 215 50 4 DACP ISTD 11 185 213 gt 231 150 12 2 6 Diamino 4 213 99 150 20 chloropyrimidine Results and Discussion Backflushing with a Purged Ultimate Union System A backflushing configuration was employed to remove higher boiling substances from the column prior to each subsequent run by flushing late eluting peaks out of the inlet split flow vent instead of driving them though the entire column and into the MSD Backflushing reduces chemical noise and the cycle time of the analysis thus increasing throughput System uptime is also increased due to reduced maintenance of the columns and MS detector The suite of Agilent Capillary Flow Technology modules comprises a proprietary solution that enables easy and rapid backflushing with small dead volumes for improved resolution and ferrules and fittings that elimi nate leaks All Capillary Flow Technology modules require the use of an Auxilia
4. 0 4 0 2 0 2 0 1 0 10 7 10 75 10 8 10 85 103 10 7 10 75 10 8 1085 10 3 MAM 328 1 gt 171 1 Formula 0 78 Std D 328 1 gt 171 1 343 1 gt 171 1 343 1 gt 214 1 y102 11 747 x10 2 Ratio 78 2 8 1 44 Ratio 16 6 C 6 B 12 3 E 4 4 lt r Ammeline 3 D d 2 le 0 11 6 11 7 11 8 11 9 11 6 11 7 11 8 11 9 MAM 345 1 gt 188 1 Formula 0 78 Std D 345 1 gt 1881 345 1 gt 2151 330 1 gt 215 1 E x102 9612 102 Ratio 88 8 o tio 68 E g 1 84 Ratio 68 9 5 1 1 6 E 1 4 x Cyanuric acid 12 E 1 3 c 0 8 0 6 E 0 4 95 955 96 965 97 975 l 35 355 96 S365 97 975 MAM 273 0 gt 99 0 Formula 3 83 Std D 273 0 gt 99 0 273 0 gt 237 1 x10 4 11 184 x x10 Flatio 42 9 o O 25 E 4 t 2 g 08 DACP 15 Q6 04 Internal coc l 0 2 standard 0 0 11 15 11 2 11 25 11 3 11 15 11 2 11 25 11 3 Quantifying Transition Relative Abundances of Quantifying and Qualifying Transitions Figure 7 The Agilent Triple Quadrupole GC MS system provides confirmation and screening in one run quantifying and normalized qualifying transitions for melamine and its analogs at 0 78 ng mL in baby formula The Agilent Triple Quadrupole GC MS system provides rapid screening and confirmation in one run MAM 327 1 gt 171 1 Soy 0 78 Std D 2 x10 12 468 8 2 1 75 1 5 Melamine 0 75 0 5 0 25 0 12 3 12 4 12 5 12 6 MRM 344 1 gt 171 1 Soy Q0 78 Std D Cou Counts Ammeline r
5. 02 2 Conclusions 12 5 12 48 99 8 Ammelide 0 78 0 72 92 9 The FDA GC MS method for screening for melamine s n id ammelide ammeline and cyanuric acid has been modified for 125 12 46 997 use on the Agilent Triple Quadrupole GC MS system in order to provide screening quantification and confirmation in one Ammeline 0 78 0 81 103 7 125 122 07 9 short run This method does not require any changes in 3 90 3 90 99 9 extraction or derivatization procedures and cycle time Is 12 5 12 50 100 0 about 15 minutes In addition this method meets the new Cyanuric acid 0 78 071 91 3 FDA requirement for sensitivity of 0 25 ppm and it demon 1 25 1 22 94 5 strates excellent linearity of quantification up to 2 5 ppm 3 90 4 49 119 1 Accuracy of quantification is greater than 97 and two SRM 12 5 12 01 96 1 transitions for each of the four compounds have been demon strated in order to provide sufficient identification points for a positive confirmation The Agilent Triple Quadrupole GC MS system provides rapid screening and confirmation in one run MAM 327 1 gt 171 1 Formula 0 78 Std D 327 1 171 1 342 1 gt 2851 342 1 gt 213 1 x103 12 467 Ratio 2 9 8 Ratio 5 1 c 2 Melamine 15 0 5 0 12 4 12 5 12 6 12 4 12 5 12 6 MAM 344 1 gt 171 1 Formula 0 78 Std D 344 1 171 1 344 1 gt 214 2 328 1 gt 171 1 y102 10 800 X x10 Ratio 3 8 8 Y Ratio 37 7 o 08 S 1 a S ag 0 6 a Ammelide 05 9 Q6 0 4 x 03
6. 2 ww AeA oO MRM 345 1 gt 188 1 Soy 0 78 StdD 2 x102 3613 2 2 3 473 Cyanuric acid 15 0 75 9 755 349 35 955 96 965 97 975 MAM 273 0 gt 99 0 Formula 3 3 Std D x10 4 11 184 25 2 DACP is Internal x standard r 11 15 11 2 11 25 11 3 Quantifying Transition Figure 8 x03 10 803 1 0 8 Ammelide 06 0 4 0 2 0 x10 11 751 6 1 0 327 1 gt 171 1 342 1 gt 2851 342 1 gt 213 1 Ratio 2 7 1 8 Ratio 4 2 Relative Abundance 12 3 12 4 12 5 12 6 x102 Ratio 11 5 Ratio 39 5 Relative Abundance 10 7 10 8 10 3 328 1 gt 171 1 3431 gt 171 1 3431 gt 214 1 x10 27 Ratio 74 4 1 44 Ratio 15 0 Relative amp bundance 11 6 11 7 11 8 11 3 345 1 gt 1881 3451 2151 3301 gt 215 1 x10 Ratio 99 6 Ratio 85 0 Relative Abundance 9 45 35 895 36 365 37 3 5 273 0 gt 930 273 0 gt 237 1 Ratio 42 9 x b e n2 Relative Abundance 2 11 15 11 2 11 25 11 3 Relative Abundances of Quantifying and Qualifying Transitions Quantifying and normalized qualifying transitions for melamine and its analogs at 0 78 ng mL in soy meal Acknowledgement The author gratefully acknowledges the guidance and materi al assistance of Greg Mercer Pacific Regional Laboratory Northwest Food and Drug Administration References 1 U S Food and Drug Administration GC MS Screen for the Presence of Melamine Ammeline Ammelid
7. MRL can be defined because of the toxicity of the compound it is banned AISUde ue id uo at all levels These compounds require four identification 1 25 1 25 99 9 3 90 379 972 points While four ions need to be monitored by GC MS to 12 5 12 52 100 2 provide four identification points only two SRM transitions pe oe 0 78 0 90 1155 need to be monitored when using triple quadrupole 1 25 1 22 97 2 GC MS MS Analysis of melamine and its analogs was per 3 90 3 78 97 0 formed using at least two SRM transitions for each com 12 5 12 52 100 3 pound on the triple quadrupole GC MS system to provide Cyanuric acid 0 78 0 67 86 1 screening and positive confirmation in the same run 1 25 1 40 111 9 3 90 3 85 90 8 Figures 7 and 8 illustrate the quantifying and qualifying transi 12 9 12 51 100 1 tion profiles for the GC separation of each of the four com pounds in both baby formula and soy meal In each case the Table 5 Calibration Data for Quantification of Melamine and its Derivatives qualifying transitions have been normalized to the quantifying in Soy Meal Based on Matrix Matched Standards transition in order to better illustrate the identical peak shape Standard Measured Accuracy of obtained from both These transitions therefore provide a pos ponicontiauon oncentation Juantication itive confirmation of each of the four compounds in each of mL mL om nm o the sample matrices Melamine 0 78 0 76 97 7 1 25 1 20 96 3 3 90 3 98 1
8. R 2 0599965084 Ro 0 99959290 Relate Fresponsez Fielabeee Respon 11 12 13 Concentration ng ml Figure 5 Calibration curves for quantification of melamine and its derivatives in baby formula based on a linear fit e 4 Levels d Levels Used 4 Fords 4 Poris U ed 0 Cx ye P0765 2 00013 y DOCS 00140 AN 099988499 Ro 0 99955761 1 ze c Relalive Responses Relate Respon n 12 13 11 Tz 13 Concertation ng ml Conceniralson ng ml l 4 Levelt 4 Levak Used 4 Foris 4 Poris Used 0 OCs y 00295 x 00022 i y OOS x 0 0064 H 2a s999ga79 A 0994827 Relate Responses Relates Fespone 11 12 13 Concentration ng ml Figure 6 Calibration curves for quantification of melamine and its derivatives in soy meal based on a linear fit 6 Table 4 Calibration Data for Quantification of Melamine and its Derivatives i 1 in Baby Formula Based on Matrix Matched Standards Confirmation Standard Measured Accuracy of The identification point system was developed by EU scien Concentration Concentration Quantification tists to define an acceptable procedure for scientifically con ng mL ng mL firming the presence of regulated substances The more iden Melamine 0 78 0 79 101 3 tification points provided by the analytical method the more 1 25 1 23 39 1 certain is the confirmation of the compound Three points are n bs qe required for compounds with an MRL When no
9. at 2 5 ppm b The quantifying transition used with the Triple Quadrupole GC MS method was m z 327 1 171 1 and the qualifying transitions were m z 342 1 3295 1 2 5 of the peak area of the quantifying transition and m z 342 1 217 1 4 8 peak area The uncertainty bands are shown in a as well The SIM ions used in the GC MS method were m z 342 2 327 2 and 171 1 b Sensitivity and Quantification Figures 5 and 6 illustrate the excellent linearity obtained for melamine and its three analogs with R values very close to 1 00 The accuracy of quantification was also very good for all four compounds in both matrices as illustrated in Tables 4 and 5 Each of the standards for melamine and its three analogs was added to matrix both baby formula and soy meal at concen trations of 0 78 1 25 3 9 and 12 5 ng mL corresponding to detection levels of 0 16 to 2 5 ppm Calibration curves were constructed for each of the four compounds in each matrix Obtain reliable quantification even below 0 25 ppm Melanie 4 Lewels 4 Lavel Used 4 Poris 4 Ponte Usad 0 Cz dunmeide 4 Level 4 Levelz Wed 4 Points 4 Pots Used 0 OCs ge O0919 s 00146 Ps l ypse00287 x 00038 A 0 89900547 AS 0 99978629 Rielalive Fiezponmes Be nd t en LR ra Cl on tcn em n ww 13 1n 172 1 Concertation ng ml Conceniralion ng ml 1 4 Levels 4 Level Used 4 Pods 4 Ports Used 0 Cs y 0028A x 00041 yig 27 y 00E 00045 z
10. e and Cyanuric Acid LIB No 4423 Volume 4 October 2008 2 H Prest C Foucault and Y Aubut Capillary Flow Technology for GC MS Efficacy of the Simple Tee Configuration for Robust Analysis Using Rapid Backflushing for Matrix Elimination Agilent Technologies publication 5989 9359EN 3 H Prest Capillary Flow Technology for GC MS A Simple Tee Configuration for Analysis at Trace Concentrations with Rapid Backflushing for Matrix Elimination Agilent Technologies publication 5989 8664EN 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 O Agilent Technologies Inc 2009 Printed in the USA June 8 2009 5990 4071EN NE Agilent Technologies
11. ection volume 1uL Transfer line 290 C temperature GC Post Run Conditions Backflush device Purged Ultimate Union p n G3186 60580 controlled by a Pressure Control Module p n G3476 60501 Backflush conditions 3 6 mL min at 300 C for 1 3 min MS Conditions Tune Autotune Delta EMV 400 V Acquisition El selected reaction monitoring parameters Solvent delay MS temperatures 6 minutes Source 230 C Quadrupoles 150 C Sample Preparation A 0 5 g amount of a representative portion of the sample was weighed into a 50 mL polypropylene centrifuge tube An extraction solvent of DEA Water Acetonitrile 10 40 50 was prepared and 20 mL added to the weighed sample Diethylamine dissociates the melamine cyanuric acid com plex thus reducing the risk of false negative measurements DEA also improves the solubility of ammelide and ammeline which have extremely low solubility in traditional extraction solvents The sample was capped vortex mixed and then sonicated for 30 minutes After the sample was centrifuged at 5 000 g or higher for 10 minutes the supernatant fluid was fil tered through a 0 45 pim nylon filter Derivatization A 160 uL amount of the filtrate was transferred to a glass GC vial The extract was evaporated to dryness under a stream of nitrogen at approximately 70 C and 600 uL of ISTD and 200 uL of BSTFA with 196 TMCS were added The sample was vortex mixed and incubated at 70 C for 45 minutes before
12. ff the second column b Bottom Subsequent blank injection showing no carryover Analysis of Melamine and its Analogs The method developed on the Triple Quadrupole GC MS sys tem provides excellent separation and analysis of melamine ammelide ammeline and cyanuric acid in one run and in less than 15 minutes Figure 3 The significant improvement in the sensitivity and selectivity of the new Triple Quadrupole GC MS method versus the GC MS SIM method is vividly illustrated in Figure 4 While the new method provides a very clean analysis of the quantifying transition of melamine at 0 25 ppm the GC MS SIM method is less effective at reduc ing chemical noise at 2 5 ppm using any of the SIM ions Analyze melamine and all of its analogs in one run DACP a m Melamine internal standard Ammelide Ammeline Cyanuric acid 11 4 11 6 Figure 3 Reconstructed Total lon Current Chromatogram RTICC resulting from SRM analysis illustrating the resolution of melamine and its analogs The Agilent Triple Quadrupole GC MS system delivers superior sensitivity and selectivity 327 1 gt 171 1 3421 gt 2851 42 1 gt 213 1 lon 327 20 326 30 to 327 50 5285155 D data ms lor 342 20 341 30 t 342 5 5285155 Ddata ms Melamine TMS demeatree ry A J i M s is a Figure 4 Comparison of detection of 0 25 ppm melamine in soy meal using the Triple Quadrupole GC MS method a versus the GC MS SIM method
13. ginning at 10 1 minutes where flow is dropped in the first 15 m column and b where the Rapid backflushing without reducing sensitivity Split splitless flow in the second column is increased The time between Injection port controller i i points a and b is the residence time of the last analyte com m Capillary flow pound in the second column The last analyte is retained but Ain P E the late eluters never enter the MS detector The bottom Quadrupole chromatogram demonstrates the lack of carryover in a subse GC MS El mode quent blank run Alternatively backflushing can begin after the last peak of interest has eluted point b This eliminates the need to experimentally determine the residence time of the last target compound in the second column while slightly increasing the cycle time 15 m HP 5ms column 15 m HP 5ms column 0 25 mm id x 0 25um 0 25 mm id x 0 25 um 7890A GC Figure 1 Schematic of the Purged Ultimate Union GC MS configuration Shorten cycle times without carryover Sample no backflush LLELELLLLE LLL LE LL LE LE LE LE LE LELEL LLL LL LE 10 00 sample with backflush a begin backflush last peak exits column 1 b increase flow 4 mL min 9 50 10 00 10 50 Solvent blank no backflush Figure 2 Backflushing with a purged ultimate union configuration Top no backflushing Middle Backflushing beginning at 10 1 minutes a until the third analyte elutes o
14. ing method with a sensitivity of 0 25 ppm for melamine and its analogs be used to assure compliance to the MRLs Therefore this method cannot be used to screen for melamine and its analogs under the new regulations and confirmation would require an additional orthogonal method This application note describes a modification of the FDA GC MS method for use on the new Agilent 7000A Series Triple Quadrupole GC MS The new method which does not require a change in sample extraction and derivatization pro cedures employs a purged union GC column configuration and backflushing to provide run times under 15 minutes Melamine and its analogs can all be detected at 0 25 ppm with highly reproducible and accurate quantification Most importantly this method provides screening quantification and confirmation of melamine and its analogs all in one short run Experimental Standards and Reagents The standards and reagents used are listed in Table 1 Stock solutions of melamine ammelide ammeline and cyanuric acid each at a concentration of 1 000 ug mL were separately prepared in a mixture of DEA H20 20 80 and stored at 4 C Internal standard 2 6 Diamino 4 chloropyrimidine or DACP was prepared at a concentration of 57 7 ng mL in pyridine The above solutions were used to prepare matrix matched standards as described in the FDA method 1 Matrix samples were generously provided by the FDA Table 1 Standards and Reagents
15. ry Electronic Pneumatic Control EPC mod ule or a Pneumatic Control Module PCM to provide a pre cisely controlled second source of gas that directs the col umn flow to the appropriate column or detector In normal operation the PCM pressure is at or slightly above the pres sure of the carrier gas through the column During backflush the inlet pressure is dropped to 1 psi and the PCM pressure is increased forcing the flow to reverse through the column and out the purged inlet A unique alternative approach to backflushing is the use of a Capillary Flow Technology device in the middle of the analyti cal column 2 3 Instead of using a 30 m column two 15 m columns are used and connected by an ultra low dead volume Purged Ultimate Union Figure 1 The PCM adds just enough makeup gas to match that from the first column Therefore there is very little flow addition and subsequent decrease in sensitivity due to sub optimal carrier gas flows into the mass spectrometer Backflushing in this configuration is accom plished by reducing the flow and pressure in the first column and increasing them in the second column Figure 2 shows an example of backflushing with the purged union configuration The top chromatogram shows six stan dards where the third peak is considered the last analyte of interest and the fourth peak is the first of the late eluting interferences The middle chromatogram shows a the same standard with backflushing be

Agilent Rapid Screening Confirmation of Melamine its Analogs in Baby Formula Soy Products Using Triple Quadrupole GC/MS Backflushing Manual

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