Chromatography Forum

I am looking for HPLC method for melamine in various matrices; we are trying to set up method for UV and LC/MS. We have few choices here including ion-pairing and HILIC. I would appreciate if somebody will share experience on sample prep and analysis.

Do you work for San Lu?. If so, you probably could test with LC-DAD, given the 2563 mg/kg reported in their product.

A literature search on "melamine and chromatography "will reveal several methods, and there are several recent ( 2007 - 2008 ) articles that describe the sample preparation in detail, eg
Determination of melamine in pet food by enzyme immunoassay, high-performance liquid chromatography with diode array detection, and ultra-performance liquid chromatography with tandem mass spectrometry. J AOAC Int. 2008 Mar-Apr;91(2):408-13

Kim B, Perkins LB, Bushway RJ, Nesbit S, Fan T, Sheridan R, Greene V.
University of Maine, Department of Food Science and Human Nutrition, 5735 Hitchner Hall, Orono, ME 04469-5735, USA.

Short abstract
" Melamine in pet food (fortified or originally contaminated) was determined by enzyme immunoassay (EIA), high-performance liquid chromatography with diode array detection (HPLC-DAD), and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). "

You may also want to look for cyanuric acid, and possibly other triazine compounds. eg Determination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromaography - tandem mass spectrometry" by Cristine M. Karbiwanyk and a whole host of FDA analysts Analytica Chimica Acta ( in press - available online )

I suspect most government laboratories currently performing testing on dairy products and other foods will be happy to share their methodology, if it's not already published.

Bruce Hamilton

We developed a LC-MS method for simultaneous separation of melamine and cyanuric acid using Acclaim Mixed-Mode WAX-1 column. Please find details in the following link. Contact person for futher questions: Leo Wang (leo.wang@dionex.com): http://www.dionex.com/en-us/webdocs/622 ... lamine.pdf

Hope this will help.

I've received several email requests for melamine in food methods, despite the apparent ease of an Internet search. The following is the simple summary of a Google search on "Melamine and chromatography ". I have not searched a science database, so could easily have missed important papers.

The obvious journal for people interested in performing such tests is Food Additives and Contaminants, which I don't have access to.

As I mentioned above, almost every food regulatory body will be conducting or arranging such tests, so talk to your local authority for details. If you want to understand some of the issues, and manufacture suitable standards, the following articles should provide a quick start..

Note that I have deleted the abstracts, but they, and at least 3 of the full articles, are freely available via Google. Also manufacturers, such as Perkin Elmer, have produced " melamine analysers " which have free data available. and there will be plenty of free information from most column and GC/HPLC manufacturers.

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FDA GC Method for petfood
www.fda.gov/cvm/GCMSMelamine.htm

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FDA HPLC-UV screening Method
www.fda.gov/cvm/melamine04022007.htm

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Determination of Melamine in Pet Food by Enzyme Immunoassay, High-Performance Liquid Chromatography with Diode Array Detection, and Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry
Byungchul Kim, Lewis B. Perkins, Rodney J. Bushway, Stephanie Nesbit, Titan Fan, Robert Sheridan, Virginia Greene
Journal of AOAC INTERNATIONAL, Volume: 91, Issue: 2, Cover date: February 2008, Page(s): 408-413

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Preparation of [13C3]-melamine and 13C3]-cyanuric acid and their application to the analysis of melamine and cyanuric acid in meat and pet food using liquid chromatography-tandem mass spectrometry
P. Varelis a; R. Jeskelis
Food Additives & Contaminants: Part A, Volume 25, Issue 10 October 2008 , pages 1210 - 1217

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High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cereal flours with melamine and related triazine by-products ammeline, ammelide, and cyanuric acid
Ehling, S.1; Tefera, S.1; Ho, I. P.2
Food Additives and Contaminants, Volume 24, Number 12, December 2007 , pp. 1319-1325(7)

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Diagnostic Determination of Melamine and Related Compounds in Kidney Tissue by Liquid Chromatography/Tandem Mass Spectrometry
Michael S. Filigenzi,* Birgit Puschner, Linda S. Aston, and Robert H. Poppenga
J. Agric. Food Chem., 56 (17), 7593–7599, 2008. 10.1021/jf801008s
Web Release Date: July 25, 2008

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The determination of melamine in muscle tissue by liquid chromatography/tandem mass spectrometry
FILIGENZI Michael S. ; TOR Elizabeth R. ; POPPENGA Robert H. ; ASTON Linda A. ; PUSCHNER Birgit ;
Rapid communications in mass spectrometry ISSN 0951-4198
2007, vol. 21, no24, pp. 4027-4032

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Rapid and Unambiguous Identification of Melamine in Contaminated Pet Food Based on Mass Spectrometry with Four Degrees of Confirmation
Teresa M. Vail, Patrick R. Jones, and O. David Sparkman
Journal of Analytical Toxicology, ISSN 0146-4760, Volume 31, Number 6, July/August, pp.304-312

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Determination and Confirmation of Melamine Residues in Catfish, Trout, Tilapia, Salmon, and Shrimp by Liquid Chromatography with Tandem Mass Spectrometry
Wendy C. Andersen, Sherri B. Turnipseed, Christine M. Karbiwnyk, Susan B. Clark, Mark R. Madson, Charles M. Gieseker, Ron A. Miller, Nathan G. Rummel, and Renate Reimschuessel
J. Agric. Food Chem., 56 (12), 4340–4347, 2008. 10.1021/jf800295z
Web Release Date: May 22, 2008

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Method development and validation for melamine and its derivatives in rice concentrates by liquid chromatography. Application to animal feed samples
Roberto Muñiz-Valencia, Silvia G. Ceballos-Magaña, Daniel Rosales-Martinez, Raquel Gonzalo-Lumbreras, Ana Santos-Montes, Angel Cubedo-Fernandez-Trapiella and Roberto C. Izquierdo-Hornillos
Analytical and Bioanalytical Chemistry, Volume 392, Number 3 October, 2008. Pages, 523-531

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Determination of Melamine and Cyanuric Acid by LC–MS Using an Acclaim Mixed-Mode WAX-1 Column and MSQ Plus MS Detection
Leo Wang, Xiaodong Liu, Mark Tracy, Bill Schnute, Ilze Birznieks, Chris Pohl
LCGC Asia Pacific, Volume 10, Issue 4, Dec 1, 2007

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Thanks Bruce, now I need to spend some time in the library

XL,

I looked at your method and noticed that melamine is barely retained, I think K' is slightly over 1. Our requirements for K' is at least two, buffer concentration is on the higher end too (55 mmol). We are also looking at UV method and it might be hard to do with acetate or formate buffer with cut-off at 230 nm. Also why melamine peak is so wide (even wider than peak at 4 and 12 minutes for CA)? I suspect that plate count is very low.

It is quite clear that HILIC is a versatile technique and particular suitable for polar and hydrophilic compounds.

By using the ZIC-HILIC column you are also able to retain other compounds related to melamine.

When using the conditions below you will get melamine retained at a retention factor (k') of 3. The conditions are suitable for all kind of detectors like UV and, in particular, MS if more sensitivity is needed.

Mobile phase
80% acetonitrile/20% ammonium acetate 20 mM

Column
ZIC-HILIC, 150 x 4.6 mm

When using a RP column, the k' of melamine can be higher than 2 but that of cyanuric acid will be very low (almost in the void). Acclaim Mixed-Mode WAX-1 column showed reversal elution order compared to RP column method - it retains cyanuric acid well while the retention for melamine is a bit lower as you pointed out. The total buffer concentration in the mobile phase was below 10 mM. I thought it was ok for MS.

If melamine is the only compound of interest, the Acclaim Mixed-Mode WAX-1 column can be used in the HILIC mode. The k' is 2.1 when the mobile phase is MeCN/ammonium acetate buffer, pH5 (total concentration is 10 mM) v/v 90/10. Further increasing MeCN content results in longer retention. Peak shape and peak efficiency were good. I only ran standard and could get low ppm detection limit by UV. But I didn't run cyanuric acid in the same condition since it had poor chromophore.

Recently - Imtakt developed an LC-MS compatible method
for melamine and cyanuric acid on Unison UK-Amino:

http://www.imtakt.com/TecInfo/TI437E.pdf

perkinelmer has a analyser for Melamine, which include GCMS method, extraction procedure and also column for melamine analysis. for more information please refer to this website: http://las.perkinelmer.com/Catalog/Fami ... e+Analyzer

With this analyser, you can pretty assure that it can run smoothly as the GCMS method is already build in as well... it is a turnkey solution.

http://www.sequant.com/melamine

Melamine and cyanuric acid is an urgent matter for a lot of countries. Therefor we provide a special webpage for the topic.

We will also dedicate time in connection to the HILIC-Day in Shanghai for this, www.seqaunt.com/hilicday.

Hello
I am currently working on determining melamine by LC MS MS using a column HILIC, someone has a reference or analytical techniques for analysis of melamine in milk.

That care must be taken with a column HILIC?

Sorry by my English

Thank you

As noted above, talk to the food regulatory authorities in your country, they should be able to provide access to procedures used.

You could also try contacting Taiwan's Department of Health, as they have chosen LC-MS-MS as the approved method, and seen to have access to English-speaking staff..

The main issue will be sample preparation, and avoiding contamination, so contacting people who are routinely using methods is agood start.

Agilent have recently published detailed information on both a GC and a HILIC LC-MS-MS method for melamine in pet-food products, freely available at their literature website.

I think the following address will get you to it, if not search their literature site for melamine. They are starting a training course in China, and they also have details of the SPE milk sample preparation used in China, so ask your local agent, or email Agilent.
http://www.agilent.com/chem/melamine

I'm sure other major LC-MS-MS and HPLC column manufacturers will have similar information available - ask the supplier of your toy.

It's fairly clear that the 2.5 ppm limit for adults is achievable by several methods, but LC-MS-MS may be required for infant formula limits, which range from Not Detected ( Taiwan, USA ) to 1 ppm ( NZ, Europe ).

LC-MS-MS is claimed to reach 1 ppb, but that's really going to depend on sample preparation.

Update - Agilent have also just released a freely-available 47 page 1.7 MB brochure entitled " Total Solution for Melamine Analysis by Agilent LC, GC/MS and Triple Quad LC/MS " which is available as a pdf from their literature library by searching on the keyword "melamine".

It describes sample preparation using SCX SPE process for LC methods, as well as instrumental conditions.

Please keep having fun,

Bruce Hamilton

Melamine itself is reportingly not very toxic. It is the combined presence of melamine and cyanuric acid (and other triazines) that may be fatal.

Consequently, simply measuring melamine may not be sufficient.

www.sequant.com/melamine

recent update www.fda.gov


I noticed that my previous link to The Hilic Day was not correct, please use www.sequant.com/hilicday

I was out on vacation and are happy to discover a lot of posts here about method for melamine. Thank you everybody for your input!

After reading some articles I decided to stick with HILIC approach for now. I wanted to order Sequant HILIC column and guards but they are VERY expensive – over $1000 for a column and set of guards! Is it high because of the weak dollar? How can people in China afford spending so much money? I have couple of HILIC columns in my lab (Altima and Inetsil) and I would try them first, I am sure that they will give similar results with slightly different conditions. I also reviewed few articles on ELISA approach……. but this is a totally different ball game.

Another question for the board: If melamine is not toxic in ppm lavels and cyanuric acid is not toxic in ppm level, why not test only melamine? As I understant complex of melamine and cyanuric acid forms coplex with precipitates, so if there is no melamine in milk there is no need to test for cyanuric acid. Also nobody adding cyanuric acid to improve protein reading, so melamine is the beast here