Michael S. Filigenzi talks with
ScienceWatch.com and answers a few questions about
this month's Fast Breaking Paper Paper in the field of
Agricultural Sciences.
Article Title: Diagnostic determination of melamine and
related compounds in kidney tissue by liquid
chromatography/tandem mass spectrometry
Authors: Filigenzi, MS;Puschner, B;Aston,
LS;Poppenga, RH
Journal: J AGR FOOD CHEM, Volume: 56, Issue: 17, Page:
7593-7599, Year: SEP 10 2008
* Univ Calif Davis, Toxicol Lab, Calif Anim Hlth & Food
Safety Lab Syst, Davis, CA 95616 USA.
* Univ Calif Davis, Toxicol Lab, Calif Anim Hlth & Food
Safety Lab Syst, Davis, CA 95616 USA.
Why do you think your paper is highly
cited?
The work done by my co-workers and I on the analysis of melamine came in
response to the pet food poisoning incident in 2007. This incident
illuminated previously unknown issues regarding melamine adulteration of
Chinese pet food additives and the toxicity of melamine and its analogs.
An incident involving melamine's use as an adulterant in infant formula in
China in 2008 resulted in a major increase in research on melamine,
particularly among Chinese researchers. Our method was the first published
method for the detection of melamine and its three analogs (ammeline,
ammelide, and cyanuric acid) in tissue samples, and it's therefore commonly
referenced in the many subsequent articles involving melamine analysis.
Does it describe a new discovery, methodology, or
synthesis of knowledge?
Our article describes a method for the analysis of melamine in kidney
tissue for use in the diagnosis of melamine poisoning. Novel aspects of
this method included the ability to detect melamine and its analogs in a
single analysis by liquid chromatography-tandem mass spectrometry
(LC-MS/MS) and the ability to detect melamine and cyanuric acid that are
present individually in the kidney tissue as well as when combined into
melamine cyanurate. This method is also capable of detecting melamine and
its analogs in archived paraffin-fixed tissue.
Would you summarize the significance of your paper
in layman's terms?
"Although this was a pet food incident, it tested the
same systems our country has in place for handling such
problems in human food."
Melamine and several related compounds can cause sudden kidney failure and
death in animals (particularly dogs and cats) which consume them. In 2007,
thousands of pets died from eating pet food which contained these toxins.
It was later determined that melamine was intentionally added to one of the
food ingredients in order to make it look as though that ingredient had
much more protein in it than it really had.
We developed a method which can determine when an animal has died from
eating melamine and/or its related compounds. This will make it much easier
to contain any future outbreaks of pet illness and death due to melamine
poisoning.
How did you become involved in this research, and
were there any problems along the way?
We became involved in this research at the beginning of the 2007 incident
involving melamine adulteration in pet food. Our group is part of a
veterinary diagnostic laboratory so when animals suddenly began dying of
what was then an unknown cause, we were involved in trying to determine
what the causative agent was.
Once it was determined that melamine and its analogs were the culprits, we
needed a method to diagnose this poisoning on a post-mortem basis. This
resulted in the development of the method we reported in our article.
We encountered a number of challenges during the development of this
method. Finding a high performance liquid chromatography (HPLC) column that
could retain all of the analytes was the first issue. These compounds are
all quite polar, which often means that the required mobile phase can't be
used with a mass spectrometer. We were fortunate in having a column on hand
that gave us the necessary retention of the analytes using a mobile phase
that was "mass spec-friendly."
A second challenge came in the sample preparation step. Other researchers
had shown that the melamine and cyanuric acid present in kidney tissue were
combined into melamine cyanurate crystals. These crystals are insoluble in
most common laboratory solvents. We experimented with several mixtures of
solvents before finding one that reliably separated melamine cyanurate into
its two constituents.
Finally, we hoped to be able to provide estimates of the concentrations of
melamine and cyanuric acid present in the kidney tissue. Quantitative
analysis is always tricky when using LC-MS/MS, particularly in complex
matrices such as kidney tissue. The use of isotopically-labeled melamine
and cyanuric acid enabled us to provide quantitative data using this
method.
Where do you see your research leading in the
future?
Because of the aforementioned problem with melamine adulteration of baby
formula, there has been an explosion of interest in these compounds, their
toxicity, their pharmacokinetics in different food animals, and in
preventing future instances of food adulteration. Our method and others
similar to it will be used to support all of these areas of research.
Future work may also involve retrospective studies to identify past
incidents of food tampering. Our lab has participated in studies involving
analysis of archived paraffin-fixed kidney tissues in Korean dogs in 2003
(Yhee, et al., Vet Pathol. 46[2]: 348-54, 2009 Mar; and
in Iberian piglets in 2003 – 2006 (Gonzalez, et al.,J.
Vet. Diagn. Invest. 21[4]:558-63, 2009 Jul).
In both cases, we were able to detect melamine and its analogs in fixed
tissue that had been stored for several years. It was concluded that the
deaths of animals in both instances were likely due to adulteration of
their feed with melamine. As researchers become more familiar with the
symptoms of melamine poisoning, we may see more such retrospective studies
identifying past incidents of feed adulteration using melamine.
Do you foresee any social or political
implications for your research?
The pet food adulteration incident of 2007 was a nightmarish situation in
several regards. All anyone knew at its outset was that pets were dying in
significant numbers from an unknown poison present in many brands of
commercial pet food.
"The globalization of our food supply has made it
increasingly difficult to assure consumers that their food
is safe"
Getting this situation figured out in a short time frame was a very
difficult endeavor. I would be remiss if I did not mention the intense
level of coordination between our group and the USFDA, other state
veterinary diagnostic labs, and private labs during this crisis. This
cooperation between groups was crucial in the rapid identification of the
toxins, their mode of action, and in the development of methods to detect
them.
Although this was a pet food incident, it tested the same systems our
country has in place for handling such problems in human food. Everyone
involved in this incident learned a lot from it and we are now much better
prepared to handle future incidents of food contamination or adulteration.
The political and social implications of melamine adulteration in food have
been far more prominent in China. As of this writing, two people have been
executed and several others jailed for their roles in the sale of infant
formula adulterated with melamine.
Consumer confidence in the Chinese dairy industry was severely shaken as
the prevalence of melamine adulteration became known and as many thousands
of infants were hospitalized with kidney stones. Many other countries which
import milk products from China were also heavily affected by this.
The methods developed by our group and by others involved with the pet food
incident enabled officials around the world to rapidly determine the extent
of the adulteration. These methods also enabled officials in our own
country to rapidly survey imported foods to insure that our own citizens
were not at risk.
In both of the above instances, illicit adulteration of a food product in
one country had severe implications for individuals in many other
countries. The globalization of our food supply has made it increasingly
difficult to assure consumers that their food is safe. Rapid, accurate
analytical methods such as ours will be increasingly important in
maintaining the safety and security of the food we eat.
Michael S. Filigenzi
Staff Research Associate/Group Leader
California Animal Health and Food Safety Laboratory
University of California at Davis
Davis, CA, USA