Melamine testing in the pharmaceutical industry: methods to ensure your products are melamine-free.
There are two primary incidents that led the FDA to scrutinize the potential impact that melamine could pose to the pharmaceutical industry. First, in 2007 numerous deaths and illnesses in both cats and dogs led investigators to discover melamine contamination in both wheat gluten and rice protein utilized as a pet food thickener (6). Then in 2008, melamine-contaminated infant formula was linked to the illness in more than 50,000 infants and to death in at least six infants in China (7).
It is suspected in both incidents that melamine was intentionally added to the pet food products and infant formula to artificially increase the apparent nitrogen content of the product. Melamine contains 66 percent nitrogen by mass, thus it's presence in various products can generate an artificially higher protein content by elevating the total nitrogen content measured. The majority of the pharmaceutical components listed in the FDA document utilize the total Kjeldahl Nitrogen test to determine nitrogen content. The total Kjeldahl nitrogen test is found in the LISP as general chapter <461> (8). In addition, the guidance document includes components that could be derived from animal-based materials.
FDA GUIDANCE DOCUMENT REQUIREMENTS
According to the guidance document, manufacturers must determine if an at-risk pharmaceutical component will be used in the manufacturing of a drug product. In addition, pharmaceutical manufacturers must evaluate and understand the supply chain of the pharmaceutical components. Manufacturers of finished products should question their suppliers to determine if documentation is available that supports that components have been tested to prove absence of melamine, or that the suppliers have other controls in place to prevent melamine contamination. It is important to recognize that if information is available from suppliers to justify that the component is melamine free, the manufacturer of the finished product would not have to repeat the testing. All decisions on the need for melamine testing or justification for not testing for melamine must be based on good scientific principles.
Melamine is not approved for use as a direct food additive but is used in the manufacture of a variety of different products ranging from various resin-based materials, including counter-tops, glues, house-wares and flame retardants (9). Melamine has a low acute toxicity and is not readily metabolized (10). Cyanuric acid is a structural analogue of melamine and is used in the manufacture of herbicides, dyes, resins and antimicrobial agents. However, it is an FDA-accepted component of feed-grade biuret and can be legally added up to 30 percent of the feed additive (11). Cyanuric acid has a low acute toxicity (12).
So if both melamine and cyanuric acid have low levels of toxicity, then why is there such a concern for melamine adulteration in pharmaceutical products? The combined presence of melamine and cyanuric acid lead to the formation of melamine cyanurate. This compound has low solubility, which can lead to the formation of crystals in the kidneys. Formation of these crystals has been documented in numerous animal studies where the ingestion of both melamine and cyanuric acid was controlled. The formation of these crystals has led to renal failure and kidney toxicity, and in some subjects, death. (13), (14)
In addition to the FDA posted methods for melamine, there are many alternatives that can be found on the internet. Methodologies range from LC-MS/MS and GC/MS methods the FDA references to alternative methods that utilize hydrophilic interaction chromatography with MS/MS, microextraction GC/MS, micellar electrokinetic capillary chromatography with amperometric detection and laser Raman spectroscopy, just to name a few. The key item to consider when determining what analytical technique to utilize is that the finalized method conditions must meet the objectives of the testing. These objectives include a method that achieves acceptable specificity and sensitivity for the compounds of interest, and also that the instrumentation selected is available to support the testing.
The FDA guidance document states that melamine should be monitored down to a concentration of 2.5 ppm in at-risk components. Therefore, any method selected should be capable of attaining this level of sensitivity. One FDA referenced method, utilizing LC-MS/MS, achieves the level of sensitivity required in the guidance document, however, the method was developed utilizing infant formula. Therefore, additional work would need to be performed to ensure this method is appropriate for use with the pharmaceutical components listed in the document.
In fact, one should evaluate all the range of sample types that will require monitoring to determine the impact that matrix may have on the analytical technique. To minimize the impact matrix may have on the response of melamine and cyanuric acid, it is good practice to develop a method that utilizes labeled reference standards of both melamine and cyanuric acid as internal standards. The use of internal standards will compensate for any impact that matrix may have on response of the compounds of interest and improve precision of the method. One must also evaluate whether the sensitivity limit listed in the guidance can be obtained for every matrix. Due to the wide variety of components, it is feasible that not all matrices will allow attainment of this sensitivity level. In these cases, documentation must display that efforts were made to determine the lowest level of sensitivity the method can support.
CRITERIA FOR CONSISTENT PERFORMANCE
To ensure the method performs consistently, system suitability criteria should also be established. These criteria may reflect precision of standard injections, resolution of various components of the standard solution, establishment that the system has adequate sensitivity to perform the method, etc. Once the method conditions have been established, the method should be qualified/validated for its intended purpose. Qualification/validation elements that are recommended include:
* Linearity - Establish a linear range from limit of quantitation to at least two times the specification limit.
* Specificity - Establish by analyzing sample blanks and sample materials known to be free of both melamine and cyanuric acid (if available).
* Limit of Quantitation - Six spiked sample solutions prepared where approximate signal to noise of compounds of interest is 10.
* Method Precision/Accuracy/Range - Establish by spiking sample matrix with compound of interest at levels ranging from limit of quantitation to two times the specification limit.
* Stability of both standard and sample solutions Precision/Accuracy/Range and Limit of Quantitation should be established for every matrix that the method will be expected to support. Following the above qualification/validation outline will ensure that the method produces valid and accurate data for all pharmaceutical components tested.
(1.) U. S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), and the Center for Veterinary Medicine (CVM), Guidance for Industry, Pharmaceutical Components at Risk for Melamine Contamination, August 2009.
(2.) GC-MS Screen for the Presence of Melamine, Ammeline, Ammelide and Cyanuric Acid (Version 2.1), May 16, 2007, www.fda.gov/AnimalVeterinary/ScienceResearch/ToolsResources
(3.) GC-MS Method for Determination of Melamine and Related Analogs (Version 2), May 7, 2007, www.fda.gov/AnimalVeterinary/ScienceResearch/ToolsResources
(4.) Determination of Melamine and Cyanuric Acid Residues in Infant Formula using LC-MS/MS, Laboratory Information Bulletin No. 4421, Volume 24, October 2008, www.fda.gov
(5.) Interim Method for Determination of Melamine and Cyanuric Acid Residues in Foods using LC-MS/MS:Version 1.0, Laboratory Information Bulletin No. 4422, October 2008, www.fda.gov
(6.) FDA FAQ: Where did the contaminated wheat gluten come from?
(7.) Scott MacDonald, "Nearly 53,000 Chinese Children Sick from Milk", Associated Press (22 September, 2008)
(8.) United States Pharmacopeia Convention, United State Pharmacopeia (USP) 31, Rockville Maryland, 2008.
(9.) Hazardous Substances Data Bank [Internet]. Bethesda (MD): National Library of Medicine (US): [Last Revision Date, 1/23/1997]. Melamine; Hazardous Substance Databank Number: 2648.
(10.) OECD (1998) Screening Information Data Set for Melamine, CAS Number 108-78-1.
(11.) "Food Additives Permitted in Feed and Drinking Water of Animals", Title 21 Code of Federal Regulations, Part 573 (Revised as of April 1, 2007).
(12.) Klaus Hutchmacher, Dieter Most, "Cyanuric Acid and Cuanuric Chloride", Ullmann's Encyclopedia of Industrial Chemistry" 2005, Wiley-VCH, Weinheim, ISBN 10.1002/114356007.a08 191.
(13.) Brown, C. A., Jeong, K. S., Popoenga, R. H., Puschner, B, Miller, D. M., Ellis, A. E., Kang, K. I., Sum, S., Cistola, A. M., Brown, S. A., (2007), Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007, J Vet Diagn Invest., 19, 525-31.
(14.) Puschner, B., Poppenga, R. H., Lowenstine, L. J., Filigenzi, M. S., Pesavento, P. A., (2007), Assessment of melamine and cyanuric acid toxicity in cats, J. Vet. Diagn. Invest., 19, 616-24.
By Dr. Thomas Lehman, Manager, Method Development and Validation, Lancaster Laboratories
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|Date:||Mar 1, 2010|
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