The scandals involving melamine as a food adulterant are valuable case studies, as they have led to extensive research efforts into analytical testing techniques and the advent of a number of commercial services and testing labs. GC-MS methods have been developed for the analysis of melamine in wheat, rice, and other gluten products, however, these methods generally include extensive sample cleanup, derivatization, and the use of hazardous solvents. The limits of detection are often suboptimal as well.
LC-MS/MS methods have also been developed for the identification and quantitation of melamine in various matrices. The benefits include reduced sample prep and run times, less sample use, lower limits of quantitation, and higher degrees of confidence with MS/MS.
One such reported method was built around fast and accurate detection of melamine and cyanuric acid in pet food, making use of simple extraction, normal phase LC separation using a HILIC column, and MS/MS MRM detection using an API 3200 System.
In this approach, liquid-liquid extraction of pet food samples was performed by addition of acetonitrile and a saturated NaCl mix followed by incubation and centrifugation. The extraction resulted in a phase separation, with an organic top layer, aqueous bottom layer, and an insoluble matrix interface layer.
The top layer was injected (5 µL) onto an Shimadzu Prominence LC System and separation was performed on a GL Science Inertsil HILIC 5 µm (150x3 mm) column at a column temperature of 40 °C. Mobile phase (A) was acetonitrile 10 mM ammonium acetate and (B) water 10 mM ammonium acetate at a flow rate of 0.5 mL/min. The gradient was as follows: 0.1 min, 97%A, 3%B; 5.0 min, 20%A, 80%B; 5.5 min, 3%A, 97%B; 10 min, 97%A, 3%B.
An AB SCIEX API 3200 LC-MS/MS equipped with Electrospray Ionization (ESI) probe and Turbo V source was used in Multiple Reaction Mode (MRM). Precursor and product ions and the corresponding declustering potentials and collision energies were determined. Melamine could be detected in positive ion mode over a linear range of 3 orders of magnitude with LOD of 1 µg/kg, <5 %CV, and good accuracy. Replicate runs of blank samples and contaminated cat food samples permitted validation and the subsequent development of dedicated standardized test method.
This example demonstrates the use of a standardized system for food safety analysis, the AB SCIEX API 3200, and the development of simple methods for extraction and LC separation based on the chemical character of melamine. The performance of the LC method combined with the sensitivity and speed of the MS/MS method led to the development of a fast and accurate test for melamine in the complex pet food background. Tests such as these illustrate the power and robustness of a relatively basic LC-MS/MS platform, one that could rather easily be implemented in a variety of testing environments.