Carefully Select Tools to Analyze Melamine, Dioxin

The European Commission and the U.S. Environmental Protection Agency (EPA) have set maximum levels for dioxins in food. Tests on some of the Irish pork products showed up to 200 times more dioxins than the recognized safety limit. Because the directives require limits of quantitation (LOQ) that are 80% lower than the lowest reported level in the US EPA Method 1613 Rev.B[3-7], laboratories require more demanding detection limits, selectivity, and sensitivity to confirm their presence, along with tools that manage data and can detect problems earlier in the process.

Melamine Analysis

Selecting the right method is critical to getting the right result. For a laboratory testing a large number of samples, throughput is a priority, and a fast screening method is ideal. If detecting a toxic or potentially lethal chemical is the goal, the selectivity of the method is paramount. Below are two timely examples, using melamine and dioxins, to illustrate the numerous variables involved in method selection and the important role the right technology can play.

The FDA has published six methods for melamine analysis using:

• Liquid chromatography-ultraviolet (LC-UV, FCC high-pressure liquid chromatography-ultraviolet [HPLC-UV] method);
• Gas chromatography-mass spectrometry (GC-MS) or GC-MS/MS (FDA LIB 4423);
• LC-MS/MS (FDA LIB 4396, 4421 and 4422); and
• Enzyme-linked immunosorbent assay (ELISA) using antigen-antibody reaction, a method developed for screening melamine in milk and milk products.

The LC-MS/MS method provides the lowest LOQ of all the FDA methods, 10-50 µg/kg. The superior LOQ is a result of highly selective reaction monitoring (H-SRM) technology, which significantly reduces matrix effects, producing cleaner mass spectrometry data and unmatched analyte specificity. The LC-MS/MS method for melamine involves solid phase extraction cleanup, which removes interfering compounds in dirty matrices such as seafood and meat. LC-MS/MS solutions can yield accuracy and precision values for FDA methods that are well within the guidelines of the FDA for analytical method development and analysis.

The GC-MS method requires a derivatization step to convert melamine and its related compounds, cyanuric acid, ammeline, and ammelide, to volatile derivatives. In selected ion monitoring mode, the GC-MS method can obtain LOQ of about 100 µg/kg for melamine. Using SRM of GC-MS/MS, the method can reach a lower LOQ of 10 µg/kg.

The LC-UV method is fast and does not need sample cleanup for melamine in milk products. If upgraded to ultra-high pressure liquid chromatography (UHPLC), the LC-UV method can detect melamine within two minutes in an LC run.

The ELISA method is fast and easy to use. Up to 96 samples can be run within two hours in a microplate using ELISA, though there is the possibility of cross-reaction with related compounds. If there is a non-detect, no further analysis is necessary. If melamine is detected, other tests may have to be run in order to meet regulatory requirements and ensure that a cross-reacting compound did not cause the response.

Dioxin Analysis

Dioxins, also called polychlorinated dibenzodioxins (PCDDs), are a group of organic compounds with one to 10 chlorines attached to biphenyl (see Figure 1, above). Theoretically, there are 209 congeners of dioxins, with TCDD (2,3,7,8-tetrachlorinated dibenzo-p-dioxin) being the most toxic dioxin to humans. Dioxins are environmental pollutants generated by the incineration of chlorinated compounds, as in trash burning, for example. Dioxins can cause cancer and severe reproductive and developmental problems in humans. Due to the toxicity of dioxins, the EPA has established MRLs for dioxins at 1 to 5 µg/kg in solids and 0.5 to 5 µg/kg in extract.

There are three analytical methods that have an LOD in the 10-12 g range, the performance criteria specified for dioxins by EU Commission Directive 2002/70/EC:

• High-resolution GC/MS (HRGC/HRMS);
• Low-resolution MS (LRMS) such as ion-trap and triple-quadrupole MS; and
• Cell-based bioassay (EPA method 4425).

For confirmation, HRGC/HRMS (EPA 1613B) is the only choice that meets the requirements of both EPA and EU regulations due to the fact that LRMS is unable to separate isomers of dioxin congeners and dioxin-like polychlorinated biphenyls (PCBs). For confirmation of dioxins, EPA method 1613B (requiring HR GC-HRMS) or its equivalent will be the choice.