Meet New Requirements for Melamine Analysis at 1µg/g in Infant Formula By Julie Kowalski, Ph.D., Innovations Chemist, Michelle Misselwitz, Innovations Chemist, and Jack Cochran, Director of New Business and Technology

 

• Easily meet FDA requirements with simple, highly reproducible GC/MS method for 1µg/g melamine in infant formula.
• FDA GC/MS method successfully transferred to meet new lower MRL requirements.
• Highly reproducible retention times on Rxi®-5Sil MS column are key to accurate compound identification.
• Leak-proof integrated guard column reduces matrix contamination and extends analytical column lifetime.

Melamine is a nitrogen-rich industrial compound used in the manufacture of plastics, flame-resistant products, and cleaning agents. It is not a legal food additive; however, it has been added to food products in order to falsely represent the amount of protein present, as protein level is often determined using simple, nonspecific nitrogen content assays (Figure 1). Melamine is not considered toxic alone at low doses; however, illnesses and deaths have been traced to exposure to melamine in the presence of cyanuric acid.

 

In response the escalating health concerns surrounding melamine, the US Food and Drug Administration (FDA) recently set the safety threshold for melamine and related compounds in infant foods at 1µg/g. This level is significantly lower than the previously published minimum reporting levels (MRLs) for other commodities (10?g/g in pet foods and 2.5?g/g in human foods) and has led to an immediate need for more sensitive methodology. Here we adapted the FDA GC/MS procedure originally for higher MRL commodities into a highly reproducible method for the low level detection required for analyzing infant formula.(1)

Figure 1: Melamine and related compounds are rich in nitrogen and have been used to misrepresent protein levels in some food products.

Detailed Procedure for Infant Formula Now Available

Matrix spikes were prepared in control infant formula at 0.5µg/g, 1µg/g, and 5µg/g (dry formula was prepared according to label instructions prior to fortification). Standards were prepared in extracted matrix at on-column concentrations equivalent to those of fortified samples, in order to minimize possible matrix effects.(2,3,4) Samples and standards were derivatized according to the FDA method; complete preparation details are available at www.restek.com/melamine.

Analyses were performed on a Shimadzu QP 2010 Plus GC/MS equipped with an AOC 20i+s auto injector and sampler. GC conditions are shown in the figures; masses analyzed are shown in Table I. An Rxi®-5Sil MS analytical column with a 5m Integra-Guard™ integrated guard column was used for analysis. The integrated guard column was chosen since it protects the analytical column from matrix contamination with no risk of leaking.

Table I: MS conditions (SIM mode).
Compound tR (min.) Quant. ion* Qual. ion Qual. ion Qual. ion cyanuric acid 10.23 345 (100) 345 (36) 346 (30) 347 (15) ammelide 11.07 344 (100) 329 (58) 345 (30) 330 (16) ammeline 11.76 328 (100) 343 (79) 329 (29) 344 (24) melamine 12.31 327 (100) 342 (53) 328 (30) 343 (17) benzoguanamine 14.54 316 (100) 331 (68) 332 (20) 330 (9) * Expected relative ion ratios from the FDA method.

Highly Reproducible Retention Times Assure Accurate Peak IDs

This method successfully detected melamine and cyanuric acid to the low levels required for the analysis of infant formula (Figure 2). Highly reproducible chromatographic separation was achieved and was critical for compound identification, since several quantitation ions were also found in other peaks. The FDA method requires retention times to be within 0.05 minutes for compound identification. This was easily achieved using the Rxi®-5Sil MS column, which produced highly reproducible results even after the approximately 150 injections made during method establishment (Table II).

Table II: Retention time is critical to accurate peak identification. Highly reproducible results were achieved using an Rxi®-5Sil MS column (n=3).
Retention time (min.)(mean±std.dev.) 0.5µg/g 1µg/g 5µg/g cyanuric acid 10.26 ± 0.05 10.23 ± 0.0006 10.23 ± 0.001 ammelide 11.08 ± 0.003 11.07 ± 0.002 11.08 ± 0.003 ammeline 11.76 ± 0.001 11.76 ± 0.003 11.76 ± 0.002 melamine 12.31 ± 0.002 12.31 ± 0.000 12.31 ± 0.004 benzoguanamine 14.54 ± 0.002 14.54 ± 0.001 14.54 ± 0.002

The analysis of melamine and related compounds in infant formula is challenging since it has the lowest MRL of all commodities and because its high sugar content results in significant matrix interferences. Due to these factors, reliable retention time identification was critical for compound identification. Using the Rxi®-5Sil MS column, highly reproducible retention times were achieved and target analytes were reliably detected at 1µg/g in infant formula. Successful establishment of this method for lower level MRL commodities and packaging of all the required components into a single kit with detailed instructions provides analytical laboratories with a simple solution for meeting new FDA food safety guidelines for melamine.

 

Figure 2: Analysis of melamine and related compounds in infant formula (1µg/g MRL spike level).

A. Solvent-only standard (0.01µg/mL injection concentration) Ret. time (min.) 1. cyanuric acid 10.238 2. ammelide 11.090 3. ammeline 11.770 4. melamine 12.318 5. benzoguanamine 14.553 B. Matrix spike (0.01µg/mL injection concentration) Ret. time (min.) 1. cyanuric acid 10.258 2. ammelide 11.073 3. ammeline 11.760 4. melamine 12.328 5. benzoguanamine 14.538 Column: Rxi®-5Sil MS, 30m, 0.25mm ID, 0.25µm, w/ 5m Integra-Guard™ (cat.# RE13623-124) Instrument: Shimadzu QP 2010 Plus Sample: A. Melamine and Related Analogs Stock Solution (cat.# RE33253) Benzoguanamine (cat.# RE33251) as tri-TMS derivatives, injection concentration: 0.01µg/mL B. infant formula fortified at 1µg/g with Melamine, Related Analogs Stock Solution (cat.# RE33253), Benzoguanamine (cat.# RE33251), analyzed as tri-TMS derivatives, injection concentration: 0.01µg/mL Inj.: 1.0µL splitless (hold 1 min.), 3.5mm splitless inlet liner with wool (cat.# RE22286-200.1) Inj. temp.: 280°C Carrier gas: helium, constant linear velocity Flow rate: 1mL/min. Oven temp.: 75°C to 320°C @ 15°C/min. (hold 4 min.) Det.: MS Transfer line temp.: 290°C Ionization: EI Mode: SIM (all method ions in table, only quantification ions were plotted)

REFERENCES

1. US Food and Drug Administration, October 2008, GC-MS Screen for the Presence of Melamine, Ammeline, Ammelide, and Cyanuric Acid, Laboratory Information Bulletin No. 4423, http://www.cfsan.fda.gov/~frf/lib4423.html.
2. C.F. Poole, J. Chromatogr. A 1158, 241-250 (2007).
3. T. ?ajka, K. Maštovská, S.J. Lehotay and J. Hajšlová, J. Sep. Sci. 28, 1048-1060 (2005).
4. K. Maštovská, S.J. Lehotay and M. Anastassiades, Anal. Chem. 77, 8129-8137 (2005).