Overcoming LC-MS/MS Matrix Effects for Maximum Reliability

As in any analytical technique, liquid chromatography-tandem mass spectrometry (LC-MS/MS) results are influenced by the matrix analyzed. These matrix effects – caused by ion enhancement or suppression – can produce overestimates or underestimates of measured values of the target analyte. Validation of routinely used methods may eliminate the most severe effects in common matrices. However, even in very simple matrices like corn, different varieties might have different influences on ionization, therefore leaving significant room for unreliability.

Common approaches to counter matrix effects include matrix- matched calibration, standard addition to each sample and the application of internal standards. The first two strategies entail additional work in terms of sample preparation or additional runs, incurring further costs. Importantly, matrix-matched calibration and standard addition cannot fully compensate for matrix effects. The effectiveness of internal standards in compensating for matrix effects depends on the choice of calibrants.

Calibrant choice matters

Internal standards can be chemically-related compounds such as derivatives (e.g. zearalanone for zearalenone) or similar compounds with identical behavior during the ionization process that differ only in terms of the mass of the atoms (stable isotopes). LC-MS/MS analyses rely on stable isotope dilution assays to overcome matrix effects by the addition of known amounts of stable isotope-labeled standards to the analyzed sample. Stable isotope labeling involves the use of non-radioactive isotopes like ²H, 13C or 15N to replace the naturally occurring atom. Using Deuterium (²H) to replace the naturally occurring 1H doubles the mass, which is the reason why deuterated labeled standards might show retention time shifts resulting in less accurate LC-MS/MS results.

Natural DON 296 amu → +15 amu → 13C DON
Natural DON 296 amu → +15 amu → 13C DON

Carbon, on the other hand, is naturally present in most compounds and mycotoxins. Replacing naturally-occurring 12C by 13C changes the total mass of the atom only slightly, unlike deuterated labeled standards. Added 13C-labeled standards, e.g. 13C-labeled mycotoxins, retain the same characteristics as their 12C analogues, eluting at the same retention time from the separation column.
The difference in mass between 12C and 13C mycotoxins, as shown for deoxynivalenol in Figure 1, allows the separation and identification of both eluted forms when the detection is performed with mass spectrometry.
The 13C peak represents the known quantity of the added standard. This peak can then be used to calculate the unknown amount of the analyte and thereby compensate for different ionization efficiencies. This explains how innovative 13C standards eliminate matrix effects.

Figure 1. Full scan MS spectra of (13C15)-Deoxynivalenol (Biopure)

Red line: Naturally-occurring DON. Blue line: BiopureTM 13C internal standard.

Reference materials

High quality reference materials are a must for accurate and reliable results in any analysis. Under the brand Biopure™, Romer Labs offers the broadest range of reference materials currently available on the market. These high quality products are available in crystalline or liquid form for mycotoxins, residues and contaminants.

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There’s more in Spot On, the free Romer Labs magazine dedicated to diagnostic solutions and quality assurance in food and feed. Topics from this issue include:

  • The top 3 challenges in external accreditation audits 
  • Traceability and certified reference materials
  • A behind-the-scenes look at creating liquid mycotoxin calibrants

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