Improving LC-MS/MS allergen testing result quantification
Accurate, precise and transparent allergen results enable you to complete meaningful quantitative allergen risk assessments and assess the possible impact on allergic consumers, and can help you to track down the source of contamination.
What are we changing?
Going forward, FACTS will quantify its liquid chromatography tandem mass spectrometry (LC-MS/MS) allergen results using Internal Quantification Standards (IQS), and move away from using external calibrants. In the past, as with ELISA results, LC-MS/MS results were quantified by comparing MS data to the calibration curve of a defined calibrant – e.g. non-fat milk powder, in the case of milk analyses.
What does it mean for you?
Accurate, precise and transparent allergen results enable you to complete meaningful quantitative allergen risk assessments, assess the impact on allergic consumers and it can help you to track down the source of contamination. All this contributes to the development and maintenance of robust, meaningful and fit-for-purpose allergen management controls.
Why are we making this change?
Processed allergenic proteins change which can give inaccurate and skewed results. For example, the casein or whey components of milk may be individually altered or depleted, resulting in distorted total milk results when compared with an external calibrant. Once the above changes are implemented a quantitative result would be a more accurate reflection of the true allergen protein content in a sample; i.e. how much total milk, but also how much whey and casein. For more information see the last section of this article.
When will this change take effect?
The new method of quantification and reporting will be phased in from February 2022. We will start with total milk, followed by egg and the other allergens.
More information for the science nerds!
Historically, quantitative allergen results were commonly expressed as a concentration of an allergen-containing commodity, e.g. defatted peanut powder or whole egg powder. These allergen-containing commodities served as external calibrants. The reason for this was that the conversion from a signal to a quantitative result required comparison to an external standard curve. This standard curve had to be constructed using something traceable, easily obtainable, and convertible to the relevant units (the concentration of allergenic protein).
When we introduced our allergen LC-MS/MS methods, initially we maintained the use of external calibration curves, to make results comparable with those from ELISA. However, it soon became clear that this approach has some significant disadvantages.
When the source of contamination differs significantly from the assay calibrant, error is introduced into the result. This is particularly acute with allergenic ingredients that can be spilt and manipulated extensively, such as milk and egg.
Additionally, a result expressed as a whole commodity, e.g. non-fat milk powder, does not take advantage of the additional depth of information and transparency offered by mass spectrometry – for example, whether the detected egg proteins are from the white, the yolk, or both. This information might provide clues as to the source of the contamination, and more accurately represent the risk to the allergenic consumer.
Having noted the advantages of moving away from commodity-based calibration, we decided to make use of the internal controls present in proteomics-based assays to provide absolute quantification.
This approach makes use of synthetic, chemically identical peptide standards added at a known concentration to each and every sample. The standard is labelled with stable heavy elemental isotopes, modifying their mass relative to the native/natural peptide that may or may not be present in the sample. The mass spectrometer can measure both simultaneously, and the ratio between them allows the precise absolute determination of the amount of the target peptide, and therefore the allergenic protein.
This change will enable the precise quantification of specific proteins and therefore simultaneous quantification of fractions of an allergenic ingredient, as well as conversion to a commodity to maintain compatibility with historical data.
We will begin changing the method of quantification and reporting from February 2022, starting with total milk, for which the error in measurement caused by the expression of results as a commodity has been most obvious. Many cases of contamination are found with primarily either the whey or the casein fraction present, which means results expressed in terms of milk powder equivalents do not reflect risk correctly or provide information regarding the nature of the contamination.
Total milk results will be expressed as total milk protein (the sum of total whey and casein protein). Results will be expressed in SI units (mg/kg) and to enable future transparency, additional values for several individual proteins can be provided where necessary.
View our testing catalogue for more information on our LC-MS/MS testing offerings.
Other articles and resources you may be interested in:
Targeted proteomics: a go-to-method
FACTS Targeted Proteomics by LC-MS/MS
Benefits of Targeted Proteomics (by LC-MS/MS) for allergen detection
Comparing test methods: PCR, ELISA & Targeted Proteomics by LC-MS/MS
Analytical advancements in Targeted Proteomics
Method selection for allergen detection
FACTS Testing Catalogue