Emerging Techniques: Liquid Chromatography Mass Spectrometry for Food Safety and Quality

In a recent overview, Yolanda Picó presented liquid chromatography mass spectrometry (LC-MS) as a rapidly developing technology with wide applications for food safety and quality assessment.¹ The flexibility of LC-MS renders it particularly suitable for the food industry with its diverse analytes in complex matrices, and Picó provided a comprehensive table summarizing recent applications. Additionally, she highlighted major uses of both targeted and untargeted analysis.

When it comes to LC separation, the author notes its importance and indicates that reversed phase (RP) LC accounts for over 95% of applications with hydrophilic interaction LC (HILIC) emerging as an option for very polar analytes. Picó recommends column miniaturization as a means to increase efficiency, sensitivity, and peak resolution. To enhance peak resolution and thus sensitivity, ultra-high performance (UHPLC) columns with smaller particle size (1.7 and 1.8 μm) can be employed, representing over 50% of the applications reported in Picó’s table despite the relative newness of UHPLC technology. LC challenges include peak coelution and poorly retained peaks. One area of interest for addressing these challenges is multidimensional chromatography, particularly the coupling of HILIC and RP-LC.

The author reports electrosray ionization (ESI) as the most common ionization source with APCI employed in some cases (like carbadox and acidic metabolites). She lists the following most common MS techniques coupled with LC: triple quadrupole (QqQ), quadrupole ion trap (QTRAP), time of flight (TOF), quadrupole time of flight (QqTOF), and Orbitrap. Picó indicates that matrix effect (signal suppression or enhancement resulting from coelution of matrix components) can impact quantitative analysis via LC-MS. Since matrix effect derives from the ionization source, it can be an issue for all mass analyzers, ultimately reducing sensitivity and selectivity as well as performance parameters like accuracy, precision, linearity, and limits of both detection and quantification. She lists the following as techniques that can improve accuracy: standard addition, isotope dilution, echo-peak technique, postcolumn standard infusion, internal standard, and matrix matched calibration.

Picó offers targeted analysis as the basic model traditionally chosen for food safety and quality applications, including:

polyphenol content	additives (and byproducts) in plastic packaging	food additives	free amino acids	mycotoxins
nucleobases, nucleosides, and nucleotides	monoacylglycerols and free fatty acids	β-sitosterol	pesticides	veterinary medicine residues
choline	folic acid and folates	ginsenosides	physalis	bisphenol A and alkylphenols
biogenic and volatile amines	isoflavones, flavonols, and triterpene glycosides	organic acids	marine lipophilic toxins	tea catechins

The requirement to optimize and validate for individual compounds rendered these protocols inefficient and expensive, leading to the evolution of targeted analysis using general extraction for a wide range of analytes plus multiscreening/multisearching techniques. Multitargeted screening is generally semi-quantitative, using standard solutions of common compounds to screen for presence/absence of a wide range of analytes, particularly useful for assessing whether contaminants approach or exceed legal limits. For example, a recent study analyzed fruits, vegetables, and related food products for 300 pesticides using an aliquot of raw acetonitrile extract followed by cleanup with fully automated multidimensional LC using a HILIC column. The team was able to identify and confirm 44 targeted compounds.² 

Picó emphasized the importance of addressing sensitivity and selectivity limits and interferences when working with complex food matrices. Toward this end, she indicates that high-resolution, full-scan MS (HRMS) offers an alternative to tandem MS, noting that Orbitrap mass spectrometers offer exceedingly high resolution and mass accuracy in full-scan mode. HRMS also allows researchers to acquire data for retrospective analysis. Picó references a recent application of HRMS to screen for plant and fungal toxins and metabolites, using a database of over 600 metabolites to create contamination profiles in food and feed. The team directly injected extracts into an automated turbulent flow clean-up system linked with an LC-Q-Orbitrap mass spectrometer. They were able to validate 15 fungal and plant metabolites in varied food/feed matrices, highlighting wide applicability.³ Another study combined UHPLC with Orbitrap Exactive HRMS to rapidly screen for multiple, varied contaminants- including pesticides, mycotoxins, and veterinary drugs- in baked goods.⁴ Their success highlights the ability to screen for a wide variety of food contaminants.

Another powerful example of LC-MS application for the detection of food contamination is the harnessing of selected reaction monitoring (SRM) and biomarker peptides to identify accidental adulteration of meat products as well as food fraud. Picó indicates that SRM methods have been able to accurately detect 0.55% horse/pork contamination and 0.13% pork contamination in a beef matrix. ⁵ Dairy matrices can pose a particular challenge due to complexity, but another study demonstrates the monitoring of a differentially phosphorylated beta-casein f33-48 peptide to sensitively and selectively detect contamination of buffalo mozzarella.⁶ Researchers also applied one-dimensional polyacrylamide gel electrophoresis, nanoflow LC separation, and LTQ-MS to map 446 unique proteins in Atlantic cod.⁷ This large-scale proteomics approach lays the groundwork for examining the effects of environmental and disease-related factors on meat quality during industrial processing.

Overall, Picó offers LC-MS as an emerging trend for the food industry. These -omics strategies applied to food products (food-omics!) for the purpose of driving production decisions and ensuring consumer safety and satisfaction could offer producers greater flexibility and confidence.

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References:

1 Picó, Y. (2015) ‘Mass Spectrometry in Food Quality and Safety: An Overview of the Current Status.’ Comprehensive Analytical Chemistry, Vol. 68, http://dx.doi.org/10.1016/B978-0-444-63340-8.00001-7.

2 Kittlaus S. et al. (2013) ‘Development and validation of an efficient automated method for the analysis of 300 pesticides in foods using two-dimensional liquid chromatography–tandem mass spectrometry.’ Journal of Chromatography A 1283 (0): 98–109.

3 Ates, E et al. (2014) ‘Screening of plant and fungal metabolites in wheat, maize and animal feed using automated on-line clean-up coupled to high resolution mass spectrometry.’ Food Chemistry 142: 276–284.

4 De Dominicis, E. et al. (2012) ‘Targeted screening of pesticides, veterinary drugs and mycotoxins in bakery ingredients and food commodities by liquid chromatography-high-resolution single-stage Orbitrap mass spectrometry.’ Journal of Mass Spectrometry 47 (9): 1232–1241.

5  von Bargen, C et al. (2013) ‘New sensitive high-performance liquid chromatography tandem mass spectrometry method for the detection of horse and pork in Halal beef.’ Journal of Agricultural Food Chemistry 61 (49): 11986–11994.

6 Russo, R. et al. (2012) ‘Detection of buffalo mozzarella adulteration by an ultra-high performance liquid chromatography tandem mass spectrometry methodology.’ Journal of Mass Spectrometry 47 (11): 1407–1414.

7  M. Gebriel, et al., Cod (Gadus morhua) muscle proteome cataloging using 1D-PAGE protein separation, nano-liquid chromatography peptide fractionation, and linear trap quadrupole (LTO) mass spectrometry, J. Agr. Food Chem. 58 (23) (2010) 12307–12312.