Quantitation in LC-MS
Credit: iStock.
Each year, there are an estimated 600 million people in the world who become sick from consuming contaminated food. With growing concerns over a multitude of food risks, accurate and sensitive analytical tools are needed to ensure food safety. With recent improvements in its technology, liquid chromatography-mass spectrometry (LC-MS) has become an indispensable method for food safety testing and quality control. It can detect and quantify trace amounts of numerous contaminants and harmful species in food, including allergens, migrants from food contact materials, pesticides and mycotoxins, and even check the authenticity of food products. Here we will discuss how LC-MS can detect the presence of even tiny quantities of harmful substances on our plates.
1. Food allergens
Food allergies have become a growing public concern as the number of people suffering from them has increased greatly. Reliable methods to detect and quantify allergens in natural and processed foods are important to prevent severe health issues.
In order to use liquid chromatography with tandem mass spectrometry (LC-MS/MS) for detection, we first need to identify the targets. The allergens in foods are always proteins,1 and for LC-MS/MS, proteins are very large compounds. Therefore, instead of using the whole proteins, peptide markers of the protein allergens are usually used as the targets. At the method development stage, several peptide markers are typically selected and undergo optimization for LC separation, charge-state selection, and compound-dependent MS parameters. The peptide markers with good resolution chromatographic peaks, satisfactory signal-to-noise ratio, that are less susceptible to matrix interference and are stable, are selected for the identification and quantification of the specific food allergens. Once the peptide markers are selected, calibration curves could be generated by preparation of the peptide markers at known concentrations. The amounts of the allergens in food samples can then be calculated.
2. Food contact materials
Whilst transitioning from harvest to delicious cuisine on our plates, our food comes into contact with many materials including plastics, paper, rubber, ceramics, and metals. Migration of chemical substances from packaging or food containers to food can pose serious health issues if unmonitored. Additives in plastics, such as plasticizers, can be dissolved in oily food products and therefore cause health concerns. The European Union (EU) provides a lengthy list of plastic monomers and food additives with specific migration limits.2 LC-MS is useful for the analysis of intermediate and low volatility additives and is often selected as the tool for simultaneous analysis of multiple migrants.
a) Per-and polyfluoroalkyl substances (PFAS)
PFAS compounds are commonly used for surface treatment in cooking papers and food packaging due to their highly repellent property. They are persistent in living organisms and accumulate easily in the body. Therefore, PFAS could aggregate in the food chain. Studies have shown that exposure to PFAS can cause kidney and testicular cancer, liver problems, hormone disruption, and neurodevelopmental disorders.3 PFAS are regulated to trace amounts. For example, perfluorooctane sulfonates (PFOS), one of the PFAS compounds, should not exceed 50 mg/kg, i.e. 0.005 % by weight, as indicated by the EU.4 LC-MS/MS is helpful to detect PFAS residues in food.
In order to identify and quantify PFAS in food contact materials, such as plastics, and the foods themselves, the samples are first cut into small pieces and ground into powder. The sample is then extracted with methanol, and the extract is cleaned up with solid phase extraction before LC-MS/MS analysis. The calibration curves of PFAS could be prepared by standards at known solutions. The amount of PFAS present in the sample is then determined by comparison to the calibration curve.5
b) Bisphenol A
Bisphenol A (BPA) is commonly used in the production of plastic materials, such as food packaging, tableware and cookware. It is a suspected endocrine disruptor, which may lead to adverse health effects if consumed in excess.6 It is regulated with a specific migration limit in many countries. LC-MS/MS is a popular technique to determine BPA in food contact materials.7
With a defined chemical structure, LC and MS conditions could be optimized for BPA with the conventional reversed-phase technique. Depending on the samples, BPA can be extracted by organic solvents,8 dissolution or reprecipitation of polymers,9 or with solid phase extraction10. Quantification is achieved by constructing a calibration curve with standard solutions of BPA at known concentrations and compared to the amounts detected in the sample.
c) Unknown migrants
There are many migrants that could be potentially harmful yet unknown to analysts. High resolution accurate MS is useful for the identification of unknown migrants based on non-targeted screening approaches. Combined with ultra-high-performance liquid chromatography (UHPLC), unexpected migrants could be unmasked. The difference between targeted and non-targeted detection is that we cannot use known chemical standards to identify or quantify the unknown compounds.
Identification of unknown compounds could be achieved by searching databases to find a possible elemental composition for the unknown compounds. Alternatively, retrospective analysis and data processing with mass spectral libraries of accurate masses, commercial or homemade databases can be used for data analysis.11 In contrast to classical quantification with known concentrations of targeted standards, semi-quantifications with a different chemical as a surrogate can be used for quantitation of unknown migrants. In theory, the surrogate chemical should have a similar measured response factor (RF) to the quantified chemical.12 By comparing the concentration of the unknown migrants and the surrogate chemical, the concentration can be determined.
3. Pesticides
Pesticides have long been a food safety issue and encompass a wide range of compounds. Whilst many of the compounds are already monitored closely with current technologies, many compounds are still yet to be covered by conventional methods. In particular, polar pesticide residues in food have been a challenge for LC-MS/MS as they have a very short retention time using conventional methods and are easily interfered with by food components present in the sample.
Polar pesticides are very soluble in water. Sample extraction of these polar pesticides usually involves acidified methanol instead of acetonitrile (used in conventional QuEChERS - Quick Easy Cheap Effective Rugged Safe method) as the extraction solvent. Anion exchange solid phase extraction (SPE) columns could be used to clean up the sample matrix before LC-MS/MS analysis. One of the problems with conventional methods is the low retention property of polar pesticide compounds with conventional reverse phase columns. Studies have suggested the use of hydrophilic interaction liquid chromatography (HILIC) columns, ion exchange columns or porous graphite carbon columns to increase the retention of the polar pesticides.13,14 As these pesticide compounds may be interfered with by sample components, calibration curves can be constructed with spiked standards onto the same sample matrix type. Quantification is then performed based on comparison with the calibration curve.
4. Mycotoxins
Mycotoxins are produced by some fungi and could pose a food safety risk. They are generally chemically stable and difficult to destroy during food processing or with heat. There are many mycotoxins with diverse physicochemical properties and therefore challenging for analysis. Pure organic solvent is not adequate to extract all mycotoxin residues as some of them are very polar. It is therefore proposed that a mixture of acetonitrile and water is used to extract most mycotoxin residues. LC-MS/MS with reversed phase columns have often been used, but studies also suggested the use of a polyaromatic column is better for polar mycotoxin residues. For each mycotoxin, one precursor and two product ions are first identified. Then the most abundant product ion is selected for quantification and the second one for confirmation. A calibration curve is constructed based on the preparation of standards at different known concentrations. The amounts of mycotoxins in samples is then determined by calculations.15
References
1. Bannon GA. What makes a food protein an allergen? Curr Allergy Asthma Rep. 2004;4(1):43-46. doi:10.1007/s11882-004-0042-0
2. Union Guidance on Regulation (EU) No 10/2011 on plastic materials and articles intended to come into contact with food as regards information in the supply chain. Accessed from: https://ec.europa.eu/food/sites/food/files/safety/docs/cs_fcm_plastic-guidance_201110_en.pdf.
3. B. Demeneix, R. Slama. Endocrine Disruptors: from Scientific Evidence to Human Health Protection. 2019 PETI Committee of the European Parliament. Accessed from: http://www.europarl.europa.eu/RegData/etudes/STUD/2019/608866/IPOL_STU(2019)608866_EN.pdf.
4. Commission Regulation (Ec) No 552/2009. Accessed from: https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:164:0007:0031:EN:PDF.
5. GB 31604.35-2016, National Food Safety Standard - Food contact materials and products - Determination of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA).
6. Konieczna A, Rutkowska A, Rachoń D. Health risk of exposure to Bisphenol A (BPA). Rocz Panstw Zakl Hig. 2015;66(1):5-11.
7. Cardama A, Rodriguez A, Sendón R. Analysis of Bisphenol A in Beverages and Food Packaging by High- Performance Liquid Chromatography. Food Nutr J. 2017;5. doi:10.29011/2575-7091.100043
8. Cheng Y, Nie X, Wu H, et al. A high-throughput screening method of bisphenols, bisphenols digycidyl ethers and their derivatives in dairy products by ultra-high performance liquid chromatography-tandem mass spectrometry. Analytica Chimica Acta. 2017;950:98-107. doi:10.1016/j.aca.2016.11.006
9. Dreolin N, Aznar M, Moret S, Nerin C. Development and validation of a LC–MS/MS method for the analysis of bisphenol a in polyethylene terephthalate. Food Chemistry. 2019;274:246-253. doi:10.1016/j.foodchem.2018.08.109
10. Gallart-Ayala H, Moyano E, Galceran MT. Liquid chromatography/multi-stage mass spectrometry of bisphenol A and its halogenated derivatives. Rapid Commun Mass Spectrom. 2007;21(24):4039-4048. doi:10.1002/rcm.3307
11.Martínez-Bueno MJ, Gómez Ramos MJ, Bauer A, Fernández-Alba AR. An overview of non-targeted screening strategies based on high resolution accurate mass spectrometry for the identification of migrants coming from plastic food packaging materials. TrAC Trends in Analytical Chemistry. 2019;110:191-203. doi:10.1016/j.trac.2018.10.035
12. Pieke EN, Granby K, Trier X, Smedsgaard J. A framework to estimate concentrations of potentially unknown substances by semi-quantification in liquid chromatography electrospray ionization mass spectrometry. Analytica Chimica Acta. 2017;975:30-41. doi:10.1016/j.aca.2017.03.054
13. M. Anastassiades; D. I. Kolberg; E. Eichhorn; A. Benkenstein; S. Lukačević; D. Mack; C. Wildgrube; I. Sigalov; D. Dörk; A. Barth. Quick Method for the Analysis of numerous Highly Polar Pesticides in Foods of Plant Origin via LC-MS/MS involving Simultaneous Extraction with Methanol (QuPPe-Method). http://www.crl-pesticides.eu/library/docs/srm/meth_QuPPe.pdf.
14. Jiang Y, Cao Z, Jia R, Qi H, Chen M. [Determination of glyphosate and aminomethylphosphonic acid in rice using hydrophilic interaction chromatography-tandem mass spectrometry]. Se Pu. 2012;30(1):39-44. doi:10.3724/sp.j.1123.2011.08040
15. De Santis B, Debegnach F, Gregori E, et al. Development of a LC-MS/MS Method for the Multi-Mycotoxin Determination in Composite Cereal-Based Samples. Toxins (Basel). 2017;9(5). doi:10.3390/toxins9050169
