Analysis of Methionine and Selenomethionine in Food Using GC–MS: An Interview with Beatrice Campanella

1. Why is it important to measure methionine (Met) and selenomethionine (SeMet) in foods?

Selenium is both a toxic element and an essential micronutrient for humans, therefore its monitoring in food is paramount. The effects of a diet with an inadequate intake of selenium are well documented and still under continuous investigation. Since in many geographical areas the content of selenium in common feedstocks is limited, consumption of Se-enriched supplements has become a valid alternative to natural sources. Selenomethionine (SeMet) is a selenoamino acid in which selenium replaces sulphur in the structure of methionine (Met). It has been estimated that almost half of all dietary selenium derives from this aminoacid, and that selenium bioavailability from selenomethionine is 1.5- to 2-fold higher with respect to that of inorganic selenium. For this reason, most of the selenium supplements are based on SeMet. However, the selenium content of these products can differ from their label claims, hence there is the need for accurate methods for monitoring SeMet in nutritional supplements.

Unlike SeMet, the ingestion of accidentally high amounts of Met isn’t per se a cause of concern, however the assessment of its concentration in food might represent a useful tool from a quality control (QC) perspective. Methionine is an essential proteinogenic amino acid, meaning that it cannot be produced by human body, but it is needed as a building block in protein synthesis. The essentiality implies that Met must be introduced in our organism from external sources, the simplest of which is food.

2. What benefits does triethyloxonium tetrafluoroborate (TEOT) offer over traditional derivatization agents for analyzing methionine and selenomethionine?

Derivatization methods gave a major contribution to the consolidation of gas chromatography as analytical technique. The analysis of amino acids (AAs) with GC techniques has involved for many years the use of silylating agents such as N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) or N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA). Despite their popularity, silylating agents require very restrictive conditions: the reaction medium tolerates little to no water, and continuous heating is needed for high derivatization yields.

A popular alternative to silylating agents is represented by alkylchloroformates (ACFs), the ester halides of carbonic acid. ACFs readily react with AAs forming carbamates and esters on the amino and on the carboxylic groups, respectively. The high reactivity towards nucleophiles makes ACFs promptly hydrolyzable in water: the by-products include hydrogen chloride (irritant) and carbon dioxide (pressure build up). Furthermore, ACFs are generally flammable and, at lower molecular weights, quite volatile. ACFs cannot be considered green reagents but, more importantly, they can be hazardous for the health.

In this regard, trialkyloxonium tetrafluoroborates (TAOTs, general formula R₃O⁺[BF₄]⁻) are a class of derivatizing agents owing the typical characteristics common to the ionic liquids lately used in green chemistry: they are water-soluble, non-volatile, and are hydrolysed in aqueous solution to safe by-products. TAOTs are powerful alkylating agents reacting accordingly to this general scheme: R₃O⁺ + X⁻ (or X:) → RX (or RX⁺) + R₂O (R = alkyl chain). Although any exposure with TAOTs should be avoided (i.e., working under fume hood with adequate PPEs accordingly to SDS), in our opinion the risk related to handling TAOTs are much lower when compared with classic alkylating agents or ACFs which are volatile.

In the current global greening scenario, the development of cleaner analytical procedures is highly encouraged and represents a hot topic in scientific literature. TAOT salts fit well in this context, and this work will hopefully contribute to their diffusion as a greener alternative to classical derivatizing agents.

Figure 1: Summary of TAOT characteristics as derivatizing agents.

3. How does TEOT derivatize Met and SeMet, and why is this beneficial for gas chromatography–mass spectrometry (GC–MS)?

Met and SeMet are polar analytes with limited volatility, therefore they cannot be analyzed directly by GC–MS. Alkylation with triethyloxonium (TEOT) resolves these issues by converting the analytes in volatile, thermally stable ethyl-derivatives. Triethyloxonium is commercial, as well as trimethyloxonium, but the former reacts slower and can be handled in water (complete hydrolysis within 80 minutes) or in acetonitrile. When kept at –20 °C, TEOT solutions in acetonitrile are stable for more than a month.Figure 2 illustrates the scheme of the reaction between TEOT and a generic AA.

Figure 2: Schematization of the alkylating action of TEOT.

Having a carboxylic and an amino group makes Met and SeMet two ideal substrates for TEOT derivatization. For each analyte, 3 different derivatives could be obtained: the ethyl ester (EtMet/EtSeMet), the N-ethyl ethyl ester (Et₂Met/Et₂SeMet) and the N,N-diethyl ethyl ester (Et₃Met/Et₃SeMet). The degree of alkylation is dependent on pH. The derivatives can be easily extracted in a low polarity organic solvent such as hexane or isooctane, and the by-products of TEOT hydrolysis do not interfere with GC–MS analysis.

4. What safety and environmental advantages does TEOT provide compared to conventional reagents?

To this day, the reference method for the determination of Met and SeMet is based on ACFs derivatization. ACFs are not green reagents as they are toxic, flammable and volatile. Furthermore, ACFs amino acids derivatization requires pyridine as a catalyst (flammable and irritant) and chloroform (irritant, highly volatile, carcinogenic, toxic) for the extraction of the slightly polar derivatives. Although the volumes of these reagents are minimal, from a laboratory safety perspective it might be reasonable to develop greener procedures. Our approach consisted in the replacement of toxic and volatile reagents will less toxic and less volatile reagents.Although TAOTs are alkylating agents, therefore potentially genotoxic, the risk of handling them are mitigated because they are water-soluble and non-volatile salts. Furthermore, the additives that increase the derivatization efficiency (Na₂SO₄, borate buffer and EtOH) are considerably less toxic then those required for ACFs.

Figure 3: ACFs vs. TEOT hazardousness.

The Green Analytical Procedure Index (GAPI) for the proposed method has been assessed and compared to the derivatization process using methyl chloroformate, finding that the newly developed method significantly reduced the environmental impact compared to the commonly used alkyl chloroformate procedure. This has been further supported by a higher Eco-Scale value (77 vs. 75), which increased to 79 when hexane is replaced with isooctane for the extraction of the alkylated derivatives. This improvement is important for the broader adoption of the method in routine analytical applications.

5. How does the new method simplify sample preparation compared to multi-step derivatization procedures?

From a sample preparation point-of-view our method offers procedural advantages. The TEOT derivatization is a one-pot reaction where the sample is simply mixed with the reagents, without the need for sample purification steps. The procedure is simple and fast and allows to detect Met and SeMet at ultra-trace level (low μg/L).

Figure 4: Scheme of the proposed procedure.

6. What challenges does TEOT face when used in complex food matrices, and how were these managed in the study?

Since the TEOT is a generic ethylating agent, the presence of the matrix can potentially suppress the derivatization yield by consuming part of the reagent. However, the TEOT reactivity can be modulated by optimizing amount of TEOT and pH in a way to compensate for potential matrix effects.

For the analysis of SELM-1, we found that signal suppression due to the presence of the digested food matrix (calculated by comparison between the area of the isotopically labeled standards in calibration blends and in the samples) was 10% for Met and between 10-55% for SeMet. These effects were managed by the use of isotopically labelled internal standard which fully accounted for the incomplete derivatization inducted by the presence of the matrix.

7. Why was methanesulfonic acid chosen for sample digestion, and what benefits does it provide?

The standard protein hydrolysis procedure requires 6 N HCl for 20-24 h at 110 °C under anoxic conditions. The HCl method works for most applications, but it is not suitable for the quantitative recovery of S- and Se-containing amino acids, which suffer oxidation within the HCl medium. In order to prevent this issue, HCl should be replaced by other organic acids, such as methanesulfonic or p-toluenesulfonic acid. These acids allow almost full recoveries (> 90 %) for most amino acids, preserving their oxidation state.

In our study, the digestion procedure was further simplified by omitting the overnight reflux step and implementing a closed-vessel digestion which has several advantages (see Figure 5): it requires only a limited amount of sample and reagents, it is safe (the closed vial and the relatively low temperature don’t allow boiling or the generation of high pressure), and it allows a large number of simultaneous digestions (only limited by the size of the oven). Plus, convective ovens set at 110 °C are very common in every analytical laboratory.

Figure 5: Comparison of different digestion strategies for proteins hydrolysis.

8. How did the design of experiments (DoE) approach help optimize the derivatization conditions?

TEOT reactivity is influenced by pH and by the composition of the medium. Buffering at pH 9 is helpful to maintain Met and SeMet in their anionic forms which are readily converted into their ethylated derivatives (mono-, di- or tri-ethylated amino acid). However, the buffer itself can react with TEOT, and its composition should be optimized. An experimental design able to consider several parameters at the same time is very useful as it allows to optimize the reaction conditions with the minimum number of experiments. In this work we adopted a face centered central composite design (CCD) to study how buffering and amount of TEOT influenced the derivatization yield of Met and SeMet. Each experimental matrix was built with k²+ 2k + n experiments, where k is the number of factors (i.e., variables) and n is the number of replicate measurements performed in the centre of the experimental domain. A quadratic model was adopted to describe the experimental data.

9. What validation steps were taken to ensure the method's accuracy when applied to the Certified Reference Material (NRC SELM-1)?

Common steps of in-house method validation were carried out for the validation of the method, including the evaluation of procedural blanks, limits of detection, working range, spike recoveries, and trueness. The last performance characteristic was evaluated using the NRC SELM-1 which is a selenium enriched yeast CRM. From a quantitative point-of-view, the strength of the method can be found in the use of isotopically enriched internal standards which allow isotope dilution mass spectrometry (IDMS) quantitation. IDMS is a ratio method able to produce accurate results and it is regarded by many scholars as a primary method of analysis. When the isotopic internal standards are spiked to the sample at the beginning of the analysis–and are allowed to equilibrate with the sample matrix–they are able to fully account for analyte losses during sample preparation, such as incomplete analyte extraction and derivatization. Although IDMS offer such great methodological advantages, one should keep in mind that IDMS is inherently a nonlinear method, and the magnitude of nonlinearity depends on the overlap between the isotopes of analyte and internal standard. In order to account for such an effect, the calibration curves for Met and SeMet were fitted with a rational function of the form y = (a₀ + a₁·x) / (1 + a₂·x) which is the “true” IDMS model equation as obtained from first principles.

In our study, IDMS was also used to monitor the digestion efficiency over time. After a 7 days-digestion the values of Met and SeMet found using the novel method were not statistically different from the certified values (t-test). Method validation is summarized in Figure 6.

Figure 6: Summary of the novel ID-GC-MS method validation for Met and SeMet analysis in food.

10. Were your results as expected?

Both at the Italian CNR and at the Canadian NRC, we have been working with trialkyloxonium salts for more than a decade and we are used to enjoy simple analytical methods with high analytical performance. In this regard, the novel method for Met and SeMet quantitation was within expectations. However, we were surprised by the high derivatization yields for these amino acids. Despite working under mild conditions, only 0.3% of Met and SeMet remained unreacted and most of the analytes were converted into the diethyl- and triethyl-derivatives.

11. What has been the feedback from other researchers regarding this work?

In general, over the years we have received positive feedback from other research groups that have chosen trialkyloxonium salts as derivatizing agents for analytical applications. The work presented here is still relatively recent but very promising. We believe that the proposed method is a valid alternative with respect to the standard ACFs method. The procedure is simple and fast and allows to detect Met and SeMet at ultra-trace level (low μg/L). In conjunction with the more performing digestion protocol, the TEOT method would definitely be suitable for metrological applications.

12. Do you plan to continue developing methods for other analytes in food based on this work?

Definitely. The real challenge is to successfully derivatize and simultaneously determine a greater number of amino acids. Since quantitation is basically matrix independent, both food and clinical samples will be studied.

Another interesting possibility would be that of using TEOT derivatization in combination with microextraction devices like SPME (solid-phase microextraction) fibers, NTDs (needle trap devices) or MEPS (microextraction by packed sorbent) syringes: aside from eliminating the use of highly volatile organic solvents, there would be a preconcentration step that would make it possible to achieve detection in sub μg/L range.

Reference

(1) Malvestio, C.; Onor, M.; Bramanti, E.; Pagliano, E.; Campanella, B. Determination of Methionine and Selenomethionine in Food Matrices by Gas Chromatography Mass Spectrometry After Aqueous Derivatization with Triethyloxonium Salts. Food Chemistry 2024, 433, 137341. https://doi.org/10.1016/j.foodchem.2023.137341

Beatrice Campanella received her Ph.D. in Chemistry from the University of Pisa in 2016. Since 2019, she has been a permanent researcher at the Institute of Chemistry of Organometallic Compounds of the National Research Council (ICCOM-CNR) in Pisa. With a background in analytical chemistry, her research focuses on the development of spectroscopic and chromatographic methods for applications ranging from clinical diagnostics to environmental monitoring and cultural heritage.

Her work is focused on the development of analytical methods for the determination of both inorganic compounds that pose a threat to the environment and human health, as well as organic compounds of diagnostic (e.g., low molecular weight metabolites, proteins) and artistic (e.g., natural and synthetic dyes) interest. The strategies she employs include direct sample analysis (using Raman and SERS spectroscopy, LIBS, FT-IR spectroscopy, liquid chromatography coupled with UV/fluorescence detectors, and inorganic mass spectrometry), as well as methods that involve chemical reactions to convert target analytes into forms suitable for analysis while simultaneously separating them from the matrix (such as chemical and photochemical vapor generation coupled with gas chromatography-mass spectrometry or atomic fluorescence).