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The Global Chemical Industries Summit 

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Determination of Organophosphate Esters in Water Samples Using GC–MS and Magnetic SPE 

Are You Sure You Are Using Your Pipette Properly?

Sweating the Small Stuff

The ancient craft of brewing beer is more than 5000 years old. As it has evolved, so have the methods of analysis employed to perfect the qualities of the brew. New research that allows for analysis and profiling of metabolites in the beer matrix is providing solutions for perfecting and maintaining beer quality. Stefan A. Pieconzonka and Philippe Schmitt-Kopplin have profiled diverse beer samples by rapid flow-injection analysis (FIA) Fourier transform ion cyclotron mass spectrometry (FTICR–MS). Here, they discuss their approach to this analysis, along with the significance of the Maillard Reaction (MR) in beer quality and analysis.

In a recent paper, you and your team profiled a set of 120 diverse beer samples by rapid flow-injection analysis Fourier transform ion cyclotron mass spectrometry (1). Why is it important to uncover and assign compositional information to what your paper describes as “thousands of yet-unknown metabolites in the beer matrix?”

Our analytical approach in this paper resolves the molecular composition and complexity of the samples in a holistic way. It gathers information of precise mass and thus sum formula that can be used to describe the intrinsic chemical compositional nature of the samples. Recognizing that 70–85% of the resolved mass signals do not have a database entry, it becomes clear that the majority of information about foods and beverages is complex and not yet unraveled. Our paper show this in a holistic analytical approach while visualizing, as well, this “dark metabolome” of unknowns.

On the other hand, the compositional information can be utilized to detect molecular patterns of interest and go deeper in structural analysis and data mining. That might be food authenticity, correlating patterns with flavor and aroma, food quality, or the description and guidance of processes. In times where “big data” frequently finds its way into everyday lives, big analytical datasets that hide deep information mirror this development.

What challenges did you encounter while profiling the compositional space of these diverse beer samples?

First, we were surprised how complex the beer matrix is. The huge molecular diversity of beer is based on various compositional factors such as the (oligo) saccharide composition, fermentation signature, Maillard reaction products, several plant-derived secondary metabolites (hops, barley, wheat, and so on). It creates an analytical spectrum of high complexity and molecular composition.

What role do small molecules (<1000 Da) for the beer metabolome play in raw material and final beer quality attributes, such as taste, aroma, yeast fermentation, foam stability, and beer aging?

Small molecules highly influence all of these aspects and are (besides proteins) major driving force of beer quality. Traditional analytical food chemistry approaches focus on what is directly linked to such quality parameters: The search for and analysis of targeted aroma compounds, taste compounds, and so on. In many cases, processes that lead to these final products or alter quality are very complex when seeing the beer as a complex mixture of (tens of) thousands of organics in an aqueous solution.

Following the big questions related to beer aging, beer stability, and flavor development, it may be advisable to have a systems approach and to consider the beer system in its whole chemistry rather than to quantify only few known targets. Underlying principles of synergistic effects, precursors, and complex reactomes can be unraveled. Whole processes can be described and guided eventually.

In another recent paper, the Maillard reaction (MR) in beer was explored using Fourier-transform ion cyclotron mass spectrometry (FTICR-MS (2). Can you describe the MR and explain its significance in beer quality and analysis?

The Maillard reaction (MR, named after Louis Maillard, who worked on thermoprocesses in the early 19th century) is commonly known as the 

reaction of reducible sugars with amino acids or amines

. It is not a single reaction as other “name-reactions,” but the term unites a whole complex reaction network with several steps and repetitive intrinsic patterns. It happens mainly at higher temperatures, meaning during malting and the brewing or boiling process. But the MR also changes the composition of the beer during storage (longer reaction times at lower temperatures), leading to unwanted off-flavors or lesser beer stability.

The Maillard reaction is also the main origin for roasty, malty, caramel, or chocolate flavors of darker beers and significantly contributes to the overall impression of the beer flavor and organoleptic.

The classical way to target the MR is to target known “marker” products. Our approach is first to describe the whole reaction network to identify novel targets and processes to be followed involving all compositions and reactions that might relate them.

How can FT-ICR-MS benefit non-targeted metabolic profiling in beer analysis?

With regard to the MR, it is clearly the holistic metabolite profiling that brings the benefits because it is not possible to describe such a complex and diverse reaction sequence by just looking at a few already-described molecular parameters. A different way to target the MR is to describe it as a molecular social network with over 2,800 different compositional individuals (which will translate to an even wider range of isomers) to find molecular interactions that are characteristic for the whole system. This enables to get insights in the compositional changes as part of the brewing process coming with it and thus to have insights into molecular fingerprints that lead to the better beer quality (shelf-life, taste, aroma). Because more than 40% of all resolved signals in beer derive from the MR, it highlights its importance in beer composition.

One of your most recent papers references the German Beer Purity Law and describes how you and your team were able to distinguish the metabolic signatures of different starch sources, such as wheat, corn, and rice in beer using two different complementary mass spectrometric methods: direct infusion Fourier transform ion cyclotron mass spectrometry (DI-FTICR-MS) for 400 samples; and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC–ToF-MS) for 100 samples (3). What were the advantages of using these two different MS techniques?

This project, besides other aspects, shows opportunities for food authenticity control. Such metabolomic approaches contributes to food quality (safety, utility, nutritional, sensory, and ecological quality) and 

. The demand for advanced analytical techniques in the field of food analysis has grown in parallel to highly industrialized food production and consumers’ concerns about food safety and 

. The trend towards non-targeted analytical approaches already is visible. An integrated system of non-targeted and high-resolving analytics is far more difficult to avoid, even when sophisticated fraud attempts aim to avoid detection with knowledge of testing programs. This is what we could provide by the FTICR-MS approach, giving a whole metabolite pattern that indicate samples’ attributes (for example corn and rice adjuncts).

On the other hand, such a sophisticated analytical approach is not yet available to food authorities (and still will not be in 10–15 years). Thus, besides showing its potential, it is important to translate the non-targeted approaches into classical marker substances that are reachable for every analytical laboratory. Knowing molecular networks from the FTICR-MS approach and adding UPLC-ToF-MS information we can specifically target the combination of molecules that – out of the holistic entity – are marker molecules for the grains, and so forth, by chemometric approaches. Ultra performance liquid chromatography-time-of-flight mass spectrometry also allows access to deeper information about the molecule’s structure (fragmentation patterns) and identification of the molecule that can then be used in other laboratories as well.

Are there other applications where employing DI-FTICR-MS with UPLC–ToF-MS would be appropriate and reveal new information? Where would you like to see these two methods further applied?

Without revealing our future projects too much, our focus will be on the role of molecular complexity and diversity during the brewing process. Beer-aging mechanisms are of interest because they enable us to have a base of knowledge that we use in archeochemical analysis of residues and historical brews.

We are also interested in a more detailed description of raw materials and their processing.

Essentially, the MR is our focus precisely because of its many bioactive compounds with their impact on human health.

What future projects lie ahead for you and your team in the exploration of chemical complexity in different foods and beverages?

We have several years of comprehensive analytical experience with various beverages and distillates. But our current milestones involve this technological know-how more towards the general fermented and thermally processed food metabolome and its impact on the gut microbiome as related to human and animal health. Our interest is the molecular coverage of the “food/gut microbiome/health continuum,” and within these aspects, functional metabolomics and bioactivity profiles are of the highest importance in terms of microbial modulators or prebiotics.

S.A. Pieczonka, M. Lucio, M. Rychlik, and P.S. Schmitt-Kopplin, 

https://doi.org/10.1038/s41538-020-00070-3

S.A. Pieczonka, D. Hemmler, F. Moritz, M. Lucio, M. Zarnkow, F. Jacob, M. Ryclik, P. Schmitt-Kopplin, 

https://doi.org/10.1016/j.foodchem.2021.130112

S.A. Pieczonka, S. Paravicini, M. Rychlik, and P. Schmitt-Kopplin, 

https://doi.org/10.3389/fchem.2021.715372

 is a Professor at the Technical University of Munich (TUM) in Freising/Weihenstephan, as head of the Comprehensive Foodomics Platform at the Chair of Analytical Food Chemistry and is director of the research unit Analytical Biogeochemistry (BGC) at the Helmholtz Zentrum München (HMGU), Germany. His research focus is on the Food/Microbiome/Health continuum using the chemical diversity analysis to study generic biotic and abiotic processes in biology and in health. His tools are a combination of (ultra)high resolution (µ)separation sciences, spectroscopy, and spectrometry for targeted and non-targeted chemical analysis.

 is a Food Chemist and doctorate student and research associate at the Technical University of Munich (TUM) in Freising/Weihenstephan, Germany. He works on the comprehensive analysis of the metabolome of beer. His expertise includes the holistic analytical description of foods/beverages, of the chemical changes during its processing, search for characteristic molecular patterns or biomarkers, and the chemometric integration of (complementary) analytical data.

Articles

Stefan A. Pieconzonka and Philippe Schmitt-Kopplin have profiled diverse beer samples by rapid flow-injection analysis (FIA) Fourier transform ion cyclotron mass spectrometry (FTICR–MS). Here, they discuss their approach to this analysis, along with the significance of the Maillard Reaction (MR) in beer quality and analysis.

Profiling Metabolites in the Beer Matrix

Tony Taylor is Group Technical Director of Crawford Scientific Group and CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LCâMS and GCâMS for materials characterization, especially in the field of extractables and leachables analysis.

I get excited when there is a new chromatographic method to implement or to transfer into the laboratory. I don’t understand why everyone doesn’t feel the same way. In this series of blog posts, I’m going to explore how the challenges of adopting methods from the literature, or from internal or external clients, can often be made easier, and more enjoyable, by taking time for some detective work prior to even entering the laboratory.

With a sound understanding of chromatography principles and taking some time to properly understand the variables within each method (all of the variables not just the “headlines”) as well as considering those variables which have not be stated in the method, we can save ourselves a lot of trouble once we don our lab coats and cross the laboratory threshold. Of course, it takes time to build the knowledge and experience necessary to spot these potential issues, but once we are aware of the possible pitfalls within a method, we can pay close attention to them during the implementation of the method, or even change those parameters, where client or regulatory guidance allows.

The sources of “problems” within chromatographic methods are too numerous to list here, however, the starting point in any method detective work is a thorough reading of the method to gain a sense of whether or not it stacks up against our previous experience and knowledge. In this series, I’ll be covering some typical examples from problematic methods to highlight specific issues and areas to focus on, but also expanding upon these a little, to give some general guidance on what does or doesn’t, in my experience, constitute a clue that may be worthy of further investigation.

I’m going to start with an HPLC method, and we will concentrate on the mobile-phase and high-performance liquid chromatography (HPLC) column combination. I will consider the issues with column geometry and stationary-phase chemistry again in later blogs, so this example will serve as an illustration of what we might look for when investigating mobile-phase conditions.

I recently came across this information in a method that was being transferred.

 Dilute sulphuric acid (approximately pH 2.8) : Methanol 50:50

 end-capped ethylene-bridged octadecylsilyl silica gel for chromatography (hybrid material) R (1.7 µm)

 Dissolve 1.36 g of potassium dihydrogen phosphate R in 900 mL of water for chromatography R. Add 0.15 g of sodium heptanesulfonate R, adjust to pH 7.0 with triethylamine R and dilute to 1000 mL with water for chromatography R.

You will note that I’ve not included any details regarding the analyte at this stage, we’ll consider this later.

For me, with over 30 years of practicing chromatography, the constituents of the mobile phase are recognizable, but certainly not what one could call “modern.” However, for some less experienced chromatographers, the mobile-phase additives and the fact that the pH is titrated to a set value using one of the reagents will be, frankly, alien.

Structures of the mobile-phase additives used in the hydroxychloroquine related substances method

Potassium dihydrogen phosphate is a traditional buffer that is used to resist small changes in pH when the sample in its diluent (approx. pH 2.8) mixes with the eluent within the instrument tubing and primarily at the head of the analytical column. As we wish to maintain a pH of 7.0 to enable reproducible separation, and because the pKa of the phosphate buffer is 7.21, this obeys the general rule that the buffer will be most effective when the desired solution pH is within 1pH unit of its native pKa. This should lead to an improvement in the robustness of retention time and peak shape. We will further discuss this particular reagent and the eluent pH later.

Sodium heptane sulphonate is a so-called 

 reagent, which has two modes of action. In the bulk mobile phase, the anionic reagent will form an ionic equilibrium with basic analyte functional moieties to form a neutral complex, which will be more highly retained on the highly hydrophobic stationary phase surface. The second mode of action of the ion pair reagent is to strongly partition into the stationary phase via the highly hydrophobic moiety of the pairing reagent which will effectively contribute anionic groups (SO3-) to the stationary-phase surface, with which any unpaired cationic analyte species may undergo electrostatic attraction, and therefore improve retention.

Apart from its use to obtain the correct mobile phase pH, triethylamine is used as a silanol group masking reagent. Figure 2 can give us further insight into the mechanism and requirement for this reagent.

 Representations of various silica surface species (note C8 ligands shown in the figure – C18 ligands are recommended in the Pharmacopeial method).

Species 5 in Figure 2 represents an acidic silanol group that has not been effectively treated with an end-capping reagent (such as that shown in species 8) which will be anionic under the mobile-phase conditions of our method (species 5 will have a pKa of around 4). If left untreated, these silanol anions will form electrostatic attractions with cationic analyte molecules and unwanted secondary interactions and can lead to issues with peak broadening and tailing as well as extended column equilibration times and possible issues with peak area reproducibility. Therefore, ionogenic masking reagents such as triethylamine were traditionally added to mobile phases to effectively “end-cap” anionic surface sites, as the triethylamine, which is fully protonated below pH 7.2, is electrostatically attracted to the ionized surface silanol residues.

Let’s now look at the column being used.

The so-called bridged ethylene hybrid columns were introduced by Waters around 2004 to fulfil several requirements including improved peak shape for charged (especially cationic) analytes, increased ruggedness under ultrahigh-pressure liquid chromatography (UHPLC) conditions and increased resistance to extremes of eluent pH (especially high pH), using organic/inorganic hybrid substrates.

However, we must go back even further to highlight some of the issues with the method under consideration. The lone (acidic) silanol groups that are masked using the triethylamine additive were particularly prevalent when manufacturers used Type A silica as stationary-phase substrate materials. This silica contained metal ions known to “activate” surface silanol groups towards interaction with basic analytes and was typically not treated to avoid the presence of lone silanol groups. The later development of Type B silica effectively reduced silanol activity by removing target metal ions from the silica matrix and used surface treatments to reduce the number of lone silanols and promote the amount of inter-hydrated or vicinal silanol groups (species 2 in Figure 2). These developments were very effective at reducing the interactions between basic analytes and surface silanol species, effectively negating the requirement for masking reagents such as triethylamine. The bridged ethylene phases (species 1 in Figure 2) are known to further reduce the number of inherent silanol species capable of interaction with basic analytes in their ionized state.

One must therefore question, given the fact that due to these new stationary-phase materials the use of silanol masking reagents has not been required for over 20 years now, why triethylamine is included in this method.

Further, triethylamine is relatively volatile and will be lost from the mobile phase to the atmosphere on standing, causing drift in analyte retention over time as the inherent pH of the mobile phase will change as the triethylamine concentration lowers. In my own experience, it is also possible for the triethylamine to “settle” within the eluent, again leading to drifting or changing analyte retention. Further problems with this reagent also exist. Because it inherently alters the stationary-phase surface, one has to be mindful when equilibrating the HPLC column because extended flushing of the phase is often necessary to obtain reproducible retention times for the first injections of a sequence. One also must question the possible interaction between the anionic ion pairing reagent and the triethylamine cation. The possibility of this interaction, forming a neutral complex, may render the masking reagent ineffective, and one needs to wonder what robustness issues this may create.

Let’s now consider the role of the ion pairing reagent in this separation. Modern stationary phases, such as those used in this method, are capable of withstanding high pH mobile-phase environments, and as such we must also call into question the use of this type of buffer in the method. In this separation, the analytes have several basic functional groups ranging from 7.25 to 10.0 and therefore it is conceivable that, by adjusting the mobile-phase pH to pH 10.5 or 11, the degree of ionization of some of the analyte basic moieties will be reduced and retention may be gained, even using the more hydrophobic C18 stationary phase. Further, there are many stationary phases available that contain more polar functional chemistry or are designed to work with very little or no organic modifier within the mobile phase at the beginning of the mobile-phase gradient to facilitate retention of more polar or ionized analytes. The use of ion pairing reagents such as sodium heptane sulphonate has not been necessary for many years, since the introduction of higher quality silica, combined with more polar (so called 

) and more pH resistant stationary phases, can be used to gain sufficient reversed-phase retention, even for more polar analytes.

The column contains 1.7 mm particles that are designed for use with UHPLC systems, given the higher inherent back-pressure created when using such small diameter particles. Intrinsically, these systems have very small internal volumes and very narrow internal diameter tubing and connectors, to reduce the effects of extra column (bed) band broadening on the separation. The use of a solid, involatile buffer with UHPLC systems is to be avoided, as any precipitation of the buffer through the use of highly organic eluents or via evaporation of the mobile phase on standing, will lead to significant problems with system blockages and overpressure situations. Further, the column end frits used with UHPLC columns containing 1.7 mm particles typically have a porosity of around 0.2 mm, which renders them even more susceptible to blockages with solid particles. It is possible to filter the eluent solution to remove any particulate materials initially present in the eluent system, however, one must again be very mindful that the triethylamine additive is relatively volatile, and this may be partially lost if filtration is achieved using vacuum apparatus. The target eluent pH of 7.0 is also relatively problematic. Given that the pKa of at least one of the ionogenic analyte functional groups in each of the analytes was close to 7.0, we might expect a relatively large change in the ionic character of the analyte, and therefore its retention, unless the pH of the mobile phase is very accurately controlled each time it is made. To ensure the method is robust, it would have been better to quote a volume or weight of triethylamine required to achieve the desired pH, which could be controlled much more reproducibly.

It should be further noted that the detection is at a fixed wavelength of 254 nm, and this can be problematical when the ionic strength of the mobile phase changes to such a large extent during the analysis, as is the case here, where the aqueous component ranges from 90% to 15% during the course of the analysis. Without the judicious set-up of the detector, for example, by employing an appropriate reference wavelength to account for differences in the refractive index of the eluent, such changes in ionic strength might well give rise to retention time shifts, peak shape changes, and unstable and rising baselines during the gradient unless sufficient time is given to allow re-equilibration of the column between sample injections. It may be far more judicious to use a ternary system in which the ionic strength is maintained using a third eluent system that contains the buffer and additives at a higher concentration (such as all reagents at 10x concentration and added at a constant 10% of mobile-phase composition during the analysis).

From this brief review it would very much appear that the current method has been “stitched together” using a very traditional mobile phase with a modern stationary phase, which should not require any of the now “exotic” mobile-phase additives to achieve a suitable separation. Without the requisite experience in critical evaluation of chromatography methods, this combination may be baffling and the resulting problems very difficult to interpret or overcome.

Let’s summarize some of the detective work that we have undertaken to start building our investigative toolkit.

1) The use of phosphate or other solid buffers suggests that the method is “older,” and these buffers usually preclude the use of mass spectrometry (MS) detection and present issues with system blockages under high organic strength mobile phases.

2) The use of ion pairing reagents such the sulphonic acids or alkyl amines again suggest the method is older. These reagents were typically used in conjunction with older stationary-phase types, and their need can often be negated by judicious selection of a more modern stationary phase that is capable of retaining ionized analytes. Further, the use of ion pairing reagents can present real challenges with column equilibration.

3) By studying the physico-chemical parameters of the analyte, it may be possible to suppress analyte ionization using pH control and modern stationary phases exist that will withstand eluent pH values between (approximately) 2 and 11.

4) The use of “exotic” additives within mobile phases such as triethylamine should ring alarm bells in terms of the robustness of the method and the potential issues with peak tailing, due to their association with older, less inert, stationary-phase chemistries and substrates.

5) One should always consider the analyte structure and the polarity (LogP, LogD) and the pKa of any ionogenic functional groups with respect to the mobile phase being used. It is a real skill to be able to visualize the degree of ionization of each analyte and anticipate any issues with retention and resolution under the mobile-phase conditions specified.

6) Eluent pH values that closely approach the pKa of any ionogenic groups will present potential robustness issues with retention and resolution of the separation. Further, methods that specify the eluent components volumetrically rather than gravimetrically should, where possible, be converted to gravimetric specification to enable more accurate mobile-phase preparation.

7) The ionic strength of the eluent additives can give clues into the quality of the method and where high ionic strength (above 25 mM) is required, this highlights several possible issues in implementation. High ionic strength can indicate that robustness can only be achieved by significantly altering the nature of the stationary phase, and there may be issues with a mismatch between the analyte diluent and the mobile phase. These phases will also require significantly extended column equilibration times. High ionic strength mobile phases that use solid buffers can present significant issues with system blockage, especially where eluent conditions reach >60% acetonitrile or >95% methanol during the gradient.

8) Eluent gradients that present a significant change in ionic strength can present issues with baseline stability, especially when using UV detection. These problems can be minimized through careful optimization of the reference wavelength, however, the general principle should be to minimize ionic strength of the eluent and the change in ionic strength throughout the gradient.

Tony Taylor is the Chief Scientific Officer of Arch Sciences Group and the Technical Director of CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LC–MS and GC–MS for materials characterization, especially in the field of extractables and leachables analysis.

In this series of blog posts, I’m going to explore how the challenges of adopting methods from the literature, or from internal or external clients, can often be made easier, and more enjoyable, by taking time for some detective work prior to even entering the laboratory.

The LCGC Blog: Critical Evaluation of Chromatography Methods—Essential Detective Skills 

Most analytical chemists believe their pipetting technique is infallible, but few of us are actually following all of the recommendations within the relevant guidance. Incognito investigates good pipetting practice and busts some of the urban myths behind what is probably the most widely used analytical tool.

At the heart of most analytical techniques is sample preparation. At the heart of most sample preparation is dilution and quantitative transfer of liquids. In many cases, these operations are performed using air displacement or positive displacement pipettes, which I’d like to focus on in this Incognito column.

You may ask why? Given that these pipettes are used daily by most practicing analytical chemists, surely we know everything there is to know? Well, the trouble is I believe many of us think we know all there is to know and that our pipetting technique is infallible. I guess I was in the same camp until recently, when a laboratory investigation caused me to question just how good I was. Had I been taught properly in the first instance? Had I become lazy over the years? Did I rely too much on the urban myths that had been relayed to me over my career about what’s good and what’s not? It turns out the answer to these questions—in my case—was a pretty emphatic “yes”. So where do I turn for a primary reference to improve my practice and bust some of these myths?

After a little searching, I came across what I believe to be two excellent reference documents on pipettes and the art of pipetting: Internal Standard (ISO) 8655-2 Piston-Operated Volumetric Apparatus–Piston Pipettes (1) and (ISO) 8655-6 Piston-Operated Volumetric Apparatus–Gravimetric Methods for the Determination of Measurement Error (2). Much of the “reference” information contained within this article, and the answers to many of my lingering questions, are drawn from these references. I have also used information and guides from several of the leading pipette manufacturers (3,4) and a useful guide from the National Physical Laboratory (UK) (5). These documents also contain a host of useful information and technical reference tables that are not directly mentioned here but which every analytical chemist should be aware of.

For those readers who may be a little sceptical as to how much difference pipetting errors can contribute to the overall uncertainty budget of an analytical measurement, I’ll be highlighting the impact of poor pipetting technique as we progress the discussion.

So, let’s start with choosing the correct pipette for the job. On the basis that I see far more air displacement pipettes in laboratories than positive displacement pipettes, lets look at the working principles of both, and the circumstances under which they should be used.

In short, an air displacement pipette has a layer of air between the head of the piston and the liquid within the pipette tip. Air pressure is used to force liquid from the tip. Positive displacement pipettes have an integral piston within the disposable tip and the piston is in contact with the liquid being dispensed. Both types are shown schematically in Figure 1.

The manufacturers recommendations align well when considering which type of pipette to use for which sample type:

Aqueous and biological samples—air displacement pipette

Viscous, volatile, or corrosive samples—positive displacement pipette

I wonder if, like me, you have been using an air displacement pipette for relatively volatile organic solvents for years?

The choice of pipette tips and correct installation also brought further surprises. First, only manufacturers tips are recommended, and all manufacturers recommend that if alternative brands are used, then a “test” for accuracy and precision of the dispensed volume should be undertaken, as per ISO 8655‑6, which I will cover in more detail later. I was a little sceptical when I read this—is it just a way of locking us into the manufacturer’s consumable? This may be true, but reference 1 does contain a detailed specification for pipette tips and the information that needs to be provided to the user—I’d recommend that anyone purchasing pipette tips reads this.

On pipette tip installation, for air displacement pipettes, rather than pressing the pipette shaft firmly down on to the tip (known as “hammering”) or pressing the tip straight onto the shaft, it is recommended that a slight twisting motion is used to create a good seal. Whilst loose tips may be purchased, it is recommended the tip is always fitted with the piston held vertically to ensure a good seal and so the tip is level on the pipette shaft, and it strikes me that tips supplied in racks are a more convenient (although more costly) option. For positive displacement pipettes, the above guidance should be followed, but once the tip is installed onto the shaft the tip should be lifted out of the rack and the piston lowered until a “click” is heard as the piston locks into the pipette body and the plunger should then be pressed to the first stop position. Figure 2 contains an explanation of the various plunger positions of a typical piston pipette.

Most manufacturers assert the importance of not cleaning and re-using pipette tips.

So now we have chosen the correct pipette type and correctly installed the tip, lets look systematically at the guidance for correct usage; note that I’ve added some notes and interpretation where this brings further clarity. I’ll deal separately with air displacement and positive displacements.

Air Displacement Pipette—Forward Pipetting Mode

a) Set Correct Volume (Variable Volume Pipettes): 

When using a variable volume pipette, some manufacturers recommend the volume is “dialled” using a clockwise motion. That is, when reducing the volume, slowly move the volume setting dial to the required volume, but when increasing the volume, overshoot the required setting by 1/3 of a turn and then reduce the volume to the desired setting. In this way, the desired volume is always reached using a clockwise motion. Always hold the volume setting at eye level to avoid parallax error.

b) Aspirate an Aliquot of the Liquid to be Dispensed, Allow to Equilibrate, and Dispense to Waste:

 Holding the pipette vertically, smoothly depress the plunger to the first stop position, immerse the tip of the pipette into the liquid to be aspirated to the depth recommended in Table 1, and aspirate the liquid by smoothly and steadily raising the plunger to the rest position. 

Notes: Jerky or rapid plunger movement will increase the error in the amounts of liquid aspirated or dispensed.If the tip is immersed too deeply, the increased hydrostatic pressure may cause too much liquid to be aspirated or liquid droplets to adhere to the outside of the pipette tip, which may be transferred to the receiving vessel. If the tip is not immersed deeply enough, then vortexing may occur or the liquid level may fall below the tip before reaching the top of the filling stroke—in both cases too little liquid will be aspirated.

Wait two seconds for the aspirated liquid to move up into the tip and to equilibrate the temperature and pressure of the headspace air above the liquid.

Notes: This step is particularly important in air displacement pipettes due to the changing compressibility of the headspace air above the aspirated liquid as a result of temperature and vapour pressure changes. It is also worthy of note that the aspiration of the liquid into the vacuum space created when the plunger is released from the first stop to the rest position is governed by the ambient atmospheric pressure, which may need to be compensated for if significantly different from those at which the pipette is calibrated (typically 20 

C, 50% relative humidity, and barometric pressure of 101 kPa).

This first aliquot should then be discarded to waste, or if the liquid is to be retained, return to the original container. Note that any contaminants present on the inside surface of the pipette tip may also be returned to the sample or reagent.

This tip wetting step can significantly reduce the error associated with pipetting and acts to reduce any interfacial effects between the liquid being aspirated and the inside surface of the pipette tip. With certain liquid types, or during pipette calibration or testing, some manufacturers and standards bodies call for up to five pre-equilibration and wetting step repeats prior to the first “real” aspiration into a new tip or when volume changes are made to adjustable pipettes. This may also be necessary in the normal course of operation when dealing with low viscosity or more volatile liquids, especially where a liquid droplet can be seen hanging from the exit orifice of the pipette tip post aspiration.

c) Repeat Step b) but Retain the Aspirate Liquid in the Pipette Tip:

 Notes: I have read guidance about, and witnessed first-hand, users wiping any excess liquid from the tip of the pipette prior to dispensing the aspirated liquid. I can see no reference to this practice in the ISO guidance, and, from experience, the use of adsorbent paper to wipe excess liquid from the outside surface of the tip risks contact with the tip orifice, which will draw out a portion of the aspirated liquid through wicking effects. If the pipette tip is immersed to the correct depth within the sample liquid, then there should be no need for wiping of the tip. The exception here will be when extremely viscous liquids are being pipetted (creams, for example), where there may be a requirement to wipe the tip. In this case, great care should be taken to avoid the tip orifice area and a resistant, lint-free cloth should be used.

Place the pipette tip gently against the wall of the receiving vessel, just above any liquid that may already be within the vessel, at an angle of between 10 and 45º. Note that this should be achieved by tilting the receiving vessel whilst the pipette remains in the upright orientation.

Notes: It is important that the pipette remains in the upright position during dispensing for the reasons outlined previously, as well as to avoid any contamination of the pipette body by aspirated liquid within the tip running up into the pipette body when tilted. The angle of contact between the pipette tip and the wall of the receiving vessel is important to ensure that the liquid is dispensed correctly and to overcome any surface tension effects. One should also note that the ISO guidance recommends a contact angle of between 30 and 45

. It is also important to note that the position of the tip should be above the level of any liquid already within the receiving vessel. If the tip is submerged, then highly inaccurate and poor irreproducible volumes will result. It may be necessary to handle the receiving vessel to create the correct contact angle with the pipette; in this case handling should be kept to a minimum (especially when the vessel is to be weighed) and lint‑free gloves should be worn.

Smoothly depress the plunger to the first stop position. Wait one second and then smoothly depress the plunger to the second stop position to purge any remaining liquid from the pipette tip.

Notes: In my opinion, this step is one of the most contentious and misunderstood areas of the pipetting technique. Both the guidance and the pipette manufacturers’ literature recommend the use of the “blow-out” or “purge” feature to dispense all liquid from within the tip, although the ISO guidance does state that this should be done “where applicable”. My question is: When is this step applicable and when is it not? I feel it is best practice to use the purge feature for all pipetting operations and some example data shown later will hopefully justify this comment.

Slowly draw the pipette tip 8–10 mm up or along the vessel wall prior to removal from the vessel and then slowly allow the plunger to return to the rest position and discard the pipette tip.

Notes: Again this last step, with particular respect to drawing the pipette tip along the vessel wall, is perhaps an area that is not properly understood. This drawing action is intended to remove any remaining liquid at or around the tip orifice to avoid it being withdrawn by the pipette. Again, some experimentation I have undertaken will be shown later to highlight the effect of not drawing the tip along the pipette wall. It is also important to note that the plunger should not be allowed to move from the second position prior to the contact with the vessel wall being broken (that is, once the tip is removed from the vessel).

Air Displacement Pipette—Reverse Pipetting Mode

Reversed pipetting is particularly useful when handling liquids with high vapour pressure (volatile liquids) or those that are highly viscous. This mode of operation follows all of the guidance that has been discussed above, with some notable exceptions.

a) Prior to aspiration the plunger is depressed to the second stop (purge) position.

b) The liquid is aspirated by allowing the plunger to move from the second stop to the rest position.

c) The liquid is dispensed by pressing the plunger to the first stop position prior to removal.

Notes: The “purge” operation is effectively carried out in the aspiration step rather than the dispensing step. During aspiration, an amount of liquid equal to the amount of air used to purge the tip is aspirated, which compensates for the amount of liquid that remains in the tip after the dispensing step.

I have seen no guidance on whether the tip should be drawn along the inner wall prior to removal in the reversed pipetting mode. I would encourage the reader to undertake their own experiments to decide if this is necessary to achieve the required accuracy when using the reverse pipetting mode.

Much of the important guidance has been discussed above, and points around tip wetting, immersion depth, and wiping are applicable. However, once again, there are some notable exceptions.

a) There is only one stop position on these pipettes, any second stop is actually an eject position for the tip.

b) In the dispense step, the piston is moved to the first stop position. The tip is not touched against the side wall of the receiving vessel and is not drawn along the vessel wall prior to removal.

Having established best practice, it is interesting to discuss sources of error and tolerance of the pipetting equipment, as again there is much debate and, frankly, nonsense spoken about the accuracy and reproducibility of these volumetric tools.

ISO 8655-2 has a useful table of permissible errors for a variety of pipette types, and I’ve shown typical examples from a range of pipette volumes in Table 2.

These figures are applicable for a series of 10-fold measurements where systematic error is the statistical deviation and random error is the coefficient of variation.

It should be noted that whilst the ISO guidance limits for each nominal volume is applicable to every selectable volume throughout the useful volume range of a variable volume pipette, manufacturers often set tighter specification limits for the nominal volume and between the nominal and lower selected volumes.

We have yet to discuss the required adjustments for non-standard environmental conditions or the buoyancy of the liquid used for these measurements, however, I thought it would be interesting to see how accurate my new-found pipetting technique was, using deionized water that meets the criteria of ISO 3696. The results and analysis are shown in Table 3 for an adjustable air displacement pipette of nominal 100 µL volume. It would appear that my technique is reasonable, and I have achieved a reasonable performance against the ISO requirements for each volume: Incognito systematic error +0.57 µL (100) / +0.36 µL (50) / +0.35 µL (10), ISO systematic error limit ± 1.5 μL (mass to volume 

 correction factor insignificant); Incognito random error +0.34 µL (100) / +0.11 µL (50) / +0.43 µL (10), ISO systematic error limit ± 0.6 µL (mass to volume 

It is worthy of note that the error associated with dispensed volume at 10 µL would not have met the ISO tolerances had I used a pipette of nominal volume 10 µL. This perhaps suggests that one needs to be reasonable about the fraction of the nominal volume that one dispenses, and I know several laboratories who specify that piston pipettes should not be used to dispense volumes less than 50% of the nominal volume for precisely these reasons.

I was also interested in finding out the magnitude of error associated with various aspects of incorrect pipetting technique. The ISO guidance suggests a wide range of estimated errors, some of which are shown in Table 4.

From Table 4 it is obvious that the total cumulative error associated with incorrect pipette technique can be very significant. I investigated the errors associated with some specific aspects of poor technique and these results are shown in Table 5; all results are from 10 replicate measurements of 50 µL of water adjusted for the 

 correction factor using a nominal 100 µL pipette.

As can be seen from Table 5, several of the poor techniques resulted in out‑of‑specification performance for the dispensed volume limits and all increased the inaccuracies relative to that obtained when using best practice. This is perhaps more salutary when considering that each error was implemented in isolation and that the cumulative error, if several of these poor techniques were combined, could be very considerable.

The reader will notice that I have made adjustments to the “expected” mass of water for these experiments in order to account for temperature and pressure differences between my local environment and the standard conditions used to calibrate (adjust) the pipettes (the 

 correction factor). This takes me onto my final topic in this discussion—that of pipette testing and calibration, which I’m often asked about, typically by end users who wish to carry out verification between annual calibration and maintenance.

There are two procedures that are defined in ISO 8655-6:2002: the test procedure for more frequent “checking” of the accuracy and reproducibility of the dispensed volume and a calibration procedure, which is typically carried out annually by the pipette manufacturer. This second procedure can also be carried out by the end user with a little experience and some basic metrology tools. Whilst the full details of the test and calibration procedures are outside the scope of this article, I’d like to note several points that are poorly understood:

Assessment of systematic and random error should be made on 10 replicate measurements.

Prior to measurement, a tip should be wetted five times and then discarded to equilibrate the air within the pipette to temperature and relative humidity.

A new tip should be installed for each of the 10 measurements and each tip should be pre-wetted once before aspirating the required volume.

For measurements below 50 µL, an assessment of evaporation of the test liquid (water to ISO 3696) should be made by measuring mass loss over the same time period as the 10 test measurements and then dividing this mass loss by 10 and  adding the resulting mass to each test weighing.

A correction for temperature and pressure (the 

 correction factor) should be made for the mass of water to account for local conditions versus those for adjustment under standard conditions (tables of water mass at various temperature and pressure values can be found in ISO 8655-6).

For variable volume pipettes, the assessment should be made at the nominal volume, as well as 50% and 10% of the nominal volume (and all must conform to the ISO 8655-6 limits required for the nominal volume). Note here that the manufacturer may have separate tolerances for the nominal, 50%, and 10% of nominal volumes.

Systematic error is defined by the variance of the 10 replicate measurements and random error by the coefficient of variation.

I hope that this article acts to stimulate pipette users to examine their own routine and to guide you in best practice as defined by an international standards organization, as well as the pipette manufacturers. I have certainly picked up many tips during the writing of this article and I feel much more confident in my own ability to not only pipette more accurately with piston pipette devices but also to sense when something is not right and to troubleshoot more effectively. To quote Henry Ford, “Anyone who stops learning is old, whether at twenty or eighty. Anyone who keeps learning, stays young”. I feel in the flush of my youth after this salutary learning experience!

Piston-operated volumetric apparatus — Part 2: Piston pipettes

Piston-operated volumetric apparatus — Part 6: Gravimetric methods for the determination of measurement error 

High Precision Micropipette, Operation Manual

Measurement Good Practice Guide No. 69: The Calibration and Use of Piston Pipettes

 (National Physical Laboratory, Teddington, United Kingdom, 2004).

Sweating the Small Stuff—Are You Sure You  Are Using Your Pipette Properly?

There has been an increased interest in monitoring food quality and safety, and their important role in relation to human life, wellness, and health. Existing regulations dictate how certain technological processes should be performed to assess food safety and quality by monitoring food components and the potential presence of contaminants. Although the “classical” targeted analytical perspective is still the major route, holistic approaches are gaining more momentum in characterizing food through “omics” technologies because this can provide novel unexpected markers of food quality.

Foodomics (1) combines food and nutrition sciences with advanced analytical techniques and bioinformatics to characterize the food composition or food consumers’ biofluids, and also to address new challenges such as authentication, traceability, improvement of food produce, food quality, and nutritional value. This holistic approach employs a cutting‑edge research infrastructure, where big data are produced that generate new knowledge and highlight previously unknown associations of biomolecules with the studied phenotype.

The potential application of foodomics is very wide and includes the following areas: food characterization and differentiation; food traceability and authenticity; adulteration control; identification of origin (geographic or genetic); monitoring of maturation; assessing freshness; linking organoleptic characteristics with metabolite composition; supporting health claims of novel functional (super)foods; understanding mechanisms of toxicity; development of a personalized dietary intervention for patients with special needs, for example, food allergies; and identifying legitimate or non-legitimate interventions (hormone treatment, use of fertilizers in crops). Ultimately, this leads to biomarker discoveries that can reveal the beneficial effects of nutrition on human health. Such data offer key elements to support marketing and brand naming. Hence, products of high economic value, such as products in the category of protected designation of origin (PDO) and protected geographical indication (PGI), can be characterized extensively and can be more effectively protected. Overall, foodomics as an omics approach can provide new knowledge and reveal biochemical mechanisms that describe the advantageous or adverse effects of food components (2). Figure 1 provides an illustration of the fields and the various subfields and applications involved in foodomics.

FoodOmicsGR_RI is a national research infrastructure that aims to perform and aid omics research in the agri-food sector in the Greek research environment. This field is of high importance for the country and its agriculture because of the unique landscape Greece exhibits that results in a diverse and rich portfolio of local products and foods. Therefore, the central scope of FoodOmicsGR_RI is to support the Greek agri-food sector by generating robust data on the composition and nutritional value of the local produce, thereby increasing a product’s position, market demand, and provide a higher revenue for the producers.

The project comprises eight Greek universities and research centres. Analytical groups and food specialist groups from the universities of Athens, Crete, the Aegean, Ioannina, the Agricultural University of Athens, the International Hellenic University, and the Biomedical Research Center of the Academy of Athens comprise a team of 60 staff scientists and 40 newly recruited researchers from 20 scientific disciplines. BIOMIC_AUTH, a new interdisciplinary laboratory of the Aristotle University Thessaloniki (http://biomic.web.auth.gr/), with great expertise in bioanalytical chemistry, is coordinating the project. More details can be found on the website (http://foodomics.gr/).

Cutting-edge research technologies include high performance liquid chromatography (HPLC), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, and inductively coupled plasma–mass spectrometry (ICP-MS) instrumentation and software. These are deployed to cover a wide area of applications such as bioanalysis, metabolomics, genomics, proteomics, and bioinformatics. The capital instrumentation of FoodOmicsGR_RI laboratories includes:

Five quadrupole time-of-flight (QTOF)-MS instruments (two trapped ion mobility mass spectrometry systems [TIMS]-TOF-MS)

Three matrix-assisted laser desorption–ionization (MALDI)-TOF-MS instruments

Four ultrahigh-pressure liquid chromatography (UHPLC)–orbital trap high resolution mass spectrometry (HRMS) instruments (one nano‑LC)

More than 10 UHPLC triple quadrupole MS/MS systems

Three HPLC–MS single quadrupole systems

Supercritical fluid chromatography (SFC)–UV/MS system

More than eight gas chromatography (GC)–MS systems (single quadrupole)

Four GC–MS/MS systems (triple quadrupoles)

NMR spectroscopy (600 MHz, 500 MHz, and 400 MHz NMR)

Two ICP-MS, two ICP-optical emission spectrometry (OES) instruments, next generation sequencing (NGS), and several other instruments.

As well as the analytical sciences, the consortium has a diverse and in-depth practical knowledge in the food sciences, namely fishery, apiculture, animal husbandry, plant growth, vinology, environmental control, natural products, food chemistry and technology, dairy product biochemistry, microbiology, and human exercise biochemistry. Researchers have gained experience from the analysis of thousands of samples of various origin by either untargeted or targeted metabolomics using HRMS; this experience includes reporting solid quantitative data in compliance with validation guidelines. FoodOmicsGR teams collaborate with the industry and the private sector (food producers, contract research organization [CRO] laboratories, life science companies, and analytical instrument vendors). In addition to that, the groups liaise with international research organizations, regulatory bodies, and consortiums (Eurachem, NIST, mQACC, Norman network, JRC-IRMM, and other key players).

Consultancy for the design of experiment (DoE) for the foodomics and nutritional studies

Genomic, proteomic, and metabolomic analysis of foods

Quantitative analysis of key metabolites (nutrients, contaminants) in foods

Big data processing and advanced statistical analysis.

FoodOmicsGR_RI aims to support R&D studies on food metabolomics, proteomics, and genomics applications, offering a “one‑stop‑shop” model to stakeholders and users.

The consortium partners offer expertise across major research lines related to the needs of the Greek agri-food domain. These can be grouped to the following research axes: 1) developing high-quality databases of Greek foods, 2) employing “omics” technologies to study local products and promote authenticity/traceability control, 3) highlighting unique traits of Greek products, 4) studying the effect of diet on health and well-being, 5) re-using and creating value from agri-food waste, and 6) studies on food safety assessment.

One striking example of the portofolio is building in-house high-quality sample libraries establishing and enhancing the nutritional value of Greek foods. The group has published a number of topical reviews on food metabolomics such as olive oil (3), grapes, marc spirits, and wine (4). The group also generates compositional databases of the molecular composition of Greek agricultural products. The tools used for this purpose are data- and text-mining of the scientific literature (developing bespoke Python algorithms). The data are soon to be made publicly available on the project webpage.

2. Food Analysis, Traceability, and Control of Geographical Origin: 

FoodOmicsGR_RI research teams have developed and practiced MS and NMR methods for a variety of foods. Untargeted LC–HRMS methods combined with chemometric approaches complement traditional targeted LC–MS analysis (5,6) to study food authenticity.

The consortium has developed MS‑based workflows for food traceability and geographical origin, such as for olive oil, wine, dairy products, and honey. For example, classification of apple varieties was achieved based on volatiles analysis by headspace–solid‑phase microextraction (HS–SPME)–GC–MS (7), while the geographical origin of grapes was assessed using a hydrophilic interaction liquid chromatography (HILIC)–MS/MS method (4).

In another study conducted in the laboratory, 63 Cretan virgin olive oils were discriminated according to their geographical origin based on HS–SPME–GC–MS volatile organic compound (VOC) analysis. A representative gas chromatogram of VOCs from the Koroneiki cultivar is shown in Figure 2. The impact of geographical origin on volatile profile is illustrated in a orthogonal projections to latent structures discriminant analysis (OPLS-DA) model (Figure 3) where samples of different origin are visually separated.

Another interesting example is an untargeted HS–SPME–GC–MS method that was applied for the classification of Greek wines based on their volatile composition. DoE assisted in the optimization of the analysis, while the sample collection consisted of 74 samples of nine different Greek grape varieties and six different regions. Preliminary results indicated that the classification of samples according to grape colour and variety is achievable (Figure 4).

3. Quality Control (QC) and Targeted MS Methods: 

FoodOmicsGR_RI laboratories have taken key steps and have extensive practical experience in the development of QC protocols for LC–MS-based metabolomics (8,9) for the validation of research findings. The group has studied the effect that sample preparation methods have on the obtained “metabolome” of different samples (10).

After two decades of metabolomics, there is a growing shift towards targeted quantitative methods, as recently reviewed by the group (11). The consortium has developed and validated targeted methods using GC–MS/MS or LC–MS/MS, providing robust and reproducible results for a set of metabolites; these include amino acids (12), organic acids, bile acids, or a wider group of up to 120 metabolites from different small molecule classes (13). Representative examples include studies of royal jelly (13,14), wine (15), and carobs, in addition to biological samples from nutritional studies.

4. Studies on Nutrition and the Nutritional Value of Foods: 

As stated previously, FoodOmicsGR_RI develops new tools to highlight the nutritional value of Greek foods; this can be attained by performing intervention studies where Greek traditional foods are used in the prevention of chronic diseases and to promote health claims. More specifically, the group monitors the effect of food ingredients at the genomic/transcriptomic/proteomic/metabolic/elemental level, followed by validation of initial findings. Ultimately the group develops proof-of-concept studies on food bioavailability on human organs and tissues. For example, the effect of a carob diet in the metabolome of biological samples of animal models has been studied (16). The facility has expertise in profiling biological fluids, such as urine, plasma, faeces, saliva, and tissues (liver, kidney, brain). Characteristic studies include the efffect of diet on human and animal models (16,17,18,19).

Other examples of nutritional studies include collaborations with partners from the private sector to improve health claims of novel superfoods from the Greek flora and marine organisms (that is, use of probiotics to maintain the balance of gut microbiota). Phenolic-rich water extracts from olives and their effect on animals and human health has also been studied. The group has investigated the role of novel animal feed enriched in chelated metals (Cu, Mn, Zn) and identified disease biomarkers in biofluids from sows after livestock intake (Project FITSOW) (20). A key goal is to offer the knowledge that could provide the base for the development of personalized diet based on observations of omics data integration. Such diets could improve health, well-being, and prevent the onset of disease.

Food safety from chemical contaminants in foodstuffs as a result of environmental contamination, packaging, and storage processes is a major concern in the food market. To this end, the group has studied the migration of an endocrine disruptor (bisphenol A, [BPA]), from baby bottles into the aqueous content of foods or canned food (21). Also, non-intentionally added substances (NIAS) incorporated into the food chain have been studied by various analytical methods, for example, monitoring phthalates (22), oligomers, and other NIAS (23).

FoodOmicsGR_RI aims to characterize unique Greek products and highlight their value and quality using cutting-edge omics technology. The facility promotes and reinforces the collaboration between consortium partners, but, more importantly, it aims to act as a liaison focal point and link the research with the private sector and the producer. Overall, the group’s activity and R&D efforts help to increase the visibility and the market value for the investigated products, support brand naming, and generate higher revenue for the producers and the national economy.

We acknowledge the project “FoodOmicsGR_RI Comprehensive Characterization of Foods” (MIS 5029057), which is implemented under the action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme Competitiveness, Entrepreneurship, and Innovation (NSRF 2014–2020) and co-financed by Greece and the European Union (European Regional Development Fund).

There has been an increased interest in monitoring food quality and safety, and their important role in relation to human life, wellness, and health. Existing regulations dictate how certain technological processes should be performed to assess food safety and quality by monitoring food components and the potential presence of contaminants. Although the “classical” targeted analytical perspective is still the major route, holistic approaches are gaining more momentum in characterizing food through “omics” technologies because this can provide novel unexpected markers of food quality. 

FoodOmicsGR_RI aims to characterize unique Greek products and highlight their value and quality using cutting-edge omics technology.