Tag Archives: liquid chromatography

Simon Hird
Ask The Expert

Plant Toxins – A Growing Global Food Safety Issue

By Simon Hird
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Simon Hird

Agriculture and food industries continue to be vulnerable to the complex problems of contamination with natural toxins. Mycotoxins, secondary metabolites produced by fungi, enter the food chain through infection of crops before or after harvest and are typically found in cereals, dried fruits, nuts and spices. Some have well established health impacts, both in humans and animals. A variety of testing solutions exist for mycotoxins, but growth in the use of methods based upon liquid chromatography with tandem mass spectrometry (LC-MS/MS) has enabled the determination of multiple mycotoxins. These methods are extremely sensitive and can be applied to the analysis of raw agricultural commodities, food ingredients and finished products.

Such LC-MS/MS techniques also serve as a powerful tool to investigate the presence of other natural toxins. They have been used for monitoring marine biotoxins[1], and to shed light on the distribution of toxins in terrestrial plants[2], highlighting them as a potentially serious food safety issue. Some terrestrial plants have evolved to produce secondary metabolites as defense mechanisms, which, while beneficial to the plant itself, cause harm to other organisms, including humans. In 2019, a humanitarian food aid product contaminated with tropane alkaloids (TAs) was distributed in Uganda, resulting in a foodborne outbreak which caused over 300 hospitalizations and five deaths[3].

Plant toxins can enter the food chain as constituents of plant products used in food processing or from the seeds and leaves of weeds mixed accidentally with the main food crop at harvest. Low levels of these toxins can be detected in cereals, herbal products, teas, salad crops and some animal products. One important class of plant toxins, pyrrolizidine alkaloids (PAs), are produced by a wide variety of plants commonly belonging to Asteraceae, Fabaceae and Boraginaceae families. Currently, there are more than 660 known PAs and metabolites. They are generally found in products such as honey, pollen, tea, herbal teas, food supplements, spices and aromatic herbs[4]. TAs are another class of plant toxins produced by plants, mostly within the Solanaceae family, and have been found in a range of agricultural cereal crops (e.g. linseed, soybean, millet, sunflower and buckwheat), tea, and herbal blends and infusions[5].

EU Legislation on Plant Toxins

The European Food Safety Authority (EFSA) has published the results of various risk assessments on those plant toxins considered to be the greatest risk to human health[6],[7], leading to the introduction of legislation on plant toxins in food by the European Commission[8]. Maximum levels have been set for PAs in herbs, spices, teas, herbal infusions and pollen products. These, which refer to the sum of 35 specified PAs (including their N-oxidized forms), vary between commodities. For example, the maximum level for PAs in most teas is 150 µg/kg, whereas the value for cumin is set at 400 µg/kg. Although there are more than 200 different TAs known, maximum levels have only been set for atropine and scopolamine (from 0.2 to 50 µg/kg, depending on the commodity). These regulations require that these plant toxins be monitored in specified foods by the Member State Food Safety Authorities and by food business operators, including those imported into the EU.

Access to data from retail surveys for PAs and TAs remains scarce when compared to that which is available for mycotoxins. However, in recent years, the number of food alerts reported on the Rapid Alert System for Food and Feed (RASFF) portal on the occurrence of PAs and TAs in different food products, exceeding maximum levels, has notably increased. The RASFF system was established to ensure the exchange of information between EU member countries to support swift reaction by food safety authorities in case of risks to public health resulting from issues with the food chain. Casado reported levels of PAs related to RASFF alerts with values ranging from 26 to 556,910 µg/kg[9], whereas the highest values of atropine and scopolamine were reported by Goncalves in tea and herbs (mean 173 and 147 µg/kg, respectively)[10]. In relation to consignments of cumin from Türkiye, a high rate of noncompliance with the relevant requirements provided for in EU legislation with respect to contamination by PAs was detected during official controls performed by the Member States[11]. The frequency of mandatory checks to be performed at border control has recently been increased to 30 %[12]. This has prompted greater awareness of the issue in other countries importing into the EU.

Techniques for Measuring Plant Toxins

Sampling plays a crucial part in precise determination of plant toxins levels in a certain lot, as contaminants within a lot may be heterogeneously distributed. It is also necessary to establish general method of analysis performance criteria to ensure that control laboratories use methods of analysis with comparable levels of performance. In December 2023, the European Commission published legislation establishing methods of sampling and analysis for the control of plant toxins levels in food[13].

Methods for PAs rely on extraction with acidified water, followed by solid-phase extraction (SPE) using a mixed-mode sorbent, which provides dual retention modes of reversed-phase and cation-exchange, followed by LC-MS/MS using alkaline or acidic chromatographic conditions[14]. The main analytical challenge is the presence of many isomers that are extremely difficult to resolve in the chromatographic dimension and exhibit the same MRM transitions. When attempting analysis in a single chromatographic run, one is left with a few pairs of coeluting isomers, which can be quantified as a sum. TAs are typically extracted with an acidified mixture of water and methanol/acetonitrile (including QuEChERS), followed by LC-MS/MS. Passing the extract through a simple ultrafiltration device or SPE cartridge can remove matrix co-extractives, enhancing method performance. To rationalize analyses in high-throughput laboratory environments, the scope of multi-mycotoxin methods can easily be extended to include the two regulated TAs, atropine and scopolamine[15].

While efforts have been made to address the food safety issue of plant toxins in Europe and reduce risk to the consumer, the number of food alerts seems to be on the rise. Fortunately, challenges with the determination of plant toxins in foods have largely been overcome, enabling testing to be carried out for checking regulatory compliance and monitoring occurrence, ensuring the safety of products for human consumption.

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References:

[1] Panda D. et al. (2022). Recent advancements in LC-MS based analysis of biotoxins: Present and future challenges. Mass Spec Rev. 41:766-803.

[2] Urugo, M. et al. (2023). Naturally Occurring Plant Food Toxicants and the Role of Food Processing Methods in Their Detoxification. Int. J. Food Sci. 2023 Article ID 9947841, 16pp.

[3] Abia W. et al. (2021). Tropane alkaloid contamination of agricultural commodities and food products in relation to consumer health: Learnings from the 2019 Uganda food aid outbreak. Compr. Rev. Food Sci. Food Saf. 20(1):501-525.

[4] Fuente-Ballesteros A. et al. (2024). Comprehensive overview of the analytical methods for determining

pyrrolizidine alkaloids and their derived oxides in foods. J. Food Compos. Anal. 125:105758.

[5] De Nijs, M. et al. (2023). Emerging Issues on Tropane Alkaloid Contamination of Food in Europe. Toxins 15(2):98.

[6] EFSA (2013). Scientific Opinion on tropane alkaloids in food and feed. EFSA Panel on Contaminants in the Food Chain. EFSA J. 11:3386.

[7] EFSA (2017). Risks for human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal infusions and food supplements. EFSA J. 15:4908.

[8] European Commission (2023). Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels for certain contaminants in food and repealing Regulation (EC) No 1881/2006. OJ L 119:103–157.

[9] Casado, N. et al. (2022). The concerning food safety issue of pyrrolizidine alkaloids: An overview. Trends Food Sci. Technol. 120:123-139

[10] Gonzalez-Gómez L. et al. (2022). Occurrence and Chemistry of Tropane Alkaloids in Foods, with a Focus on Sample Analysis Methods: A Review on Recent Trends and Technological Advances. Foods 11:407.

[11] https://webgate.ec.europa.eu/rasff-window/screen/notification/651495

[12] European Commission (2024). Commission Implementing Regulation (EU) 2024/286 of 16 January 2024 amending Implementing Regulation (EU) 2019/1793 on the temporary increase of official controls and emergency measures governing the entry into the Union of certain goods from certain third countries. OJ L 2024/286.

[13] European Commission (2023). Commission Implementing Regulation (EU) 2023/2783 of 14 December 2023 laying down the methods of sampling and analysis for the control of the levels of plant toxins in food and repealing Regulation (EU) 2015/705. OJ L 2023/2783.

[14] Method Development and Validation for the Determination of Pyrrolizidine Alkaloids in a Range of Plant-Based Foods and Honey Using LC-MS/MS. Waters Application Note 720007624.

[15] Development of a Multi-Toxin UPLC-MS/MS Method for 50 Mycotoxins and Tropane Alkaloids in Cereal Commodities. Waters Application Note 720007476.

 

Katie Banaszewski, NOW Foods
In the Food Lab

Making Supplements Safer: Tackling the Pesticide Problem

By Katie Banaszewski
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Katie Banaszewski, NOW Foods

Precise, accurate contaminant analysis is crucial to ensure that dietary supplements are of high quality and free from potentially harmful chemicals, such as heavy metals or pesticide residues. As supplements become an increasingly prevalent part of global health culture, with their global market forecast to reach a value of more than $230 billion by 2027, there is an urgent need to ensure their safety for consumers—but manufacturers face many challenges in this area.

Assuring that dietary supplements are free of pesticide contamination is especially difficult given their botanical ingredients, which can be more complex than other analytes. A prominent obstacle is matrix interference. As most botanical ingredients exist in the form of concentrated extracts, smaller sample sizes are needed to overcome heavy matrix interference, in turn requiring highly sensitive instrumentation to detect minute amounts of pesticide residues.

With this in mind, we adopted an analytical workflow comprising both gas and liquid chromatography (GC and LC) systems for orthogonal residue analysis. GC-MS/MS can achieve fast, robust separation of ~300 pesticide residues, while LC-MS/MS enables analysis of ~280 residues. The GC and LC instruments are sufficiently sensitive to allow dilution of samples to mitigate matrix interference— essential to determine potentially low residue levels in complex matrices, and ensure dietary supplements can confidently be certified safe.

Clearing Analytical Hurdles

Matrix complexity is only increased by the fact that botanical ingredients are sourced from across the world and, therefore, exposed to many different agricultural practices. As a wide range and great many of these botanical ingredients are used to produce supplements, it is challenging to develop sample preparation procedures that are suitable for all products.

To prevent frequent iterations of analytical procedures, we developed one sample preparation workflow for GC-MS/MS and another for LC-MS/MS. In both, samples are hydrated and extracted (using acetonitrile:water and the salts anhydrous magnesium sulfate and sodium chloride) before cleanup by solid-phase extraction (SPE). For LC, various defined combinations of dispersive SPE analysis are used to accommodate different matrices (pigmented, high-fat or high-protein, for example) before samples are diluted prior to analysis. Doing so allows us to optimize sample preparation for particular groups of botanical matrices and target specific matrix mitigation without needing to change the entire workflow.

In addition to the aforementioned analytical hurdles, some lesser-defined commodities lack maximum residue limits, complicating the interpretation of results and specification of acceptable criteria. To mitigate these difficulties, we opted to streamline our data processing and reporting by implementing integrated chromatography data system software for both LC-MS/MS and GC-MS/MS. This enables on-the-spot evaluation of QC criteria and rapid assessment of component presence (or absence) in data review and facilitates swifter and easier cGMP compliance.

Keeping Supplements Safe

Our chosen analytical approach has created robust, sensitive processes for optimized multi-residue analysis of dietary supplement samples in a regulated QC environment.

With uptake of supplements fast increasing, guaranteeing product safety is more important than ever. Improved pesticide screening, and quality control of food ingredients, holds great value for both individual organizations and the industry as a whole, while—crucially—enabling consumers to rest assured about the safety of the products available to them.

Vitamins

Revamped Liquid Chromatography Enhances Analysis of Vitamins and Beyond

By Maria Grübner
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Vitamins

Vitamins play a critical role in the regulation of key physiological processes, such as blood clotting, metabolism and maintaining our vision. These biologically important compounds can be divided into two broad classes based on their solubility and differ in the way they are handled in the body—and in food safety laboratories. While excess amounts of water-soluble vitamins (including B1, B2, B3, B6 and B12) are excreted, fat-soluble vitamins (including vitamin A, D, E and K) can be stored in the liver or fatty tissue for later use. The simultaneous analysis of water- and fat-soluble vitamins in traditional liquid chromatography is difficult, and is compounded by the presence of biologically important vitamin isomers, which exist at lower concentrations and demand greater sensitivity from analytical techniques.

Food analysis laboratories support food manufacturers by assessing food safety and authenticity, and have a responsibility to produce precise and reliable data. Vitamins are among a number of compounds assessed in infant formulas, energy drinks and other supplements, and are added to fortify the nutritional value of these products. Given the critical nutritional role of vitamins, especially during early developmental periods, their characterization is highly important. This, along with the challenging and cumbersome nature of vitamin analysis, has spurred the development of innovative high-performance liquid chromatography (HPLC) methods for food safety testing.

Unique Challenges of Vitamin Analysis

The simultaneous analysis of water- and fat-soluble vitamins is difficult to achieve with reversed-phase high-performance liquid chromatography, due to the wide range of hydrophobicity among vitamins. Highly hydrophobic fat-soluble vitamins are retained strongly by chromatography columns and are only eluted with high-strength mobile phases. In contrast, water-soluble vitamins are usually poorly retained, even with very weak mobile phases. As the ideal conditions for chromatographic separation are very different for the two vitamin classes, there have been efforts to explore the possibility of operating two columns sequentially in one system. The early versions of this approach, however, were not well suited to high-throughput food safety laboratories, requiring complex hardware setup and even more complicated chromatography data system programming.

Prior to liquid chromatography analysis, food samples must be purified and concentrated to ensure target analytes can be detected without matrix interference. Liquid-liquid extraction is one purification method used to prepare for the analysis of vitamins and other compounds; it was one of the first methods developed for purification and enables compounds to be separated based on their relative solubilities in two different immiscible liquids.1 It is a simple, flexible and affordable method, yet has several major disadvantages.2 Liquid-liquid extraction consists of multiple tedious steps and requires the use of large volumes, therefore the time for completion is highly dependent on the operator’s skills and experience. Consequently, the duration of sample exposure to unfavorable conditions can vary greatly, which compromises reproducibility and efficiency of the method. This is of concern for vitamins that are particularly prone to degradation and loss when exposed to heat and light, such as vitamin D in milk powder.

Two-Dimensional Liquid Chromatography Enables Deeper and Faster Analysis

Analysts in the food industry are under pressure to process high volumes of samples, and require simple, high-throughput and high-resolution systems. Fortunately, two-dimensional liquid chromatography (2D-LC) systems have evolved markedly in recent years, and are ideally suited for the separation of vitamins and other compounds in food and beverages. There are two main types of systems, known as comprehensive and heart-cutting 2D-LC. In comprehensive 2D-LC, the sample is separated on the first column, as it would be in 1D-LC. The entire eluate is then passed in distinct portions into a second column with a different selectivity, enabling improved separation of closely eluting compounds. In contrast, heart-cutting 2D-LC is more suited to targeted studies as only a selected fraction (heart-cut) of the eluate is transferred to the second-dimension column.

Recently, another novel approach has emerged which utilizes two independent LC flow paths. In dual workflows, each sample is processed by two columns in parallel, which are integrated in a single instrument for ease of use. The columns may offer identical or different analyses to enable a higher throughput or deeper insights on each sample. This approach is highly suited to vitamin analysis, as the two reversed-phase columns enable simultaneous analysis of water- and fat-soluble vitamins. A simple, optimized preparation method is required for each of the two vitamin classes to ensure samples are appropriately filtered and concentrated or diluted, depending on the expected amount of analyte in the sample. The dual approach enables a broad range of ingredients to be assessed concurrently in supplement tablets, energy drinks, and other food and beverages containing both water- and fat-soluble vitamins. For analysts working to validate claims by food vendors, these advances are a welcome change.

Refined Detection and Extraction Methods Create a Boost in Productivity

Analysts in food analysis laboratories now have a better ability to detect a wide range of components in less time, due to improved detection and extraction methods. Modern LC systems utilize a wide range of analytical detectors, including:

  • Mass spectrometry (MS)
  • Diode array detection (DAD)
  • Multi-wavelength detection
  • Charged aerosol detection (CAD)
  • Fluorescence detection (FLD)

The optimal detector technology will depend on the molecular characteristics of the target analyte. Infant formula, for example, can be analyzed by DAD and FLD, with detection and separation powerful enough to accurately quantify the four isomers of vitamin E, and separate vitamin D2 and D3. Highly sensitive 2D-LC methods are also particularly favorable for the trace level quantitation of toxins in food, such as aflatoxins in nuts, grains and spices.

Given the limitations of liquid-liquid extraction, an alternative, simplified approach has been sought for 2D-LC analysis. Liquid-liquid extraction, prior to chromatography analysis, involves many tedious separation steps. In contrast, the use of solid phase extraction for infant formula testing reduces pre-treatment time from three hours to one hour, while improving detection. This is of great significance in the context of enterprise product quality control, where a faster, simpler pre-treatment method translates into a greater capacity of product testing and evaluation.

HPLC Toolkit for Food Safety Analysis Continues to Expand

Several other HPLC approaches have also been utilized in the field of food safety and authentication. For example, ultra-high-performance liquid chromatography (UHPLC) with detection by CAD followed by principal component analysis (PCA) can be used to investigate olive oil purity. In contrast to conventional approaches (fatty acid and sterol analysis), this revised method requires very little time and laboratory resources to complete, enabling companies to significantly reduce costs by implementing in-house purity analysis. With a reduced need for chemicals and solvents compared with fatty acid and sterol analyses, UHPLC-CAD provides a more environmentally friendly alternative.

Analyzing amino acid content in wine is an important aspect of quality control yet requiring derivatization to improve retention and separation of highly hydrophilic amino acids. Derivatization, however, is labor-intensive, error-prone, and involves the handling of toxic chemicals. To overcome these limitations, hydrophilic interaction liquid chromatography (HILIC) combined with mass detection has been identified as an alternative method. While HILIC is an effective technique for the separation of small polar compounds on polar stationary phases, there still may be cases where analytes in complex samples will not be completely separated. The combination of HILIC with MS detection overcomes this challenge, as MS provides another level of selectivity. Modern single quadrupole mass detectors are easy to operate and control, so even users without in-depth MS expertise can enjoy improved accuracy and reproducibility, while skipping derivatization steps.

Conclusion

Recent innovations in 2D- and dual LC technology are well suited to routine vitamin analysis, and the assessment of other components important in food safety evaluation. The concurrent and precise assessment of water- and fat-soluble vitamins, despite their markedly different retention and elution characteristics, is a major step forward for the industry. Drastic improvements in 2D-LC usability, flexibility and sensitivity also allows for biologically important vitamin isomers to be detected at trace levels. A shift towards simpler, high-throughput systems that eliminate complicated assembly processes, derivatization and liquid-liquid extraction saves time and money, while enabling laboratories to produce more reliable results for food manufacturers. In terms of time and solvent savings, solid phase extraction is superior to liquid-liquid extraction and is one of many welcome additions to the food analysis toolkit.

References

  1. Schmidt, A. and Strube, J. (2018). Application and Fundamentals of Liquid-Liquid Extraction Processes: Purification of Biologicals, Botanicals, and Strategic Metals. In John Wiley & Sons, Inc (Ed.), Kirk-Othmer Encyclopedia of Chemical Technology. (pp. 1–52).
  2. Musteata, M. and Musteata, F. (2011). Overview of extraction methods for analysis of vitamin D and its metabolites in biological samples. Bioanalysis, 3(17), 1987–2002.

 

magnifying glass

How Workflow Advances Raise the Bar in LC-MS/MS Veterinary Drug Quantitation

By Ed George
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In the modern world, it’s often taken for granted that consumers can head to their local grocery store and fill their baskets with a broad range of meat, poultry, fish and dairy produce. Yet the plentiful availability of these products is possible, to a large extent, thanks to modern farming methods that rely on veterinary drugs to promote healthy animal growth, protect livestock from contracting diseases, and in some cases provide aesthetic qualities to food.

Despite the important role veterinary drugs play in farming and food production, usage must be carefully controlled, as their inappropriate administration can have adverse effects on animals, the environment and human health. A particular concern is the growing problem of antimicrobial resistance, which can be promoted in the environment by the overuse of some of these veterinary drugs.

As a result, the analysis of veterinary drugs forms an important part of routine food safety and quality control testing. However, the wide range of residue concentrations required to be quantified, along with the diverse sample matrices and chemical properties of multiple classes of veterinary drugs placed in a single analytical method, pose significant analytical challenges. The latest multi-residue, multi-class analytical workflow solutions using a generic sample preparation method and liquid chromatography tandem mass spectrometry (LC-MS/MS) are overcoming these issues to provide a robust, sensitive method for the extraction, detection, confirmation, and quantitation of veterinary drugs below their required maximum residue limits (MRLs).

Meeting the Needs of Veterinary Drug Analysis Workflows

Given the need to accurately and reliably quantify veterinary drugs in food, testing workflows must be both sensitive and operationally robust. Importantly, workflows must be amenable to a variety of different matrices, including meat, fish and dairy, and should be capable of screening for drug molecules with a broad range of physicochemical properties. The sample preparation protocols that are employed must minimize the loss of analytes and be sufficiently simple and cost effective to enable routine laboratory use. Additionally, the separation steps that are employed must be sufficiently rugged and should ideally be able to handle any analyte and matrix. Finally, the methods used to identify and quantify samples must be sufficiently selective and sensitive to detect and confirm drug molecules and their metabolites at trace levels.

Developing methods that can meet all of these criteria for a wide range of drug molecules and food matrices, while minimizing the potential for false positive and negative results, is not straightforward and has proven challenging for the industry. As a result, many analytical methodologies have emerged that are typically limited in scope to a limited number of residues or specific chemical classes, are labor intensive, and require extensive sample preparation and clean-up. Fortunately, ongoing advances in veterinary drug analysis workflows are helping to drive the adoption of standardized protocols that have universal applicability.

QuEChERS: Making Sample Preparation Quick, Easy and Reliable

Sample preparation is a key first step in veterinary drug analysis workflows, but its importance is often overlooked. Even with the most advanced downstream separation and detection technologies, workflows are liable to generate poor quantitative results without reliable residue extraction methods.Having robust sample preparation protocols is especially important given the heterogeneous nature of the sample matrix and the different physicochemical properties of the residues that must be extracted.

Traditional approaches, based on sample homogenization and multi-step solvent extraction procedures, were time-consuming and did not always produce consistent results. The loss of residues during sample grinding or through the formation of insoluble drug-matrix complexes would often impact the accuracy of measurements. Moreover, the need for labor-intensive sample cleanup steps, based on separation methods such as gel permeation chromatography, added additional complexity to workflows.

The widespread adoption of universal sample preparation protocols based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) methods has simplified the process of extracting veterinary drugs from matrix samples. These approaches have been specifically designed to be quick and easy to implement, and enable high extraction efficiency with a very broad range of chemical properties from a variety of matrices. As a result, QuEChERS has proven to be a very reliable means of preparing samples for veterinary drug analysis.

The universal suitability of the QuEChERS approach has reduced the complexity of sample preparation workflows to such an extent that many suppliers now offer kits containing pre-weighed reagents that can be used straight from the box. Moreover, because they only require small amounts of sample and solvent, and little in the way of equipment, these easy-to-use methods are helping laboratories minimize waste and make workflows more cost-effective.

Triple Quadrupole MS: Design Improvements Driving Exceptional Sensitivity

LC-MS/MS has rapidly established itself as the go-to technique for sensitive and reliable veterinary drug analysis, with set-ups based on ultra-high performance liquid chromatography (UHPLC) systems and triple quadrupole mass spectrometers proving to be particularly effective. With drug residues typically on the parts per billion scale, these systems have proven to be more than capable of delivering the level of performance that’s required when working with analytes that require low detection limits.

What’s more, recent advances in triple quadrupole mass spectrometer technologies are pushing the limits of quantitation even further. Improved instrument designs based on segmented quadrupoles, more powerful electron multipliers and enhanced ion transmission optics are enabling food analysis laboratories to achieve even greater levels of experimental sensitivity, mass accuracy, selectivity and precision. These performance improvements are allowing analysts to make more confident decisions around every sample.

ion chromatogram, salmon extract sample
Figure 1. Total extracted ion chromatogram of salmon extract sample at 1× STC. Results obtained using a Thermo Scientific Vanquish Flex Binary UHPLC system and a Thermo Scientific TSQ Altis triple quadrupole mass spectrometer. (Click to enlarge)

The capabilities of the latest generation of triple quadrupole LC-MS/MS systems for quantitative veterinary drug analysis were put to the test in a recent study. More than 170 veterinary drugs were added directly to a variety of homogenized matrices, including bovine muscle, milk, and salmon fillet using a QuEChERS sample preparation protocol to create a series of matrix extracted spikes (MES). The concentration of residues in the MES samples referenced a chosen screening target concentration (STC), which was typically one-third to one-quarter of the defined European Union MRL for each residue/matrix combination. Figure 1 presents the total extracted ion chromatogram for an MES sample of salmon fillet at the STC, obtained with a binary UHPLC system and a triple quadrupole mass spectrometer.

For each analyte, calibration curves were constructed using replicate measurements of each of the MES samples at seven concentrations ranging from one-fifth to five times that of the STC. Figure 2 highlights the calibration curve constructed for ethyl violet, a therapeutic dye used in aquaculture, in

Calibration curve, salmon extract
Figure 2. Calibration curve generated for ethyl violet in salmon extract (0.2–5.0 ng/g). (Click to enlarge)

the range 0.2 to 5.0 ng/g (STC = 1 ng/g). The calculated method detection limit of 0.03 ng/g for this compound in salmon fillet demonstrates confidence in the results well below the minimum required performance limit (MRPL).

LC-MS/MS: Leading the Way in Workflow Robustness

With potentially hundreds of samples to analyze every week, veterinary drug analysis workflows not only demand the highest levels of sensitivity, but also exceptional speed and robustness.
One way in which greater throughput can be achieved is by using shorter instrument dwell times, an experimental optimization that allows more compounds to be analyzed within a given timeframe during a chromatographic separation. Traditionally, the use of shorter dwell times would typically require sacrificing some measurement sensitivity. However, the latest advances in triple quadrupole instrument design are ensuring short dwell times no longer come at the expense of analytical performance.

Timed selected dreaction monitoring (SRM) is an effective strategy that allows analysts to overcome this challenge to achieve sensitivity with high throughput. Using timed SRM, data acquisition occurs within a short retention time window. This reduces the number of transitions that are monitored in parallel for each residue peak, while ensuring consistent quantitation even at low concentrations. Instrument control system software can automatically optimize the SRM conditions across the chromatographic run, maximizing operational efficiency with minimal need for manual input.

Instrument uptime is another factor that is of paramount importance for veterinary analysis workflows. With large workloads and tight turnaround times, regular instrument recalibration and frequent maintenance can be a major frustration for busy food testing laboratories. UHPLC is renowned for its operational robustness and suitability for fast-paced routine screening workflows, and the latest instruments are taking this reputation to an even higher level.

Comparison of injections of bovine muscle extract
Figure 3. Comparison of injections of bovine muscle extract MES at 3× STC over 500 injections (A: injection #20; B: injection #260; C: injection #500). Analytes shown: cyromazine (black), ciprofloxacin (red), sulfamethoxazole (green) and flunixin (blue). (Click to enlarge)

Figure 3 compares injections of bovine muscle extract at 3× STC over a 500-injection run that took place over a period of one week, obtained using the experimental set-up described earlier. Despite continuous operation over this extended period, the peak shape, intensity and retention time stability are maintained. These results further highlight the robustness of the LC-MS/MS system for routine veterinary drug testing.

Conclusion

Enforcing the responsible use of veterinary drugs in farming and food production depends upon comprehensive, sensitive, robust and reliable workflows capable of delivering quantitative results. Advances in sample preparation techniques and LC-MS/MS technologies are setting new standards when it comes to confident multi-residue veterinary drug analysis. From the development of reliable easy-to-use QuEChERS protocols, through to robust UHPLC separation methods and sensitive triple quadrupole mass spectrometers, improvements across the workflow are driving exceptional performance—whatever the matrix, whatever the residue.