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Fields & Applications Food, Beverage & Agriculture, Thin Layer Chromatography, Gas Chromatography, Liquid Chromatography, Mass Spectrometry, Metabolomics & Lipidomics

Gurus of Pesticide Residue Analysis

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The Experts

Finbarr O’Regan

Finbarr O’Regan (FO) is technical manager at the Pesticide Control Laboratory at the Department of Agriculture, Food and The Marine in Ireland. The laboratory is the national reference laboratory for pesticide residues in food of plant origin, cereals and feeding stuffs, food of animal origin, and also for single residue methods. Finbarr assesses methods to ensure they are fit for purpose, deals with technical issues, and works with the quality manager to ensure continued accreditation of the lab and that the scope is expanded where necessary. In 2014, Finbarr was the chair of the European Pesticide Residue Workshop (EPRW), leading the scientific organizing committee.

lutz alder

Lutz Alder (LA) has been employed by the German Federal Health Office – now the Federal Institute for Risk Assessment (BfR) – since 1991. At the BfR, he is responsible for the evaluation of monitoring methods for pesticide residues provided for pesticide registration. He is the chairman of a German Working Group that publishes Germany’s official methods for pesticide residues and the convenor of two working groups of the European standardization body, CEN, which are responsible for European methods for pesticide residues. Lutz has been on the EPRW Scientific Organizing Committee since its inception.

André de Kok

André de Kok (AK) is senior analytical chemist for the NVWA - Netherlands Food and Consumer Product Safety Authority, which is the national reference laboratory for pesticide residues in food and feed. André is responsible for method development and validation, and the implementation of new methods and improvement of existing methods in the routine analysis team. André was also on the scientific organizing committee of the 10th EPRW.

How would you describe the impact of pesticide residue analysis on society?

Finbarr O’Regan: A 2010 European Food Safety Authority (EFSA) food barometer study showed that, across the 27 EU member states, some 72 percent of Europeans are concerned about the presence of pesticide residues in food. In conventional agricultural production, the effective and safe use of plant protection products is critical to mitigate the negative effects of pests and plant diseases. It is important that consumers can be confident that pesticides are being used in accordance with good agricultural practice.

André de Kok: Well, the analysis of pesticide residues ensures safety of consumer products and, therefore, indirectly but positively affects the health of consumers. What could be more important than that?

Lutz Alder: Like Finbarr, I’d like to draw on the 2010 food barometer survey report on risk perception for EU consumers. The survey was carried out on a representative sample of 26,691 individuals, aged 15 or over. Those European consumers who are concerned about possible food-related risks tend to worry more about chemical contamination of food rather than bacterial contamination or health and nutrition issues – in fact, the number one concern was pesticide residues!

How has pesticide residue analysis changed over the past few decades?

LA: Let me offer a potted history... Back in the 1960s, pesticide analysis began with methods based on paper and thin layer chromatography that covered no more than 5–20 analytes. The 1970s focused on gas chromatography (GC) methods with packed columns and specific detectors, such as the electron capture detector (ECD), nitrogen phosphorus detector (NPD), and flame photometric detector (FPD), which eventually allowed the detection of up to 200 volatile pesticides (25 percent of the produced active substances) – but only when using a number of column and detector combinations. The commercial availability of capillary columns changed GC analysis in the 1980s, offering the measurement of up to 100 analytes with a single column that also offered much improved selectivity. GC-MS methods for about 400 pesticides, contaminants or their derivatives using the selected ion monitoring (SIM) mode dominated the 1990s, which was made possible by the availability of data systems for routine use.

The most dramatic change was made by atmospheric pressure ionization for LC-MS/MS at the turn of the millennium, offering the measurement of up to 600 pesticides (volatile and non-volatile without derivatization) in a single run with unknown selectivity. It was a little bit surprising that we had to wait many further years for the availability of GC-tandem-MS/MS instruments, which similarly improved selectivity of GC methods…

Over the last decade, the introduction of LC-MS/MS and GC-MS/MS triple-quad instruments has increased the selectivity and sensitivity of multiresidue methods tremendously

FO: Lutz has covered the milestones nicely. As noted, we’ve moved on from methods, such as GC-ECD and GC-FPD, which gave no structural information and relied heavily on retention time and a second analysis with a different column. Moreover, the typical standard mix contained 30 or so compounds – any more than that tended to cause problems with co-elution. HPLC was used for just a small number of compounds – carbendazim and thiabendazole only in our lab. Today, we use GC-MS/MS and LC-MS/MS, which give structural information leading to much higher confidence in the result. Co-elution is no longer an issue. The number of compounds in our GC method is now 170, and for LC it’s 300.

ADK: Pesticide residue analysis has traditionally been done – and still is being done – via multiresidue analysis, covering as many pesticides as possible in a single chromatographic run. As noted by Lutz and Finbarr, the most important change has been the move from selective detectors to MS detectors for both GC and LC. Over the last decade, the introduction of LC-MS/MS and GC-MS/MS triple-quad instruments has increased the selectivity and sensitivity of multiresidue methods tremendously – and the scope of methods has been continuously expanded through increasing scan speeds.

What are the major milestones that have brought us to the current state-of-the-art?

FO: I guess we’ve already highlighted the impact of MS (GC-MS, LC-MS/MS, GC-MS/MS, LC-time-of-flight (TOF), GC-TOF are all milestones). But I think cryogenic milling deserves a special mention as a major advance; sample preparation is sometimes forgotten when dealing with analysis, as noted by the “Three Wizards of Sample Preparation” (tas.txp.to/0914/samplewizards). Without cryogenic milling, results variation for some samples can be as high as 25 percent – with it, the result variation drops to less than 5 percent.

ADK: In addition to the introduction of GC-MS(MS) ion trap, single quad and triple quad and LC-MS/MS triple quad instruments for multiresidue analysis, I’d like to highlight the introduction of miniaturized extraction methods, such as the Dutch mini-Luke acetone method (1983), QuEChERS acetonitrile method (2002) and the Swedish ethylacetate (SwEet) method (2007). They have all increased the efficiency of pesticide residue analysis, resulting in increased sample throughput and decreased turn-around and reporting terms. The more recent introduction of GC- and LC-High Resolution MS instrumentation offers a new era of pesticide analysis, further increasing selectivity and reliability of identification and offering an indefinite scope of the methods. When sensitivity of these instruments is further improved, they are likely to finally replace triple-quad instruments, especially for wide-scope analyses, combining analysis of pesticides with, amongst others, mycotoxins, other contaminants and veterinary drugs.

LA: For me, the major milestones have been electrospray ionization (ESI) and tandem mass spectrometry, which feature selected and multiple reaction monitoring (SRM/MRM) modes. Without this universal, very sensitive, selective and robust detection technique, the current success of very simple extraction and cleanup methods, such as QuEChERS, that provide rather dirty extracts would not be possible.

What big challenges remain?

ADK: I think that the biggest challenge is still the further improvement of software for the newest generation LC- and GC-MS instrumentation to speed up data processing and reporting times. In terms of sample analysis, the so-called “difficult” matrices, such as cocoa, tobacco, spices, herbs, dietary supplements, and animal products, are the new challenge.

LA: Today, most analysis is targeted. Such methods seek previously specified pesticides, of course. However, outdated, nationally unregulated or very new pesticides are often not covered. Non-targeted methods based on sufficiently sensitive high mass resolution mass spectroscopy must be developed and validated.

A second point is the analysis of very polar pesticides, which are typically covered by single residue methods or methods that cover small groups of analytes. Because of the high price of such analyses per analyte (often 100 times higher than the price of multi-residue analysis), simpler, more effective methods are needed. A promising solution could be screening with flow injection analysis in combination with HR-MS/MS.

For now, the relevance of cumulative or combined exposure is vague and scientifically based legal limits are not defined. One of the rare outliers is the maximum residue limit (MRL) for the sum of dithiocarbamates. However, this type of regulation was needed because routine individual analysis of different dithiocarbamates is currently impossible.

A more targeted approach will identify and flag problem compound/crop combinations at an EU level

FO: People talk of increased sampling, but I think that is unlikely. Rather, a more targeted approach will identify and flag problem compound/crop combinations at an EU level. I also believe that single residue methods will become a more significant part of routine analysis. The challenge is to have robust methods for difficult compounds that do not fit into multi-residue methods, as noted by Lutz.

Do you believe that these challenges are being adequately addressed by regulatory agencies/research programs/industry?

FO: The regulations state that, where possible, compounds that are new to the market should be amenable to analysis using multi-residue methods. This puts an onus on industry to investigate the possibility of adding new compounds to existing multi-residue methods. However, because this is not always possible, the EU Reference Laboratory (EURL) for single residue methods in Stuttgart is constantly working on improving existing methods and developing new methods where necessary. I feel that more could be done in terms of ring trials on single residue methods before they are put up on the website to iron out robustness or transferability issues.

LA: Regulatory agencies work adequately on the problem of cumulative/combined pesticide exposure, but fast results or simple answers should not be expected. In addition, some human drugs with similar modes of action are sometimes simultaneously used in much higher amounts than pesticide residues (for example, antifungal drugs with triazole moiety). The resulting cumulative effect has not yet been discussed…

ADK: In short, yes. I do believe that many of the challenges are being addressed by instrument vendors in cooperation with leading research and regulatory laboratories.

What is the current regulatory landscape – are there any gaps?

LA: It seems that several MRLs are arbitrarily set for the parent pesticide, especially for foodstuffs of animal origin. Such MRLs exist without the detection of residues in feeding studies or when feeding studies are conducted with the parent pesticide, even though the pesticide is significantly metabolized in plants. In other cases, metabolism studies of relevant feeding crops are not provided by pesticide producers. Therefore, agencies are unable to calculate the intake of such pesticides and their metabolites, which is necessary to derive a correct residue definition. Incorrect definitions can waste a great deal of analytical effort on residues that will never be found.

Another issue is the existence of unclear residue definitions. For example, unspecified conjugates are included in several residue definitions. In such cases, the appropriate procedures to liberate the analytes from conjugates remain vague. In addition, validation of methods is impossible.

ADK: EU MRLs for pesticides in food have been harmonized since 2008 (via Regulation 396/2005). This regulation has eased trade between EU countries and made trade with third countries more transparent in terms of MRLs. Worldwide harmonization of MRLs (EU, USA, Australia/NZ, Latin-America, Africa, Asia) to break down further trade barriers is still a major challenge; pesticide companies and regulatory agencies are working on initiatives to tackle this huge task. The introduction of an EU default-“zero” tolerance of 0.01 mg/kg for forbidden or non-registered pesticides has proven to be very practical for both enforcement agencies and traders. This value will likely be introduced by the EU as a practical “zero”-level for biological products.

FO: The coordinated program for pesticide residue analysis across the EU deals with the compounds and commodities to be covered. It’s a useful piece of legislation that sets out the expectations for the following three years, which gives laboratories a chance to transfer or develop methods as necessary.

How does today’s practice match the regulatory criteria?

FO: The use of techniques that allow a high degree of structural information to be gleaned gives us huge confidence in the data being produced. The criteria laid down in regulations insist on the type of information that must be achieved using LC-MS/MS and GC-MS/MS techniques.

ADK: Both trade and enforcement agencies apply or adhere to the present legislation. The cumulative exposure of consumers to multiple pesticides (and other toxic compounds) will attract more attention.

LA: I will be bold and state that no analytical lab (not even the best) is able to analyze residues of every regulated pesticide in every sample. Each lab has to find its own balance between completeness of scope and costs – a fact that is known and accepted by agencies. The problematic areas are the most polar pesticides, residues in animal materials, and pesticides with complex residue definitions; for example, those that include conjugates. In these cases, the overall sampling rate in Europe is 100 to 1000 times lower compared with residues of “simple” pesticides.

On the up side, the sensitivity of methods for the majority of pesticides (the simpler ones), is now much better than 10 years ago and generally sufficient to fulfill all regulatory demands.

How do you see the field developing over the next 10-20 years?

FO: The field will develop by scanning against libraries using TOF technology. At the moment, this technique is still not sensitive enough. There will still be standards for those compounds that we expect to find, so I believe LC-MS/MS and GC-MS/MS will still be used to test for expected compounds; TOF will be used as a tool to determine future additions to the main scope. And although TOF may only be used by countries across the EU that can afford the capital investment, the data generated can be used to add to existing laboratory scopes.

ADK: GC- and LC-high resolution (HR) MS instrumentation will probably be the preferred choice for (pesticide) residue analysis. Sample preparation will be further miniaturized, simplified and automated. Direct flow-injection MS is a promising option, especially for pesticides otherwise requiring very specific chromatographic conditions that do not match multiresidue methods. Ever increasing selectivity and sensitivity of new instruments will assist these developments. Instruments may be further miniaturized, thereby requiring less lab space or even transferring instruments to sampling locations, such as border control points.

LA: I would say an increasing proportion of method development will be conducted by instrument companies. I would also expect more analytical automation, which is today only really found in clinical analysis.

Big instrument companies will no longer sell just instruments; they will provide the chemicals and consumables needed for extraction and cleanup. Sophisticated acquisition methods will be developed by vendor applications chemists and delivered to fulfil bespoke requests from clients.

Which analytical techniques are driving the field forward towards this future?

ADK: GC- and LC-HRMS technology. Metabolomics can become very important for studying the toxicological effects of pesticides on humans, which could have an effect on the registration and re-registration of new or existing pesticides, respectively. Nano-flow LC will reduce the consumption of hazardous solvents and fast GC will further lower analysis times.

LA: I agree with André and expect increasing use of HRMS (resolution > 50,000 FWHM) in combination with LC and GC analysis. This technique offers analogous selectivity compared with tandem mass spectrometers – and without the limitations of targeted analysis.

The actual Achilles heel of ESI – matrix effects – will probably become less important through the use of micro HPLC columns.

FO: As noted earlier, TOF will be a useful screening technique along with GC-MS/MS and LC-MS/MS for confirmation and quantitation.

Which techniques are likely to fall by the wayside?

ADK: Easy. All non-MS based techniques!

FO: Difficult to say, as LC-MS/MS and GC-MS/MS are excellent techniques with excellent sensitivity. Right now, GC-MS is the only technique that I could see becoming obsolete.

LA: Except for GC-ECD, GC methods using specific detectors are no longer important in developed countries. In the future, GC-MS will be completely replaced by GC-MS/MS instruments. And the actual workhorses – LC-MS/MS instruments – will lose their top position as soon as LC-HR-MS/MS offers comparable sensitivity.

Pesticide Industry Insider

Sergio Nanita is principal investigator at DuPont Crop Protection R&D, where he leads analytical and environmental chemistry projects that support the discovery, development, registration and launch of novel agricultural products. Here, he adds an industry perspective to the mix.

The global population outlook makes agricultural efficiency and food security more important than ever. Pesticide analysis is central in the enforcement of regulations to ensure compliance in the use of agricultural products. Pesticide analysis is also essential across all stages of discovery and development of novel agrochemicals, which, together with other modern tools and practices, will address the agricultural challenges of the future. Novel agricultural chemicals are designed to meet modern standards. Today, new active ingredients are expected to replace obsolete chemistries and provide farmers with pest control options that are better in several aspects, including environmental profile, risk reduction and efficacy. Pesticide analysis benefits the greater scientific community and society in many ways, and contributes to fulfilling global food security needs.

Driving analytical science
An often overlooked contribution made by pesticide analytical research is the fact that it has consistently led to the development of broadly-applicable methods; thus, contributing to the broader field of analytical chemistry. I believe that this has occurred in part due to the complexity of the samples typically encountered in pesticide residue analysis. Methods developed to provide reliable analysis of difficult matrices, such as canola seed, dried tea leaves and spices, often work well for simpler systems. In other words, methodologies that pass “the pesticide residue analysis test” are often suited for other purposes. For example, methods by Hans Mol and coworkers have allowed multi-scope analysis to be possible, covering mycotoxins, veterinary drugs and pesticides in a single analytical run, and the QuEChERS method originally introduced by Anastassiades, Lehotay and coworkers has expanded far beyond pesticide residue laboratories.

On the other hand, pesticide residue analysis often demands the best technology. We enjoy today’s state-of-the-art thanks to several milestones by many analytical researchers. In my opinion, (i) the use of electrospray ionization in commercial mass spectrometers, (ii) large-scale multiresidue methods, and (iii) the routine use of sensitivity in analytical instrumentation toward simplification of sample preparation, represent key milestones in the 1990s, 2000s and 2010s, respectively. Electrospray ionization was an important milestone because it allowed LC-MS to be a reliable and highly sensitive technique for analyzing non-volatile pesticides that are not amenable to GC.

Regarding instrumentation sensitivity: the well-known “dilute-and-shoot” approach was made possible by the sub-part-per-billion sensitivity that can be obtained routinely with modern mass spectrometers. Therefore, dilution (a straightforward and high-throughput procedure) has become the preferred method for reduction of matrix in complex extracts prior to analysis.

Current trends
As kindly noted by Lutz Alder, over the past few decades, we have seen the addition of new analytical techniques employed in pesticide residue analysis, as well as the evolution and improvement of classical methods. Today, LC-UV has nearly disappeared from pesticide residue analysis, but continues to be an important technique in process chemistry and manufacturing quality control (for example, active ingredient purity assays and impurity analysis). On the other hand, GC-MS is more than 50 years old and continues to be a useful technique for trace-level analysis. A common trend with all techniques is that instruments are more sensitive and rugged today, which has allowed for simpler sample preparation methods. Similarly, analytical instruments rely more on sophisticated software applications to acquire and process data, making chemical analysis more efficient overall – from sampling to reported result. Another clear trend also continues to be seen; simpler, faster procedures that consume fewer resources (solvents, energy, laboratory personnel time) and generate less chemical waste are highly preferred. Consequently, the use of automation in sample preparation has widened (see page 16 for the continued discussion on the importance of sample prep). Test kits for sample preparation, such as commercially available QuEChERS, have improved laboratory efficiency and also reduced the frequency of operational errors.

In summary, instrumentation is more complex, but the procedures are simpler, thanks to advances in computing and sample preparation techniques. The result? A single analytical chemist can do more today than ever before.

Future drivers
I believe that the primary trend of faster and simpler methods for pesticide residue analysis will continue, likely driven by several factors, such as increasing demand for chemical analysis, judicious/selective spending at laboratories across all sectors, and the need to reduce the environmental footprint of testing facilities.

Collaborative and interdisciplinary research efforts are at an all-time high. Consequently, access to (and the advancement of) emerging techniques will continue to accelerate – especially methods that are practical and directly address the needs of laboratories and society. The prevalence of counterfeit materials is a global concern and a risk to society across many markets. To combat this trend, in-situ analysis could become routine as instrumental methods employed for product quality and authenticity analysis advance, and as miniaturized analytical instrumentation improves and becomes more affordable in the next 10–20 years.

Novel direct MS analysis techniques, such as ambient MSn and flow injection MSn, have already started a revolution in quantitative screening methods. They allow analysis in seconds rather than minutes, representing an excellent tool for laboratories with high analytical demand. I believe that we will see many more methods implemented and published that use and improve these techniques in the next few years. In addition, kits that employ immunoassay technology are very common in clinical testing. This technology could continue to find applications in pesticide residue analysis, particularly in settings where a small number of analytes need to be measured. Various spectroscopic techniques (not just mass spectrometry) will likely play a key role in advancing in-situ analysis. For example, there are several commercially-available portable instruments based on ion mobility, infrared spectroscopy, raman spectroscopy and x-ray fluorescence. I expect that these instruments will get better over time and improve in-situ analysis.

The core analytical chemistry principles will likely remain in the analytical toolbox forever. Many techniques evolve, rather than disappear. Advances in liquid chromatography are an example of an evolutionary track; LC > HPLC > UPLC. It is likely that, as the “dilute-and-shoot” approach continues to expand, methods that involve hands-on sample preparation will be used less. Similarly, it is possible that several laboratories pursue implementation of ambient MS and flow injection MS to benefit from high throughput, which could decrease the use of chromatography-MS, particularly for screening purposes. That said, I believe that chromatography-MS combined with elaborate sample preparation will continue to allow the most selective and sensitive chemical analysis. Therefore, all the aforementioned techniques will play important roles for decades to come.

Challenges within the regulatory landscape
Some challenges in industry are different from those encountered in government and academic laboratories. Agrochemical industry R&D efforts have changed over the years with technology, as well as the regulatory landscape. For example, the number of analytes (active ingredient and metabolites) covered in pesticide residue methods in industry has increased significantly because of changes in regulation. Moreover, metabolites are often very difficult to analyze because of their physicochemical properties (polarity, stability, volatility). Consequently, the development of a multiresidue method in industry that covers about a dozen analytes (for example, one active ingredient with eleven metabolites) could require significantly greater effort than a pesticide screening method designed for more than 100 active ingredients. Additional challenges arise from the need to measure very low exposure levels, resulting in methods with detection limits significantly below the MRL. Therefore, novel techniques and methods that are broadly-applicable for both active ingredients and metabolites are needed. Another challenge is to maintain the simplicity of procedures to minimize resources utilized in laboratory operations – that’s a challenge we all appear to share!

Of course, agrochemical industries must adequately address the scientific challenges encountered during the development of novel pesticides to comply with current regulations. In fact, industry stewardship practices and sustainability initiatives represent standards that are often more stringent than regulatory requirements. I believe the combination of stewardship, sustainability and regulatory compliance are absolute conditions for the existence and success of industry.

Industry relies on regulatory guidelines to direct long-term research efforts. The review of registration applications can have significant duration and, in the meantime, regulations can change or evolve. Regulations (and their amendments) impact all sectors at once: the agrochemical industry, farmers, consumer protection and commerce. Such a large impact emphasizes the importance of advanced communication of regulatory changes and phase-in periods for implementation. In other words, clarity in the regulatory landscape benefits society in general.

Unharmonized regulations represent a hurdle that, for the most part, has a negative impact by creating inefficiencies and confusion in the affected sectors. The good news is that regulatory harmonization, legislature and emerging topics of interest are under open discussion. International forums, such as EPRW, have been successful at bringing together scientist and experts from government, academia, industry and NGOs for debate and consideration of the various perspectives.

Regulatory compliance is intrinsic in industry laboratories dedicated to pesticide residue analysis and studies that are part of pesticide registration applications. But there are other research settings where novel analytical techniques can be explored, developed and implemented prior to broad acceptance, consideration or inclusion in regulatory guidelines. For example, novel methods for pesticide residue screening based on flow injection/mass spectrometry have been developed at DuPont. These methods have recently gained popularity; academic, government and industry research groups currently have active research programs running to further improve the technique. And so, while current analytical methods used in agrochemical product registration meet the regulatory criteria, novel and emerging techniques often contribute to the future regulation of analytical chemistry.

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About the Authors
Finbarr O’Regan
Finbarr O’Regan

Finbarr O’Regan is technical manager at the Pesticide Control Laboratory at the Department of Agriculture, Food and The Marine in Ireland. The laboratory is the national reference laboratory for pesticide residues in food of plant origin, cereals and feeding stuffs, food of animal origin, and also for single residue methods. Finbarr assesses methods to ensure they are fit for purpose, deals with technical issues, and works with the quality manager to ensure continued accreditation of the lab and that the scope is expanded where necessary. In 2014, Finbarr was the chair of the European Pesticide Residue Workshop (EPRW), leading the scientific organizing committee.


Lutz Alder
Lutz Alder

Lutz Alder has been employed by the German Federal Health Office – now the Federal Institute for Risk Assessment (BfR) – since 1991. At the BfR, he is responsible for the evaluation of monitoring methods for pesticide residues provided for pesticide registration. He is the chairman of a German Working Group that publishes Germany’s official methods for pesticide residues and the convenor of two working groups of the European standardization body, CEN, which are responsible for European methods for pesticide residues. Lutz has been on the EPRW Scientific Organizing Committee since its inception.


André de Kok
André de Kok

André de Kok is senior analytical chemist for the NVWA - Netherlands Food and Consumer Product Safety Authority, which is the national reference laboratory for pesticide residues in food and feed. André is responsible for method development and validation, and the implementation of new methods and improvement of existing methods in the routine analysis team. André was also on the scientific organizing committee of the 10th EPRW.


Sergio Nanita
Sergio Nanita

Sergio Nanita is principal investigator at DuPont Crop Protection R&D, where he leads analytical and environmental chemistry projects that support the discovery, development, registration and launch of novel agricultural products.

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