1.0 Abstract
An Environmental Risk Assessment or ERA needs to clearly identify hazard and exposure to evaluate risk. Having good analytical chemistry methods is essential in measuring environmental concentrations in field samples as well as in hazard based hazard-based testing to determine the effects of a specific chemical. These methods are also needed to determine physical chemical properties used in models to predict environmental concentrations of chemicals.

Developing methods can be challenging enough for one test chemical, however, multi-component or multi-constituent substance, substances present an even greater significant challenge. According to the European Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) definition, multi-constituent substances are defined by their quantitative composition in which more than one main constituent is present in a concentration ≥10% (w/w) and <80% (w/w). The definition also states that components or constituents with a concentration <10% (w/w) should be identified as impurities, and it requires that impurities ≥1% (w/w) be specified by at least one classifier, such as name, CAS Registry number, etc. In addition, the U.S. Toxic Substances Control Act or TSCA regulates the introduction of new or already existing chemicals.

The successful analysis of multi-component or multi-constituent substances requires a combination of in-depth knowledge of the chemical process used to manufacture multi-component or multi-constituent substances. As a result, analytical techniques used for multi-constituent substance characterization must have the capacity to distinguish between the various components present and generate direct evidence of their chemical structure and concentration. In order to be successful, a number of questions need to be answered before the start of the analytical process: What toxicity and physical/chemical data are available? What models are available? What are the matrices of interest (water, soil, sediment, air, animal or plant tissue)? What range of concentrations do we need to achieve in matrices of interest? What are the limiting factors in achieving good recoveries in matrices on interest? Stability and homogeneity of the test substance is key in many of the matrices (e.g., diet, water, sediment).

In order to meet the analytical requirements of regulatory agencies, scientists depend on more sensitive and rapid analytical techniques than the traditional technologies of gas chromatography (GC), gas chromatography mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC). These more sensitive analytical technologies may include liquid chromatography coupled to mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS), allowing quantitative analysis according to regulatory requirements as well as aiding in compound screening, identification and confirmation.

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In this article, the authors discuss some of the key aspects involved in the analytical methods involving multi-component or multi-constituent substances, as well as a number of applicable regulatory requirements.

2.0 Introduction
The number of multi-component or multi-constituent substances found in the environment is rapidly increasing. At the same time, our understanding of both regulated and unregulated chemicals, often with highly diverse structures and broad biological activities, and the (medicinal) (bio)pharmaceuticals and personal-care products involved, is growing.

The increased occurrence as well as the added complexity of multi-component substances presents a major challenge for environmental analytical chemists. This challenge can be met with extensive analytical methods and comprehensive characterization using a variety of techniques and methods to confirm the occurrence of a particular chemical in the environment.

Definition: What are multi-component or multi-constituent substances
Multi-component or multi-constituent substances are, according to the European Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulatory framework, defined by their quantitative composition in which more than one main constituent is present in a concentration ≥10% and <80% (w/w). The definition also states that components or constituents with a concentration <10% (w/w) should be identified as impurities, and it requires that impurities ≥1% (w/w) be specified by at least one classifier, such as name, CAS Registry number, etc. [1]

In addition to REACH, the manufacturers of Fertilizers and Related Materials (FARM) have joined forces for their REACH compliance activities by launching the FARM REACH consortium in December 2008. They define multi-constituent substances as preparations. In their view, multi-constituent substances, irrespective of the manufacturing method of a final chemical product used, always contain a combination of a few (simple) ionic components. [1]

In the United States, the U.S. Toxic Substances Control Act of 1976 or TSCA regulates the types of chemicals that can be used in manufacturing, as well as the introduction of new chemicals. The act, which specifically mandates the United States Environmental Protection Agency (U.S. EPA) to protect the public from “unreasonable risk of injury to health or the environment” by regulating the manufacture and sale of chemicals, is essential given the large number of chemicals and multiple -constituent constituent substances subject to control, each with myriads of multiple and synergistic (toxic) effects and multiple pathways of exposure.

3.0 Significant environmental risk
A key question, asked by analytical scientists and regulators alike, is whether the combined low concentrations of chemicals, (medicinal) (bio)pharmaceuticals and personal-care products found in multi-component substances in the aquatic environment have a significant, recognizable effect on ecologic function. A second, equality equally important, question asks if multi-component substances pose a long-term risk to human and veterinary health. [2]

These questions are complicated by the fact that while the concentrations of these chemicals, (medicinal) (bio)pharmaceuticals and personal-care products may be relatively low – measured on the sub–part per billion or sub-nanomolar level – a number of these substances may share a specific mode of action (MOA), which, in turn, could lead to possible significant environmental effects through additive exposure. The concern is that these substances, which may enter our environment from a variety of routes, may escape detection if they are present at concentration concentrations below detection, while still having hazardous long-term cumulative effects. This may especially be the case with residues of specific pharmaceuticals or personal care personal-care products found in the aquatic environment. [2]

The reason for their concern is that a number of these pharmaceuticals are specifically designed to modulate endocrine and immune systems and cellular signal transduction. They have the potential to function as endocrine disruptors. Other agents found in the aquatic environment, including antibiotics for the treatment of human and veterinary disease, may contribute to the potential of resistance to human pathogens. [3]

While anti-cancer drugs and antibiotics are generally administered to “cure” disease, other pharmaceuticals are largely designed to manage or control symptoms of chronic disease, leading to long-term use. Analytical scientists and regulators are concerned about the (in many cases) still-unknown effects of unintentional and long-term exposure to these agents in a healthy population. [4]

Complementing this concern is the fact that select multi-component substances “surviving” various steps of metabolism and other degradative or sequestering actions may create an environmental risk. In some cases, these products may even be more bioactive than the original compound, adding to the potential risk. [2]

4.0 Regulated and unregulated chemicals
Analytical scientists have, for many decades, centered their research on regulated chemicals included in various legislation in the United States and Europe. With the availability of more sensitive analytical methods such as liquid chromatography coupled to mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS), which allow the detection of chemicals used in everyday life, such as surfactants and surfactant residues, pharmaceuticals and personal-care products, and gasoline additives, as well as determining their biological effects, scientists now are able to analyze unregulated contaminants that either went undetected before or were not considered a risk. A number of the chemicals in high-risk multi-component substances, including detergent metabolites, steroids, and prescription and non-prescription drugs, are among the compounds most frequently found in high concentrations in the aquatic environment. [4]

5.0 Human health and regulations
In addition, due to the potential implications of these compounds on human and veterinary health, environmental analysis, as part of the regulatory approval process related to the manufacturing of novel (bio) pharmaceuticals, usually includes rigorous quality assurance and quality control (QA/QC) metrics designed to confirm the reliability of the analytical data.

Overall, the regulatory expectations to better understand product impurities and degradants in biopharmaceutical products continue to increase, making environmental risk assessment infinitely more complex, especially if it involves multi-component substances.

6.0 Why so complex?
Analyzing multi-component or multi-constituent substances is complex because of the difference between perceived facts and reality. For example, in analyzing multi-component or multi-constituent substances, it may be assumed that the materials or substances looked for are of a well-known toxicology and physiology, with well-known chemical and biological properties and known concentrations. In reality, specific data may be missing, chemical properties may be unknown or poorly understood, and finding the range of the concentration may be challenging. At the same time, questions may arise about the kind of mixture to be tested (what are we testing?), the formulation (do co-solvents alter the formulation?), insoluble materials (how low do we need to go?), polymers (differences in chain lengths) and testing near solubility (stock volumes).

Hence, successful analysis of multi-component substances requires in-depth knowledge of the chemical process used to manufacture multi-component or multi-constituent substances.

Characterizing multi-constituent substances is also complex because the analytical methods used must have the capacity to distinguish among all the substances present and must be able to generate direct evidence of their chemical structure and concentration.

According to REACH, this includes impurities down to a level of ≤1% (w/w) of the substance impacting the overall hazard classification. Because REACH defines multi-constituent substances as the product of a chemical reaction, comprehensive knowledge of the actual chemical process used to prepare the multi-constituent substance is essential when selecting analytical techniques that are meaningful for each substance, as well as when deciphering the test results. [1]

In the development of these meaningful analytical procedures, different analytical technologies designed to provide the means to acquire structural and compositional data, can be applied. This choice largely depends on the chemical nature and the complexity of the multi-constituent substance, but it should include either simultaneous separation-analysis techniques or an analysis of the reaction mass without physical separation of each of the constituents. [1]

7.0 Analytical methods
In their Guidance for Identification and Naming of Substances under REACH, the European Chemicals Agency (ECHA) offers limited details concerning spectroscopic and chromatographic methods that can be used to characterize multi-constituent substances. Among the methods they list as techniques that can be used to confirm the composition of multi-constituent substances are mass spectroscopy spectrometry (MS); gas chromatography (GC); gas chromatography–mass spectrometryspectroscopy (GC-MS); high-performance liquid chromatography (HPLC); ultra-performance liquid chromatography (UPLC), a relatively new technique offering new possibilities in liquid chromatography; and liquid chromatography–tandem mass spectrometry, or LC–MS/MS. [1]

Overall, the Guidance explains that methods offering simultaneous separation and analysis have the potential to significantly contribute to the characterization process. [1]

8.0 Gas chromatography
Gas chromatography is a technology often used in detecting volatile organic compounds (VOCs). This chromatography technique, extensively used in the analysis of pharmaceutical products, allows the analysis of impurities in pharmaceuticals as well as the identification of residual solvents listed by the International Conference of Harmonisation (ICH), making accurate quantitative determination of complex mixtures possible. This includes traces of multi-component or multi-constituent substances down to parts per trillion.

9.0 Gas chromatography–mass spectrometry
Gas chromatography is generally a reliable and effective method for separating compounds into their various components. However, it may not always be used for reliable identification of specific substances in the analysis of multi-component or multi-constituent substances.

In these cases, gas chromatography–mass spectrometry (GC-MS) may be used for conclusive proof of identity. By heating a mixture to separate the elements, GC-MS separates the chemical elements of a certain (unknown) compound to identify its molecular-level components. After vaporizing this mixture, the effluent of gas chromatography is fed into a mass spectrometer, where the chemical elements can be separated. This, in turn, leads to the identification of the components through the mass of the analyte.

GC-MS is commonly used for confirmation testing of substances in pharmaceutical drug testing, quality control and environmental assessment.

10.0 High-performance liquid chromatography
One of the primary analytical tools to assess environmental occurrence of multi-component or multi-constituent substances is high-performance liquid chromatography or HPLC. High-performance liquid chromatography is an advanced form of liquid chromatography used to separate, identify and quantify the components in complex mixtures in both chemical and biological environments. This analytical technique is commonly used to determine the molecular species present in a specific multi-component sample.

The underlying principles guiding HPLC are based on the Van Deemter equation, an empirical formula describing the relationship between linear velocity (flow rate) and plate height (height equivalent of theoretical plate [HETP] or column efficiency). According to the Van Deemter equation, a decrease in particle size not only allows significant gain in efficiency, but this efficiency does not diminish at increased flow rates or linear velocities. [5]

Although HPLC helps analytical chemists answer many questions, it lacks, as a result of the proprietary nature of column packing, long-term reproducibility.

11.0 Ultra-performance liquid chromatography
Ultra-performance liquid chromatography (UPLC; Waters) is based on the same principles as HPLC and used in similar applications, focusing on small-molecule analysis. The use of smaller columns that are tightly packed with smaller particles (up to sub 2-m) increases speed, resolution and sensitivity. As a result, UPLC significantly improves chromatographic separations. But this may also present unique challenges. UPLC systems and columns require higher levels of care and attention compared with traditional HPLC. To gain these benefits requires adding fittings and pumps designed to support high system back-pressure. [5][6][7][10]

Depending on the actual system used, system back-pressures used in UPLC may reach values of 100 MPa. In contrast, in HPLC the maximum is between 35 and 45 MPa. This means, for example, that an analysis performed using UPLC can withstand back-pressures of about 90 MPa, which is not possible when using conventional HPLC. [10]

Although UPLC is generally used for small-molecule analysis, the technique is also used for the analysis of proteins. While protein chromatography has generally suffered from problems related to carryover, peak splitting, peak broadening and poor peak shape, using smaller particles at a higher pressure and flow rate – as in the case of UPLC – has remedied many of these problems. Furthermore, when combined with mass spectrometry, UPLC becomes a powerful tool used to separate, identify, characterize and quantify (intact) proteins. [7]

12.0 Liquid chromatography–mass spectrometry
One of the first applications of liquid chromatography–mass spectrometry, also known as LC-MS, was the detection of specific pharmaceuticals and their metabolites within biological fluids in pharmacokinetic studies. While this is still a major application today, LC-MS has also become a powerful and invaluable technique adapted for the detection and trace analysis of polar compounds in aqueous samples from the environment. [8]

Used for many applications, LC-MS combines the physical separation capabilities of high-performance liquid chromatography (HPLC) with the mass analysis capabilities of mass spectrometry, offering a range of advantages due to its high sensitivity and mass selectivity.

Liquid chromatography coupled to mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) has become a technique of choice for the analysis of high-risk chemicals and (bio) pharmaceuticals found in multi-component substances. This includes endocrine disruptors, causative agents of bacterial resistance (as a result of antibiotic use) and alkylphenolic surfactants.

LC-MS has many specific benefits. Because it enables the identification and quantification of substances without derivatization, this technique is faster, more convenient and more sensitive compared with other methods. In some cases, LC-MS has been used to analyze multi-component or multi-constituent substances that could not be determined before by using different technologies and methods. Furthermore, because of superior sensitivity, selectivity, flexibility and a wide range of metabolite detection, LC-MS has been a promising platform in the analysis of low-abundant metabolites. Overall, the use of LC-MS results in lower detection limits.

The advancement of LC-MS has largely been made possible by changed interface designs. Over the last decade, they have become more sophisticated and efficient. Among the most widely used interfaces for LC-MS analysis of steroids, drugs and surfactants in the aquatic environment are electrospray ionization or ESI (for analysis of polar compounds) and atmospheric pressure chemical ionization or APCI (for the analysis of medium- and low-polarity substances). [9]

However, although LC–MS profiling has become more advanced, it may not always be sensitive enough to detect and characterize metabolites at trace levels. Hence, liquid chromatography–tandem mass spectrometry, or LC–MS/MS, has been developed.

13.0 Liquid chromatography–tandem mass spectrometry
Liquid chromatography–tandem mass spectrometry or LC-MS/MS is widely used for highly selective and sensitive bioanalysis of small molecules and offers higher sensitivity and selectivity in the trace analysis of multi-component or multi-constituent substances. In contrast to conventional photodiode array detection (PDA), analytes do not have to be fully resolved to be identified and quantitated. Furthermore, chemical derivatization is not required nor needed, as, for example, in the case of gas chromatography-mass spectrometry (GC-MS).

LC-MS/MS has evolved into a vital, strategic and powerful qualitative and quantitative analytical technique with a wide range of clinical applications, including therapeutic drug monitoring (TDM), toxicology, microbiology, the emerging field of proteomics and other applications.

One of the benefits of LC-MS/MS is that it allows analytical chemists to multiplex, helping them to identify and quantify multiple analytes simultaneously, reducing cost per test. Cost savings are further realized by increased throughput via simplified or minimal sample preparation for a variety of applications, including dilute-and-shoot or protein crash, compared with more time-consuming and expensive sample preparation methods like solid phasesolid-phase extraction (SPE) or derivatization.

14.0 A different strategy
A different analytical strategy is the analysis of the reaction mass without physical separation of each of the constituent substances. This characterization process may be adopted when physical separation using standard technologies to unequivocally confirm the composition of the multi-component or multi-constituent substance may be difficult or impossible.

This alternative strategy uses light in the infrared (IR) spectrum, which interacts with the bonds in molecules and resonates at particular frequencies. And while the “light” absorption signatures of some of the molecules of interest can be quite weak, if relevant signals can be sufficiently detected and identified, this non-separation approach may involve the use of so-called “spectral fingerprints.” [1] However, when comparing these spectral fingerprints with reference data, special care needs to be given to the fact that the data used may have been obtained using a number of different instruments, conditions or methods, limiting reproducibility. [1][11]

15.0 Matrix effect
In the analysis of multi-component or multi-constituent substances, the matrix effect, historically associated with bioanalytical methods, refers to the specific effects caused by all other components in a sample except for the target compound to be quantified. These effects may be caused by either endogenous or exogenous composites in the sample.

In the analysis of very complex matrices, even when using selected reaction monitoring or SRM detection, false negative results (as a result of matrix ionization suppression effects) and false positive results (due to insufficient selectivity) can occur. [12]

The incidence of matrix effects in LC-MS/MS methods has led to an increased understanding of the factors contributing to the occurrence of these effects and how to handle them. Improvement in the instrumentation and analytical methodology, including modified ionization, ionization switching and extraction modification, has improved the reproducibility and robustness of LC-MS/MS. [4][13]

16.0 Increased selectivity
Due to its inherent selectivity, sensitivity, flexibility and multi-component capability, the application of LC-MS/MS has rapidly expanded in recent years. Using LC-MS/MS, analytical chemists can solve challenging clinical and biomedical research problems more thoroughly and efficiently than was previously possible. Novel technology also offers analytical chemists reproducibility, selectivity and sensitivity often unattainable with immunoassays.

17.0 Sample preparation
However, with all the advanced analytical methods and techniques available, there remains one crucial aspect to be considered: sample preparation. LC-MS and LC-MS/MS analysis of multi-component or multi-constituent substances requires sensitive and robust assays. Analysis often involved involves very complex samples, requiring expert preparation protocols designed to remove unwanted components as well as selectively extract components of interest. Hence, before selecting a specific testing method, analytical chemists need to answer a number of key questions, including:

  • How high do we need to test?
  • What are the limiting factors in achieving nominal concentrations?
  • Is it necessary for test concentrations to be between 80% and 120% of nominal?
  • What toxicity and physical-chemical data are available?
  • What are the test concentrations in aquatic testing? and
  • What models are available?

Some answers may be found as recommendations and (regulatory) guidelines. For example, the guideline recommendations for the selection of test concentrations for aquatic testing limit the concentration of pesticides to 100 mg/L and industrial chemicals to 1,000 mg/L. Likewise, in the development of aquatic tests, a number of important questions related to functional solubility need to be considered, including stability vs. degradation (hydrolysis and photolysis) and absorption vs. volatility. With multi-component substances in aquatic tests it may be necessary to run Water Accommodated Fraction (WAF) trials, followed by toxicity tests. One could then develop methods to identify the key analytical components in the WAF to determine which components are likely causing the toxicity. If there are no effects, than then additional testing would not be warranted.

18.0 Conclusion
The use of advanced LC-MS and LC-MS/MS technologies for the environmental assessment of multi-component substances has allowed analytical chemists to define a large number of compounds, especially polar compounds, that previously were either difficult or even impossible to analyze. [3]

Moreover, the introduction of novel interfaces and triple quadrupole analyzers has improved the sensitivity and selectivity of detection. Consequently, the analysis of steroids, many pharmaceuticals and alkylphenolic surfactants in the environment is possible at the ng/L and ng/g level, and even at the pg/L and pg/g level. [8]

Although enhanced selectivity and sensitivity and rapid, generic gradients have made LC–MS the predominant technology for both quantitative and qualitative analyses, the most important value and application of current LC-MS techniques is the determination of known target compounds, because the capacity of these techniques for screening and identification of unknowns is relatively low. [15]

Despite the high selectivity of LC-MS–based methodologies, and in particular of LC-MS/MS, false negative findings can still occur due to the often high complexity of multi-component substances. To solve this problem, rigorous confirmation and identification criteria in terms of retention time, base peak and diagnostic ions, and relative abundances remain crucial. [8] [14]

In routine analyses it is important to consider speed, sensitivity and resolution. However, the costs associated with the analysis and the associated column maintenance also should also be considered. This, in turn, involves choosing the appropriate mobile phases, careful system washing, and application of adequate flow rates with regard to the column and system properties.

Finally, a proper approach to sample pretreatment remains an indispensable part of the analytical workflow. In recent decades, important progress has been made with regard to the preparation of samples, including compensation for matrix effects. This is important when considering the nature of matrix interference in LC-MS analysis. [6]

The development of new technologies, including fully automated LC-MS/MS assays, is expected to significantly impact the environmental assessment of multi-component substances. In turn, these technologies can help analytical scientists expand the knowledge about the presence, fate and persistence of known and newly identified multi-component substances and their degradation products found in the environment, allowing them to assess potential risks and develop, if necessary, remediation strategies and actions. [15]

February 26, 2017 | Corresponding Authors: Duane B. Huggett, Ph.D. and Hank Krueger | DOI: 10.14229/jadc.2017.29.04.004

Received: April 27, 2017 | Accepted for Publication: April 27, 2017 | Published online May 16, 2017 |

Last Editorial Review: May 16, 2017

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How to Cite


Duane B. Huggett, Ph.D., 1, Hank Krueger. 1
Challenges in Environmental Testing of Multi-component Substances – ADC Review / Journal of Antibody-drug Conjugates – May 16, 2017. DOI: 10.14229/jadc.2017.29.04.004.
1 EAG laboratories, LLC

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