How Do Stable-Isotope-Labelled Reference Compounds Enable Human Biomonitoring?

Short answer: Stable-isotope-labelled reference compounds enable specific substances and their metabolites to be reliably identified and quantified by LC–MS/MS. They behave very similarly to the compounds being measured but can be distinguished by their defined mass difference. This makes them particularly valuable as internal standards in complex biological samples such as urine.

Efficacy and toxicity are the two antagonistic terms in the evaluation of any active chemical compound.

This was already known at a time when synthetic chemistry had not yet been “invented”: Each active “agent” can both be a remedy or a poison – this just depends on its dosage. Or – with the famous words of Paracelsius: “Sola dosis facit venenum”.

Today, with the availability of not just natural extracts and minerals but also with tens of thousands artificial compounds, this basic finding has become even more important. With the best of intentions, chemists have been synthesising new molecules for more than 200 years aiming to cure diseases, to support plant growth, to fight parasites, to improve the quality of life.

Right from the beginning, it was known that finding an active compound structure is a challenging and tedious business. Trial and error approaches have been supplemented by high throughput strategies and by more or less sophisticated and scientifically based structure activity relations.

Today, there do exist proven avenues to develop new active compounds for a given target. In the light of the higher profit margins, these approaches are more sophisticated for pharmaceutical applications than for crop sciences or cosmetics – but finally both industry and academics manage to discover dozens of new active structures per year.

So, is everything OK so far? When reverting to the beginning of this article – we know: The emergence of new active compounds is only one side of the coin. Every substance can be toxic at a sufficiently high dose – even water. This can not be denied, but the actual risk depends on dosage and application.

Consequently, we have to address this other side of the coin: How to determine the safety profile? How to avoid damaged to humans, animals, plants, the environment?

First, there is the safest but nihilistic idea to not use (artificial) agents (drugs, fungicides, cosmetics, dyes, materials) at all. The propagators can be sure to always be on the “safe side” of the discussion. However – this also means to abstain from any the new, helpful, promising properties of the newly developed compounds. For a humanity of more than 8 billion people, this is certainly not an option.

So, what needs to be done to benefit from new agents without putting people and nature at risk?

What role plays a custom synthesis company like ChiroBlock in that process?

How can a company that doesn’t develop APIs, cosmetic ingredients or other functional molecules contribute to the safety of such compounds?

There are several answers to these questions:

• Identifying the safest synthetic route to make these substances
• Developing strategies to avoid toxic by-products
• Utilisation of nature by using benign natural matter as starting materials
• Making of less toxic derivatives that are still active enough
• Developing of analytical methods for monitoring the “fate” of the compound after its application
• Synthesizing analytical standards for these methods
• Synthesising reference compounds of any kind

…

Today, I would like to focus on the last point on “reference compounds”. This term regularly causes confusion – especially with respect to “analytical standard” and regarding the relation of the reference compound to the original active molecule.

Reference compounds also comprise “analytical standards” but their meaning is much broader.

Definition: A reference compound is a chemically defined substance used to identify, compare or quantify another substance. It may represent the target compound itself, a metabolite, an impurity, a degradation product or an isotope-labelled analogue. An analytical standard is a reference compound that has been characterised and qualified for a defined analytical purpose.

What types of “reference compounds” do exist?

• There are compounds of completely different molecular structure showing comparable activity. Such compounds are regarded as “reference” when looking for new compounds that address the same target. As an example – these can be custom made samples derived from a competitor’s patent.
• There are “analytical standards”. These are compounds having the same (or a related) structure of the target. They furthermore are qualified by a very detailed and tough specification. Their identity and purity are certified by experienced analytical labs adhering to strict regulations and using validated analytical methods.
• There are reference compounds of “impurities”. These are custom made substances of impurities that have been found in the production process.
• There are reference compounds of “degradation products” – synthesised to monitor compounds that occur during long time storage.
• There are reference compounds showing the molecular structure of metabolites in humans or deriving from degradation processes in nature.
• There are reference compound that are labelled by “cold” or “hot” isotopes at defined moieties of the molecule.
• And, finally, there also exist reference compounds that bear more than just one of the features mentioned above.

Why stable-isotope labelling matters in LC–MS/MS: A stable-isotope-labelled reference compound has nearly the same chemical behaviour as the corresponding non-labelled analyte but a different molecular mass. Used as an internal standard, it can help compensate for losses during sample preparation, matrix effects and variations in mass-spectrometric detection. This improves the reliability of quantitative biomonitoring data.

What do these reference compounds have in common?

They need to be synthesised, purified, analysed. And exactly this is the task of CRO labs like ChiroBlock. A very limited number of reference compounds can be found in catalogues of dedicated “analytical standards” or “reference compounds” vendors. However, most of such substances – especially when looking for answers to new questions, safety threads and environmentally concerns have to be exclusively synthesised.

These tasks require thorough expertise in the field of synthetic chemistry. They normally can not be addressed at catalogue vendors or at the manufacturer of a new API, cosmetic agent or functional material.

Why are specialised synthetic chemistry companies required to make such new reference compounds?

• Many of such compounds are hardly accessible and need sophisticated synthetic approaches.
• The known synthetic route to the active agent can not be used in most cases.
• The requirements differ completely from those of a final product: small amount, one-time synthesis, high purity, very detailed specification with sophisticated analytical methods, low yields and a lengthy synthetic route are acceptable, other regulatory and IP-requirements apply, the costs per gram are much higher than with products or catalogue standards.
• An unbiased assessment of all synthesis options is helpful.
• Timelines and flexibility are extremely important.
• Consulting often also is a major part of projects where special reference compounds are needed.

An example often helps to clarify a complex topic. It also will answer the initial question of the value of such specific reference compounds for safety issues.

Let’s have a closer look at a recently published biomonitoring study of the compound “Climbazole”. Originally developed as a fungicide, it also is being used as anti-dandruff agent in rinse-off cosmetics such as shampoos.

Climbazole:

There have been observations that this agent may show endocrine-disruptive potential.¹ As a consequence, this quite effective compound was banned from many cosmetic products. Is that justified? What is the acceptable balance between benefits for people suffering from dandruffs (e.g. roughly 50% of all teenagers) and real toxicity in humans? So how safe is Climbazole formulated in rinse-off cosmetics in reality?

Answers only can be given in the course of long-term biomonitoring studies. While there are strict regulatory safety investigations required already during the development of such new active agents, the real effects only can be checked much later and after years of application.

So, the authors of a recently published paper analysed the urine of 300 students between 2002 and 2022. They actively were looking for two Climbazole metabolites, namely (OH)2-climbazole and cx–OH–climbazole:

“Exposure of young German adults to the anti-dandruff agent climbazole from 2002 to 2022: Analysis of specific biomarkers in urinary samples”²

The idea behind: If there was any up-take of this compound by the students’ bodies, these metabolites would have to be detectable and its concentration is expected to correlate with the amount of Climbazole that had entered the body.

So, the study concept was clear, the urine samples available and even a sophisticated analytical method was developed.

What was missing to conduct the biomonitoring?

The required reference compounds!

They did not belong to the commercially available group of “analytical standards”. They had not been made in the process of the product development. Their chemical synthesis was not known.

Exactly at this point, specialised CROs come into play. ChiroBlock designed, tested and executed the synthesis of both metabolites, labelled by several stable isotopes to facilitate the mass spectroscopy analytics.

It was necessary to:

• identify several possible synthesis approaches
• prioritise these approaches regarding technical risks, feasibility
• design a labelling strategy, selecting both the best isotopes and the suitable positions within the molecule
• perform test reactions
• optimise reaction conditions
• develop and apply specific analytical methods monitoring the synthesis as well as the products’ identity and purity

So, these reference compounds became available and the study could be initiated.

It became clear, that the metabolites concentration in the urine samples was very low (if detectable at all). The figures are far below all safety thresholds set by regulatory authorities. The study provided strong evidence that Climbazole does not pose the anticipated risk in rinse-off cosmetics.

The Climbazole case in brief: The biomonitoring study analysed urine samples from 300 students collected between 2002 and 2022 for the metabolites (OH)2-climbazole and cx–OH–climbazole. ChiroBlock designed, tested and executed the synthesis of the stable-isotope-labelled reference compounds required for the LC–MS/MS analysis. The measured metabolite concentrations were very low, if detectable at all, and the reported values remained far below the relevant safety thresholds.

What is the overall benefit of this biomonitoring study?

• The ban of Climbazole in anti-dandruff formulations might not be justified.
• The benefits of Climbazole seem to be more distinct than its safety issues (at humans).

As a result, there is less pressure to develop new (potentially more toxic?) agents. This not only saves costs but also other resources such as testing facilities and, hence, serves the environment. By the way, it always makes sense to look for other, additional applications of known agents than to develop new ones from scratch…and Climbazole is a good example.

There are no such findings without carefully executed biomonitoring studies.

And there are no sound biomonitoring studies without the support of chemical CROs as competent service providers.

#ChiroBlock #CRO #ConvertingIdeasIntoMolecules #ReferenceCompounds #Biomonitoring

Footnotes

1. Matthiessen, P., & Weltje, L. (2015). A review of the effects of azole compounds in fish and their possible involvement in masculinization of wild fish populations. Critical Reviews in Toxicology, 45(5), 453–467. https://doi.org/10.3109/10408444.2015.1018409 ↩
2. https://doi.org/10.1016/j.chemosphere.2024.143611 ↩

Last edited by Dr. Oliver Seidelmann (author)