Bioanalytical Method Development: Isomers
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Isomers are molecules that share the same molecular formula but have different structures. This means they contain the same number of atoms of each element, but these atoms are arranged differently. There are two main forms of isomerism: structural (or constitutional) isomerism, where the bonds between atoms differ, and stereoisomerism (spatial isomerism), where the bonds are the same, but the relative positions of the atoms differ.
Enantiomers are a type of stereoisomer. These molecules are mirror images of each other but cannot be superimposed, no matter how they are rotated. If molecules are not superimposable and are not mirror images, they are referred to as diastereomers. Chirality describes a molecule that is non-superposable on its mirror image, leading to the formation of enantiomers. Epimers, a specific type of diastereomer, are typically found in carbohydrates and differ at only one location of the -OH group.
The phenomenon of isomerism was first recognized by the German chemist Friedrich Wöhler in 1827. Despite sharing the same molecular formula, isomers can have vastly different physical properties, such as melting points, boiling points, densities, and solubilities. These differences are particularly pronounced when the functional groups associated with each molecule vary. Isomerization refers to the process where one molecule transforms into another with the same atoms but a different arrangement. This transformation can occur spontaneously or may require a specific reaction to take place.
Isomerization is critical in clinical pharmacology and pharmacotherapeutics because isomers can have different pharmacokinetic (how drugs move through the body) and pharmacodynamic (how drugs affect the body) properties. Research in stereoisomerism has opened new avenues and challenges for clinical pharmacology. Currently, a significant number of clinical trials are underway to compare the efficacy and safety of single enantiomers and racemic mixtures. Many single-enantiomer drugs are expected to enter the market soon due to their improved safety and efficacy profiles.
The knowledge of isomerism has enabled the development of safer, more effective new drugs and alternatives to existing therapies. For example, D-methadone, which makes up the currently approved racemic methadone containing both D and L enantiomers, has distinct pharmacological properties from L-methadone and does not produce significant opioid effects at therapeutic doses due to its NMDAR antagonism. Recent evaluations of D-methadone in two phase I clinical studies with healthy, opioid-naive subjects have demonstrated its safety and supported its ongoing development as a potential treatment for depression and other central nervous system disorders.
The Importance of Isomers in Pharmacology:
The ability of isomers to have different physical and chemical properties despite having the same molecular formula is what makes them attractive for industrial applications. Isomers in pharmacology are used in the production of various products such as fuels, plastics, and pharmaceuticals. In pharmaceutical industries, isomers are particularly important for controlling drug properties and enhancing their effectiveness. Isomers in pharmacology with different biological activities can be developed to target specific therapeutic effects, and the pharmacokinetic profiles of different isomers can significantly influence their safety and efficacy.
Approximately 50% of currently produced drugs are optically active, and isomers of optical compounds typically exhibit different pharmacokinetic properties. About 50% of chiral drugs are produced as racemic mixtures, as this is more cost-effective than producing individual isomers. However, the two enantiomers within a racemic mixture can interact differently with biological targets. For example, enantiomers may engage distinct receptors or molecular targets, with one or both interactions being either desirable or undesirable, depending on the therapeutic goal. Furthermore, enantiomers often exhibit different metabolic pathways, leading to variations in their pharmacokinetics and overall efficacy or safety profiles.
One of the key challenges in isomer bioanalytical method development is the separation and quantitation of isomers in complex biological matrices. In clinical science and drug analysis, non-stereoselective assays that analyze racemic mixtures without distinguishing between enantiomers can lead to misleading pharmacokinetic and therapeutic data. Therefore, early stereoselective pharmacokinetic analysis is crucial for clinical drug development. Differences in target engagement, metabolism, and pharmacokinetics between enantiomers highlight the importance of stereochemistry in drug study design and therapeutic outcomes.
Another critical role of isomers in pharmacology is their involvement in enzymatic reactions. The three-dimensional structure, functional groups, and overall orientation of a molecule can significantly affect its ability to bind to enzymes. Enzymes are highly selective, typically recognizing only specific molecular shapes, much like a lock and key. Therefore, even though isomers share the same molecular formula, their differing physical structures can prevent them from binding to the same enzymatic sites.
How Isomers Affect Bioanalytical Method Development
Isomer analysis plays a crucial role in clinical science, but the separation, qualitative, and quantitative analysis of isomers—particularly in complex biological samples—remains a significant challenge in bioanalytical method development. These methods are employed to determine the concentration of drugs and their metabolites in biological matrices (such as plasma, urine, saliva, and serum), which is critical for evaluating bioavailability, bioequivalence, and pharmacokinetic data.
One of the key challenges in bioanalytical method development is the separation and quantitation of isomers in these complex matrices. When attempting to link plasma concentrations to pharmacological or therapeutic effects, non-stereoselective assays that fail to distinguish between enantiomers in racemic mixtures can lead to misleading metabolic and pharmacokinetic data. Therefore, early stereoselective pharmacokinetic evaluation of enantiomers can significantly improve our understanding of the safety and efficacy of new drug candidates.
Chiral separations are particularly important for drug safety. Enantiomers often interact with different receptors or molecular targets, with one enantiomer engaging in desirable therapeutic effects while the other may cause less desirable or even adverse outcomes. The pharmacokinetic and metabolic pathways for each enantiomer can also differ significantly, leading to variations in efficacy and toxicity. An early understanding of these differences through stereoselective assays allows for better therapeutic design and optimization.
A classic example of this is the distinction between omeprazole and esomeprazole, where the latter, a single-enantiomer version, was developed to provide more consistent therapeutic outcomes with fewer side effects. Similarly, citalopram and escitalopram highlight how a more active enantiomer can offer improved efficacy while reducing adverse effects. Such examples emphasize the importance of resolving isomeric mixtures during drug development.
Mass spectrometry (MS) is a powerful analytical technique used to qualitatively and quantitatively identify a wide range of clinically relevant analytes. However, mass spec alone cannot distinguish between structural or conformational isomers with the same mass. When coupled with gas or liquid chromatography, mass spec enables the separation of isomers, allowing for accurate quantification and identification. This combination has significantly expanded its use in various clinical applications. Supercritical fluid chromatography (SFC) and chiral high-performance liquid chromatography (HPLC) are two emerging technologies offering enhanced resolution and faster separation of chiral compounds in bioanalytical studies. These advancements provide more precise data for complex biological matrices.
The regulatory environment has also evolved to reflect the importance of stereoselective analysis. Regulatory bodies like the FDA and EMA emphasize the need for distinguishing between enantiomers in new drug submissions. Accurate pharmacokinetic and pharmacodynamic data on each enantiomer are required to meet these guidelines and ensure that any safety or efficacy concerns associated with one enantiomer are addressed. This highlights the necessity for bioanalytical methods capable of resolving and quantifying individual enantiomers during drug development.
Additionally, interferences from isobaric or isomeric metabolites—compounds that share the same mass but differ structurally—can complicate LC/MS/MS analysis. These metabolites may display the same mass-to-charge (MRM) transitions as the parent drug, making it difficult to quantify them accurately without proper separation techniques. For certain drugs, the presence of such metabolites can obscure accurate pharmacokinetic data, leading to incorrect therapeutic conclusions. This is why further development of isomer chromatographic separation methods remains essential in bioanalytical method development.
Case Study from BioPharma Services: Midostaurin and Its Metabolites
At BioPharma Services, we have developed and validated a sensitive, rapid, and economical LC-MS/MS method for the simultaneous determination of Midostaurin and its metabolites CGP62221 and CGP52421 in human plasma. Sample pretreatment was performed using supported liquid extraction (SLE), and the separation was achieved on a Phenomenex Luna PFP column. Find all our generic drug compounds we have worked on here.
This method improved the sensitivity and linearity of Midostaurin and its metabolites within their respective concentration ranges. Notably, CGP52421 exists as a mixture of two epimers, and we successfully separated and quantified these epimers by optimizing various chromatographic conditions, including the mobile phase composition, temperature control, and gradient optimization.
Our method demonstrated excellent sensitivity, specificity, recovery, precision, accuracy, and stability. This approach has been used in bioequivalence studies, with promising results.
Why Choose BioPharma Services for your Next Drug Development Project?
The accurate separation and quantification of isomers are crucial in bioanalytical method development. Insufficient separation during liquid chromatography can result in poor peak shape and inaccurate data. At BioPharma Services, our experienced R&D team develops robust methods with high chromatographic resolution, repeatable results, and rapid run times to meet the specific needs of our sponsors.All methods are validated in accordance with FDA, EMA, and ICH M10 guidelines to ensure the accuracy, reliability, and integrity of all data.
At BioPharma Services Inc., our team have successfully developed and validated approximately 300 bioanalytical assays in biological matrices, including whole blood, plasma, serum and the urine bioanalysis. These bioanalytical method validations include prodrugs, therapeutic peptides and polar compounds. Based on our extensive experience, we always strive to develop the right method for the right sample matrix. Meeting and exceeding the satisfaction of our customers is our top priority.
Written By:
Hongzhi Liu
Principal Research Scientist
BioPharma Services, Inc., a HEALWELL AI and clinical trial services company, is a full-service Contract Clinical Research Organization (CRO) based in Toronto, Canada, specializing in Phase 1 clinical trials 1/2a, Human Abuse Liability(HAL) and Bioequivalence clinical trials for international pharmaceutical companies worldwide. BioPharma Services conducts clinical research operations from its Canadian facility, with access to healthy volunteers and special populations.