Solutions to Analyte Stability Issues in Preclinical Bioanalysis

In preclinical pharmacokinetic studies, it is crucial to determine the stability condition of the analyte in the matrix when its physicochemical properties and biological activity are not fully characterized. This is related to the ability to accurately measure its concentrations and correctly calculate the related pharmacokinetic (PK) parameters. This article offers suggestions and solutions for addressing analyte instability in preclinical bioanalysis, drawing on the experiences of the bioanalysis team in the WuXi AppTec DMPK.

Causes of instability and proposed solutions

The instability of the analyte can occur at any stage of the sample collection, storage, extraction, and analysis. This can result in either a lower or higher concentration than the real one for the analyte or its metabolites. During the method development, the stability of the analyte should be fully evaluated. This article will primarily explore two aspects: enzymatic types and activities of biological factors and the active moieties in chemical factors.

In the screening preclinical study, as it’s in the early stage of R&D, the biological properties of the analyte are not fully understood. Instability can arise from both biological and chemical factors. Biological factors mainly include the type and activity of enzymes in the biological matrix, anticoagulants, and protein concentration. Chemical factors encompass active groups of the analyte, oxidation reactions, interconversion between analyte isomers, and transformation between the analyte and its metabolites. Other factors such as light exposure, temperature, pH, and time will also affect the stability of the analyte.

Enzymes

Analytes undergo metabolism in the body through changes in chemical structure (hydrolysis, oxidation, dehydrogenation, hydrogenation, conjugation, etc.), most of which require the participation of enzymes. Some enzymes retain their activity in the ex vivo matrix, which is a major cause of the degradation of analytes or prodrug analytes ex vivo. For example, esterases, are important enzymes involved in the hydrolysis of some ester bonds in analytes. Under the action of esterases, ester bonds will be hydrolyzed and broken, such as acetylcholine will hydrolyze into acetic acid and choline under the action of esterase (Figure 1). Other types of enzymes in the ex vivo matrix, such as deaminases and proteases, may also cause ex vivo degradation of some analytes. The expression and activity of enzymes vary greatly in the biological matrices of different species. For example, the esterase activity in rodent matrices is much greater than that in non-rodents ^[1], so instability is more likely to occur in rodent samples and requires greater attention during method development.

Figure 1. Esterase-mediated hydrolysis of ester bonds

Given the biological activity of enzymes, it is essential to implement solutions to address enzyme-related instability issues.

(1) Use of specific enzyme inhibitors

Specific enzyme inhibitors can target active centers or essential groups of enzymes, thereby reducing their activity or even completely inactivating them, which effectively intervenes in the occurrence of enzymatic reactions.

For carboxyl esterases or phosphoesterases, the inhibitors include phenylmethylsulfonyl fluoride (PMSF), dichlorvos (DDVP), and bis(p-nitrophenyl) phosphate (BNPP). In addition, PMSF and DDVP are also inhibitors of cholinesterase. Using diisopropyl fluorophosphate (DFP), PMSF, and the anticoagulant heparin together can effectively inhibit the activity of serine protease. Tetrahydrouridine can inhibit cytidine deaminase activity.

EDTA and potassium oxalate can not only combine with Ca²⁺ ions in the blood to form chelates, interrupting the coagulation process, but can also chelate with Mg²⁺, Mn²⁺, and Fe²⁺, inhibiting the activity of some nucleases and proteases that require metal ion activation. Additionally, sodium fluoride, except acting as an anticoagulant, is also a non-specific inhibitor of esterases ^[1].

Table 1. Common enzymes and corresponding enzyme inhibitors in isolated matrices

The mechanisms behind enzyme-catalyzed degradation are complex, and enzyme sensitivity can vary across different species’ matrices. Therefore, it is necessary to select appropriate enzyme inhibitors. Table 1 summarizes the common types of enzymes in the ex vivo matrix and the corresponding types of inhibitors. If the analyte is affected by multiple enzymes at the same time and the introduction of a single enzyme inhibitor cannot achieve the desired stability effect, it is recommended to use multiple enzyme inhibitors together.

(2) Dried blood spot (DBS) method

This method involves collecting a drop of whole blood on a cellulose card, drying it at room temperature to form a blood spot, and then performing the test. During the DBS preparation process, the three-dimensional structure of the enzyme protein is destroyed, thus inactivating it. The DBS method not only solves the instability issue of the analyte in whole blood but also significantly reduces the volume of blood samples required for analysis and simplifies the sample collection and storage process. However, this method is only applicable to whole blood, so it has limitations when testing with other matrices.

(3) Control pH

pH significantly influences enzyme activity by altering the dissociation state of groups associated with the enzyme’s active site. At optimal pH levels, the active groups are best suited for substrate binding. Deviations from this optimal pH can hinder enzyme-substrate interactions, leading to reduced enzyme activity. Furthermore, pH affects enzyme stability; extreme pH levels can alter the conformation of the enzyme’s active center and potentially denature the entire enzyme molecule, rendering it inactive.

Active groups

The chemical structure of the analyte may contain some unstable active groups (Figure 2). Due to the influence of their specific environment, these active groups will undergo structural changes in the ex vivo matrix, thereby affecting the accuracy of the measurements.

Figure 2. Unstable reactive groups in the analyte structure

(1) Analytes containing thiol groups, or similar to thiol groups, are prone to forming disulfide bonds with their thiol groups or those in amino acids, peptides, and proteins, causing deviations in the detection results. For example, the thiol group of cysteine can bind with itself in the ex vivo matrix to form cystine .

Tris-2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) can solve the instability problem caused by the thiol group. They can inhibit the formation of disulfide bonds and break disulfide bonds, reducing them into the corresponding original analytes (Figure 3).

Figure 3. Tris (2-carboxyethyl) phosphine (TCEP) is involved in the thiol reduction reaction

During sample collection, unstable analytes can undergo derivatization reactions. Unstable analytes react with derivatization reagents to form stable derivatization products. By quantitatively analyzing the derivatization products, the original analytes can be indirectly determined (Figure 4) ^[2]. Analytes containing thiol groups are generally unstable, so they can be converted into stable cysteine analogs through derivatization.

Figure 4. Generation of stable derivatives by derivatization

(2) In solutions and biological samples, analytes containing carbonyl groups, nitro aromatic structures, N-oxides, C=C double bonds, and aryl chlorides are light-sensitive and prone to oxidation. During the analysis process, operations are usually carried out in the dark, and antioxidants such as vitamin C, sodium metabisulfite, or hydrochloric acid are added to the biological samples, extraction solutions, or reconstitution solutions. Alternatively, they can be used in combination with other reagents such as sodium citrate, glutathione, heparin, or EDTA to achieve stability (Figure 5).

Figure 5. Handling of unstable and oxidizable analytes

(3) Analytes containing Z/E isomers, chiral groups, and groups that readily form lactones can undergo Z/E isomerization, chiral conversion, and lactonization during storage, thawing, extraction, drying of sample extracts, reconstitution, and MS detection, especially in neutral or suitable pH environments. This leads to deviations from the actual concentration values in detection ^[3]. This instability is related to pH or specific substances contained in the matrix, especially in excretion and tissue homogenate samples which are more prone to instability.

Adding formic acid, acetic acid, hydrochloric acid, phosphoric acid, citric acid, and various buffer solutions can help shift the pH away from instability ranges, preventing further structural changes and stabilizing the analyte. However, caution is necessary during pH adjustments to avoid extreme acidity or alkalinity, which could introduce additional instability issues.

(4) Analytes containing N-oxides, S-oxides, glucuronides, or sulfates can undergo cleavage or conversion in the ESI or APCI source during LC-MS/MS analysis. Molecules with weak chemical bonds can also easily fragment during the ionization process before entering the Q1 chamber of the tandem mass spectrometer.

Changing the ion source type or adjusting MS parameters can effectively reduce the in-source degradation of the original analyte and its metabolites. N-oxide metabolites are heat unstable and easily convert to the original analyte at higher ion source temperatures. Using an ESI source can effectively reduce the conversion of N-oxides compared to an APCI source ^[4]. Figure 6 summarizes the abovementioned strategies for resolving analyte instability during bioanalysis.

Figure 6. Measures to prevent instability issue

Concluding remarks

Evaluating and controlling stability conditions is essential in bioanalytical development, and understanding the underlying causes of instability is critical for selecting effective solutions. During the early stages of method development and validation, assessing the short-term stability of unstable analytes using real samples can reveal hidden instability issues. The exact causes of instability issues vary, and the corresponding solutions differ among different analytes, species, and matrices. It is necessary to investigate these on a case-by-case basis according to the actual situation. Currently, analytes such as PROTACs, PDCs, peptides, chiral compounds, and nucleic acids encountered in bioanalysis may have potential instability in the matrix, so it is particularly important to pay attention to and investigate their stability during the method development process.

Author: Dan Chen, Zhengzhen Gao, Jinlian Lu, Lingling Zhang

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