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Summary of the Development and Verification of Analytical Methods for Stability Indicators in New Drug Research

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In the Non-Clinical stage of new drug development, why develop methods with stability indicators?

What is the difference between the non-Clinical stage degradation test and the later stage? What precautions need to be considered?

Have the difficult problems in method validation trouble you for a long time?

Drug Non-clinical Research and Stability Indication Method

Non-clinical research preparations (also called preclinical or non-GLP preparations) play a key role in the early development of drugs. This preparation can bring drugs (raw materials) into experimental animals for pharmacokinetic and toxicological studies. Since these studies will ultimately be used to support human dosage and safety, it is important to understand the safety data of drug concentration, PK/PD, process impurities and degradable impurities. Therefore, people in the industry have started to develop and verify methods that can accurately detect and quantify active pharmaceutical ingredients, impurities, and degradable impurities. This method can often provide stable trend results, so it is called stability indicator method. This article reviews the development and verification of stability indicator methods for non-clinical research formulations, and provides guidance for teams engaged in related research.

Drug development includes many stages as needed. Although it is not necessary to analyze degradation products in the non-clinical stage, many companies still need to learn more about active ingredients, for example, to study degradation products at this stage. The toxicological data obtained at this stage can understand the safety of drugs, impurities and degradation products, so which impurities will appear in the subsequent stages and how much each impurity is very important. The current guidelines emphasize later verification and do not involve stability indication methods in the non-clinical phase. This article reviews several methods for the development and verification of stability indicator methods and quantitative determination of degradation products. These suggestions are not regulations that must be followed technically, but provide basic ideas for those who want to introduce this method into non-clinical research. This article focuses on the development and verification of analytical methods for impurities and degradation products in non-clinical preparations of small molecule drugs.

Analysis of Important Issues in the Development and Verification of Stability Indicator Methods

All analytical methods used in the research should be in line with the purpose of use, and reliable results can be obtained without significant deviation. The analysis methods used in these non-clinical studies are mainly to provide quantitative results of trace impurities and degradation products. The development and verification of the stability indicator method should be designed for this purpose to ensure sufficient selectivity and sensitivity. There are several important parts to consider:

(1) Degradation test

In non-clinical studies, if there is sufficient time and samples, degradation test studies should be considered in the development of stability indicator methods. Usually, at this stage, the process impurities of the drug have been determined, and the forced degradation study can provide a multi-dimensional analysis of the results of the drug due to chemical or physical instability. The degradation test in method development should be selective to potential oxidation, light, and hydrolysis impurities. It should be noted that the degradation products produced in the degradation test may or may not be produced under normal storage conditions. If degradation products may cause safety or toxicity issues, necessary studies should be conducted in preclinical studies.

All degradation tests can be performed using stock solutions of the expected concentration of active ingredients. The experiment should be carried out with the original solution at the expected dose concentration of the active ingredient, and can be prepared with a definite prescription. If the prescription is not finalized, it can be carried out with a solution.

Considering the degradation conditions, the stock solution can be prepared with acetonitrile-water and methanol-water as solvents. It is recommended to prepare quality control samples. The UV spectra of all degradation products should be measured with a PDA detector. If there is no PDA detector, the UV/Vis detector should use the detection wavelength of 210-220nm, but at this time, the peak purity data cannot be obtained. The UV spectrum should be compared with the active ingredient to determine whether the relative response of impurities and degradation products is consistent with the main peak. For example, if the detection wavelength of the method is 235nm, and the degradation product does not absorb at this wavelength, then the impurity will not obtain a mass balance. Any degradation test should be carried out under short-term or non-critical conditions.

The sensitivity of the main degradation pathways can be obtained by observing the degree of degradation of the active ingredients. If there is 10% or less, it means that the possibility of appearing in the preclinical preparation is small. If the drug loss is large, it means that the possibility of potential degradation products in the preclinical preparation is high. The degradation products should be checked in the method validation. Resolution and quantitative investigation. Determining the key degradation pathway helps to determine the preclinical prescription and storage conditions. The following are several common degradation test designs:

  • Free radical oxidation can be studied using Azobisisobutyronitrile (AIBN). Take about 10mg of AIBN, add it to a 20ml volumetric flask containing 10ml stock solution, and react at 40°C for 1-3 days.

  • The nucleophilic oxidation reaction can be carried out by adding dilute hydrogen peroxide solution to the stock solution or recipe, and reacting at room temperature for 24 hours. May produce N-oxide or sulfoxide impurities.

  • Acid hydrolysis can be carried out by adding dilute hydrochloric acid solution to the stock solution or prescription. The degradation solution can be placed at room temperature or 60°C for 3 days. In the same way, alkaline hydrolysis can be obtained in the same way by dilute sodium hydroxide solution. The two can be neutralized with the corresponding acid-base dilute solution.

  • The photodegradation test can use acetonitrile/water or methanol/water stock solution or planned prescription, and carry out a 20h test of 10w/m2 in a light box specified by ICH.

  • The thermal degradation test can be carried out at a high temperature using a stock solution. Typical conditions are at 60°C for 24–48 h. Based on storage conditions, other conditions above room temperature can also be used.

  • Mass balance should be considered. There are many factors that can cause mass imbalance, such as: degradation products have no emission groups, eluted in dead volume, are too polar, or due to strong hydrophobicity, especially if dimers are formed Or adducts without being eluted under gradient conditions.

For non-clinical analysis, it is generally considered that all impurities have the same response factor. If the response factor of the degradation product at the detection wavelength is significantly different from the fire active ingredient (more than 20%), the mass balance may not be obtained. If the mass balance is not achieved due to the above-mentioned reasons, the results should be recorded and further research should be carried out in the subsequent development phase.

(2) Answers to questions about degradation test

As with any method development project, what actually happens is often different from what was planned. Here are some ways to deal with the problem.

Co-solvent: When acetonitrile is used as a co-solvent, formic acid may be formed. Formic acid and secondary amine will form adducts in solution. In this case, other solvents such as DMSO can be considered when conducting hydrolysis studies.

pH value: When using extreme pH values for degradation studies, if HPLC/UV detection is used or the peak shape is found to be poor, the pH value of the solution injected into the HPLC should be close to the pH value of the mobile phase. It is recommended that the mobile phase or buffer solution should have a stronger ionic strength to control the pH value of the solution so that the peak shape is not affected. It is strongly recommended to check the pH of the sample solution before HPLC. Generally, a pH range of 2-8 is acceptable for most chromatographic columns, but the manufacturer’s recommended range should be paid attention to.

Suspension: For suspension preparation samples, homogeneity must be guaranteed before any analysis. This usually involves vortexing or vigorous mixing of the sample to ensure representativeness. If the solution is not uniform, it will result in inaccurate determination of impurities and degradation products.

Impurity level: The concentration of API injected into the HPLC should be high enough to ensure that all relevant impurity/degradation product peaks can be peaked and quantified. The impurity/degradation product peaks are not separated from each other and can be adjusted by adjusting the mobile phase composition, pH, column temperature, column length, and gradient slope.

Chromatographic method: Gradient elution is better than isocratic. It should start with high proportion of aqueous phase and end with high organic proportion. Be careful not to make the buffer turbid in the high proportion of organic phase. When using UV detection, it is recommended to use the lowest possible wavelength to ensure that the impurity/degradation product peaks can be detected. Using the maximum absorption wavelength of API may not detect degradation product peaks that are not available at this wavelength. The PDA detector can detect peak purity and spectra to increase specificity. It is recommended to use a chromatographic column with a column length of 15 cm or more and a particle size of ≥ 5 µm to increase the HPLC resolution. For UPLC, short, fine-particle columns should be used. Column packing should ensure durability against pH and high proportion of aqueous solutions. C18 is a commonly used chromatographic column packing. The goal of chromatographic separation is to separate the impurity/degradation product peaks by using an appropriate stationary phase. The choice of chromatographic column is essential for successful method development and validation.

Things to note are as follows:

  • Non-specific adsorption with glass or plastic

  • The peak width/peak shape difference is due to the intensity of the injected solution being greater than the mobile phase

  • Unacceptable leading/tailing peaks are due to pH incompatibility

  • Poor resolution between degradation products in degradation test

  • Degradation products or active substances are not sufficiently retained

  • All samples should be thoroughly mixed before analysis to ensure homogeneity

Recommended method for method validation

The purpose of method validation is to prove the reliability and accuracy of non-clinical preparation test results through qualitative and quantitative data. The sensitivity and specificity of the stability indicator method are very important for the determination of trace impurities and degradation products. Verification parameters and acceptable ranges should be determined based on the purpose of use, requiring a certain degree of accuracy and precision. Each parameter and acceptable range are set in the method verification scheme. The method should be able to distinguish between active ingredients, known degradation products and other components in the formulation or API-related ingredients (such as residual starting materials, intermediates, by-products, auxiliary materials, etc.) and ensure that the results are accurate and reliable.

After the method is validated, any changes to the method will require partial validation. Partial verification usually includes an evaluation of any parameters in the method. In addition, if the validated method is transferred to another laboratory, a method transfer plan must be drafted.

Method validation parameters

(1) Exclusiveness/selectivity

Specificity/selectivity refers to the ability of a method to distinguish API from other ingredients. Other ingredients include excipients, dissolution from the container, residual solvents, impurities or degradation products related to the test substance. The specificity/selectivity should be evaluated by the interference of those potentially interfering ingredients and active ingredients (such as excipients, blank solvents) and degradation tests. In chromatography, other peaks should be separated from the main peak to ensure the accuracy and repeatability of the integrated chromatographic peaks. In addition, for degraded samples, PDA or MS should be used to evaluate the peak purity of the active ingredients and compare with the newly prepared solution to ensure that no chromatographic peaks are co-eluted with the main peak. Known impurities or degradation products should be added to the newly prepared solution containing active ingredients to ensure complete separation from the measured peak.

(2) Accuracy

Accuracy indicates how close the measurement result is to the theoretical result. If possible, degradable impurities can be added to the sample at different levels of active ingredients. If not available, degraded samples can be used to determine the accuracy of the method. The accuracy is expressed by the percentage of recovery rate, relative error or deviation from the theoretical value of the test object. The accuracy of the method is related to the purpose of the method.

(3) Precision

Precision represents the closeness of each measurement result of the component to be tested under the same instrument conditions. The precision of low-level impurities should be expressed in absolute difference rather than %RSD or %CV, because both will be negatively affected by low-level impurities. The precision should be evaluated using at least three (5-6 recommended) samples.

(4) Linearity and range

The linear range of the method should be included in the entire analytical concentration range. The detection capability of the stability indicator method is critical. The analyte concentration is linearly regression with the detector response value, and the linear relationship is good when the correlation coefficient is ≥0.99. Because the impurity monomers separated separately in the early development stage may not be obtained, the relative response factor (RRF) of each impurity to the main peak is considered to be 1 in the non-clinical stage. In the later stages of development, these values should be determined experimentally.

(5) Detection limit and quantification limit

The limit of detection (LOD) is the lowest analytical concentration of the analyte and the background response ratio of the blank sample, usually 3-5 times the response value of the blank sample. The limit of quantification (LOQ) is the lowest detection concentration with a certain degree of accuracy and precision. Both can be measured by different methods. No matter which method is used, the method used should be clearly and accurately recorded in the record. Generally, the method used is based on the availability of the analyte. LOD sometimes does not need to be determined, unless the impurity or degradation product is a component of interest or its concentration is lower than the LOQ, in which case obtaining LOD data can help assess the concentration of the analyte.

(6) Durability

Robustness is evaluated by small changes in method parameters. Including flow rate, mobile phase composition, pH, column temperature, different use time chromatographic columns, different batch numbers of chromatographic columns, different personnel, etc. These changes can be evaluated by the following parameters: tailing factor, resolution, number of theoretical plates, accuracy, precision, LOD or LOQ.

(7) Stability

The stability of the sample during storage, preparation and analysis should be evaluated. Including API stability, formulation stability, process formulation stability, freeze-thaw stability, and active stock solution stability. Each solution should be stored under appropriate storage conditions (such as room temperature, refrigerator, freezing, etc.) for a certain period of time to evaluate the stability. The quantitative analysis method of impurities in degradation products examines the amount of each impurity. Therefore, these methods provide powerful predictive capabilities. Several experimental designs involved in these methods are described below.

Material stability

Generally, the reference substance of the drug substance should be stored under long-term conditions lower than that of the drug substance. For example, if the API is required to be stored in the refrigerator, then the reference substance will be stored in the freezer. The stability of the drug substance is analyzed by comparison with the reference substance, and the increased degradation products are quantitatively analyzed. It usually needs to be measured at the planned time interval.

Formulation stability

The stability of the formulation is to ensure the stability during use. It is determined by comparison with a freshly prepared control solution. Knowing the impurity profile and degradation degree of the preparation will help the toxicological identification of each impurity.

Process stability

The sample may be re-injected due to instrument failure or power failure. At this time, the stability during use should be investigated, for example, under storage conditions (such as in an autosampler). It usually needs to be investigated for 2-3 days under the storage conditions of the samples.

Freeze-thaw test

According to expected storage conditions and sample analysis methods, it is necessary to evaluate freeze-thaw stability. If an evaluation is performed, the number of freeze-thaw cycles and conditions of the sample should be included. The frozen and thawed samples should be compared with a freshly prepared control solution of the same concentration of active ingredient.

Stock solution

The stability of the active ingredient stock solution should be evaluated within the expected storage conditions and storage time (such as 6h). During the evaluation, the response value of the stock solution should be compared with the newly prepared solution.


Understanding the toxicity of new drugs and their impurities is an important part of preclinical research . Analysis methods for impurities in non-clinical studies should be appropriately developed and verified, including accuracy, precision, linearity, range, selectivity, sensitivity, and durability, and should comply with the relevant FDA guidelines. These methods help to evaluate the stability and purity of preparations before and during non-clinical studies. Following the methods recommended in this article will ensure that the analytical methods used are durable and reliable.

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