The entire result of a bioequivalence study rests on a single measurement: the concentration of the active ingredient in the blood. No matter how well the study is designed, if this measurement is not reliable, the result obtained is not reliable either. In the previous articles of this series we addressed study design, variability, and the regulatory framework. In this final article we examine bioanalytical method validation, which stands at the silent foundation of the entire process, and the LC-MS/MS technique with which these measurements are predominantly made today.
What is a bioanalytical method?
A bioanalytical method is the analytical method that measures the concentration of an active ingredient in a biological sample. In a bioequivalence study, the blood samples taken from participants at defined time intervals are measured one by one with this method. Each measurement forms one point on the concentration-time curve described in the earlier articles. The method is therefore the source of all the data the study produces.
A method this decisive must be proven to be reliable. This process of proof is called method validation and is defined by a common guideline on an international scale.¹ This guideline sets out under which conditions a method is to be considered reliable and which of its characteristics must be tested.
Why LC-MS/MS?
The concentrations of an active ingredient in the blood are often extremely low, and the sample is a complex environment containing thousands of other components. To measure accurately in such an environment, both high sensitivity and high selectivity are needed.
LC-MS/MS brings these two requirements together. Liquid chromatography separates the components of the mixture from one another; tandem mass spectrometry then recognizes and measures the target molecule according to its mass. This combination makes it possible to measure even very low concentrations reliably, distinguishing them from the other substances in the sample. For this reason the method preferred in modern bioequivalence studies is largely this one.
The core validation parameters
Validating a method means testing it from several angles.² Each of these tests secures a particular dimension of the measurement’s reliability.
Selectivity shows that the method can distinguish the target substance from the other components in the sample. The calibration curve defines the relationship between the measured signal and the actual concentration and allows the concentration of unknown samples to be calculated. Accuracy describes how close the measured value is to the true value; precision describes how close repeated measurements are to one another.
The lower limit of quantification defines the lowest concentration the method can measure reliably; in studies where low concentrations matter, this limit is decisive. Recovery shows the extent to which the substance can be retrieved from the sample. Carry-over checks whether one sample affects the next. Stability confirms that the substance remains intact in the sample under storage and processing conditions.
Matrix effect
Among these parameters there is one of special importance for methods based on mass spectrometry: the matrix effect. Other components in the biological sample may enhance or suppress the measurement signal of the target substance. If this effect is not taken into account, the measurement may give systematically incorrect results. For this reason, the evaluation of the matrix effect is a separate and critical step in the validation of LC-MS/MS methods.
Analysis of study samples
Validation is not only a test carried out at the setup stage of the method. The method must also be shown to maintain its reliability while the actual study samples are being analyzed. For this, some samples are measured again at a different time and checked for consistency with the first result. This check confirms that the measurement is reliable not only under ideal conditions but also within the real flow of the study.
A single international framework
In the past, different regions had separate guidelines for bioanalytical method validation.²³ These guidelines were used in parallel for a long time. The common international guideline prepared later brought these approaches together within a single framework and has today become the common standard adopted by the major authorities.¹ Türkiye, too, takes internationally accepted principles as its basis in this field; in this way, the data produced in a Turkish center speaks the same language on an international scale.
NanoTox’s role
Throughout this series, one sentence recurred: even the best-designed study loses its meaning if the measurement is not reliable. Bioanalytical method validation is precisely the step that provides this reliability. As a center authorized to conduct these studies, NanoTox uses its LC-MS/MS infrastructure with methods validated in accordance with international guidelines. Accurate, selective, and reproducible measurement even at low concentrations is, for us, not a goal but a precondition of the study. The scientific robustness behind a bioequivalence result is born from exactly this foundation.
References
¹ ICH M10. Bioanalytical Method Validation and Study Sample Analysis (Step 4, 24.05.2022). https://database.ich.org/sites/default/files/M10_Guideline_Step4_2022_0524.pdf
² U.S. Food and Drug Administration. Bioanalytical Method Validation — Guidance for Industry (May 2018). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalytical-method-validation-guidance-industry
³ European Medicines Agency. Guideline on bioanalytical method validation (EMEA/CHMP/EWP/192217/2009 Rev. 1). https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf