LC MS Method Development And GLP Validation For Human Plasma PK Sample Analysis

Background

LC MS method validation is crucial for ensuring the reliability and accuracy of analytical results in pharmacokinetic and bioanalytical studies. LC MS Sample preparation and liquid chromatography are sometimes ignored during LC MS method development and validation, given the highly selective Multiple Reaction Monitoring (MRM) in LC-MS/MS assay. For a rugged method, however, we must pay attention to all sample cleanup and chromatography steps. Additionally, this approach safeguards reliability and minimizes tweaking while transitioning from study-to-study all the way up to clinical development.

In LC MS method development and validation, limited information on drug metabolism is typically available at the early stages of drug development. The drug and its metabolite can transition similarly in the Mass Spectrometer if highly metabolized. This introduces complexity as the Mass Spectrometer can’t distinguish between two entities with the same mass and daughter ion. Therefore, separating these analytes by LC-MS/MS assay becomes essential to avoiding metabolite interference. Using the wide range of LC MS method validation protocols confers the method’s durability, protecting its credibility and, in turn, reducing the need to compromise with the technique when one transition step from a clinical trial to another, especially at the clinical development stages.

In contrast, while valuable in certain contexts, ELISA assay methods or immunoassay services may not offer the same level of specificity and selectivity as LC-MS/MS assays. Immunoassays are highly sensitive to specific antigens or antibodies and are beneficial for quantifying large molecules like proteins or peptides. However, immunoassay services might not be suitable for quantifying small molecules such as drugs and their metabolites, particularly in complex biological matrices where cross-reactivity and interference pose challenges. Thus, LC-MS/MS assays are mainly applied to their capacity to deliver accurate and reliable quantification data, which is especially in demand for early drug development processes when little-known information about drug metabolism is available.

Study

In this case, one of our clients sought help with LC MS method development, optimization, and validation. During sample analysis, we noticed a small peak appearing as a shoulder to the analyte peak. The analyte at hand had a retention time of 1.5 minutes. Our experimental approach increased that and separated the analyte peak from endogenous material eluting earlier, as LC MS sample preparation was based on protein precipitation. We aimed to achieve this by changing chromatographic conditions, including change of mobile phase and columns. Our scientists confirmed this was not a split peak due to improper chromatography or interference in control plasma but rather due to another compound with the same MS transition. Thus, we hypothesized and started investigating whether this issue was due to a metabolite with a similar transition.

During this investigation, we also explored the possibility of utilizing an ELISA assay method or immunoassay services to complement the LC-MS/MS assay. These methods could provide additional specificity and sensitivity, particularly in detecting and quantifying the target analyte and potential interfering compounds. However, we initially prioritized optimizing the LC MS method development to address the observed peak interference, as it offered direct quantification and identification of the analyte and potential metabolites. Nonetheless, incorporating complementary techniques like the ELISA assay method or immunoassay services could be considered in future studies to enhance the analytical robustness and accuracy of the analysis.

LC MS method validation and LC MS sample preparation are essential components of our approach to ensure the reliability and accuracy of our analytical methods.

In our process of separating the two peaks through LC MS method validation, we found that the intensity of the interfering peak was changing from sample to sample. This established that the noise was emanating from a metabolite. We further estimated this metabolite to be a glucuronide conjugate, given the peak under consideration was eluting earlier than the analytes. However, we had no available reference standard for glucuronide then. Finally, we confirmed that this peak was a glucuronide conjugate after examining the plasma samples using enzymatic hydrolysis. Consequentially, we successfully validated and applied the method to analyze plasma samples.

Although we focused on LC MS method development, optimization, and validation, we also provide ELISA assay method development and immunoassay services to cater to diverse client needs in bioanalytical research.