Method validation is a process to demonstrate the reliability of a bioanalytical method. Method validation is necessary for all bioanalytical methods. However, the extent of method validation differs between techniques. A sophisticated technique will require a higher amount of validation and vice versa.
LC-MS is a powerful and sophisticated bioanalytical technique. Over time, it has become the backbone of the drug development life cycle. LC-MS systems have several operation modes, parameters, and numerous ionization possibilities. These features make LC-MS systems a crucial piece used at every phase of drug development. However, these characteristics make LC-MS a complex technique. Therefore, sponsors must thoroughly conduct LC-MS method development and validation.
Recent technological advancement has continuously upgraded LC-MS testing, and these innovations have positively influenced LC-MS method validation. The current article will highlight the impact of technology on lc ms ms analysis. Moreover, each validation parameter is discussed individually for better clarity.
Technology and LC-MS method validation
Selectivity and specificity of LC-MS systems aim at streamlining the method to differentiate and measure analytes present in study samples. Method development and validation studies have helped researchers understand that focusing on LC-MS specificity and selectivity during method development help identify limitations early during analysis and confirm them later during method validation.
Matrix effect is an alteration in assay response due to interfering substances present in the study matrix. Advancements in synthetic peptides helped elevate most issues of matrix effects. Researchers can easily evaluate matrix effects by spiking regent and matrix blank samples with SIL-peptide internal standards and synthetic surrogate peptides. However, these spiking materials must be rigorously processed according to protocols specified for sample preparation. These protocols include reduction, enrichment, digestion, and alkylation.
Therapeutic proteins can be affected due to several factors, such as chemical or environmental stress. As most LC-MS analyses quantify selected peptide surrogates, modifications in the therapeutic proteins may go undetected if they do not affect the monitoring of surrogate peptides. Hence, stability studies are crucial in drug development. A protein analyte is considered stable under the experimental condition until the responses of surrogate peptides are under acceptable criteria. Stability studies are required under certain circumstances, which may include:
- Stock solutions prepared in different solvents or from a powder
- Change in storage conditions
- Extension of storage duration beyond the expiry date.
Emerging solutions can help demonstrate stability in these circumstances. Researchers can compare diluted working solution or stored stock solution with a freshly prepared solution for stability studies. However, this solution must be derived from an unopened vial of material or freshly weighed. Moreover, researchers can find another efficient alternative, such as the HPLC-UV system, to estimate the relative content of peptide solutions and non-matrix proteins.
Today LC-MS has become an integral component of the drug development process for quantifying analytes in complex biological samples. Besides, enhanced hyphenated techniques such as LC-MS/MS systems are helping researchers quantify analytes in samples that were not possible before. Moreover, specific modifications in LC-MS settings can quantify both large and small molecule drug products. Thus, technological advancements have profoundly impact on LC-MS method development and validation.
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