Large molecule quantification requires a lot of sample extraction typically involving a wide range of immunocapture approaches. Such quantification methods can be target specific (involving the use of receptor-specific antibodies for the complementarity of the drug’s molecule); drug-target specific (involving the use of developed antibodies for specific pharmaceutical drug of interest); or semi-targeted capture with the antibodies designed specifically for the fragment crystallizable region of the molecule of interest.
Sampling technique for LC-MS/MS analysis requires extensive workflows and are not always intended for the MS-MS analysis. Thus, analyte quantification should be scheduled for just a small protein fraction with a signature peptide possessing unique m/z ratio thereby providing a representative concentration for the entire protein. The targeted drug therefore should undergo both proteolytic digestion as well as peptide quantification.
Furthermore, targeted protein digestion results in variable peptide release thereby generating a detrimental impact on the accuracy of the peptide quantification data. Such sample preparation steps therefore are required to be highly optimised in accordance with a robust and thoroughly validated protocol. This aids in achieving reproducible data with lower quantitation limits and involves both enrichment and semi-purification of the analytes involved.
LC-MS/MS dependent biological analysis requires excellence in the lc ms sensitivity and selectivity for quantifying both the peptides and proteins accurately below the lowest ng/ml range. Several LC/MS/MS technologies advances involves enhancement of the ionization efficiency as well as the ion transmission with improved sensitivity thereby making it possible to detect biologics at the lowest levels.
The coupled bioanalytical tool, LC-MS/MS proteomic approaches can routinely be employed in the detection of around thousands of proteins and PTMs for uncovering all the complex proteomes. This further helps in drug discovery possible candidate biomarkers.
Selection of the overall approach of LC-MS/MS analysis involves a wide range of factors including matrix type and its selection, choice as well the reagent availability. Since 1980s, this technique has been widely employed in the bioanalysis of the small molecules for its increased sensitivity, high throughput, selectivity and accuracy. It further offers similar advantages to biologics as well as it is not a subject to any antibody cross-reactivity as this technique takes a direct dig into the chemical properties of the analyte under evaluation. LC-MS/MS analysis offers similar advantages for the biologics as well as it is not a subject to any sort of antibody cross-reactivity. Since it easily distinguishes and quantifies highly homogenous forms isoforms at their lowest levels, LC-MS/MS analysis offers excellent selectivity, accuracy as well as precision over a wide range.
Validation of the LC-MS/MS method –
Validation is a critical step to be performed for demonstrating the reliability of the bioanalytical method. This is a pre-requisite of all the analytical or the bioanalytical methods. It follows a general or ground rule principle that the more the complex and sophisticated a method is, the more stringent is its validation. In case of the LC-MS/MS analysis, both the instrument as well as the drugs to be tested are complex in nature and thus, validation becomes an utmost care to be employed in the entire process.