Development and Validation of an LC/MS-MS method for Drug and Biomarkers in Clinical Samples

In recent times, Liquid Chromatography tandem mass spectrometry (LC-MS/MS), especially triple-quadrupole mass spectrometry, has been the latest alternative technique for ligand binding assays for therapeutic monoclonal antibody quantification in biological samples. An improvised LC-MS/MS method for quantifying infliximab from human serum was both developed and validated in this current study. An alternate peptide as a representative was used and was cleaved for infliximab quantification on LC-MS/MS assay.

Liquid Chromatography-mass spectrometry (LC-MS) analysis

It is the most widely used bioanalytical tool in the industry. LC is a highly preferred technique due to much-needed features such as rapidity, sensitivity, and isotopic-specificity of MS analytes. Its other aspects, such as stability and tandem mass, yield sensitive and accurate sample analyses. The LC MS technique can be divided into two parts, such as sample extraction method development and LC-MS/MS instrument method development.

The sample extraction

This process is conducted employing protein precipitation (PPT), liquid-liquid extraction (LLE), or solid-phase extraction (SPE) methods. PPT is a sample extraction method during the drug discovery stage. This technique is quick, easier for small molecule analysis. The disadvantage of PPT is unlike LLE or SPE; the matrix effect or ion interference might be noticed in chromatograms. LLE is another majorly used sample extraction technique. This technique can produce pure sample extract solutions. All three different sample extraction techniques can be moved to robotic systems to enhance the sample preparation efficiency.

LC-MS/MS instrument development method

It needs suitable settings of the LC system and MS system. The crucial factors for LC system setup are mobile phase, LC column, gradient, flow rate, column temperature dependent on method development time, analysis time, and a considerable number of samples per day.

HPLC-MS/MS analysis technique

It is also known as HPLC testing, is developed to complete amino/phenol metabolites. It is a technique here differential isotope labeling with dansyl chloride is applied. The method was reliably validated as per the guidelines of the FDA and EMA. Novel development and validation methods are opted to determine this reproducible method.

The Characteristics required in LC-MS/MS method development and validation

This Include accuracy, precision specificity, quantification limit, linearity, recovery, matrix effect, and stability. All LC-MS/MS techniques are developed and validated according to the protocols or according to the Standard Operation Procedures (SOPs) to abide by the regulatory guidelines. These techniques are reliable for high-grade screening analysis, pharmacokinetic study sample analysis.

The objective of method development

The objective is to provide a quick, reliable method through which an analyte’s exact resolution can be obtained. In the MS system setup, ion source parameters, mass transitions supervised in multiple reactions monitoring (MRM), and collision cell parameters are significant factors that are dependent on analyte concentration and response. The goal of this technique is to optimize MRM response with free interference.

The Bioanalytical services

These services include method development and validation (LC-MS/MS analysis and HPLC assay), method transfer and optimization (LC-MS/MS method and HPLC analysis), pre-clinical TK/PK studies, high throughput analysis for discovery studies (TK/PK/PD), analytical methods for clinical trials, pharmacokinetic studies (PK) studies, bioequivalence studies, and metabolite identification, etc. However, the sample analysis will be performed to determine whether the results from the samples are accurate and reliable. 

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