7 Crucial Criteria Every LC-MS Method Validation Must Follow

LC-MS method is an HPLC assay with MS detector units. With an enhanced option of analyte detection, LC-MS methods are increasingly used in routine analysis of biological samples. T LC-MS method can be readily upgraded to LC-MS/MS analysis. A robust LC-MS method will require adequate method validation, similar to HPLC method validation.

Increasing complexity around drug interactions require sufficient advancements in method development. LC-MS is an inherently complex instrument, and are mainly used with complex samples. 

We highlight the seven crucial FDA criteria every LC-MS method validation must follow.

Reference Standards and Critical Reagents 

Documentation of reference standards and critical reagents are the primary requirements of robust method validation. Pure reference standards can influence the study results. Researchers must use authenticated reference standards for preparing QCs and calibrators. 

The reference standard must be similar to the analyte of interest. In some cases, researchers can substitute it with a chemical form of known purity. Any changes in critical reagents must be duly documented for robust method validation.

Calibration Curve 

The concentration range and calibration standard must reflect the values of that particular study. Ligand binding assays (LBA) need additional anchor points to fit the calibration curve. LC-MS assays require fewer calibration standards compared to LBAs.

A continuous and reproducible calibration curve is required for method validation. Calibration standards must be prepared in the same biological matrix. A study may contain multiple analytes where researchers must generate an individual curve for each sample analyte.

Must Read: Applications of LC-MS Assays in Drug Discovery and Development

Quality Control Samples 

Quality control samples assess the accuracy and precision of an LC-MS assay. QC samples should be prepared for adequate method validation in the same sample matrix. Freshly prepared QC samples are best for accurate and precise results during method development. In method validation, performance QCs evaluate the accuracy of an LC-MS method, while stability QCs assess the stability of analytes under various conditions.

Selectivity And Specificity

It is crucial to identify whether the assessed substance is the intended analyte. Such confirmation prevents any interference. 

Researchers usually evaluate selectivity by testing blank samples in several biological matrices. The interrelation between matrix effects on ion suppression or enhancement must be determined early during method validation.


Sponsors assess assay sensitivity by defining the lower limits of quantification. The method developed should be sensitive enough to meet all the detection criteria. It should demonstrate that it is fit for the intended purpose. Sponsors can assess sensitivity separately or during the assessment of the calibration curve.

Accuracy And Precision 

Assessment of accuracy and precision is crucial to confirm LC-MS assays’ suitability for method validation. Sponsors analyze QCs at a wide assay range for determining assay accuracy and precision. This range involves assessing low, mid, and high QC levels.


Stability validation studies involve the assessment of the sample condition before and after the receipt at the analytical site. The stability of a drug depends on the storage conditions, the container system, and the physicochemical properties of the drug product. The lifespan of a particular drug product depends on its stability. 

olsen anderson

5 years experience in business and financial adviser in USA

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