The Guiding Principles Of LC-MS/MS Analysis

Today in the 21st century, LC-MS bioanalysis has reached laboratories all around the world. Moreover, they have started penetrating large and medium-sized laboratories and regional hospitals. Their applications now range from drug toxicology studies and newborn screening analysis to more specific in vivo bioequivalence studies. The LC component separates the mixture, while the MS unit detects individual elements based on their mass-to-charge ratios.

Moreover, advanced hyphenated techniques such as LC-MS/MS have also seen tremendous growth in the past two decades. This technique offers superior specificity compared to conventional HPLC systems and immunoassays for low molecular weight analytes and higher throughput than GC-MS analysis. Though, it needs robust development and validation similar to LC-MS validation. The current article provides the principles that guide LC-MS/MS analysis. It also highlights the working of LC-MS/MS assays.

The guiding principle of LC-MS/MS analysis

An LC-MS/MS instrument consists of six critical components. Each of these components is vital for a robust LC-MS/MS analysis.

1. An ion source such as atmospheric pressure ionization source and atmospheric pressure chemical ionization source
2. Ion-inlet component providing transition of ions 
3. The first mass-filtering unit
4. Collision chamber for collision-induced dissociation
5. The second mass filtering unit
6. Ion impact detector

A combination of this instrumentation offers a wide range of experiments. The five main types of experimentation include full scan, product ion scan, precursor ion scan, neutral loss scan, and selective reaction monitoring. 

• Full scan assesses the entire mass range of both Q1 and Q3 filters allowing the identification of all ions in the sample.
• In the product ion scan, one m/Q is selected in Q1, while Q2 is filled with collision gas, and the entire Q3 is scanned. This experiment identifies all fragments and product ions.
• The precursor ion scan assesses the entire range of Q1. Q2 is filled with the collision gas to fragment the ions, and one specific m/Q is selected in Q3. This approach helps researchers identify the m/Q precursor ion that has generated the selected product ion.
• A neutral loss scan assesses the entire Q1 range while the Q2 is filled with the collision gas. Then the Q3 is scanned to assess the loss of one specific mass occurring in the precursor scan range. This setup identifies all lost precursors with a common chemical group.
• Finally, selective reaction monitoring is a highly specific detection tool. In this experiment, one specific m/Q is selected in Q1, and Q2 is filled with collision gas while selecting one specific m/Q in Q3. This setup identifies the analyte that has specifically fragmented into the product ion. The ion-pair detection sensitivity is also immensely increased as the detector only processes a single analyte-specific ion-pair.

The first three are used widely during LC-MS/MS method development. These experiments can determine the m/Qs, identify the precursor m/Q and confirm that the analyte of interest produces the product ion. Generally, the final method is SRM. Besides, a neutral loss scan can also be an ideal option for common analytes or related compounds.

Must Read: 5 Steps For Selecting The Right LC-MS/MS Assay When Conducting Drug Research