Mass Spectrometry (MS) is among the most powerful tools in analytical chemistry—equally vital for small-molecule identification and the structural study of complex biologics like monoclonal antibodies. In this post, we share highlights from Venture Center’s Only One Analytical Technique series, covering its core principles, applications, and practical insights.
From the "Only One Analytical Technique" talk series by Venture Center. Watch the full video here -
https://www.youtube.com/watch?v=wESefpsHVcI
What is Mass Spectrometry?
Mass Spectrometry measures the mass-to-charge ratio (m/z) of ions to identify and quantify molecules. However, Mass Spectrometry isn’t used in isolation—it is almost always paired with High Performance Liquid Chromatography (HPLC), forming an LC-MS or LC-MS/MS setup. This hybrid enables both separation and detection, making it especially useful in analyzing complex mixtures.
Components of an LC-MS System
- HPLC Module:
- Degasser: Removes dissolved gases to ensure pressure stability
- Pump: Drives mobile phrase through the column
- Auto Sampler: Automates injection of samples
- Column: Seperates components based on size, charge or hydrophobicity
- Coulmn Thermostat: Maintains optimal temperature for consistent seperation
2. Mass Spectrometer:
- Ion Source: Converts analytes into gas-phase ions (e.g., ESI, APCI)
- Mass Analyzer: Seperates ions by m/z (e.g., quadrupole, TOF, Orbitrap)
- Dectector: Captures ion signals and converts them into data
- Software Suite: Controls instrumentation, data acquisition, and reporting
Choosing the Right HPLC Mode
The selection of a chromatography method depends on the physicochemical properties of
the molecule:
- Reverse Phase HPLC (RP-HPLC) : Separates based on hydrophobicity; most common method in LC-MS
- Size Exclusion Chromatography (SEC) : Separates based on molecular size
- Ion Exchange Chromatography (IEX) : Separates based on charge differences
- Hydrophobic Interaction Chromatography (HIC) : Focuses on surface hydrophobicity
Mobile phases should be volatile and MS-compatible (avoid phosphate buffers).
Mass Spectrometry Principles
Ionization is crucial—only charged molecules are detectable. Common ionization techniques include:
- Electrospray Ionization (ESI) : Suitable for proteins and peptides
- Atmospheric Pressure Chemical Ionization (APCI) : Ideal for lipids and small molecules
- MALDI, EI, FAB : Used in specific applications like GC-MS or tissue imaging
Mass Analyzers : How Separation Happens
- Quadrupole : Gold standard for quantitation; stable ions pass through oscillating fields
- Time-of-Flight (TOF) : Separates ions based on flight time; higher m/z ions take longer
- Orbitrap : High resolution and accurate mass; excellent for proteomics
- Triple Quad (QqQ) and Q-TOF : Hybrid analyzers for advanced workflows
Key Metrics in Mass Spectrometry
- PPM Accuracy : Difference between theoretical and observed mass (lower is better)
- Resolution: Ability to distinguish closely spaced peaks
- Charge State (z): MS detects m/z, not mass directly; deconvolution is required for large biomolecules
Protein Analysis Workflows
- Top Down : Inject intact protein; detect charge envelope
- Middle Down : Digest into larger fragments
- Bottom Up : Digest into peptides; analyze and reassemble sequence
Each approach offers different insights; CBA employs all three depending on the analytical
question.
Deconvolution and Fragmentation
- Deconvolution : Converts observed m/z data into neutral mass
- MS/MS or Fragmentation : Confirms identity by breaking ions into characteristic fragments (fingerprinting)
Two acquisition strategies include :
- DDA (Data Dependent Acquisition) : Fragments top intense peaks
- DIA (Data Independent Acquisition) : Fragments entire m/z windows
Real World Applications
- Small Molecule Analysis : Confirm identity and purity
- Protein Characterization : Mass, glycosylation, PTMs
- Peptide Mapping : Sequence confirmation
- Regulatory Submissions : Required for biosimilar comparability
Practical Tips :
- Always degas mobile phase or sonic it before use
- Use slitted caps and avoid over-tightening vials
- Ensure proper cleaning between injections to avoid carryover
- Choose detectors (UV, fluorescence, MS) based on your analytical goal
- Monitor pressure stability and troubleshoot based on deviation patterns (e.g., leaks,
clogs, air bubbles)
CBA at Venture Center : Your Mass Spectrometry Partner in India
The Center for BioPharma Analysis (CBA) offers:
- Access to high-end systems like Orbitrap and Q-TOF
- Skilled analysts with experience in protein and small molecule workflows
- NABL-accredited, GLP-compliant lab infrastructure
- Hands-on training sessions and method development support
Need support for your LC-MS project or biosimilar characterization? Reach out to CBA
at Venture Center to get started.
Email : cba@venturecenter.co.in
Learn More At: - https://bioanalysis.in/
Book our analytical services today