The Role of Internal Standards In Mass Spectrometry
Why does mass spectrometry analysis require an internal standard?
Mass spectrometry analysis requires an internal standard (IS) to ensure accurate quantitation and to correct for various sources of variability that can affect the results.
In mass spectrometry, the sample preparation process is often more complex than for other analytical techniques. Methods like solid-phase extraction (SPE), liquid-liquid extraction (LLE), or QuEChERS can introduce variability through steps like matrix removal, extraction, or derivatization. Since mass spectrometry is highly sensitive, any fluctuation or error introduced during sample preparation can significantly impact the results. An internal standard helps correct for this variability, providing a more accurate and consistent measurement.
More Variability in Sample Preparation:
The sample preparation process for mass spectrometry (MS) is often more complicated than for other analytical methods. The most commonly used sample preparation techniques for Mass spectrometry include Solid-phase extraction (SPE), liquid-liquid extraction (LLE), or QuEChERS. These techniques often involve steps like matrix removal, cleanup, extraction, or derivatization.
Due to the high sensitivity of MS (especially in SIM Mode), additional dilution or concentration of the sample may sometimes be required. The more complex the sample preparation, the more variability that may be introduced into the result. Internal standards help correct for this variability and ensure consistent results.
Complexity of Mass spectrometry:
Mass spectrometry is also inherently more complex compared to other detectors like an FID (Flame Ionization Detector) MS involves ionization, mass analysis, and detection, each of which can introduce sources of variability. The ionization process can be affected by sample composition, leading to matrix effects (where certain compounds enhance or suppress ionization). By using an internal standard with a similar chemical structure to the analyte, it can undergo similar ionization, helping to correct matrix effects and ensuring more reliable results.
Higher sensitivity:
Due to the high sensitivity of mass spectrometry, even small fluctuations can have a large impact on results. The internal standard provides a stable reference point that compensates for these small errors, which can be magnified in a highly sensitive MS setup. Additionally, mass spectrometers can experience slight instrumental drift over time, with changes in sensitivity. Since the internal standard and analyte experience the same conditions, the IS can help correct for these variations, ensuring more consistent data.
Accounting for Instrumental Drift:
Additionally, mass spectrometers can experience slight instrumental drift over time, with changes in sensitivity. Since the internal standard and analyte experience the same conditions, the IS can help correct for these variations, ensuring more consistent data.
Complexity of Data Processing:
Mass spectrometry generates complex data that includes not only retention time but also the mass-to-charge ratio (m/z) and signal intensity of different ions. The internal standard helps with data processing by providing a stable reference point for interpreting changes in ion fragmentation patterns, thereby enhancing the reliability of the quantitation process.
What Internal standards can be used in MS?
Choosing the right internal standard for mass spectrometry depends on the analyte, matrix, and analysis type. Common types of internal standards used in MS include:
Stable Isotope-Labeled Internal Standards
- Examples: Deuterated (D), ^13C-labeled, or ^15N-labeled versions of the analyte.
- Uses: These are ideal for quantitative MS methods because they are nearly identical to the analyte but differ in m/z. They closely mimic the analyte’s behaviour, including ionization efficiency, and are thus widely used in bioanalysis, environmental studies, and pharmaceuticals.
Structural Analogues
- Examples: Compounds with a similar chemical structure but a different m/z (e.g., using caffeine as an internal standard for theophylline analysis).
- Uses: These can be used when isotope-labeled standards aren’t available. Although they don’t mimic the analyte as closely, structural analogs can compensate for variability in sample preparation and instrument drift.
Surrogate Compounds
- Examples: Compounds that are not structurally related but are added to all samples to monitor extraction or processing efficiency (e.g., triphenylphosphate as an IS in pesticide analysis).
- Uses: These are often used in environmental and food testing, where matrix effects can vary widely. They are especially helpful for correcting errors due to sample handling or instrument performance.
Commercial Internal Standards Mixer
- Examples: Commercially available mixtures containing multiple isotope-labeled standards or another type of Internal Standards Mixer related to the method (e.g., Method 8280 Internal Standard solution for the analysis of dioxins and furans).
- Uses: These are convenient for a wide range of applications, such as PAHs and Pesticide analysis in environmental testing, where multiple analytes need to be quantified.
When selecting an internal standard, it’s crucial to confirm that it does not interfere with the analyte’s ionization and does not introduce background noise at the analyte’s m/z.