[Reader Insights] Setting Key Parameters in Mass Spectrometry Analysis

This article is written by an expert chromatographer under the pen name of Chromatography Mound. Welch Materials, Inc. is authorized to translate this article to English and publish it on behalf of the author.

During the process of developing mass spectrometry (MS) methods, numerous parameters of the mass spectrometer need to be configured, such as the ion source temperature, nebulizer gas pressure, curtain gas pressure, declustering voltage, and collision energy.

When building an MS library, we often find that many of these parameters are set to default values or are carried over from a previous method; in fact, the only parameters we need to adjust are typically the precursor ion m/z (mass-to-charge ratio), product ion m/z, declustering voltage, and collision energy.

But are these the optimal values for our analysis? In this article, we will share insights on how to obtain and optimize these key parameters.

First, it's important to understand what may be overlooked when building an MS library.

During the optimization of declustering voltage and collision energy, the voltage and collision energy curves obtained in Multiple Reaction Monitoring (MRM) mode are continuous. Therefore, even if there is some deviation from the actual optimal values, we are often inclined to assume that the parameters we selected are optimal. After all, this result was obtained using MRM mode, which seems logical and well-supported, and no significant issues arise during the actual analysis.

However, this approach often neglects factors that contribute to discrepancies between the measured results and the optimization process. For example, factors like the composition and flow rate of the mobile phase, other ions present in the system, and matrix effects can all influence the outcome.

To build an MS library, we can prepare a 1 mg/L standard solution using a stock solution and directly inject it into the ion source via flow injection. Then, identify the precursor ion m/z, followed by deriving the product ions from the precursor ion. Finally, using the MRM mode, we optimize the declustering voltage for the precursor ion and the collision energy for the product ions.

However, this approach may not be representative of the actual analysis conditions. Firstly, the ion source temperature can be set to a low value, and even room temperature, because the injection flow rate is much lower than in an actual analysis (even when the ion source temperature is low, organic solvents can still be efficiently nebulized). Additionally, if a pure solvent is used as the carrier solution, the ionization process might lack suitable ions, which could prevent us from selecting the highest-responding precursor ion. This makes the subsequent optimization futile.

To achieve the best parameters, we must be meticulous and follow a step-by-step approach. Here are the author's suggestions:

This is particularly important for analytes containing nucleophilic groups. Such compounds can typically form precursor ions by associating with H+, NH₄+, Na+, K+, and other ions.

To optimize ionization, prepare an ion solution of approximately 2 mmol/L and use it to prepare a 1 mg/L standard solution. This ensures a stable ion source during optimization, allowing us to assess the response intensity of different ion forms of the target analyte, rather than relying on residual ions from the system (which can introduce significant errors, potentially resulting in missing the optimal precursor ion).

After roughly determining parameters such as m/z, declustering voltage, and collision energy, and finalizing the mobile phase, optimize the gradient elution program to obtain the ion chromatogram. Then, calculate the mobile-phase composition at the peak of the target analyte. Using this ratio, perform isocratic elution and optimize the declustering voltage and collision energy through flow injection with the mixed mobile phase.

This step provides more reliable MRM results since the ionization environment now more closely matches that of the actual analysis.

With the introduction of the mobile phase, the nebulization temperature needs to be increased to ensure complete droplet nebulization. Otherwise, incomplete nebulization can prevent the target analyte from being fully released into the environment. Typically, higher flow rates or higher aqueous phase content require higher nebulization temperatures.

In multi-component analysis, if target analytes reach the ion source at different times and with varying mobile-phase compositions, set the nebulization temperature higher for mobile phases with more aqueous content. However, other compounds' tolerance to high temperatures must also be considered to avoid in-source fragmentation.

Generally, it is acceptable to optimize declustering voltage using either the precursor or product ions, and the results often converge. However, for certain compounds with multiple ionization sites, different fragments may correspond to different precursor ions. In such cases, the optimal declustering voltage for different product ions may differ.

For example, in the case of a compound (see Figure below) that can lose hydrogen from either the carboxyl group or the phenolic hydroxyl group, generating different precursor ions, the response differences in the fragments can be substantial. If we wish to optimize the declustering voltage for the fragment at m/z 152, we should optimize it for the precursor ion corresponding to this fragment rather than the optimal voltage for both precursor ions. Therefore, the optimization should focus on the 152 fragment, not the 167 precursor ion.

In our pursuit of efficiency in MS analysis, we often overlook many details, especially when the results appear "problem-free". This tendency can lead to difficulties in identifying issues later on, resulting in many "unexplained phenomena".

Therefore, it is essential to understand the logic behind the experiments and develop good laboratory practices. This approach ensures that when problems arise, we will be equipped with strategies to resolve them.