Calibration of the Mass Axis and System Suitability

The ‘mass-to-charge ratio’ measured by mass spectrometry is essentially derived from the ion’s flight time or deflection path; the conversion relationship depends on the instrument’s condition. Mass calibration involves establishing a ‘measured value–true value’ mapping using a set of standards with known, accurate m/z values (such as mixed peptides, PEG and perfluorotributylamine), so that the m/z reported by the instrument closely approximates the theoretical value. Calibration directly determines mass accuracy, which in turn affects the reliability of molecular formula identification.

The condition of instruments in long-term operation is subject to drift: temperature, vacuum level and high-voltage sources all undergo gradual changes, causing shifts in the mass axis, a decline in resolution and fluctuations in sensitivity. System suitability refers to the process of using standard samples before each day’s or batch’s experiments to check key parameters—whether mass accuracy remains within tolerance, whether resolution meets requirements, and whether the response of target compounds is stable—to confirm that ‘the instrument is fit for purpose today’ before sample analysis begins. It is akin to a daily health check for the instrument and serves as a safeguard to ensure data comparability.

Calibration and suitability checks are mutually reinforcing: calibration provides an accurate mass axis, whilst suitability checks verify the instrument’s overall condition following calibration. For the new Ceyuan ProC series of online time-of-flight mass spectrometers, basic calibration is completed at the factory; however, regular recalibration using standard samples is still required during operation, particularly following significant temperature fluctuations or prolonged continuous monitoring (such as catalytic reactions or flue gas analysis). When performing MSI imaging quantification, a stable mass axis is also essential to ensure consistent molecular identification across different pixels and batches.

In practice, a combination of ‘internal standards and external standards’ is commonly used: external standards are used for initial mass axis calibration, whilst internal standards monitor drift in real time within each batch of samples. If suitability parameters exceed tolerance limits, the instrument should first be recalibrated or the vacuum and temperature checked, rather than relying directly on the data from that batch.

To summarise in a single sentence: Without a reliable mass axis, even the most beautiful spectral peaks are merely numbers; system suitability is the process of turning ‘reliability’ into a verifiable daily routine.

Calibration also has ‘degrees of importance’: following new instrument installation, relocation or prolonged downtime, a complete two-point or multi-point calibration is required to reconstruct the entire mass axis; for day-to-day operations, an internal standard can be used to continuously monitor drift, with local corrections applied upon detection of deviations. For projects requiring the merging of data across instruments and batches (such as multi-centre imaging studies), standardised calibration criteria and calibration standards are particularly important; otherwise, the m/z values reported for the same molecule by different instruments will exhibit systematic deviations, affecting both clustering and statistical analysis. The New Ceyuan ProC and MSI instruments are factory-calibrated upon delivery; consistent daily suitability checks by the user are key to transforming ‘factory accuracy’ into ‘long-term accuracy’.