LC-MS · Calibration procedure
Mass accuracy calibration
Calibrates the m/z scale of the mass spectrometer with a reference solution, then verifies accuracy on known compounds.
PURPOSE
Why this is checked
Identification by MS depends on measuring the right mass. A drifting mass scale can mis-assign ions, lower sensitivity in MRM methods or give a wrong elemental formula on high-resolution instruments.
Frequency
High-resolution instruments are typically calibrated or checked daily or before each batch; quadrupole instruments typically every few months and after maintenance or venting, as defined in your SOP and the manufacturer's guide.
Learn how the LC-MS worksYOU WILL NEED
Standards & tools
- Manufacturer's calibration solution covering the working mass range (e.g. sodium formate or caesium iodide clusters, or a proprietary tuning mix)
- Check compounds with known exact masses, e.g. caffeine, leucine enkephalin, reserpine
- LC-MS grade solvents
BEFORE YOU START
Prerequisites
- Vacuum stable and the instrument at operating temperature for the time the manufacturer specifies
- Ion source clean; spray stable when infusing calibrant
- Room temperature within the instrument's specified range
PROCEDURE
Step by step
- 1Infuse the calibration solution in the polarity (positive and/or negative) to be calibrated.
- 2Run the manufacturer's calibration routine; it fits the measured peaks to their known m/z values.
- 3Review the calibration report: number of ions used, residual error of each and overall fit.
- 4Inject or infuse the check compounds and record their measured m/z.
- 5Calculate mass error for each check ion and compare with the limit.
RECORD
Example observation table
| Check ion | Theoretical m/z | Measured m/z | Error (ppm) |
|---|---|---|---|
| Caffeine [M+H]⁺ | 195.0877 | 195.0874 | −1.5 |
| Leucine enkephalin [M+H]⁺ | 556.2766 | 556.2771 | +0.9 |
| Reserpine [M+H]⁺ | 609.2807 | 609.2815 | +1.3 |
CALCULATION
How to calculate the result
Mass error (ppm) = (Measured m/z − Theoretical m/z) ÷ Theoretical m/z × 10⁶; for unit-resolution instruments, error (Da) = Measured − Theoretical
- Reserpine [M+H]⁺: theoretical 609.2807, measured 609.2815
- Difference = 609.2815 − 609.2807 = 0.0008
- Error = 0.0008 ÷ 609.2807 × 10⁶ = +1.3 ppm
ACCEPTANCE CRITERIA
Pass if
- High-resolution MS (Q-TOF, Orbitrap): typically within ±5 ppm with external calibration (often ±2–3 ppm, or better with a lock mass), as per manufacturer and SOP.
- Unit-resolution quadrupole MS: typically within ±0.1–0.2 Da of the theoretical value, as per manufacturer and SOP.
IF IT FAILS
What to do
- Clean the ion source and check spray stability, then recalibrate.
- Check room temperature and allow the analyser to stabilise.
- Do not use the instrument for identification or quantitation until it passes.
- Contact service if the calibration repeatedly fails.
In LimsPulse · Calibration Management
LimsPulse schedules this calibration, records the result against limits, and blocks the instrument everywhere if it fails or goes overdue.