Hello Roshanak Khandanlou,

You write that you do not see chromatographic peaks for your impurities.

However, during optimization, these can be seen as signals in the MS spectrum.

I can imagine that you are looking at the “wrong” chromatogram.

The standard chromatogram for LC-MS is the Total Ion Chromatogram (TIC),

alternatively, there is the Base Peak Chromatogram (BPC) when you load your data into the analysis software. I expect the impurities to be in much lower concentrations than the active ingredient. This means that the masses of the impurity do not show up in the BPC because they never become the base peak of the spectrum. In the TIC, the masses “disappear” in the sum of all masses. Have you tried to visualize your target masses as an Extracte Ion Chromatogram (EIC)? Since I don't know what kind of MS you are working with, I can recommend selective measurements as a general strategy, but these can only be performed with triple-quad MS or ion traps.

In our laboratory, we also see our analytes (degradation products of pharmaceuticals) exclusively in EIC, as their concentration is not sufficient for TIC and BPC.

In order to reduce the noise signal in TIC, you can also reduce the mass range, as fewer “interfering masses” are then measured.

Best regards and good luck

Joachim Horst

The absence of impurity peaks in your celecoxib standard dilutions during LC-MS analysis could be due to sensitivity issues, matrix effects, or suboptimal separation conditions. Ensure that the impurity concentrations are within the detectable range, the sample matrix is properly accounted for, and the LC conditions are optimized for better separation and detection of the impurities. Conduct recovery studies and adjust method parameters as needed to enhance the specificity and accuracy of the impurity detection.

1. The analyte may be present in quantities that are below the detection threshold. The sample may not have a enough concentration. 2. Configuring the Instruments: Temperature, mass spectrometer parameters, and flow rate are examples of improperly installed LC-MS settings.

The absence of peaks in LC-MS chromatograms can stem from multiple interrelated factors spanning sample preparation, instrumental parameters, and data processing. Sample-related issues may include insufficient analyte concentration due to improper sample handling, degradation of light-sensitive compounds, or adsorption losses to container surfaces, which can be mitigated by using amber vials, cold storage, and silanized containers. LC method problems often involve suboptimal mobile phase conditions where incorrect pH fails to properly ionize the analyte, requiring adjustment to ±2 units from the compound's pKa, or elution failures where analytes remain trapped in the column, necessitating increased organic solvent percentages or alternative stationary phases like HILIC for polar compounds. MS detection failures commonly occur from incorrect ionization mode selection (ESI+ vs ESI-), requiring testing of both polarities, or from source contamination that demands regular cleaning with 50:50 MeOH:H2O flushes. Instrumental issues range from flow path blockages visible in pressure traces to nebulizer failures observable through absent spray patterns, while data system errors might involve improperly set acquisition channels or processing thresholds that miss legitimate peaks. A systematic troubleshooting approach should begin with injecting system suitability standards to isolate hardware problems, followed by concentrated standards to check sensitivity, while examining raw data for injection volume verification and reviewing total ion chromatograms before extracted ion filtering. Quick corrective measures include increasing injection volumes 5-fold, switching to full scan mode (50-1000 m/z) for broader detection, or performing direct injections to bypass potential column issues. Persistent challenges typically indicate deeper instrumental problems requiring ion source maintenance or method redevelopment with modified chromatographic conditions, always ensuring solvent compatibility with the MS interface to prevent buffer precipitation in the ion source. This comprehensive perspective integrates all critical aspects of LC-MS operation while maintaining technical precision and practical applicability for troubleshooting chromatographic peak absence.