CLN readers are invited to test their liquid chromatography-tandem mass spectrometry (LC-MS/MS) troubleshooting skills with this real-world technical challenge. Four possible solutions appear at the end of the challenge. Once you've read the article, learn about the solution to this challenge.

Presenting Symptoms

During an overnight LC-MS/MS production run, several samples had two to five missing peaks (no internal standard peak and no associated analyte peak—Figure 1). The assay was a validated, 11-component, targeted multiple reaction monitoring method on an ultra performance liquid chromatography-tandem­ quadrupole instrument.


The missing peaks were those with expected retention times (RT) in the middle of the LC gradient (peaks four to eight between 2–4 minutes—Figure 3). Peaks one to three with early (<2 minutes) RTs and peaks nine to 11 with late (>4 minutes) RTs in the LC gradient appeared as expected for most of the problem samples. 


Samples one through seven, injected early in the batch, showed all expected peaks. But as the run progressed from samples eight through 40, more samples had asymmetric and/or missing peaks. Examples of normal and abnormal composite total ion chromatogram overlays and LC pressure traces are shown in Figure 1, Figure 2, Figure 3, and Figure 4


What do you think is the most likely cause of the missing peaks? Browse the answers below and read the solution explained online.

Possible Solutions

a.         Post-column leak in the LC system

b.         Mistakes made when adding internal standard during sample preparation

c.         LC pump check valves need cleaning or replacement

d.         Error made when preparing new mobile phase B 

Think you know the answer? Read the solution now.

Judith Stone, PhD, is the senior clinical laboratory scientist specialist at the University of San Diego toxicology laboratory in the Center for Advanced Laboratory Medicine. +Email: [email protected]

CLN's Focus on Mass Spectrometry is supported by Waters Corporation.

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