Troubleshooting liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems can be intimidating because the technique is complex, instrument operation and sample processing are still quite manual, and the majority of assays are laboratory-developed tests (LDTs). Vendor service and support has improved, but it is unrealistic to expect that a vendor can have the same degree of understanding for an LDT as for a Food and Drug Administration (FDA)–approved test.

Another challenge is the natural history of LC columns and MS/MS instruments. Chromatography and MS/MS response degrade incrementally with every injection, as residual matrix from extracted sample deposits on the LC column and the MS/MS interface region. Labs replace the LC column when it becomes unusable, and clean the interface region when MS/MS sensitivity falls too low. This pattern recurs regularly; the only variable is the length of time that the MS/MS can operate before cleaning is needed, called the maintenance-free interval. Although most laboratories find LC failures much more often than MS/MS problems, it is often faster to diagnose the root cause and return the LC to normal operations than it is to recover MS/MS sensitivity (Figure 1).

The key to minimizing instrument unavailability and producing reliable patient results is implementing sample preparation and maintenance protocols that deliver predictable maintenance-free intervals. Although needing to clean the MS/MS less often does mean that more samples can be tested per instrument, it is the unexpected instrument failure that compromises productivity more than the regularly scheduled preventative maintenance. Therefore, successful troubleshooting skills are vital to diagnose instrument decline early and intervene before there is a batch failure. Of course, troubleshooting will be of little value unless the laboratory knows its instrument well (Table 1).

I recommend the following steps to enhance your troubleshooting abilities, outlined below: implement a robust production infrastructure, build troubleshooting skills, and divide and conquer problems to reduce complexity.

Set Up and Support a Robust Production Infrastructure

1. Create detailed maintenance charts—with practical language—and insist on complete documentation, including data-derived action limits and corrective actions. Post images to make compliance easier (Table 2).

2. Incorporate a System Suitability Test (SST) in daily maintenance. An SST is the injection of neat standards to check LC and MS/MS reagents, parameters, and status (Table 3). Think of the SST like a patient’s vital signs—pulse, blood pressure, temperature—for evaluating the health of your LC-MS/MS.

3. Derive realistic practices for adding and discarding residual mobile phase, for changing mobile phase and autosampler wash containers and filters, and for cleaning and storing the containers. Avoiding contamination when performing trace MS/MS analysis at pg/mL concentrations requires a different mindset compared to practices appropriate for the mg/dL concentrations tested on automated, routine chemistry analyzers.

4. Have spare, clean, MS/MS interface parts ready to install. When the MS/MS interface needs cleaning, this decreases from hours to minutes the length of time the instrument is vented to atmospheric pressure, and as a result, less time elapses until the MS/MS is pumped down again to operating vacuum pressure (Figure 2).

5. Track column and lot changes for as many chemicals, reagents, and solvents as possible, and avoid using plastic containers and parafilm.

Strap on Your Troubleshooting Tool Belt

1. If signal to noise is low, increased noise as well as decreased signal may be a contributor. Compare the baseline to your archived image: elevated baselines suggest contamination of mobile phases, mobile phase containers, or reagents.

2. If peaks are missing or retention time (Rt) shifts, compare pressure traces to archived images—a rapid way to detect LC leaks, overpressure, and pump problems.

3. For almost any problem, review the SST results. The SST can distinguish between an instrument problem versus a sample preparation failure because the sample preparation phase is bypassed by using neat standards.

4. Compare composite extracted ion chromatogram (XIC) overlays from questionable injections to your archived images. Changes in the pattern of relative peak abundance, Rt, or resolution may be immediately apparent with XIC overlays when review of individual peaks is unrevealing.

5. When you question your MS/MS response, compare peak heights from post-column syringe infusions to your historical values to isolate a loss of sensitivity to the MS/MS rather than LC. However, infusion abundance sometimes appears acceptable even when the MS/MS needs service, so always evaluate infusions in the context of the SST.

6. Peruse the maintenance chart and accept that every human intervention (including your own) is suspect when there is a subsequent change in instrument performance. As long as good records are kept, you can appear to be a genius with the quick fix of an operator mistake or non-compliance with the standard operating procedure.

7. Ask the best vendor service representative to teach you his or her protocol for detecting leaks and finding the source of overpressure.

Divide and Conquer

1. First rule out false alarms from common mistakes. For example, an error yields bad data, such as injecting the wrong vial (Table 4).

2. Any maintenance, component, or reagent replacement is susceptible to human error. Don’t hesitate to check, reconnect, repeat, and replace even though it may feel insulting or redundant.

3. Distinguish between sample preparation, LC, and MS/MS problems using the SST and MS/MS infusion.

4. Sample Prep: a normal SST indicates a sample preparation problem, but casual inspection of the SST can lead to a false conclusion that the instrument is working correctly. Evaluate SST results for trends over time as well as for out-of-range values. Verify that the vial or plate cap was pierced by the autosampler needle and confirm that the liquid contents appear as expected. Re-inject a stable extracted sample from a previous batch to confirm acceptable instrument performance. Also, check for lot changes. Finally, a step-by-step review with the analyst who performed the sample preparation may be the only remaining option before repeating the extractions (See Table 5 for a list of common mistakes categorized by type of sample preparation).

5. MS/MS: If infusion of standards or calibration solutions indicates the MS/MS signal is decreased, first rule out interventions that do not require venting. For example, confirm that the MS/MS detector voltage is appropriate, mass resolution and calibration are correct, and no ion source maintenance (e.g. new capillary electrode) is needed. Consulting the pattern of MS/MS maintenance-free intervals for the instrument may be helpful (SST records), although one bad sample or a change in the assays performed on the instrument can cause a dramatic loss of response. Most clinical laboratories consult vendor technical support and request onsite service at this stage, although competent end users can change interface components followed by mass resolution and calibration.

6. LC: LC-related problems are the most common reason for SST and batch failures. Review of the SST report, XIC overlays, and pressure traces are the best tools to detect and solve an LC problem. If you suspect a leak, look at and touch every tubing connection from the pump to the MS/MS source while the instrument is pumping, and look for buffer deposits below a connection or discoloration of metal fittings (green) as indication of a slow leak. Leaks often occur following over pressure.

7. Selected distortions in peak shape and pressure traces are diagnostic for specific injection matrix, LC plumbing, LC pump, and column problems (Table 6).

Even if you can’t solve the problem, taking the first steps and sharing your troubleshooting data with the vendor service representative can get the instrument back in to service more quickly. Discussions with and learning from the service representative also boosts your troubleshooting skills, although nothing is more rewarding than finding the root cause of an obscure LC problem on your own. Grab your wrenches and ¬≠troubleshoot!

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

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